Fluorescence quenching immunoassay

Abstract

The present invention relates to methods and reagents for determining the presence or amount of an analyte in a sample.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits as of the filing dates of U.S. Provisional Patent Application No. 63 / 011403, filed on 17 April 2020, and U.S. Provisional Patent Application No. 63 / 152365, filed on 23 February 2021, the entirety of which is incorporated herein by reference.

[0002] This invention relates to a fluorescence quenching immunoassay for determining the concentration of an analyte in a sample. The invention also relates to a novel class of fluorescein derivatives that can be used as tracers in immunoassays. [Background technology]

[0003] Immunoassays are techniques for measuring the presence or concentration of substances in test samples, typically solutions, which often contain complex mixtures of substances. Typically, test samples are biological fluids such as serum or urine. Immunoassays are based on the unique ability of antibodies or other proteins to bind with high specificity to one or a very limited group of molecules. The molecules that bind to antibodies are called antigens. Immunoassays can be performed to measure the presence or concentration of either an antigen or an antibody (i.e., either the antigen or the antibody can be the analyte). In either case, the specificity of the assay depends on the extent to which the analyte can bind to its specific binding partner, excluding other substances that may be present in the sample being analyzed. In addition to the need for specificity, the binding partner must be selected that has a sufficiently high affinity for the analyte to enable accurate measurement.

[0004] The requirement for an immunoassay is a means of generating a measurable signal in response to a specific binding event. This can be achieved by measuring changes in certain physical properties, such as light scattering or refractive index, that occur when an analyte binds to its binding partner. Most immunoassays rely on the use of a binding partner associated with a detectable label. A binding partner associated with a detectable label is often called a tracer. A wide variety of detectable labels are used, including radioactive elements (used in radioimmunoassays); enzymes; fluorescent, phosphorescent, and chemiluminescent dyes; latex and magnetic particles; dye crystallites; gold, silver, and selenium colloid particles; metal chelates; coenzymes; electroactive groups; oligonucleotides, stable radicals, and more. Such detectable labels enable the detection and quantification of binding events after separation of the free tracer from the bound tracer, or by designing the system so that the binding event results in a change in the signal generated by the tracer.

[0005] Immunoassays requiring a separation step, often called separation immunoassays or heterogeneous immunoassays, frequently involve multiple steps, such as careful surface washing to separate unbound tracers from tracers bound to their binding partners. Immunoassays in which the signal is affected by binding can often be performed without a separation step. Such assays can often be performed simply by mixing reagents and samples and performing physical measurements. Such assays are called homogeneous immunoassays and are easier to perform than heterogeneous immunoassays.

[0006] Regardless of the method used, interpretation of the signal generated in an immunoassay requires reference to standards that mimic the properties of the sample medium. In the case of qualitative assays, standards may consist of a reference sample containing no analyte and a positive sample having the lowest concentration of analyte considered detectable. Quantitative assays require additional standards with known analyte concentrations. Comparison of the assay reaction of a test sample with the assay reaction generated by a standard allows interpretation of signal intensity with respect to the presence or concentration of the analyte in the sample.

[0007] Immunoassays may be either competitive or non-competitive. In a competitive immunoassay, an antibody in a sample competes with a tracer (i.e., an antibody linked to a detectable label) for binding to an antigen. Therefore, the amount of tracer bound to the antibody is measured. In a competitive immunoassay, the amount of tracer bound to the antibody is inversely proportional to the concentration of antibody in the sample. This is because when a large amount of antibody is present in the sample, more antigen binds to the antibody in the sample, leaving less antigen available to bind to the tracer.

[0008] In a non-competitive immunoassay, an antigen in a sample specifically binds to an antibody, or an antibody in a sample specifically binds to an antigen. Therefore, the amount of antigen or antibody is measured. Unlike competitive immunoassays, in non-competitive immunoassays the reaction is directly proportional to the concentration of antigen in the sample. This is because if no antigen is present in the sample, no detectable label on the secondary antibody will bind.

[0009] Fluorescence polarization is used, for example, as a detection technique to determine the concentration of antibodies or antigens in a sample. The fluorescence polarization technique is based on the principle that compounds containing a group that fluoresces when excited by linearly polarized light (i.e., fluorescent tracers) emit fluorescence with a degree of polarization inversely proportional to their rotation speed. Therefore, because the size of a fluorescent antigen-antibody complex limits its rotation between the time the light is absorbed and the time it is emitted, when a fluorescent antigen bound to an antibody is excited by linearly polarized light, the emitted light remains highly polarized. When an unbound fluorescent antigen (i.e., not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than that of the corresponding fluorescent antigen-antibody complex, the unbound fluorescent antigen molecule is more randomly oriented, and therefore the emitted light is depolarized. Fluorescence polarization provides a quantitative means for measuring the amount of fluorescent antigen-antibody complex produced in an immunoassay.

[0010] Fluorescence quenching technology also involves the use of a fluorescent tracer, but it is based on the principle that when a fluorescent antigen is bound to an antibody, the fluorescence of the fluorescent antigen is quenched. Therefore, when a fluorescent antigen bound to an antibody is excited by light, the intensity of the emitted light decreases compared to the intensity of light emitted from an unbound fluorescent antigen. Thus, fluorescence quenching also provides a quantitative means of measuring the amount of fluorescent antigen-antibody complex produced in an immunoassay. In a typical assay, a test sample containing an antibody whose concentration is to be determined is mixed with a fluorescent antigen, the resulting mixture is excited by light, and the intensity of the emitted light is measured. The antibody present in the sample binds to the fluorescent antigen. The decrease in the intensity of light emitted from the mixture compared to a control (without antibody) is inversely proportional to the concentration of the antigen in the sample.

[0011] In another fluorescence quenching assay, a test sample containing an antigen whose concentration is to be determined is combined with a mixture of a fluorescent antigen and an antibody specific for the antigen portion of the fluorescent antigen. The antigen present in the test sample and the fluorescent antigen compete for a limited number of antibodies. A higher concentration of antigen in the test sample results in more antibodies binding to the antigen from the test sample and fewer antibodies binding to the fluorescent antigen (i.e., results in more unbound fluorescent antigen). By maintaining constant concentrations of the fluorescent antigen and the antibody, the ratio of sample antigen-antibody complex to fluorescent antigen-antibody complex is directly proportional to the amount of antigen present in the sample. Similarly, the amount of unbound fluorescent antigen is directly proportional to the amount of antigen present in the sample. Therefore, the amount of antigen in the sample can be quantitatively determined by exciting the mixture with light and measuring the intensity of emitted light. The concentration of antigen in the test sample is directly proportional to the intensity of the emitted light.

[0012] These techniques are not limited to antibody-antigen binding partners. A similar assay can be performed using a protein (not an antibody) to determine the presence or concentration of a substrate that specifically binds to the protein. The tracer in these assays can be, for example, a detectable label linked to the protein or a detectable label linked to a molecule that binds to the protein.

[0013] Immunoassays are advantageous over other analytical methods for measuring the presence or concentration of a substance in a test sample, such as gas chromatography (GC) and high performance liquid chromatography (HPLC), because they often avoid extraction and other complex post-sample processing procedures and long assay times associated with these other methods.

[0014] However, there remains a need in the art for immunoassays that exhibit higher sensitivity, are easier to perform, and are less expensive to perform.

[0015] Citation of any reference in the present application shall not be construed as an admission that such reference is prior art to the present application.

[0016] These and other features and advantages of the present invention will become apparent from the remainder of this disclosure, in particular from the following detailed description of preferred embodiments, all of which illustrate the principles of the present invention as examples. [Overview of the Initiative] [Means for solving the problem]

[0017] This invention relates to a method for determining the presence or amount of an analyte in a sample. (i) The step of preparing a sample suspected to contain the analyte; (ii) A step of contacting a sample with a fluorescent tracer and a binding partner to obtain an assay composition, wherein the binding partner is specific to the analyte and the fluorescent tracer; (iii) Irradiating the assay composition with light of a first wavelength that is not linearly polarized; (iv) The step of measuring the intensity of light emitted at a second wavelength accompanied by, The intensity of the light emitted at the second wavelength is proportional to the concentration of the analyte in the sample.

[0018] This method can be carried out in solution or as a dry slide assay. In one embodiment, the analyte is SDMA. In one embodiment, this method does not involve a separation step.

[0019] The present invention also includes a fluorescent tracer that can be used in the method of the present invention. In one embodiment, the fluorescent tracer is

[0020] [ka] (In the formula, X = -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, or -SO3H) It is selected from the group consisting of the following.

[0021] The present invention also includes slides that can be used in this method.

[0022] The present invention also includes derivatized fluorescein molecules that are useful as intermediates for synthesizing substituted fluorescent tracers that can be used in the methods of the present invention. [Brief explanation of the drawing]

[0023] [Figure 1] This is a schematic diagram illustrating the general principle of this method. [Figure 2] This is a schematic diagram showing a synthetic scheme for functionalizing the 4' position of the fluorescein molecule. [Figure 3] This is a plot of the fluorescence change percentage as a function of the [anti-Mel-Ab] / [Mel-tracer] ratio for various Mel-tracers, as described in Example 15. [Figure 4] This is a plot of fluorescence intensity versus melamine concentration when melamine is added to the solutions of Mel-F and anti-Mel-Ab, as described in Example 16. [Figure 5] This is a plot of fluorescence intensity for Mel-F, Mel-F + anti-Mel-AB, and Mel-F + anti-Mel-AB + Mel solutions in various liquids, as described in Example 17. [Figure 6] This is a fluorescence plot of a mixture of Mel and anti-Mel-Ab with added milk containing melamine, as a function of melamine concentration, as described in Example 18. [Figure 7] This is a plot of the percentage of fluorescence change versus the molar ratio of streptavidin to tracer when streptavidin is added to solutions of biotin-F and biotin-ED, as described in Example 19. [Figure 8] This is a plot of relative fluorescence for solutions of T3-F (conjugate only); T3-F and anti-T4-Mab (conjugate and monoclonal antibody); and T3-F, anti-T4-Mab (conjugate and monoclonal antibody), and T4, as described in Example 20. [Figure 9]The graphs in Example 20 show the percentage change in fluorescence as a function of time when T3-F is added to PBS and PBS + anti-T4-Mab (i.e., AB), as well as the quenching recovery after T4 addition (PBS + Ab + T4). [Figure 10] This is a plot of fluorescence intensity versus T4 concentration when various concentrations of T4 are added to T3-F and anti-T4-Mab solutions, as described in Example 21. [Figure 11] This is a plot of fluorescence intensity for various solutions of T3-F or T3-F with anti-T4-Mab before and after freeze-drying, as described in Example 22. [Figure 12A] This is a plot of fluorescence intensity in a solution containing T4-5 and Ab-Cy5 as a function of the ratio of T4-5 to Ab-Cy5, as described in Example 22. [Figure 12B] This is a plot of the percentage change in fluorescence of solutions containing T4-5 and Ab-Cy5 as a function of the ratio of T4-5 to Ab-Cy5, as described in Example 23. [Figure 13A] This is a plot of fluorescence intensity versus T4 concentration for T4-F and Ab-Cy5 solutions with added T4, as described in Example 23. [Figure 13B] This is a plot of fluorescence recovery percentage versus T4 concentration for T4-F and Ab-Cy5 with added T4, as described in Example 23. [Figure 14A] This is a plot of fluorescence intensity versus T4 concentration for various solutions containing T3-F and Ab-Cy3 with added T4, as described in Example 24. [Figure 14B] This is a plot of fluorescence intensity versus T4 concentration for various solutions containing T4-F and Ab-Cy3 with added T4, as described in Example 24. [Figure 14C] This is a plot of fluorescence intensity versus T4 concentration for various solutions containing T4-added T4-F and Ab-Cy3, as described in Example 24, when the solution is excited at 490 nm and the emission is measured at 615 nm. [Figure 15]Example 25 shows plots of the percentage change in fluorescence for solutions of various beta-lactam antibiotics linked to a fluorescent tracer; solutions of the tracer and antibody (i.e., Ab); and solutions of the tracer, antibody, and ampicillin or cefotaxime. [Figure 16] This shows the solutions of SDM-F or SDM-Su-F described in Example 26; the solutions of SDM-F or SDM-Su-F and the anti-sulfadimethoxine monoclonal antibody (Ab); and plots of the relative fluorescence percentages of the solutions of SDM-F or SDM-Su-F, Ab, and SDM. [Figure 17] This is a plot of the fluorescence intensity of various solutions of T3-F and monoclonal anti-T4-Mab as described in Example 27, versus the molar ratio of monoclonal anti-T4-Mab to T3-F. [Figure 18A] This figure shows the structure of various embodiments of slides used in the dry slide assay method described herein. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A] This figure shows an example of the assay liquid used in the dry slide assay method described herein. [Figure 19B] Same as above. [Figure 20] This figure shows how to perform the dry slide assay method described herein using the dry slide shown in Figure 18C. [Figure 21] This is a plot of fluorescence quenching percentage against the antibody:cortisol-4-Fl ratio, as described in Example 28. [Figure 22] This is a plot of fluorescence quenching percentage against the antibody:cortisol-4-Fl ratio and the antibody:cortisol-5-Fl ratio, as described in Example 28. [Figure 23] This figure shows the decrease in fluorescence when fluorescent tracer A3 forms a complex with an antibody against SDMA, as a function of the equivalent weight of the BHQ10 quencher conjugated to the antibody. [Figure 24A] This figure shows the results of an assay (using slides with a filtering layer) to determine the concentration of SDMA in a sample containing a certain amount of SDMA (μg / dL), where the sample contains compounds that may potentially interfere with the assay at various concentrations (i.e., interfering substances). The interfering substances are (A) hemolysis (0-500 mg / dL), (B) bilirubin (0-30 mg / dL), (C) intralipid (0-1000 mg / dL), and (D) whole blood (0-10%). [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 25A] This figure shows the absorption spectra of DFF, SDMA-DFF, DCF, SDMA-DCF, Fl, and SDMA-FL as described in Example 33. [Figure 25B] This figure shows the emission spectra of SDMA-DFF, SDMA-DCF, Fl, and SDMA-FL as described in Example 33. [Figure 26] This figure shows the fluorescence quenching percentage for different SDMA tracers as a function of increasing antibody concentration, as described in Example 34. [Figure 27] This figure shows the fluorescence quenching percentage for different SDMA tracers as a function of increasing concentrations of anti-SDMA antibody conjugated to BHQ10 (4 equivalents), as described in Example 34. [Figure 28] This figure shows the increase in fluorescence intensity as a function of SDMA concentration when SDMA is combined with the SDMA-DFF tracer / SDMA antibody solution, as described in Example 34. [Figure 29] This figure shows the increase in fluorescence intensity as a function of SDMA concentration when SDMA is combined with an SDMA-DCF tracer / SDMA antibody solution, as described in Example 34. [Figure 30] This figure shows the increase in fluorescence intensity as a function of SDMA concentration when SDMA is combined with an SDMA-DCF tracer / SDMA antibody solution, as described in Example 34. [Figure 31] This figure shows riboflavin fluorescence in milk as a function of riboflavin-binding protein, as described in Example 36. [Figure 32] This figure shows the output from SMDA analysis using the dry slide described in Example 37. [Figure 33] This is a plot of the fluorescence quenching percentage versus antibody:CA-Fl ratio described in Example 40. [Figure 34A] This is a plot of fluorescence intensity versus cholic acid concentration when cholic acid is added to a PBS solution of CA-Fl and α-cholic acid antibody, as described in Example 40. [Figure 34B] This is a plot of fluorescence intensity versus cholic acid concentration when taurocholic acid is added to a PBS solution of CA-Fl and α-cholic acid antibody, as described in Example 40. [Figure 35A] This is a plot of the fluorescence versus bile acid concentrations of CA-Fl and α-cholic acid antibody solutions containing different concentrations of cholic acid, as described in Example 42. [Figure 35B] This is a plot of the fluorescence versus bile acid concentrations of CA-Fl and α-cholic acid antibody solutions containing different concentrations of taurocholic acid, as described in Example 42. [Figure 36] This is a plot of fluorescence intensity (λex=470nm, λem=520nm) versus CysB concentration (μg / mL), as described in Example 47. [Figure 37] This is a plot of the rate of increase in fluorescence intensity (measured over time intervals of 30 to 100 seconds) versus the concentration of CysB, as described in Example 48, using a dry slide format. [Figure 38] This is a plot of fluorescence quenching percentages against the ratio of anti-CysB antibody to CysB peptide P6-Fl and the ratio of antibody to P7-Fl, as described in Example 51. [Figure 39] This is a plot of fluorescence quenching percentages against the ratio of anti-CysB antibody to CysB peptide P6-DFF and against the ratio of antibody to P7-DFF, as described in Example 51. [Figure 40] This is a plot of fluorescence quenching percentages against the ratio of anti-CysB antibody to CysB peptide P6-Fl or P6-DFF, and against the ratio of antibody to P6-4-Fl or P-4-DFF, as described in Example 52. [Figure 41] This is a plot of fluorescence quenching percentage against the ratio of anti-CysB antibody (Ab) to CysB peptide P6-Fl, and against the ratio of antibody-BHQ10 (Ab-BHQ10) to P6-Fl. [Figure 42] This is a plot of fluorescence quenching percentage against the ratio of anti-CysB antibody to CysB peptide P6-DFF, and against the ratio of antibody-BHQ10 (Ab-BHQ10) to P6-DFF. [Figure 43] This is a plot of fluorescence recovery percentage as a function of CysB peptide P6, P7, or P14 concentration when P6, P7, or P14 is added to a solution of P6-Fl and anti-CysB antibody, or to a solution of P6-DFF and anti-CysB antibody, as described in Example 51. [Figure 44A] This is a plot of fluorescence recovery percentage as a function of CysB protein concentration when CysB full-length protein is added to a solution of P6-Fl and anti-CysB antibody in the presence of the washing agent sarcosyl, as described in Example 51. [Figure 44B] This is a plot of fluorescence recovery percentage as a function of CysB protein concentration when CysB full-length protein is added to a solution of P6-DFF and anti-CysB antibody in the presence of the washing agent sarcosyl, as described in Example 51. [Figure 45] This is a plot of fluorescence recovery percentage as a function of CysB protein concentration, as described in Example 51, when CysB full-length protein added to serum is added to a solution of P6-Fl and anti-CysB antibody (Ab) or a solution of P6-Fl and anti-CysB antibody (Ab-BHQ10) conjugated to BHQ10 in the presence of the washing agent sarcosyl. [Figure 46]This is a plot of fluorescence recovery percentage as a function of CysB protein concentration, as described in Example 51, when CysB full-length protein added to serum is added to a solution of P6-DFF and anti-CysB antibody (Ab) in the presence of the washing agent sarcosyl, or to a solution of P6-DFF and anti-CysB antibody (Ab-BHQ10) conjugated to BHQ10. [Figure 47] This is a plot of fluorescence quenching percentages against the ratio of anti-NT-proBNP antibody to NT-proBNP peptide P1-Fl(Ab) and against the ratio of anti-NT-proBNP antibody-BHQ10 to peptide P1-Fl, as described in Example 52. [Figure 48] This is a plot of fluorescence quenching percentage against the ratio of anti-NT-proBNP antibody to NT-proBNP peptide P1-DFF (Ab) and against the ratio of anti-NT-proBNP antibody-BHQ10 to P1-DFF (Ab-BHQ10), as described in Example 52. [Figure 49] This is a plot of fluorescence recovery percentage as a function of P1 concentration when NT-proBNP peptide P1 is added to a solution of P1-Fl and anti-NT-proBNP antibody (Ab), or to a solution of P1-Fl and antibody-BHQ10 (Ab-BHQ10), as described in Example 52. [Figure 50] This is a plot of fluorescence recovery percentage as a function of P1 concentration when NT-proBNP peptide P1 is added to a solution of P1-DFF and anti-NT-proBNP antibody (Ab), or to a solution of P1-DFF and anti-NT-proBNP antibody-BHQ10, as described in Example 52. [Figure 51] This is a plot of fluorescence recovery percentage as a function of P1 concentration when NT-proBNP peptide P1 added to charcoal-striped serum is added to a solution of P1-Fl and anti-NT-proBNP antibody, as described in Example 52. [Figure 52]This is a plot of fluorescence recovery percentage as a function of P1 concentration when NT-pro-BNP peptide P1, added to charcoal-treated serum, is added to a solution of P1-DFF and anti-NT-pro-BNP antibody, as described in Example 52. [Figure 53] This is a plot of fluorescence recovery percentage as a function of NT-proBNP protein concentration when NT-proBNP full-length protein in PBS is added to a solution of P1-Fl and anti-NT-proBNP antibody (P1-Fl / Ab=1:1), as described in Example 52. [Figure 54] This is a plot of fluorescence recovery percentage as a function of NT-proBNP protein concentration when NT-proBNP full-length protein in PBS is added to a solution of P1-DFF and anti-NT-proBNP antibody (P1-DFF / Ab=1:1), as described in Example 52. [Figure 55] This is a plot of fluorescence recovery percentage as a function of NT-proBNP protein concentration when serum NT-proBNP full-length protein is added to a solution of P1-Fl and anti-NT-proBNP antibody (P1-Fl / Ab=2:1), as described in Example 52. [Figure 56] This is a plot of fluorescence recovery percentage as a function of NT-proBNP protein concentration when serum NT-proBNP full-length protein is added to a solution of P1-DFF and anti-NT-proBNP antibody (P1-DFF / Ab=2:1), as described in Example 52. [Figure 57] This figure shows an exemplary embodiment of a slide used in the dry slide assay method described herein. [Figure 58] This figure shows an exemplary embodiment of a slide used in the dry slide assay method described herein. [Modes for carrying out the invention]

[0024] The present invention relates to a method for determining the presence or amount of an analyte in a sample.

[0025] The present invention also includes slides that can be used in this method.

[0026] The present invention also includes substitutional fluorescent tracers that can be used in the methods of the present invention.

[0027] The present invention also includes derivatized fluorescein molecules that are useful as intermediates for synthesizing substituted fluorescent tracers that can be used in the methods of the present invention.

[0028] 1.Definition The term "analyte," as used herein, refers to a molecule (e.g., an antibody or antigen) present in a sample, such as a biological fluid, that binds to (i.e., forms a complex with) a binding partner (e.g., an antigen or antibody) and whose presence or concentration in the sample is to be determined. The analyte may be, for example, a protein, glycoprotein, sugar, polysaccharide, amino acid, substituted amino acid, methylated amino acid, hormone, antibiotic, nucleic acid, metabolite, or a derivative of any of the foregoing.

[0029] When used herein, the term "complex" refers to a species formed by the association of two or more molecular entities (which may be ionic or uncharged) without covalent bonds between them. Examples of complexes include the association of an antibody with an antigen (or antigen derivative) and the association of a peptide with a receptor.

[0030] The term "hapten," as used herein, refers to a molecule that, when injected into an animal, does not induce antibody formation but provides an antigen (immunogen) that, when injected into an animal, binds to a carrier protein and triggers an immune response, thereby resulting in antibody formation. The resulting antibody can be isolated by known antibody isolation techniques. The hapten binds to the resulting antibody.

[0031] When used herein, the term "antigen" has the meaning recognized in the art, namely, a substance that, when introduced into the body, stimulates the production of antibodies.

[0032] The term “antibody,” as used herein, has the meaning recognized in the art, namely, a protein produced for the introduction of an antigen into the body. Antibodies can be produced in vivo, in vitro, recombinantly, or synthetically. The term “antibody,” as used herein, includes polyclonal antibodies, monoclonal antibodies, single-chain antibodies (scFv), or antigen-binding fragments of antibodies. An antigen-binding fragment of an antibody is a portion of an intact antibody that includes the antigen-binding site or variable region of the intact antibody, but does not include the constant heavy chain domain of the Fc region of the intact antibody. Examples of antigen-binding antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and F v A fragment is an example. The antibody may be any antibody class, including, for example, IgG, IgM, IgA, IgD, and IgE.

[0033] The term "mAb" or "Mab" is an abbreviation for monoclonal antibody, as used herein.

[0034] The phrase “binding partner” means, as used herein, a molecule that specifically binds to a second molecule. For example, the second molecule may be an antigen / antibody and the “binding partner” may be an antibody / antigen. Similarly, the second molecule may be a receptor substrate and the “binding partner” may be a receptor, or the second molecule may be a receptor and the “binding partner” may be a substrate. Furthermore, the binding partner may be an aptamer. The binding partner may be a lectin and the second molecule may be a carbohydrate. The second molecule may be an analyte.

[0035] The phrases “specifically,” “specifically binds,” “specifically binds to the analyte,” and “specific to the analyte,” and similar phrases, when used herein, have the meanings recognized in the art, namely, that the binding partner recognizes the analyte (or class of analytes) and binds to it with a higher affinity than other nonspecific molecules. For example, an antibody produced against an antigen that binds to the antigen more efficiently than other nonspecific molecules may be described as specifically binding to the antigen. Binding specificity can be tested using methodologies known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay. A nonspecific molecule is a molecule that binds to the binding partner with less than 25%, preferably less than 10%, more preferably less than 5%, and most preferably less than 1% of the reactivity shown between the binding partner and its corresponding analyte.

[0036] The term "analyte-conjugate," as used herein, refers to a molecule (e.g., an antigen or antibody) that is recognized and binds to a binding partner (e.g., an antibody or antigen) that is linked to a second molecule. A characteristic of an analyte-conjugate is that it has sufficient structural similarity to the analyte of interest so that it is recognized and binds to the analyte by its binding partner.

[0037] For example, if the analyte is an antigen, a significant proportion of analyte-conjugates will have substantially the same structure, spatial, and polar configuration as the antigen, defining one or more determinants or epitope sites (hereinafter referred to as "epitope sites"), so that the analyte-conjugate can compete with the antigen for the antibody binding site(s). In most cases, the analyte-conjugate will have the same or substantially the same structure and charge distribution (spatial and polar configuration) as the analyte being assayed in the sample, with respect to a significant proportion of its molecular surface.

[0038] The analyte-conjugate may be an epitope portion that is linked to a detectable label to provide a "tracer" (i.e., the second molecular portion is the detectable label).

[0039] The analyte-conjugate may be a hapten conjugated to a detectable label to provide a tracer. In other words, the epitope portion conjugated to the detectable label to provide the tracer is the hapten portion, i.e., the portion of the hapten used to produce an immune response. Functional groups on the hapten can be chemically modified to conjugate the hapten to a detectable label.

[0040] The analyte-conjugate can be a conjugating protein, such as an antibody, linked to a detectable label to provide a tracer.

[0041] "Tracer" is, as used herein, an analyte-conjugate containing a detectable label.

[0042] Fluorescein has the following structure:

[0043] [ka] It is a molecule that possesses the following: As shown below, fluorescein can exist in both open and closed forms, and the open form will be recognized as existing as a pair of rotational isomers:

[0044] [ka] As used herein, the term fluorescein includes both open and closed forms, as well as both rotational isomers of the open form. As used herein, the term fluorescein also includes ionic and / or salt forms. The numbering of carbon atoms in fluorescein molecules varies in the art depending on whether the open or closed form of the molecule is considered. Therefore, the literature relating to fluorescein and its derivatives is not uniform with respect to carbon atom numbering. The numbering shown above is used for the purposes of this disclosure.

[0045] The term "fluorescent tracer," as used herein, means an analyte-conjugate in which the epitope moiety is linked directly or via a linking group to a fluorescent molecule. In some embodiments, the epitope moiety is linked to a molecule derived from fluorescein. Preferably, the epitope moiety is linked to fluorescein or difluorofluorescein.

[0046] The phrase “epitope moiety,” as used herein, means a portion of an analyte having a structure, space, and polar arrangement that defines one or more determinants or epitope sites of the analyte so that the analyte can be recognized by its binding partner and specifically bind to it. The phrase “epitope moiety,” as used herein, means an epitope moiety or a molecular entity containing an epitope moiety.

[0047] In one embodiment, the fluorescent tracer is

[0048] [ka] (In the formula, T is a bond or divalent group (i.e., a linking group) that connects the epitope to the fluorescein molecule, and the curved line indicates that the T-epitope can be replaced by any hydrogen atom bonded to the carbon of the fluorescein molecule.) That is the case.

[0049] As used herein, the term "quencher" refers to a portion in a fluorescence quenching immunoassay that, when bound to a binding partner, reduces the intensity of light emitted by the fluorescent tracer that is complexed with the same binding partner compared to the intensity of light emitted by the fluorescent tracer when it is not bound to the quencher.

[0050] The term "approximately" as used herein means ±10%, preferably ±5%, more preferably ±2%, and most preferably ±1%.

[0051] When used herein, the phrase “substantially does not contain” means less than 10%, preferably less than 5%, more preferably less than 2%, and most preferably less than 1%.

[0052] The phrase "proportional," as used herein, means that there is a correspondence or relationship between a measured value and some quantity. For example, the phrase "the intensity of light emitted at a second wavelength is proportional to the concentration of the analyte in the sample" means that the intensity of light emitted at a second wavelength corresponds to the concentration of the analyte in the sample. Measured values ​​and quantities may be linearly correlated.

[0053] As used herein, the term "polymer" means a compound having a molecular weight greater than about 500 daltons. In one embodiment, the molecular weight is greater than about 2200 daltons. In one embodiment, the molecular weight is greater than about 5500 daltons. In one embodiment, the molecular weight is greater than about 11000 daltons. The molecular weight of a polymer is typically less than about 200 kilodaltons. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 3 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 5 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 10 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 20 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 50 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 100 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 200 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 300 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length greater than about 400 amino acids. In one embodiment, the polymer is a polypeptide with an amino acid length of more than about 500 amino acids. In another embodiment, the polymer is a polypeptide with an amino acid length of more than about 600 amino acids. In yet another embodiment, the polymer is a polypeptide with an amino acid length of more than about 700 amino acids. In yet another embodiment, the polymer is a polypeptide with an amino acid length of more than about 800 amino acids. When the polymer is a polypeptide, the amino acid length is typically less than about 1000 amino acids. The polypeptide may be glycosylated. The polypeptide may be an antibody. In yet another embodiment, the polymer may be a carbohydrate, polysaccharide, lipid, or nucleic acid.

[0054] The term "SDMA" refers to symmetric dimethylarginine, also known as free SDMA, and its derivatives. Examples of SDMA derivatives include alkylated SDMA and acylated SDMA. Certain antibodies specific to SDMA are described in U.S. Patent No. 9970927.

[0055] 2. Explanation of Method The method of the present invention is, (i) The step of preparing a sample suspected to contain the analyte; (ii) A step of contacting a sample with a fluorescent tracer and a binding partner to obtain an assay composition, wherein the binding partner is specific to the analyte and the fluorescent tracer; (iii) Irradiating the assay composition with light of a first wavelength that is not linearly polarized; (iv) The step of measuring the intensity of light emitted at a second wavelength Includes, The intensity of the light emitted at the second wavelength is proportional to the concentration of the analyte in the sample.

[0056] In another embodiment of the present invention, this device (i) The step of preparing a sample suspected to contain the analyte; (ii) A step of contacting a sample with a fluorescent tracer and a binding partner to obtain an assay composition, wherein the binding partner is specific to the analyte and the fluorescent tracer; (iii) Irradiating the assay composition with light of a first wavelength that is not linearly polarized; (iv) The step of measuring the intensity of light emitted at a second wavelength Includes, The intensity of the light emitted at the second wavelength is proportional to the concentration of the analyte in the sample.

[0057] Therefore, in one embodiment, the light of the first wavelength is not linearly polarized, while in another embodiment, the light of the first wavelength is linearly polarized.

[0058] In one embodiment, a linear relationship exists between the intensity of light emitted at a second wavelength and the concentration of the analyte in the sample.

[0059] While we do not wish to be constrained by theory, the basis of this method is that the fluorescence of a fluorescent tracer is quenched when the tracer is bound to a binding partner. Therefore, when a fluorescent tracer is bound to a binding partner and excited by light of a first wavelength, the intensity of the light emitted at the second wavelength is reduced (quenched) compared to the intensity emitted when the tracer is not bound to the binding partner. The analyte and fluorescent tracers present in the sample compete for a limited number of binding sites on the binding partner, resulting in the formation of analyte-binding partner complexes and tracer-binding partner complexes. The higher the concentration of the analyte in the sample, the fewer fluorescent tracer molecules can bind to the binding partner, and therefore, the more unbound fluorescent tracer molecules there are. Thus, the intensity of the light emitted at the second wavelength is directly proportional to the amount of analyte in the sample. This general principle is shown in Figure 1. The affinity of the binding partner to the analyte and the affinity of the binding partner to the fluorescent tracer may differ.

[0060] In one embodiment, the analyte is an antigen, the fluorescent tracer is an analyte-conjugate containing the epitope portion of the antigen linked to a fluorescent label, and the binding partner is an antibody against the antigen.

[0061] In one embodiment, the fluorescent tracer is selected from the group consisting of a 4'-substituted fluorescent tracer, a 4'-substituted fluorescent tracer derivative, a 5-substituted fluorescent tracer, and a 5-substituted fluorescent tracer derivative.

[0062] In one embodiment, the fluorescent tracer is a 4'-substituted fluorescent tracer.

[0063] In one embodiment, the fluorescent tracer is a 4'-substituted fluorescent tracer derivative.

[0064] Preferably, the fluorescent tracer is a 4'-substituted fluorescent tracer or a 4'-substituted fluorescent tracer derivative, and most preferably, the fluorescent tracer is a 4'-substituted fluorescent tracer.

[0065] In one embodiment, the 4'-substituted fluorescent tracer is structure A1.

[0066] In one embodiment, the 4'-substituted fluorescent tracer has structure A2.

[0067] In one embodiment, the 4'-substituted fluorescent tracer has structure A3.

[0068] In one embodiment, the fluorescent tracer is a 5-substituted fluorescent tracer.

[0069] In one embodiment, the fluorescent tracer is a 5-substituted fluorescent tracer derivative.

[0070] In one embodiment, the 5-substituted fluorescent tracer has structure A4.

[0071] In one embodiment, the 5-substituted fluorescent tracer has structure A5.

[0072] In one embodiment, the 5-substituted fluorescent tracer is structure A6.

[0073] In one embodiment, the tracer has the following structure:

[0074] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) It is a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative having [a specific characteristic].

[0075] In one embodiment, the tracer has the following structure:

[0076] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) It is a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative having [a specific characteristic].

[0077] In one embodiment, the tracer has the following structure:

[0078] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) It is a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative having [a specific characteristic].

[0079] In one embodiment, the tracer has the following structure:

[0080] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) It is a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative having [a specific characteristic].

[0081] In one embodiment, the tracer has the following structure:

[0082] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) It is a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative having [a specific characteristic].

[0083] In one embodiment, the tracer has the following structure:

[0084] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) It is a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative having [a specific characteristic].

[0085] The molecular weight of the analyte can vary over a wide range. Typically, the molecular weight of the analyte is greater than 50 daltons. For low molecular weight analytes, the molecular weight is typically between about 50 and about 4000 daltons, preferably between about 100 and about 2000 daltons. If the analyte is a larger molecule such as a protein, the molecular weight may be greater than 2000 daltons. If the analyte is a larger molecule such as a protein, the molecular weight may be greater than 4000 daltons.

[0086] A wide variety of analytes can be assayed using the method of the present invention. Exemplary analytes include, but are not limited to, low molecular weight substances (e.g., symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), monomethylarginine (MMA), melamine, antibiotics, T4, β-lactam antibiotics (such as penicillin), sulfonamides, cephalosporins, and steroids (e.g., progesterone and cortisol)), as well as high molecular weight substances such as proteins (e.g., cystatin-B) and antibodies.

[0087] In one embodiment, the analyte is SDMA.

[0088] In one embodiment, the analyte is melamine.

[0089] In one embodiment, the analyte is T4.

[0090] In one embodiment, the analyte is cortisol. In another embodiment, the analyte is bile acid.

[0091] In one embodiment, the analyte is progesterone.

[0092] In one embodiment, the analyte is cystatin-B. In a specific embodiment, the analyte is canine or feline cystatin-B. In one embodiment, the analyte is NT-proBNP. In a specific embodiment, the analyte is canine or feline NT-proBNP.

[0093] In one embodiment, the analyte is an antibiotic.

[0094] Examples of antibiotics include, but are not limited to, amoxicillin, ampicillin, cefacetril, cefquinome, cefazolin, cefoperazone, ceftiofur, cephalexin, cephalonium, cloxacillin, desacetylcefapirin, dicloxacillin, nafcillin, oxacillin, cefapillin, desfroil-ceftioflu, cefuroxime, and penicillin.

[0095] In one embodiment, the analyte is one or more antibiotics selected from the group consisting of antibiotics that must be tested in milk as required by the European Union.

[0096] In one embodiment, the analyte is one or more antibiotics selected from the group consisting of penicillin G (benzylpenicillin), ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, cefapillin, desacetylcefapirin, ceftiofur, desfroilceftioflu, cefquinome, cephalonium, cefazolin, cepacetril, cephalexin, cefuroxime, and cefoperazone.

[0097] In one embodiment, the analyte is one or more antibiotics selected from the group consisting of antibiotics that must be tested in milk as required by the U.S. Food and Drug Administration.

[0098] In one embodiment, the analyte is one or more antibiotics selected from the group consisting of penicillin G (benzylpenicillin), ampicillin, amoxicillin, cloxacillin, cefapillin, ceftiofur, and desfroil-cefttiofur.

[0099] In one embodiment, the analyte is a polymer. In one embodiment, the polymer is a polypeptide.

[0100] In one embodiment, the assay composition is a solution. In another embodiment, the assay composition is an aqueous solution.

[0101] The sample is typically a liquid, but is not limited to, biological fluids such as urine, serum, milk, saliva, plasma, whole blood, sweat, tears, and cerebrospinal fluid. Although the sample is typically a liquid, solid samples can also be used in the method of the present invention. Solid materials include, but are not limited to, fecal samples and solid tissue samples, extracts of solid materials, and dried liquid samples. In one embodiment, the solid sample is dissolved or suspended in a liquid.

[0102] In one embodiment, the sample is an aqueous solution.

[0103] In one embodiment, the sample is urine.

[0104] In one embodiment, the sample is serum.

[0105] In one embodiment, the sample is milk.

[0106] In one embodiment, the sample is saliva.

[0107] In one embodiment, the sample is plasma.

[0108] In one embodiment, the sample is whole blood.

[0109] In one embodiment, the sample is sweat.

[0110] In one embodiment, the sample is tears.

[0111] In one embodiment, the sample is cerebrospinal fluid.

[0112] In one embodiment, the sample is a fecal sample or fecal extract.

[0113] Typically, the sample size is in the range of about 1 μL to about 5 mL. In one embodiment, the sample size is preferably about 2 μL to about 20 μL, more preferably about 2 μL to about 15 μL, and most preferably about 2 μL to about 10 μL. In one embodiment, the sample size is in the range of about 50 μL to about 200 μL. In one embodiment, the sample size is about 75 μL to about 150 μL. In one embodiment, the sample size is about 100 μL.

[0114] The concentration of the analyte in the assay solution can vary over a wide range. Generally, the concentration of the analyte in the assay solution ranges from approximately 0.1 nM to approximately 1000 nM, typically from approximately 10 nM to approximately 100 nM. It will be readily apparent that a more concentrated sample can be easily diluted with a suitable diluent to obtain a diluted sample with a concentration suitable for the assay. The method of the present invention is extremely sensitive and, for some analytes, can be used to detect analytes at concentrations as low as 4 ppb.

[0115] The concentration of the binding partner in the assay solution is typically in the range of about 0.1 nM to about 2000 nM. In one embodiment, the concentration of the binding partner in the assay solution is in the range of about 1 nM to about 1000 nM. In one embodiment, the concentration of the binding partner in the assay solution is in the range of about 5 nM to about 500 nM. In one embodiment, the concentration of the binding partner in the assay solution is in the range of about 10 nM to about 200 nM.

[0116] In various embodiments, the binding partner is linked to a solid support. Examples of solid supports include, but are not limited to, reaction vessels, particles, microparticles, microbeads, barcode beads, magnetic particles, or magnetic barcode beads.

[0117] The concentration of the fluorescent tracer in the assay solution is typically in the range of about 0.1 to about 1000 nM. In one embodiment, the concentration of the fluorescent tracer in the assay solution is in the range of about 1 nM to about 500 nM. In another embodiment, the concentration of the fluorescent tracer in the assay solution is in the range of about 10 nM to about 200 nM.

[0118] Typically, the ratio of binding partner to fluorescent tracer in the assay solution is in the range of about 0.05 to about 10. In one embodiment, the ratio of binding partner to fluorescent tracer in the assay solution is in the range of about 0.1 to about 8. In another embodiment, the ratio of binding partner to fluorescent tracer in the assay solution is in the range of about 0.5 to about 5. However, it will be understood that the accuracy of the assay depends on having the correct ratios for binding partner to tracer and for binding partner to analyte. The appropriate ratio depends on the affinity of the analyte (and tracer) to the binding partner. Those skilled in the art will be able to easily determine what the appropriate ratio is.

[0119] In one embodiment, the ratio of the binding partner to the fluorescent tracer is approximately 1:1. In one embodiment, the binding partner and the tracer are provided as a complex in which the ratio of the binding partner to the fluorescent tracer in the complex is approximately 1:1. In one embodiment, the complex is provided as a solid. For example, the binding partner and the fluorescent tracer are combined in an aqueous solvent to obtain an aqueous solution of the complex, and the water is removed from the solution by freeze-drying to provide the complex as a solid.

[0120] Typically, the assay is performed by mixing the sample with a pre-combined binding partner and fluorescent tracer. Typically, about 25 μl of the binding partner solution is combined with about 25 μl of the fluorescent tracer solution, and then the resulting solution is added to about 50 μl of the sample solution suspected to contain the analyte to obtain the assay composition. However, the volumes of the binding partner solution, fluorescent tracer solution, and sample solution can vary widely. The assay can also be performed by mixing the fluorescent tracer with a pre-combined binding partner and sample. The assay can be further performed by mixing the binding partner with a pre-combined fluorescent tracer and sample.

[0121] In one embodiment, the assay composition is prepared as a solution by mixing the sample, fluorescent tracer, and binding partner by gently swirling or shaking. After mixing the sample, fluorescent tracer, and binding partner, the resulting assay solution must typically be equilibrated for a short period before being irradiated with light of a first wavelength. Generally, it is necessary to equilibrate the assay solution for less than one minute. In most cases, it is necessary to equilibrate the assay solution for less than 15 seconds.

[0122] The first and second wavelengths depend on the excitation and emission spectra of the fluorescence tracer. Those skilled in the art will be able to easily determine what the appropriate first and second wavelengths are. The term "first wavelength," as used herein, can encompass a range of wavelengths. Similarly, the term "second wavelength," as used herein, can encompass a range of wavelengths. Typically, the first and second wavelengths are between approximately 200 nm and approximately 900 nm, respectively. In one embodiment, the first wavelength (λ) ex ) is approximately 490 nm, and the second wavelength (λ em ) is approximately 520 nm.

[0123] In one embodiment, the sample is milk. In one embodiment, the sample is milk that has been contacted with riboflavin-binding protein (commercially available from Sigma Aldrich, St. Louis, Missouri). Although we do not wish to be constrained by theory, it is thought that treating milk with riboflavin-binding protein reduces the autofluorescence associated with the milk sample. Although we do not wish to be constrained by theory, it is thought that the autofluorescence observed in the milk sample is caused by riboflavin in the milk, and that riboflavin-binding protein reduces autofluorescence by binding to riboflavin. By reducing autofluorescence, a more accurate measurement of light emitted at a second wavelength is advantageously provided. Typically, riboflavin-binding protein is added to the milk in an amount sufficient to provide a concentration of riboflavin-binding protein in the assay solution higher than about 100 μg / mL, preferably about 100 μg / mL to about 300 μg / mL.

[0124] In one embodiment, the sample is milk, and the milk is irradiated with light of a first wavelength on a first side of the sample, and the intensity of the light emitted at a second wavelength is measured from a second side of the sample that is different from the first side.

[0125] In one embodiment, the sample is milk, and the milk is irradiated with light of a first wavelength on one side of the sample, and the intensity of the light emitted at a second wavelength is measured from the opposite side of the sample.

[0126] In one embodiment, the sample is milk, and the milk is irradiated with light of a first wavelength on one side of the sample, and the intensity of the light emitted at a second wavelength is measured from the same side of the sample.

[0127] If the sample is milk, it may be whole milk (raw milk) or skim milk (i.e., milk from which the fat has been removed). If the sample is milk, there is no requirement for a pretreatment step to remove cream from the milk, for example, by centrifugation.

[0128] In one embodiment, the sample is milk and the analyte is melamine.

[0129] In one embodiment, the sample is milk and the analyte is an antibiotic. Exemplary antibiotics include, but are not limited to, beta-lactam antibiotics (such as penicillin), cephalosporins, sulfonamides, fluoroquinolones, chloramphenicol, and fluoramphenicol.

[0130] In one embodiment, the sample is milk, and the analyte is one or more antibiotics selected from the group consisting of antibiotics that must be tested in milk as required by the European Union.

[0131] In one embodiment, the antibiotic is selected from the group consisting of penicillin G (benzylpenicillin), ampicillin, amoxicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, cefapillin, desacetylcefapirin, ceftiofur, desfroylceftioflu, cefquinome, cephalonium, cefazolin, cefacetril, cephalexin, cefuroxime, and cefoperazone.

[0132] In one embodiment, the sample is milk, and the analyte is one or more antibiotics selected from the group consisting of antibiotics that must be tested in milk as required by the U.S. Food and Drug Administration.

[0133] In one embodiment, the sample is milk, and the analyte is one or more antibiotics selected from the group consisting of penicillin G (benzylpenicillin), ampicillin, amoxicillin, cloxacillin, cefapillin, ceftiofur, and desfroil-cefttiofur.

[0134] Suitable instrumentation that can be used to perform the assay includes any commercially available fluorescence spectrometer, such as the Fluoromax+ spectrometer (commercially available from Horiba Instruments Inc. in Japan) and the Synergy 4 Microplate Reader (commercially available from Biotek Instruments Inc. in Winooski, Vermont).

[0135] This method involves only one step (i.e., combining the sample with the fluorescent tracer and its binding partner) and, advantageously, does not require any separation steps such as washing steps (i.e., steps to separate the tracer bound to its binding partner from the unbound tracer).

[0136] 3. Dry slide assay method In one embodiment, the method involves the step of determining the presence or amount of an analyte in a sample on a dry slide, also referred to herein as a “slide.” The method simply involves the steps of applying the sample to the slide, irradiating the slide with light of a first wavelength, and measuring the intensity of light emitted at a second wavelength. The present invention also encompasses slides used in the method. The slide comprises two or more layers.

[0137] The general structure of the slide is shown in Figure 18A. The slide includes a support layer on which an indicator layer is applied.

[0138] The support layer is a solid that provides support to the slide. The support layer is optically transparent to a first and second wavelength, i.e., it substantially does not absorb light of the first and second wavelengths. Preferably, the support layer is water-insoluble and water-impermeable. Preferably, the support layer is mechanically and thermally stable and scratch-resistant. The support layer can be any suitable polymer that meets these criteria.

[0139] Examples of supporting layers include, but are not limited to, glass, polystyrene, polyester, polycarbonate, cellulose derivatives (such as cellulose acetate), polyethylene terephthalate, and mixtures thereof.

[0140] In one embodiment, the support layer is a polyethylene terephthalate layer.

[0141] In one embodiment, the support layer is a polyester layer. In one embodiment, the support layer is a commercially available polyester under the trade name Melinex® (commercially available from Tekra, a division of EIS, Inc. in Berlin, Wisconsin). In one embodiment, the support layer is a commercially available polyester under the trade name ESTAR® (commercially available from Eastman Kodak Company in Rochester, New York).

[0142] The thickness of the support layer is typically in the range of about 15 μm to about 200 μm, preferably about 50 μm to about 150 μm, and more preferably about 70 μm to about 130 μm. In one embodiment, the thickness of the support layer is about 125 μm. In another embodiment, the thickness of the support layer is about 75 μm.

[0143] The indicator layer contains a fluorescent tracer and a binding partner dispersed in a polymer. The fluorescent tracer and binding partner may exist as a complex.

[0144] In one embodiment, a fluorescent tracer and a binding partner are combined in a liquid, the liquid is removed to obtain a solid, the obtained solid is dispersed or dissolved in a polymer, and this is used to obtain an indicator layer. In another embodiment, a fluorescent tracer and a binding partner are combined in water, the water is removed by freeze-drying, and the obtained freeze-dried solid is dissolved or dispersed in a polymer to obtain an indicator layer. In yet another embodiment, the fluorescent tracer and the binding partner are provided as a pre-formed composite.

[0145] In one embodiment, a fluorescent tracer and a binding partner (preferably as a pre-formed composite) and a polymer are dissolved or suspended in a solvent to obtain a coating solution, the coating solution is coated onto a support layer to obtain a wet indicator layer, and then the solvent is removed to obtain a dry indicator layer in which the fluorescent tracer and binding partner are dispersed in the polymer, thereby obtaining an indicator layer. Preferably, the solvent is an aqueous solvent.

[0146] The thickness of the wet indicator layer is typically in the range of from about 30 μm to about 200 μm, preferably from about 50 μm to about 150 μm, more preferably from about 80 μm to about 120 μm. When dry, the thickness of the indicator layer is typically in the range of from about 15 μm to about 100 μm, preferably from about 10 μm to about 75 μm, more preferably from about 20 μm to about 60 μm. In one embodiment, the thickness of the dry indicator layer is from about 30 μm to about 40 μm. In one embodiment, the thickness of the dry indicator layer is about 50 μm. The thickness of the indicator layer depends, in part, on the sample volume. For example, if the sample is a very dilute solution of the analyte, the assay requires a larger sample volume, and a thicker indicator layer is preferred to accommodate the larger sample volume.

[0147] In one embodiment, the binding partner is an antibody. The concentrations of the fluorescent tracer and the antibody in the wet indicator layer are typically about 4×10 -6 % by weight to about 4×10 -2 % by weight and about 4×10 -4 % by weight to about 4% by weight, based on the layer. In one embodiment, the concentrations of the fluorescent tracer and the antibody in the wet indicator layer are about 8×10 -6 % by weight to about 2×10 -2 % by weight and about 8×10 -4 % by weight to about 2% by weight, based on the layer. In one embodiment, the concentrations of the fluorescent tracer and the antibody in the wet indicator layer are about 1.6×10 -5 % by weight to about 1×10 -2 % by weight and about 1.6×10 -3 % by weight to about 1% by weight, based on the layer. In one embodiment, the concentrations of the fluorescent tracer and the antibody in the wet indicator layer are about 4×10 -5 % by weight to about 4×10 -3 % by weight and about 4×10 -3 % by weight to about 4×10 -1 % by weight, based on the layer. The concentrations of the fluorescent tracer and the antibody in the dry indicator layer are typically about 1×10 -5 % by weight to about 1×10 -1Weight % and approximately 1 x 10 -4 The range is from wt% to approximately 1 wt%. In one embodiment, the concentrations of the fluorescent tracer and antibody in the dried indicator layer are, respectively, approximately 2 × 10⁻⁶ of the layer. -5 Weight% ~ approx. 5×10 -2 Weight % and approximately 2 x 10 -4 Weight% ~ approx. 5×10 -1 This is in the range of weight percent. In one embodiment, the concentrations of the fluorescent tracer and antibody in the dry indicator layer are, respectively, about 4 × 10⁻⁶ of the layer. -5 Weight% ~ approx. 2.5×10 -2 Weight % and approximately 4 x 10 -4 Weight% ~ approx. 2.55×10 -1 This is in the range of weight percent. In one embodiment, the concentrations of the fluorescent tracer and antibody in the dry indicator layer are, respectively, about 1 × 10⁻⁶ of the layer. -4 Weight% ~ approx. 1×10 -2 Weight % and approximately 1 x 10 -3 Weight% ~ approx. 1×10 -1 It is within the range of weight percent.

[0148] The indicator layer is water-permeable. Suitable polymers for the indicator layer include, but are not limited to, cellulose and cellulose derivatives (e.g., hydroxypropylcellulose, methylcellulose, and hydroxypropylmethylcellulose), polysaccharides (such as dextran, gum arabic, agarose, and pullulan), gelatin and gelatin derivatives, polyvinyl alcohol, polyvinylpyrrolidone, acrylamide polymers, polyurethanes, alginates, xanthams, and mixtures thereof.

[0149] In one embodiment, the indicator layer contains a polysaccharide. In another embodiment, the indicator layer contains a linear polyglucan. In yet another embodiment, the polysaccharide contains maltotriose units. In a preferred embodiment, the indicator layer contains pullulan. While we do not wish to be constrained by theory, the polysaccharide is thought to act as a binder that provides stability and integrity to the indicator layer.

[0150] Polysaccharides are typically present in the wet indicator layer in amounts ranging from about 0.5% to about 40% by weight, preferably about 1% to about 30% by weight, more preferably about 5% to about 20% by weight, and most preferably about 6% to about 15% by weight. In the dry indicator layer, polysaccharides are typically present in amounts ranging from about 1% to about 60% by weight, preferably about 5% to about 50% by weight, more preferably about 10% to about 40% by weight, and most preferably about 20% to about 35% by weight.

[0151] Preferably, the indicator layer contains pullulan. In one embodiment, the indicator layer contains pullulan, cellulose, hydroxypropylcellulose, and mixtures thereof.

[0152] In one embodiment, the indicator layer comprises pullulan, cellulose, a surfactant, a buffer, and a fluorescent tracer and binding partner.

[0153] Pullulan is structured as follows:

[0154] [ka] It is a polysaccharide polymer consisting of maltotriose units having [a specific characteristic].

[0155] Pullulan is typically present in the wet indicator layer in an amount ranging from about 0.5% to about 40% by weight, preferably about 1% to about 30% by weight, more preferably about 5% to about 20% by weight, and most preferably about 6% to about 15% by weight. In the dry indicator layer, pullulan is typically present in an amount ranging from about 1% to about 60% by weight, preferably about 5% to about 50% by weight, more preferably about 10% to about 40% by weight, and most preferably about 20% to about 35% by weight.

[0156] While not wishing to be constrained by theory, pullulan is thought to provide stability to the indicator layer and, advantageously, act as a binder that does not result in the degradation of the complex of the fluorescent tracer and its binding partner, the fluorescent tracer, or the binding partner. Pullulan is a particularly advantageous polymer for forming indicator layers because it is water-soluble and therefore allows for the formation of indicator layers using solutions that are primarily aqueous and contain minimal amounts of organic solvents (e.g., ethanol) that could result in the degradation of the fluorescent tracer and / or binding partner. Pullulan stabilizes bioreagents such as proteins. In one embodiment, pullulan has a molecular weight of about 25 kDa.

[0157] The surfactant is optional. Preferably, the indicator layer contains a surfactant. Preferably, the surfactant is a nonionic surfactant. Exemplary surfactants include, but are not limited to, Igepal (commercially available from Sigma Aldrich in St. Louis, Missouri) or Merpol A (commercially available from Stepan Company in Northfield, Illinois). The surfactant is typically present in the wet indicator layer in an amount ranging from about 0.05% to about 2.0% by weight, preferably about 0.1% to about 1.0% by weight, more preferably about 0.2% to about 0.6% by weight, and most preferably about 0.3% to about 0.5% by weight. In the dry indicator layer, the surfactant is typically present in an amount ranging from about 0.1% to about 4.0% by weight, preferably about 0.2% to about 3.0% by weight, more preferably about 0.4% to about 2.5% by weight, and most preferably about 0.5% to about 2.0% by weight. While we do not wish to be constrained by theory, surfactants are thought to provide better wetting of the indicator layer, resulting in a more uniform layer.

[0158] Examples of buffers include, but are not limited to, phosphate-buffered saline (PBS) and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The buffer is typically present in the wetting indicator layer in an amount ranging from about 10 mM to about 300 mM, preferably about 25 mM to about 250 mM, more preferably about 50 mM to about 200 mM, and most preferably about 75 mM to about 175 mM. Preferably, the buffer is one that provides an aqueous solution pH value between about 4.5 and about 9.5, more preferably about 5 to about 9, and most preferably about 6 to about 8.5. In one embodiment, the buffer is one that provides an aqueous solution pH value of about 8.

[0159] Cellulose is typically present in the wet indicator layer in an amount ranging from about 2% to about 40% by weight, preferably about 5% to about 35% by weight, more preferably about 10% to about 30% by weight, and most preferably about 15% to about 25% by weight. In the dry indicator layer, cellulose is typically present in an amount ranging from about 30% to about 90% by weight, preferably about 40% to about 85% by weight, more preferably about 45% to about 85% by weight, and most preferably about 50% to about 75% by weight. In one embodiment, the cellulose is microcrystalline cellulose having a molecular weight of about 350 kDa. Preferably, the particle size of the cellulose is in the range of about 5 μm to about 30 μm, preferably about 10 μm to about 25 μm. In one embodiment, the particle size of the cellulose is about 20 μm. Although we do not wish to be constrained by theory, the amount and particle size of the cellulose polymer are considered important for maximizing the sensitivity of the method.

[0160] The indicator layer may further contain hydroxypropyl cellulose (HPC). If present, HPC is typically present in the wetted layer in an amount ranging from about 2% to about 10% by weight, preferably about 3% to about 7.5% by weight, and more preferably about 5% by weight. While not intended to be constrained by theory, it is thought that HPC increases the viscosity of the wetted layer to improve layer diffusion. It is also thought that HPC improves the integrity of the indicator layer.

[0161] In one embodiment, an indicator layer was formed by applying a solution / suspension containing approximately 18 wt% cellulose, approximately 9 wt% pullulan, approximately 0.4 wt% Merpol A, approximately 52 wt% HEPES aqueous solution (250 mM deionized water, pH 8), approximately 20 wt% antibody conjugated to a quencher (2.31 mg / mL in 250 mM HEPES buffer, pH 8), and approximately 1.5 wt% fluorescent tracer solution (0.25 mg / mL in deionized water) to a support layer and removing the solvent from the solution / suspension. The resulting dried indicator layer contained approximately 57.0 wt% cellulose, approximately 28.5 wt% pullulan, approximately 1.2 wt% Merpol A, approximately 0.14 wt% antibody conjugated to a quencher, and approximately 1.1 × 10⁶ -3 It contains wt% of a fluorescent tracer and about 13.2 wt% of HEPES buffer. In one embodiment, the fluorescent tracer has structure A3, the antibody is an antibody against SDMA, and the quencher is BHQ10. The antibody against SDMA is described, for example, in U.S. Patent No. 8,481,690.

[0162] The solution / suspension used to form the indicator layer may further contain an amount of preservative suitable for preventing bacterial growth in the solution / suspension. In one embodiment, the preservative is Proclin® 300 (MilliporeSigma, Burlington, Massachusetts) at a concentration of 0.05% by weight in the solution / suspension. Those skilled in the art will readily be able to identify other suitable preservatives.

[0163] Typically, the indicator layer is prepared by combining the components in a solvent, preferably an aqueous solvent, to allow them to be mixed. The order of addition is not particularly important. Generally, after adding each component, the resulting solution / suspension is mixed. Typically, the final solution is mixed for about 1 / 2 hour before coating it onto the support layer. The solution is then applied to the support layer, and the solvent is removed to provide the indicator layer on top of the support layer. The fluorescent tracer and binding partner can be added to the solution individually or as a pre-formed complex of the fluorescent tracer and binding partner. Preferably, the pre-formed complex of the fluorescent tracer and binding partner is added to the solution.

[0164] In one embodiment, the slide further includes a diffusion layer. The diffusion layer is coated on top of the indicator layer. The diffusion layer is water permeable. The diffusion layer is water permeable, isotropic, and porous, i.e., porous in all directions within the layer.

[0165] In one embodiment, the slide further includes a filtering layer, which is coated on top of the indicator layer.

[0166] In one embodiment, the slide further includes a diffusion layer and a filtering layer. When the slide further includes a diffusion layer and a filtering layer, the filtering layer is coated on top of the indicator layer, and then the diffusion layer is coated on top of the filtering layer. A general structure of a slide further including a diffusion layer and a filtering layer is shown in Figure 18B.

[0167] The diffusion layer and filtering layer are provided in a similar manner to the indicator layer, i.e., the components of the layer are mixed together in a solvent to obtain a solution / suspension, the solution / suspension is applied to the appropriate layer on the slide, and the solvent is removed to provide the diffusion layer or filtering layer on top of the appropriate layer.

[0168] The thickness of the wet diffusion layer is typically in the range of about 100 μm to about 500 μm, preferably about 150 μm to about 450 μm, more preferably about 200 μm to about 400 μm, and most preferably about 250 μm to about 350 μm. In the case of dry, the thickness of the diffusion layer is typically in the range of about 25 μm to about 250 μm, preferably about 50 μm to about 200 μm, and more preferably about 75 μm to about 150 μm. The thickness of the diffusion layer depends in part on the sample volume. For example, if the sample is a very dilute solution of the analyte, the assay requires a larger sample volume, and a thicker diffusion layer is preferred to accommodate the larger sample volume.

[0169] The diffusion layer is typically a mixture of cellulose in a hydrophilic polymer matrix. Suitable hydrophilic polymers include, but are not limited to, polyacrylic acid, polyvinylpyrrolidone, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyacrylamide, and polyethyleneimine. The diffusion layer advantageously allows for uniform diffusion and dispersion of the liquid sample on the slide.

[0170] The amount of cellulose in the wet diffusion layer is typically in the range of about 1% to about 20% by weight of the layer, preferably 5% to about 15% by weight, and more preferably about 10% by weight. In the dry state, the amount of cellulose in the wet diffusion layer is typically in the range of about 50% to about 98% by weight of the layer, preferably about 60% to about 95% by weight, and more preferably about 70% to about 90% by weight. In one embodiment, the diffusion layer is 100% cellulose. In one embodiment, the cellulose has a particle size of about 300 μm.

[0171] The diffusion layer may contain polyvinylpyrrolidone (PVP). The amount of PVP by weight in the wet diffusion layer is typically in the range of about 0.5% to about 10% by weight of the layer, preferably about 1% to about 5% by weight, and more preferably about 1.5% to about 3% by weight. In one embodiment, the amount of PVP in the wet diffusion layer is about 2% by weight. The amount of PVP by weight in the dry diffusion layer is typically in the range of about 1% to about 30% by weight of the layer, preferably about 5% to about 25% by weight, and more preferably about 10% to about 20% by weight. In one embodiment, the amount of PVP in the dry diffusion layer is about 17% by weight.

[0172] The diffusion layer may contain tetramethylammonium hydroxide (TMAH) or other suitable bases. TMAH may help adjust the pH of the diffusion layer. While we do not wish to be constrained by theory, TMAH is thought to increase the diffusion layer's ability to rapidly absorb and diffuse the sample across the entire slide. The amount of TMAH by weight in the wet diffusion layer is typically in the range of about 0.01% to about 0.5% by weight, preferably about 0.025% to about 0.25% by weight. In one embodiment, the amount of TMAH in the wet diffusion layer is about 0.05% by weight. The amount of TMAH by weight in the dry diffusion layer is typically in the range of about 0.08% to about 4% by weight, preferably about 0.2% to about 2% by weight, more preferably about 10% by weight. In one embodiment, the amount of TMAH in the dry diffusion layer is about 0.4% by weight.

[0173] In one embodiment, a solution / suspension containing about 10% by weight of cellulose, about 2% by weight of polyvinylpyrrolidone (PVP), about 68% by weight of water, about 20% by weight of ethanol, about 0.06% by weight of polyacrylic acid (PAA), and about 0.05% by weight of tetramethylammonium hydroxide (TMAH) is applied, and a diffusion layer is formed by removing the solvent from the solution / suspension. The resulting dry diffusion layer contains about 82.6% by weight of cellulose, about 16.5% by weight of PVP, about 0.5% by weight of PAA, and about 0.4% by weight of TMAH.

[0174] The filtering layer binds to compounds in the sample other than the analyte that could potentially interfere with the assay, preventing these other compounds from diffusing into the indicator layer.

[0175] The thickness of the wet filtering layer is typically in the range of about 50 μm to about 250 μm, preferably about 75 μm to about 225 μm, and more preferably about 100 μm to about 200 μm. In the dry case, the thickness of the filtering layer is typically in the range of about 5 μm to about 50 μm, preferably about 7 μm to about 40 μm, and more preferably about 10 μm to about 30 μm. The thickness of the filtering layer depends in part on the sample volume. For example, if the sample is a very dilute solution of the analyte, the assay requires a larger sample volume, and a thicker filtering layer is preferred to accommodate the larger sample volume.

[0176] The filtering layer typically comprises polyurethane in combination with another hydrophilic polymer(s) that retains moisture. In one embodiment, the filtering layer comprises polyurethane and cellulose. A suitable polyurethane is HydroMed D4 (commercially available from AdvanSource Biomaterials Corp. in Wilmington, Massachusetts). In one embodiment, the polyurethane is a mixture of low-viscosity HydroMed D4 and high-viscosity HydroMed D4. In another embodiment, the polyurethane is a mixture of approximately equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4.

[0177] The amount of polyurethane in the wet filtering layer is typically in the range of about 2% to about 40% by weight of the layer, preferably about 5% to about 30% by weight, and more preferably about 10% to about 20% by weight. In one embodiment, the amount of polyurethane in the wet filtering layer is about 15% by weight of the layer. When dry, the amount of polyurethane in the filtering layer is typically in the range of about 40% to about 95% by weight of the layer, preferably about 50% to about 80% by weight, and more preferably about 55% to about 75% by weight. In one embodiment, the amount of polyurethane in the dry filtering layer is about 65% by weight of the layer.

[0178] The amount of cellulose in the wet filtering layer is typically in the range of about 2% to about 60% by weight of the layer, preferably about 4% to about 50% by weight, and more preferably about 6% to about 40% by weight. In one embodiment, the amount of cellulose in the wet filtering layer is about 8% by weight. When dry, the amount of cellulose in the filtering layer is typically in the range of about 10% to about 60% by weight of the layer, preferably about 20% to about 50% by weight, and more preferably about 25% to about 45% by weight. In one embodiment, the amount of cellulose in the dry filtering layer is about 35% by weight of the layer.

[0179] Figure 24 shows the results of an assay to determine the concentration of SDMA in a sample containing a fixed amount of SDMA (μg / dL), where the sample contains compounds that may potentially interfere with the assay at various concentrations (i.e., interfering substances). (Using slides with a filtering layer containing approximately 20 wt% cellulose, approximately 42 wt% titanium dioxide, and approximately 38 wt% HydroMed D4, as described below.) The interfering substances are (A) hemolysis (0-500 mg / dL), (B) bilirubin (0-30 mg / dL), (C) intralipid (0-1000 mg / dL), and (D) whole blood (0-10%). The experiment is described in Example 39.

[0180] In one embodiment, the filtering layer further comprises titanium dioxide (TiO2). Typically, titanium dioxide is present in the wet filtering layer in an amount ranging from about 2% to about 30% by weight, preferably about 5% to about 20% by weight, and more preferably about 10% to about 20% by weight. In one embodiment, titanium dioxide is present in the wet filtering layer in an amount ranging from about 14% by weight. Typically, titanium dioxide is present in the dry filtering layer in an amount ranging from about 20% to about 60% by weight, preferably about 25% to about 55% by weight, and more preferably about 30% to about 50% by weight. In one embodiment, titanium dioxide is present in the wet filtering layer in an amount ranging from about 42% by weight.

[0181] The average particle size of titanium dioxide particles is typically less than about 10 μm, preferably less than about 5 μm. In one embodiment, the average particle size of titanium dioxide particles is about 0.35 μm.

[0182] If the slide includes a carbon black layer as described below, a filtering layer containing titanium dioxide is particularly advantageous because the carbon black layer absorbs scattered light. The titanium dioxide layer advantageously prevents the carbon black layer from absorbing scattered light and reflects light away from the carbon black layer back to the detector, resulting in improved sensitivity.

[0183] In one embodiment, a filtering layer is formed by applying a solution / suspension containing about 7 wt% cellulose, about 14 wt% titanium dioxide, and about 79 wt% D4 hydrogel solution (containing a mixture of about 16 wt% equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4, about 90 wt% ethanol, and about 10 wt% water), and removing the solvent from the solution / suspension. The resulting dried filtering layer contains about 20 wt% cellulose, about 42 wt% titanium dioxide, and about 38 wt% HydroMed D4.

[0184] In one embodiment, the slide further includes a primer layer. The primer layer is laminated on top of the support layer and positioned between the support layer and the indicator layer. A general structure of the slide, further including a diffusion layer, a filtering layer, and a primer layer in addition to the support layer and indicator layer, is shown in Figure 18C.

[0185] The primer layer advantageously facilitates the coating of the indicator layer onto the support layer. Although we do not wish to be constrained by theory, since the support layer is hydrophobic and the indicator layer is hydrophilic, the two are considered to be particularly incompatible as a result. The primer layer overcomes this incompatibility. The primer layer preferably contains a polyurethane such as HydroMed D4. In one embodiment, the polyurethane is a mixture of low-viscosity HydroMed D4 and high-viscosity HydroMed D4. In another embodiment, the polyurethane is a mixture of approximately equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4.

[0186] The thickness of the wet primer layer is typically in the range of about 5 μm to about 60 μm, preferably about 10 μm to about 55 μm, and more preferably about 20 μm to about 50 μm. In one embodiment, the wet thickness of the primer layer is about 40 μm. When dry, the thickness of the primer layer is typically in the range of about 1 μm to about 20 μm, preferably about 1.5 to about 15 μm, and more preferably about 2 μm to about 10 μm. In one embodiment, the dry thickness of the primer layer is about 4 μm.

[0187] In one embodiment, a primer layer is formed by applying a solution / suspension containing a mixture of approximately 10% by weight equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4 in a solvent of approximately 90% by weight of ethanol and approximately 10% by weight of water, and then removing the solvent from the solution / suspension. The resulting dried primer layer contains approximately 100% by weight of HydroMed D4.

[0188] In one embodiment, the slide further includes a carbon black layer. The carbon black layer is coated on top of the filter layer and positioned between the filter layer and the diffusion layer. The carbon black layer acts as a light barrier, advantageously filtering out stray light from the environment that interferes with the measurement of emitted light of a second wavelength. The carbon black layer also functions to bind to compounds present in the sample other than the analyte that could potentially interfere with the assay, preventing these other compounds from diffusing into the indicator layer. Thus, the function of the carbon black layer is similar to that of the filtering layer.

[0189] Carbon black can be replaced with other materials for filtering stray light, or used in combination with them. Other materials, though not limited to these, include, but are: black latex beads, black silica beads, activated carbon, and C 60 This may include graphene or other colored materials such as red latex beads.

[0190] The carbon black layer contains carbon black dispersed in a polymer. Suitable polymers for the carbon black layer include, but are not limited to, polyurethane (such as HydroMed D4), polyethylene oxide, silicone, polyvinyl alcohol, and polyacrylamide. In one embodiment, the polymer is HydroMed D4.

[0191] The amount of carbon black in the wet carbon black layer is typically in the range of about 1% to about 20% by weight of the layer, preferably about 1.5% to about 15% by weight, and more preferably about 2% to about 10% by weight. In one embodiment, the amount of carbon black in the wet carbon black layer is about 5% by weight of the layer. When dry, the amount of carbon black in the carbon black layer is typically in the range of about 10% to about 40% by weight of the layer, preferably about 15% to about 35% by weight, and more preferably about 20% to about 30% by weight. In one embodiment, the amount of carbon black in the wet carbon black layer is about 25% by weight of the layer.

[0192] The thickness of the wet carbon black layer is typically in the range of about 50 μm to about 300 μm, preferably about 75 μm to about 250 μm, and more preferably about 100 μm to about 200 μm. In one embodiment, the thickness of the wet carbon black is about 140 μm. When dry, the thickness of the carbon black layer is typically in the range of about 5 μm to about 30 μm, preferably about 7.5 μm to about 25 μm, and more preferably about 10 μm to about 20 μm. In one embodiment, the thickness of the wet carbon black is about 14 μm.

[0193] In one embodiment, a carbon black layer is formed by applying a solution / suspension containing about 4.7 wt% carbon black, about 95.3 wt% D4 hydrogel solution (containing a mixture of about 16 wt% equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4, about 90 wt% ethanol, and about 10 wt% water), and removing the solvent from the solution / suspension. The resulting dried carbon black layer contains about 24 wt% carbon black and about 76 wt% HydroMed D4.

[0194] As described above, if the slide contains a carbon black layer, the slide preferably contains titanium oxide in the filtering layer.

[0195] Alternatively, if the slide contains a carbon black layer rather than titanium dioxide in the filtering layer, the slide may include a separate titanium dioxide layer positioned between the indicator layer and the filtering layer, or between the filtering layer and the carbon black layer. If the slide includes a separate titanium dioxide layer, the thickness of the wet titanium dioxide layer is typically in the range of about 50 μm to about 250 μm, preferably about 75 μm to about 225 μm, and more preferably about 100 μm to about 200 μm. In the dry case, the thickness of the titanium dioxide layer is typically in the range of about 5 μm to about 50 μm, preferably about 7 μm to about 40 μm, and more preferably about 10 μm to about 30 μm.

[0196] The titanium dioxide layer contains titanium dioxide dispersed in a polymer. Suitable polymers for the titanium dioxide layer include, but are not limited to, polyurethane polymers (e.g., HydroMed D4), polyethylene oxide, silicone, polyvinyl alcohol, and polyacrylamide. In one embodiment, titanium dioxide is dispersed in a polyurethane polymer such as HydroMed D4. In one embodiment, the polyurethane is a mixture of low-viscosity HydroMed D4 and high-viscosity HydroMed D4. In one embodiment, the polyurethane is a mixture of approximately equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4.

[0197] If the slide contains a separate titanium dioxide layer, titanium dioxide is typically present in the wet titanium dioxide layer in an amount ranging from about 2% to about 30% by weight, preferably about 5% to about 25% by weight, and more preferably about 10% to about 20% by weight. In one embodiment, titanium dioxide is present in the wet layer in an amount ranging from about 14% by weight. Typically, titanium dioxide is present in the dry layer in an amount ranging from about 20% to about 60% by weight, preferably about 25% to about 55% by weight, and more preferably about 30% to about 50% by weight. In one embodiment, titanium dioxide is present in the dry layer in an amount ranging from about 42% by weight.

[0198] The average particle size of titanium oxide particles is typically less than about 20 μm, preferably less than about 15 μm, more preferably less than about 10 μm, and most preferably less than about 5 μm.

[0199] Titanium oxide may be replaced with other reflective materials, or may be used in combination therewith. Exemplary other reflective materials include, but are not limited to, barium sulfate, zinc oxide, clay, and aluminum silicate. In one embodiment, the reflective material is fully or partially metal-coated particles. Suitable metals include, but are not limited to, aluminum, gold, nickel or silver. Silver is a preferred coating. These particles can be used alone or in combination with other light-reflective materials such as TiO₂. The particles (i.e., the core to be coated) can be various materials including, but not limited to, solid and hollow glass, poly(methyl methacrylate) (PMMA), and silica. Suitable metal-coated particles are commercially available, for example, from Cospheric LLC (Santa Barbara, California, USA). Preferred particles are, for example, silver-coated silica microspheres manufactured by Cospheric LLC.

[0200] In another embodiment, the slide does not include a separate carbon black layer. Rather, carbon black is included in the diffusion layer. The amount of carbon black included in the diffusion layer is similar to the amount of carbon black included in a separate carbon black layer.

[0201] A slide is prepared by starting with the support layer and sequentially coating each layer on top of the support layer. Components of each layer are dissolved or suspended in a suitable solvent to obtain a solution or suspension, then the obtained solution or suspension is applied onto the previous layer to obtain a desired wet layer, and the solvent is removed to provide a desired dry layer. Preferably, the solution is an aqueous solution. Any coating method can be used to provide each layer of the slide at a desired thickness. Suitable coating techniques are described in "Liquid Film Coating:Scientific principles and their technological implications", edited by Stephan F. Kistler and Peter M. Schweizer, 1st edition, copyright 1997 Springer Science+Business Media Dordrecht.

[0202] Typically, the thickness of the slide does not exceed about 300 μm, preferably, the thickness of the slide does not exceed about 250 μm, more preferably, the thickness of the slide does not exceed about 200 μm. In one embodiment, the thickness of the slide is about 185 μm.

[0203] Figure 19 shows an exemplary embodiment of the slide.

[0204] Figure 19A shows an embodiment in which the Melinex support layer is sequentially coated with a primer layer, an indicator layer, a filtering layer including titanium dioxide, a carbon black layer, and a diffusion layer. In one embodiment, as shown in Figure 19A, an aqueous ethanol solution / suspension containing about 10 wt% cellulose, about 2 wt% polyvinylpyrrolidone (PVP), about 0.1 wt% tetramethylammonium hydroxide (TMAH), about 0.01 wt% polyacrylic acid (PAA), about 68 wt% water, and 20 wt% ethanol is applied, and then the water and ethanol are removed from the solution / suspension to form a diffusion layer. The wet diffusion layer has a thickness of about 310 μm. The resulting dry diffusion layer contains about 82.6 wt% cellulose, about 16.5 wt% PVP, about 0.42 wt% TMAH, and about 0.46 wt% PAA. The dry diffusion layer has a thickness of about 100 μm.

[0205] An aqueous solution / suspension containing approximately 95 wt% D4 hydrogel solution (containing a mixture of approximately 16 wt% equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4 dissolved / suspended in a solvent of approximately 90 wt% ethanol and approximately 10 wt% water) and approximately 5 wt% carbon black is applied, and a carbon black layer is formed by removing the solvent from the solution / suspension. The wet carbon black layer has a thickness of approximately 140 μm. The resulting dry carbon black layer contains approximately 76.3 wt% HydroMed D4 and 23.7 wt% carbon black. The dry carbon black layer has a thickness of approximately 14 μm.

[0206] A filtering layer is formed by applying a solution / suspension containing approximately 79 wt% D4 hydrogel solution (containing a mixture of approximately 16 wt% equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4 dissolved / suspended in a solvent of approximately 90 wt% ethanol and approximately 10 wt% water), approximately 7 wt% cellulose, and approximately 14 wt% titanium dioxide, and removing the solvent from the solution / suspension. The wet filtering layer has a thickness of approximately 130 μm. The resulting dry filtering layer contains approximately 37.9 wt% HydroMed D4, approximately 20.0 wt% cellulose, and approximately 42.1 wt% titanium dioxide. The dry filtering layer has a thickness of approximately 20 μm.

[0207] Approximately 18.7 wt% cellulose, approximately 9.3 wt% pullulan, approximately 0.4 wt% Merpol A, approximately 4.3 wt% HEPES buffer (added as a 52 wt% 250 mM aqueous solution), approximately 4.0 × 10 -4 An indicator layer is formed by applying an aqueous solution / suspension containing wt% of a fluorescent tracer and approximately 0.04 wt% of antibody conjugated to a quencher, and then removing the solvent from the solution / suspension. The wet indicator layer has a thickness of approximately 93 μm. The resulting dry indicator layer is composed of approximately 57.0 wt% cellulose, approximately 28.5 wt% pullulan, approximately 1.2 wt% Merpol A, approximately 13.2 wt% HEPES buffer, approximately 0.14 wt% antibody conjugated to a quencher, and approximately 1.1 × 10⁻¹ -3 It contains a fluorescent tracer by weight. The dried indicator layer has a thickness of approximately 30-40 μm. In one embodiment, the fluorescent tracer is A4, and the antibody is an antibody against SDMA conjugated to BHQ10, which is obtained by combining A4 with 20 equivalents of BHQ10.

[0208] A primer layer is formed by applying an aqueous solution / suspension containing approximately 10% by weight of D4 hydrogel (containing equal amounts of low-viscosity HydroMed D4 and high-viscosity HydroMed D4) dissolved / suspended in approximately 90% by weight of ethanol and approximately 10% by weight of water. The wet primer layer has a thickness of approximately 40 μm. The dry primer layer is 100% by weight of HydroMed D4. The dry primer layer has a thickness of approximately 4 μm.

[0209] Figure 19B shows an embodiment in which the Melinex support layer is sequentially coated with a primer layer, an indicator layer, a titanium dioxide-containing layer, a filtering layer, a carbon black layer, and a diffusion layer. In one embodiment, as shown in Figure 19B, an aqueous solution / suspension containing about 10 wt% cellulose, about 1 wt% PVP, about 1 wt% TMAH, about 0.01 wt% PAA, and about 88 wt% water is applied, and the diffusion layer is formed by removing the water from the solution / suspension. The wet diffusion layer has a thickness of about 350 μm. The resulting dry diffusion layer contains about 83.2 wt% cellulose, about 8.3 wt% PVP, about 8.3 wt% TMAH, and about 0.1 wt% PAA.

[0210] A carbon black layer is formed by applying a 10% solution of HydroMed D4 (in 90% water and 10% ethanol) containing approximately 95% by weight and a solution / suspension containing approximately 5% by weight of carbon black, and then removing the solvent from the solution / suspension. The wet carbon black layer has a thickness of approximately 250 μm. The resulting dry carbon black layer contains approximately 65.5% by weight of HydroMed D4 and 34.5% by weight of carbon black.

[0211] A filtering layer is formed by applying an aqueous solution / suspension containing approximately 95% by weight of HydroMed D4 (in 90% by weight of water and 10% by weight of ethanol) and approximately 5% by weight of cellulose, and removing the solvent from the solution / suspension. The wet filtering layer has a thickness of approximately 250 μm. The resulting dry filtering layer contains approximately 65.5% by weight of HydroMed D4 and approximately 34.5% by weight of cellulose.

[0212] A titanium dioxide-containing layer is formed by applying a 9.13% solution of HydroMed D4 (in 90% water and 10% ethanol) containing approximately 95% by weight and an aqueous / suspension containing approximately 5% by weight of titanium dioxide, and removing the solvent from the solution / suspension. The wet titanium dioxide-containing layer has a thickness of approximately 100 μm. The resulting dry titanium dioxide-containing layer contains approximately 63.5% by weight of HydroMed D4 and approximately 36.5% by weight of titanium dioxide.

[0213] An indicator layer was formed by applying an aqueous solution / suspension containing approximately 14 wt% cellulose, approximately 14 wt% pullulan, approximately 0.1 wt% Igepal CA-630, approximately 58 wt% aqueous PBS solution (100 mM), and approximately 14.5 wt% of the complex between the fluorescent tracer and its binding partner, and removing the solvent from the solution / suspension. The wet indicator layer has a thickness of approximately 100 μm. The resulting dry indicator layer contains approximately 32.8 wt% cellulose, approximately 32.8 wt% pullulan, approximately 0.2 wt% Igepal CA-630, approximately 34.0 wt% of the complex, and phosphate buffer. In one embodiment, the complex is a complex of A4 and an antibody against SDMA conjugated with BHQ10, obtained by combining the antibody with 4 equivalents of BHQ10.

[0214] The wet primer layer is approximately 35 μm thick. The dry primer layer is 100% HydroMed D4.

[0215] The assay is performed by simply adding the sample containing the target analyte to the top of the slide (i.e., the slide layer furthest from the support layer), illuminating the slide with light of a first wavelength from the bottom of the slide (i.e., the slide layer with the support layer), and measuring the intensity of the light emitted from the bottom of the slide at a second wavelength. Figure 20 shows the assay method using the slide shown in Figure 18C.

[0216] Typically, the sample is added to the top of the slide as a liquid containing the analyte dissolved within it. Typically, the sample size is in the range of approximately 0.5 μL to approximately 30 μL. In one embodiment, the sample size is in the range of approximately 1 μL to approximately 20 μL. In one embodiment, the sample size is in the range of approximately 2 μL to approximately 15 μL. In one embodiment, the sample size is in the range of approximately 3 μL to approximately 12 μL.

[0217] In one embodiment, the slide further includes a “pop-off” layer. The pop-off layer contains a reagent that releases the analyte when the analyte complexes with another molecule, such as a binding protein. The analyte (e.g., cortisol) often complexes with a binding protein (e.g., cortisol-binding protein). The pop-off layer contains a reagent that releases the analyte from the binding protein. In one embodiment, the reagent that releases the analyte from the binding protein is sarcosyl. The structure of sarcosyl is,

[0218] [ka] Sarcosyl is particularly useful when the analyte is cortisol. Other reagents that can potentially be used to release cortisol from cortisol-binding proteins include prednisolone, prednisone, 11-deoxycorticosterone, cortisone, and 11-deoxycortisol. In another embodiment, the reagent for releasing the analyte from the binding protein is doxart (dioctyl sodium sulfosuccinate). Sarcosyl is preferred when the analyte is cortisol.

[0219] In one embodiment, the slide does not include an additional pop-off layer, but one or more of the layers already present on the slide, for example a diffusion layer, a filtering layer, and / or an indicator layer, comprises a reagent that releases an analyte from a binding protein.

[0220] Illustrative examples of slide configurations comprising a reagent that releases an analyte from a binding protein are described below. These embodiments are particularly useful when the analyte is cortisol.

[0221] In one embodiment, shown in FIG. 57, a support layer (e.g., Melinex) is sequentially coated with a primer layer, an indicator layer (comprising a fluorescent tracer and a binding partner), a filtering layer (optionally comprising titanium dioxide or other reflective material), a pop-off layer, and a diffusion layer.

[0222] In one embodiment, the primer layer is formed by applying a solution / suspension containing about 9 to 10% HydroMed D4 (commercially available from AdvanSource Biomaterials Corp, Wilmington, Massachusetts, USA) to the support layer and removing the solvent from the solution / suspension; the indicator layer is formed by applying a solution / suspension containing about 12% polyvinylpyrrolidone, about 6% cellulose, about 1% Tween-20, a fluorescent tracer and a binding partner to the primer layer and removing the solvent from the solution / suspension; the filtering layer is formed by applying a solution / suspension containing about 10% HydroMed D4, about 10% TiO2, and about 5% cellulose to the detection layer and removing the solvent from the solution / suspension; the pop-off layer is formed by applying a solution / suspension containing about 12% pullulan, about 6% cellulose, about 1% reagent that releases an analyte from a binding protein, and about 1% Merpol A to the filtering layer and removing the solvent from the solution / suspension; and the diffusion layer is formed by applying a solution / suspension containing components suitable for a diffusion layer to the pop-off layer and removing the solvent from the solution / suspension.

[0223] In one embodiment, the reagent that releases the analyte from the binding protein is sarcosyl. In another embodiment, the analyte is cortisol, the binding partner is an antibody against cortisol, and the reagent that releases the analyte from the binding protein is sarcosyl.

[0224] In one embodiment, as shown in Figure 58, a support layer (e.g., Melinex) is sequentially coated with a primer layer, a detection layer, a filtering layer (optionally containing titanium dioxide or other reflective material), an indicator layer, and a diffusion layer. In this embodiment, the diffusion layer contains a reagent that releases the analyte from the binding protein.

[0225] In one embodiment, a primer layer is formed by applying a solution / suspension containing approximately 9-10% HydroMed D4 (commercially available from AdvanSource Biomaterials Corp., Wilmington, Massachusetts) to a support layer and removing the solvent from the solution / suspension; a detection layer is formed by applying a solution / suspension containing approximately 12% polyvinylpyrrolidone, approximately 6% cellulose, and approximately 1% Tween-20 to the primer layer and removing the solvent from the solution / suspension; a filtering layer is formed by applying a solution / suspension containing approximately 10% HydroMed D4, approximately 10% TiO2, and approximately 5% cellulose to the detection layer and removing the solvent from the solution / suspension; an indicator layer is formed by applying a solution / suspension containing approximately 12% pullulan, approximately 6% cellulose, approximately 1% Tween-20, and a fluorescent tracer and binding partner to the filtering layer and removing the solvent from the solution / suspension; and approximately 1% of a reagent to release the analyte from the binding protein, and approximately 1% Merpol A solution / suspension containing A and appropriate diffusion layer components is applied to the indicator layer, and the solvent is removed from the solution / suspension to form the diffusion layer.

[0226] In one embodiment, the reagent that releases the analyte from the binding protein is sarcosyl. In another embodiment, the analyte is cortisol, the binding partner is an antibody against cortisol, and the reagent that releases the analyte from the binding protein is sarcosyl.

[0227] In another embodiment, as shown in Figure 58, the indicator layer contains a reagent that releases the analyte from the binding protein.

[0228] In one embodiment, a primer layer is formed by applying a solution / suspension containing about 9-10% HydroMed D4 to a support layer and removing the solvent from the solution / suspension; a detection layer is formed by applying a solution / suspension containing about 12% polyvinylpyrrolidone, about 6% cellulose, and about 1% Tween-20 to the primer layer and removing the solvent from the solution / suspension; a filtering layer is formed by applying a solution / suspension containing about 10% HydroMed D4, about 10% TiO2, and about 5% cellulose to the detection layer and removing the solvent from the solution / suspension; an indicator layer is formed by applying a solution / suspension containing about 12% pullulan, about 6% cellulose, about 1% sarcosyl, about 1% Merpol A, as well as a fluorescent tracer and binding partner to the filtering layer and removing the solvent from the solution / suspension; and a diffusion layer is formed by applying a solution / suspension containing appropriate diffusion layer components to the indicator layer and removing the solvent from the solution / suspension.

[0229] In one embodiment, the reagent that releases the analyte from the binding protein is sarcosyl. In another embodiment, the analyte is cortisol, the binding partner is an antibody against cortisol, and the reagent that releases the analyte from the binding protein is sarcosyl.

[0230] In another embodiment, as shown in Figure 58, a reagent for releasing the analyte from the binding protein is included in both the diffusion layer and the detection layer. In one embodiment, a primer layer is formed by applying a solution / suspension containing about 9-10% HydroMed D4 to the support layer and removing the solvent from the solution / suspension; a detection layer is formed by applying a solution / suspension containing about 12% polyvinylpyrrolidone, about 6% cellulose, and about 1% Tween-20 to the primer layer and removing the solvent from the solution / suspension; a filtering layer is formed by applying a solution / suspension containing about 10% HydroMed D4, about 10% TiO2, and about 5% cellulose to the detection layer and removing the solvent from the solution / suspension; an indicator layer is formed by applying a solution / suspension containing about 12% pullulan, about 6% cellulose, about 0.5% sarcosyl, about 0.5% Merpol A, and a fluorescent tracer and binding partner to the filtering layer and removing the solvent from the solution / suspension; and appropriate diffusion layer components, about 0.5% sarcosyl, and about 0.5% Merpol A diffusion layer is formed by applying a solution / suspension containing A to an indicator layer and removing the solvent from the solution / suspension.

[0231] In one embodiment, the reagent that releases the analyte from the binding protein is sarcosyl. In another embodiment, the analyte is cortisol, the binding partner is an antibody against cortisol, and the reagent that releases the analyte from the binding protein is sarcosyl.

[0232] In a further embodiment, the primer layer and the detection layer are combined. A solution / suspension containing approximately 9-10% HydroMed D4 and approximately 10% cellulose is applied to the support layer, and the combined primer / detection layer is formed by removing the solvent from the solution / suspension. In one embodiment, the primer layer and detection layer of the slide shown in Figure 58 are combined.

[0233] In some embodiments, one or more layers may contain glass beads. To form a layer containing glass beads, the solution / suspension used to form this layer contains approximately 6% glass beads. In preferred embodiments, the glass beads are included in the indicator layer and / or filtering layer.

[0234] One or more layers may contain Merpol A. The addition of Merpol A is preferable when the reagent for liberating the analyte is sarcosyl. When sarcosyl is used as the reagent for liberating the analyte, it is preferable to include Merpol A in at least the layer containing sarcosyl.

[0235] The sample may be any of the above-mentioned samples, but is not limited to, urine, serum, milk, saliva, plasma, whole blood, sweat, tears, feces, and spinal fluid.

[0236] The analytes may include, but are not limited to, any of the above analytes, including SDMA, melamine, antibiotics, T4, β-lactam antibiotics (such as penicillin), sulfonamides, cephalosporins, progesterone, cortisol, bile acids, proteins, NT-proBNP, and cystatin-B.

[0237] In one embodiment, a dry slide is part of a device that applies a liquid sample containing the analyte of interest to an opening in the device and then transports the liquid to the dry slide via a capillary transport zone. Exemplary examples of such devices are described in U.S. Patents 4,323,536 and 5,726,010. The slide may be part of a device in which the slide is located within an opening or cavity defined by a frame, housing, or case. In one embodiment, the slide is located within a housing or case as disclosed in U.S. Patent 9,933,428. Other examples of dry slide devices including a housing or case are Catalyst® slides, e.g., Catalyst® Fructosamine slides or Catalyst® Total T4 (TT4) slides (commercially available from IDEXX Laboratories, Inc., Westbrook, Maine).

[0238] In one embodiment, the assay is performed using a dry chemical analyzer, such as a Catalyst One® or Catalyst Dx® analyzer (commercially available from IDEXX Laboratories, Inc. in Westbrook, Maine).

[0239] 4. Fluorescent tracer A fluorescent tracer can be obtained using any molecule that emits fluorescence and can be linked to the T-epitope. Suitable fluorescent molecules, but not limited to, include fluorescein, coumarin, and rhodamine dyes.

[0240] In one embodiment, the fluorescent tracer has a T-epitope moiety linked to a fluorescein molecule. Preferably, the T-epitope moiety is linked to the 4' or 5' position of the fluorescein molecule. More preferably, the T-epitope moiety is linked to the 4' position of the fluorescein molecule.

[0241] Specifically, the present invention aims to provide a 4'-substituted fluorescein tracer, i.e., a fluorescent tracer in which the T-epitope is attached to the 4' position of the fluorescein molecule. The general structure of a 4'-substituted fluorescein tracer is:

[0242] [ka] (In the formula, T is a bonding or linking group.) That is the case.

[0243] The present invention also aims to provide 4'-substituted fluorescein tracer derivatives. The phrase “4'-substituted fluorescein tracer derivative” means, as used herein, a 4'-substituted fluorescein tracer molecule in which one or more hydrogen atoms bonded to the carbon atoms of the fluorescein core structure are replaced by another functional group. In one embodiment, one or more hydrogen atoms bonded to the carbon atoms of the fluorescein core structure are replaced by electron-donating groups such as, but not limited to, -CH3, -OCH3, and -OH. In one embodiment, one or more hydrogen atoms bonded to the carbon atoms of the fluorescein core structure are replaced by electron-withdrawing groups such as, but not limited to, -F, -NO2, -CN, -COOH, -SO3H, and -Cl. In one embodiment, the functional group replaces the hydrogen atoms at the 2' and / or 7' positions of the fluorescein molecule.

[0244] Exemplary 4'-substituted fluorescein tracer derivatives are,

[0245] [ka] That is the case.

[0246] The present invention also envisions a 5-substituted fluorescein tracer, i.e., a fluorescein tracer in which the T-epitope moiety is attached to the 5-position of the fluorescein molecule. The general structure of a 5-substituted fluorescein tracer is:

[0247] [ka] That is the case.

[0248] The present invention also aims to provide 5-substituted fluorescein tracer derivatives. The phrase “5-substituted fluorescein tracer derivative” means, as used herein, a 5-substituted fluorescein tracer molecule in which one or more hydrogen atoms bonded to the carbon atoms of the fluorescein core structure are replaced by another functional group. In one embodiment, one or more hydrogen atoms bonded to the carbon atoms of the fluorescein core structure are replaced by electron-donating groups such as, but not limited to, -CH3, -OCH3, and -OH. In one embodiment, one or more hydrogen atoms bonded to the carbon atoms of the fluorescein core structure are replaced by electron-withdrawing groups such as, but not limited to, -F, -NO2, -CN, -COOH, -SO3H, and -Cl. In one embodiment, the functional group replaces the hydrogen atoms at the 2' and / or 7' positions of the fluorescein molecule.

[0249] Exemplary 5-substituted fluorescein tracer derivatives are,

[0250] [ka] That is the case.

[0251] The epitope portion may be directly bonded to the fluorescein core structure, or it may be separated from the fluorescein core structure by a linker T. Generally, the epitope portion is separated from the fluorescein core structure by a linker. Generally, the linker is less than 8 atomic lengths, preferably less than 6 atomic lengths, more preferably less than 4 atomic lengths, and most preferably less than 2 atomic lengths. In one embodiment, the linker is 1 atomic length.

[0252] In one embodiment, the fluorescent tracer has a T-epitope moiety linked to a coumarin molecule.

[0253] Examples of linkers include, but are not limited to, -CH2-, -C(O)-, -CH2-NH-CH2-CH2-, -NH-CH2-CH2-, -CH2-NH-C(O)-CH2CH2-C(O)-, -NH-, -CH2-NH-, -CH2N(CH3)-, and -NH-C(O)-CH2CH2-C(O)-.

[0254] In one embodiment, a 4'-substituted fluorescein tracer is obtained by functionalizing the 4' position of fluorescein with an aldehyde group, as shown below, and then reacting this with a functional group (such as an amine) on the epitope moiety to obtain a 4'-substituted fluorescein tracer in which the linking group is a -CH2- moiety:

[0255] [ka]

[0256] In another embodiment, the aldehyde group is converted to another functional group using chemistry well known to those skilled in the art, as shown in Figure 2, and then the resulting 4'-substituted fluorescein molecule is reacted with a functional group on the epitope moiety, such as an amine or carboxylic acid group, using chemistry well known to those skilled in the art, to obtain a 4'-substituted fluorescein tracer.

[0257] In another embodiment, as shown below, an aldehyde is converted to a carboxylic acid, the resulting carboxylic acid is reacted with N-hydroxysuccinimide, and then the resulting N-hydroxysuccinimide ester is reacted with a functional group (such as an amine) on the epitope moiety to obtain a 4'-substituted fluorescein tracer in which the linking group is the -C(O)- moiety:

[0258] [ka] Suitable reagents for converting aldehydes to carboxylic acids include, but are not limited to, potassium permanganate and sodium dichromate.

[0259] In another embodiment, as shown below, the amine group on the epitope moiety is reacted with 2,2-dimethoxyacetaldehyde to obtain an acetal-derivative epitope moiety, the acetal group of the acetal-derivative epitope moiety is converted to an aldehyde, and the resulting aldehyde-derivative epitope moiety is then condensed with a fluorescein derivative whose 4' position is substituted with a -CH2-NH2- or -NH2 group to obtain a 4'-substituted fluorescein tracer whose linking group is a -CH2NHCH2CH2- or -NHCH2CH2- moiety:

[0260] [ka]

[0261] A fluorescein molecule substituted at the 4' position with a -NH2 group can be prepared by first protecting the carboxylic acid group at the 3 position of fluorescein (prepared as described in Example 1) which is substituted at the 4' position with an aldehyde group (e.g., as an ester), and then converting the aldehyde group to a carboxylic acid to obtain fluorescein having a carboxylic acid group at the 4' position and a protected carboxylic acid at the 3 position. Next, the carboxylic acid at the 4' position is converted to an acid chloride (e.g., by reaction with thionyl chloride), the acid chloride is reacted with ammonia to obtain an amide, and the amide is reacted with Br2 / NaOH to obtain an amine (i.e., Hofmann-bromoamide decomposition). Then, the acid protecting group is removed. A general reaction scheme is shown below.

[0262] [ka] [ka]

[0263] In another embodiment, the amine group on the epitope molecule is reacted with succinic anhydride (dihydrofuran-2,5-dione) to obtain a -C(O)-CH2CH2C(O)OH derivatized epitope moiety, then the derivatized epitope moiety is reacted with N-hydroxysuccinimide to obtain an anhydride, and then the anhydride is reacted with a fluorescein derivative whose 4' position is substituted with a -CH2-NH2- group or an -NH2 group to obtain a 4'-substituted fluorescein tracer whose linking group is -CH2NHC(O)CH2CH2C(O)- or -NHC(O)CH2CH2C(O)-:

[0264] [ka]

[0265] In another embodiment, as shown below, an acid group on the epitope molecule is reacted with N-hydroxysuccinimide to obtain an anhydride, and then the anhydride is reacted with a fluorescein derivative whose 4' position is substituted with a -CH2-NH2- group (commercially available from AAT Bioquest, Sunnyvale, California) or an -NH2 group to obtain a 4'-substituted fluorescent tracer whose linking group is a -CH2-NH2- group or an -NH2 group:

[0266] [ka]

[0267] When synthesizing 4'-substituted fluorescein tracers, it is understood that conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. The selection of suitable protecting groups for specific functional groups, as well as suitable conditions for protection and deprotection, is known in the art. For example, numerous protecting groups, as well as their introduction and removal, are described in TW Greene and PGMWuts, *Protecting Groups in Organic Synthesis*, 2nd edition, Wiley, New York, 1991, and the references cited therein.

[0268] Similar chemistry can be used to obtain 4'-substituted fluorescein tracer derivatives.

[0269] In one embodiment, the epitope molecule having an -NH2 group is symmetric dimethylarginine (SDMA). The structure of SDMA is:

[0270] [ka] That is the case.

[0271] In one embodiment, the present invention has the following structure:

[0272] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) This invention relates to a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative having [a specific characteristic].

[0273] In one embodiment, the present invention relates to a conjugate comprising a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative selected from the structures identified above, and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0274] In one embodiment, the present invention has the following structure:

[0275] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) This invention relates to a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative having [a specific characteristic].

[0276] In one embodiment, the present invention relates to a conjugate comprising a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative selected from the structures identified above, and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0277] In one embodiment, the present invention has the following structure:

[0278] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) This invention relates to a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative having [a specific characteristic].

[0279] In one embodiment, the present invention relates to a conjugate comprising a 4'-substituted fluorescein tracer or a 4'-substituted fluorescein tracer derivative selected from the structures identified above, and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0280] In one embodiment, the present invention has the following structure:

[0281] [ka] This relates to a 4'-substituted fluorescein tracer having the following properties.

[0282] In one embodiment, the present invention has the following structure:

[0283] [ka] This relates to a 4'-substituted fluorescein tracer having the following properties.

[0284] In one embodiment, the present invention has the following structure:

[0285] [ka] This relates to a 4'-substituted fluorescein tracer having the following properties.

[0286] In one embodiment, the present invention relates to a complex comprising a 4'-substituted tracer of structure A1 and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0287] In one embodiment, the present invention relates to a complex comprising a 4'-substituted tracer of structure A2 and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0288] In one embodiment, the present invention relates to a complex comprising a 4'-substituted tracer of structure A3 and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0289] Similarly, a 5-substituted fluorescein tracer can be obtained by functionalizing the 5-position of fluorescein with an aldehyde group, and then reacting this with a functional group (such as an amine) on the epitope moiety to obtain a 5-substituted fluorescein tracer in which the linking group is a -CH2- moiety, as shown below:

[0290] [ka]

[0291] Fluorescein functionalized at the 5th position with an aldehyde group can be obtained by reducing the carboxylic acid group of a fluorescein molecule substituted at the 5th position with a carboxylic acid group to an alcohol (-CH2OH), and then oxidizing the resulting alcohol to an aldehyde. Fluorescein functionalized at the 5th position with a carboxylic acid group is commercially available from Thermo Scientific in Waltham, Massachusetts.

[0292] In one embodiment, the present invention has the following structure:

[0293] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) This invention relates to a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative having [a specific characteristic].

[0294] In one embodiment, the present invention relates to a complex comprising a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative selected from the structures identified above, and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0295] In one embodiment, the present invention has the following structure:

[0296] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) This invention relates to a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative having [a specific characteristic].

[0297] In one embodiment, the present invention relates to a complex comprising a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative selected from the structures identified above, and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0298] In one embodiment, the present invention has the following structure:

[0299] [ka] (In the formula, X is selected from the group consisting of -H, -F, -CH3, -OCH3, -Cl, -OH, -NO2, -CN, -COOH, and -SO3H) This invention relates to a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative having [a specific characteristic].

[0300] In one embodiment, the present invention relates to a complex comprising a 5-substituted fluorescein tracer or a 5-substituted fluorescein tracer derivative selected from the structures identified above, and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0301] In one embodiment, the present invention relates to a 5-substituted fluorescein tracer of structure A4.

[0302] [ka]

[0303] In one embodiment, the present invention relates to a 5-substituted fluorescein tracer of structure A5.

[0304] [ka]

[0305] In one embodiment, the present invention has structure A6:

[0306] [ka] Regarding the 5-substituted fluorescein tracer.

[0307] In one embodiment, the present invention relates to a complex comprising a 5-substituted fluorescein tracer of structure A4 and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0308] In one embodiment, the present invention relates to a complex comprising a 5-substituted fluorescein tracer of structure A5 and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0309] In one embodiment, the present invention relates to a complex comprising a 5-substituted fluorescein tracer of structure A6 and an antibody against SDMA. In one embodiment, the antibody is conjugated to a quencher.

[0310] An exemplary 4'-substituted fluorescein tracer that can be used in assays for melamine is:

[0311] [ka] That is the case.

[0312] In one embodiment, the present invention relates to a complex comprising Mel-F or Mel-Su-F and an antibody against melanin. In one embodiment, the antibody is conjugated to a quencher.

[0313] An exemplary 4'-substituted fluorescein tracer that can be used in assays for biotin is:

[0314] [ka] That is the case.

[0315] In one embodiment, the present invention relates to a complex comprising biotin-F and an antibody against biotin. In one embodiment, the antibody is conjugated to a quencher.

[0316] An exemplary 4'-substituted fluorescein tracer that can be used in assays for thyroxine is:

[0317] [ka] That is the case.

[0318] In one embodiment, the present invention relates to a complex comprising T2-F, T3-F, T4-F, or T3-EF and an antibody against thyronine. In one embodiment, the antibody is conjugated to a quencher.

[0319] An exemplary 4'-substituted fluorescein tracer that can be used in assays for amoxicillin is:

[0320] [ka] That is the case.

[0321] In one embodiment, the present invention relates to a conjugate comprising AMO-F and an antibody against amoxicillin. In one embodiment, the antibody is conjugated to a quencher.

[0322] An exemplary 4'-substituted fluorescein tracer that can be used in assays for ampicillin is:

[0323] [ka] That is the case.

[0324] In one embodiment, the present invention relates to a conjugate comprising AMP-F and an antibody against ampicillin. In one embodiment, the antibody is conjugated to a quencher.

[0325] An example of a 4'-substituted fluorescein tracer that can be used in assays for cefotaxime is:

[0326] [ka] That is the case.

[0327] In one embodiment, the present invention relates to a conjugate comprising CEF-F and an antibody against cefotaxime. In one embodiment, the antibody is conjugated to a quencher.

[0328] An exemplary 4'-substituted fluorescein tracer that can be used in assays for sulfadimethoxine is:

[0329] [ka] That is the case.

[0330] In one embodiment, the present invention relates to SDM-Su-F or a complex comprising SDM-F and an antibody against sulfadimethoxine. In one embodiment, the antibody is conjugated to a quencher.

[0331] An example of a 4'-substituted fluorescein tracer that can be used in an assay for cortisol is:

[0332] [ka] That is the case.

[0333] In one embodiment, the present invention relates to a complex comprising cortisol-4-Fl and an antibody against cortisol. In one embodiment, the antibody is conjugated to a quencher.

[0334] An exemplary 4'-substituted fluorescein tracer that can be used in assays for progesterone is:

[0335] [ka] That is the case.

[0336] In one embodiment, the present invention relates to a complex comprising progesterone-4-Fl and an antibody against progesterone. In one embodiment, the antibody is conjugated to a quencher.

[0337] An exemplary 4'-substituted fluorescein tracer that can be used in assays for bile acids is:

[0338] [ka] That is the case.

[0339] In one embodiment, the present invention relates to a complex comprising CA-Fl and an antibody against bile acids. In one embodiment, the antibody is conjugated to a quencher.

[0340] Examples of bile acids include, but are not limited to, cholic acid and taurocholic acid.

[0341] [ka]

[0342] In one embodiment, the fluorescent molecule is a fluorescein functionalized at both the 4' and 5' positions with, for example, an aldehyde group, a -COOH group, or a -CH2NH2 group, as shown below:

[0343] [ka] (In the formula, X is -C(O)H, -COOH, or -CH2NH2). The T-epitope moiety can attach to the fluorescein core structure at either the 4' or 5' position, or at both the 4' and 5' positions. The T-epitope moiety can attach to the fluorescein core structure using the chemistry described above.

[0344] In one embodiment, the present invention relates to a complex comprising AMO-F, AMP-F, or CEF-F and a penicillin-binding protein. In one embodiment, the penicillin-binding protein is conjugated to a quencher.

[0345] 5. Integration Partners A binding partner specific to the analyte and fluorescent tracer may be, for example, an antibody. Antibodies can be obtained by inducing an immune response in animals to the analyte using techniques recognized in the art. Typically, a hapten (having an epitope moiety common to the analyte of interest) is conjugated to a carrier protein such as bovine serum albumin to obtain an immunogen (antigen), which is administered to animals such as rabbits, mice, or sheep by a series of injections, and the resulting antibody is then isolated using conventional techniques. Other exemplary protein carriers that can be used to form immunogens include, but are not limited to, keyhole limpet hemocyanin, egg ovalbumin, bovine gamma globulin, and thyroxine-conjugated globulin. Alternatively, antigens can be formed by conjugating a hapten to a synthetic or natural polymer material containing a functional group reactive with the hapten.

[0346] The binding partner may also be a protein other than an antibody. The protein can be isolated by conventional techniques. The analyte is a protein-specific substrate. For example, the analyte may be selected from the group consisting of penicillin, estradiol, and progesterone, and the binding partner may be selected from the group consisting of penicillin-binding proteins, estradiol-binding proteins, and progesterone-binding proteins, respectively.

[0347] In one embodiment, the coupling partner is connected to a magnetic barcode bead.

[0348] In one embodiment, the binding partner is conjugated to the quencher. While we do not wish to be constrained by theory, if a fluorescent tracer is bound to a binding partner, and the binding partner is conjugated to the quencher, and the fluorescent tracer is excited by irradiation with light of a first wavelength, then it is thought that energy is transferred from the fluorescent tracer to the quencher, and then the quencher loses energy thermally or by emitting light of wavelengths other than the second wavelength. Thus, the quencher results in a greater decrease in fluorescence intensity than would be observed if the binding partner were not conjugated to the quencher.

[0349] The decrease in fluorescence of a fluorescent tracer when it forms a complex with a binding partner conjugated to a quencher is a function of the equivalent weight of the quencher molecule conjugated to the binding partner. The more quencher molecules that bind to the binding partner, the greater the decrease in fluorescence. This is shown in Figure 23 as a plot of fluorescence vs. antibody:fluorescent tracer ratios for antibodies conjugated to 2, 4, 6, and 8 BHQ10 quencher molecules. Figure 23 shows that when fluorescent tracer A3 forms a complex with an antibody against SDMA, the fluorescence decreases, and the maximum decrease in fluorescence is proportional to the equivalent weight of the quencher molecule (BHQ10) conjugated to the antibody. Figure 23 shows that when the antibody is conjugated to 2 equivalents of BHQ10, the maximum decrease in fluorescence is approximately 50%, and when the antibody is conjugated to 8 equivalents of BHQ10, the maximum decrease in fluorescence is approximately 70%.

[0350] Suitable quenchers that can bind to a binding partner include, but are not limited to, the following:

[0351] [ka] (In the formula, the counterion is not shown in the above structure, and the binding partner is,

[0352] [ka] (It is connected to the quencher.)

[0353] Additional suitable quenchers include, but are not limited to, DABCYL and DABCYL-Plus® (commercially available from AnaSpec Inc. in Fremont, California); Procion® MX-5B, Reactive Red 4, and Reactive Red 120 (commercially available from Sigma Aldrich in St. Louis, Missouri); Dylight Q543 (commercially available from Thermo Scientific in Waltham, Massachusetts); TIDE QUENCHER® 2WS (commercially available from AAT Bioquest in Sunnyvale, California); PADA (pyridine-2-azo-p-dimethylaniline) (commercially available from TCI America in Cambridge, Massachusetts); and QSY® quenchers (including QSY7, QSY9, and QSY21, from Thermo Fisher in Waltham, Massachusetts). This includes commercially available products from Scientific; QXL® Quencher (including QXL490, QXL570, QXL610, and QXL670, commercially available from AnaSpec, Inc., Fremont, California); Iowa Black® Quencher (including Iowa Black FQ and Iowa Black RQ, commercially available from Integrated DNA Technologies, Inc., Coralville, Iowa); and juroridine derivatives (including BlackBerry® Quencher 650, commercially available from Berry & Associates, Inc., Dexter, Michigan).

[0354] The binding partner-quencher conjugate is prepared using commercially available reagents. For example, the binding partners bound to Cy3 and Cy5 are prepared using commercially available reagents from GE Healthcare in Chicago, Illinois.

[0355] [ka] (Counterions are not shown) Prepared by reacting with; the binding partner bound to IRDyeQC1 is commercially available from Li-Cor Biosciences in Lincoln, Nebraska.

[0356] [ka] (Counterions are not shown) Prepared by reacting with; the binding partner bound to BHQ1 is commercially available from LGC Biossearch Technologies in Petaluma, California.

[0357] [ka] (Counterions are not shown) Prepared by reacting with; the binding partner of BHQ10 is commercially available from LGC Biossearch Technologies in Petaluma, California.

[0358] [ka] (Counterions are not shown) It is prepared by reacting with [the specified substance].

[0359] The binding partner is conjugated into the quencher by reacting it with an N-hydroxysuccinimide ester according to the supplier's instructions. Alternatively, the N-hydroxysuccinimide ester can be prepared from the corresponding carboxylic acid.

[0360] The ratio of N-hydroxysuccinimide to its binding partner is typically in the range of about 1 to about 30, preferably about 1 to about 20, and more preferably about 1 to about 12. This ratio varies depending on the choice of fluorescent molecule, quencher, analyte, and binding partner used to prepare the fluorescent tracer. The preferred ratio is determined by measuring the quenching of the fluorescence of each of these molecules at different concentrations.

[0361] While we do not wish to be constrained by theory, it is thought that N-hydroxysuccinimide esters acylate one or more groups on their binding partners, such as amine groups, to yield structures for quencher CY3 as shown below:

[0362] [ka]

[0363] Preferably, the quencher is CY3, CY5, BHQ1, or BHQ10. More preferably, the quencher is BHQ10.

[0364] The quencher is selected such that the light emitted from the fluorescent tracer can either excite the quencher or be absorbed by the quencher. The quencher is selected such that the wavelength of the light emitted by the quencher does not substantially overlap with the wavelength of the irradiated light.

[0365] In one embodiment, the analyte is an antigen, the fluorescent tracer is an analyte-conjugate containing the epitope portion of the antigen linked to a fluorescent label, and the binding partner is an antibody against the antigen.

[0366] In one embodiment, the analyte is SDMA, the fluorescent tracer is an analyte-conjugate containing the epitope portion of SDMA, and the binding partner is an antibody against SDMA conjugated to BHQ10. Preferably, the ratio of BHQ10 to antibody is 20:1. In another embodiment, the ratio of BHQ10 to antibody is 12:1.

[0367] In one embodiment, the analyte is SDMA; the fluorescent tracer is selected from the group consisting of A1, A2, A3, A4, A5, and A6; and the binding partner is an antibody against SDMA conjugated to BHQ10. In one embodiment, the analyte is SDMA; the fluorescent tracer is selected from the group consisting of A1, A2, and A3; and the binding partner is an antibody against SDMA conjugated to BHQ10. In one embodiment, the analyte is SDMA, the fluorescent tracer is A3; and the binding partner is an antibody against SDMA conjugated to BHQ10.

[0368] 6. Exemplary Embodiments of Fluorescence Quenching Assays Where the Analyte is a Polymer In one embodiment, the analyte is a macromolecule such as a protein. An exemplary protein is cystatin.

[0369] In one embodiment, the analyte is cystatin-B (i.e., Cys-B or CysB); the fluorescent tracer is a fluorescent moiety attached to CysB; and the binding partner is an anti-cystatin-B antibody (i.e., an anti-Cys-B or anti-CysB antibody) conjugated to one or more quenchers.

[0370] In one embodiment, the fluorescent tracer is CysB conjugated to fluorescein through one or more lysine residues. To provide a fluorescent tracer conjugated to fluorescein through one or more lysine residues, CysB is reacted with various equivalents of fluorescein-NHS (N-hydroxysuccinimide ester of fluorescein, i.e., 5-(and 6-)carboxyfluorescein, succinimidyl ester, commercially available from Sigma Aldrich in St. Louis, Missouri) to obtain CysB-Fl conjugated to FL through lysine residues. Although we do not wish to be constrained by theory, it is thought that fluorescein-NHS reacts with lysine residues on CysB to yield CysB-FL conjugated to FL through lysine residues. Typically, CysB-FL conjugated to FL through lysine residues is obtained by reacting CysB with fluorescein-NHS between 1 and 4 equivalents. In one embodiment, CysB-Fl, conjugated to FL through a lysine residue, is obtained by reacting CysB with 2 equivalents of fluorescein-NHS. In another embodiment, CysB, conjugated to FL through a lysine residue, is obtained by reacting CysB with 4 equivalents of fluorescein-NHS.

[0371] In one embodiment, the fluorescent tracer is CysB conjugated to fluorescein through one or more cysteine ​​residues. To provide a fluorescent tracer conjugated to fluorescein through one or more cysteine ​​residues, CysB is reacted with various equivalents of fluorescein-5-maleimide (commercially available from Sigma Aldrich, St. Louis, Missouri). While we do not wish to be constrained by theory, it is thought that fluorescein-5-maleimide reacts with cysteine ​​residues on CysB to yield CysB-FL, which is conjugated to fluorescein through cysteine ​​residues. Typically, CysB conjugated to FL through cysteine ​​residues is obtained by reacting CysB with fluorescein-5-maleimide between 1 and 4 equivalents. In one embodiment, CysB-FL conjugated through cysteine ​​residues is obtained by reacting CysB with 2 equivalents of fluorescein-5-maleimide.

[0372] In one embodiment, the fluorescent tracer is CysB-peptide 9 conjugated with fluorescein, i.e., CysB-peptide 9-FL. CysB-peptide 9-FL is obtained by reacting CysB-peptide 9 with fluorescein-NHS. Although we do not wish to be constrained by theory, it is thought that fluorescein-NHS reacts with lysine residues on CysB-peptide 9 to yield CysB-peptide 9-FL. Typically, CysB-peptide 9-FL is obtained by reacting CysB-peptide 9 with between 1 and 4 equivalents of fluorescein-NHS. In one embodiment, CysB-peptide 9-FL is obtained by reacting CysB-peptide 9 with 4 equivalents of fluorescein-NHS.

[0373] In one embodiment, the binding partner is an anti-cystatin-B antibody (i.e., an anti-CysB antibody) conjugated to one or more BHQ10 molecules, i.e., anti-CysB-BHQ. Anti-CysB-BHQ can be obtained by reacting an anti-CysB antibody with BHQ10-NHS (N-hydroxysuccinimide ester of BHQ10, commercially available from LGC Biosearch Technologies in Petaluma, California). While we do not wish to be constrained by theory, it is thought that BHQ10-NHS reacts with lysine residues on the anti-CysB antibody to yield anti-CysB-BHQ. Typically, anti-CysB-BHQ is obtained by reacting CysB with BHQ10-NHS in amounts between 1 and 16 equivalents. Exemplary examples of CysB antibodies include, but are not limited to, 3H4-anti-cystatin-B antibody, Ra355 polyclonal-anti-CysB antibody, and 7C2-anti-CysB antibody (each of these antibodies is custom-made; Ra355 is a rabbit polyclonal anti-CysB antibody produced by SDIX, Newark, Delaware 19702, and the 3H4-anti-cystatin-B antibody and 7C2-anti-CysB antibody are mouse monoclonal anti-CysB antibodies produced by Immunoprecise Antibodies LLC, Vancouver, British Columbia, Canada). In one embodiment, anti-CysB-BHQ is obtained by reacting 3H4-anti-cystatin-B antibody with 2 equivalents of BHQ10-NHS. In one embodiment, anti-CysB-BHQ is obtained by reacting 3H4-anti-cystatin-B antibody with 4 equivalents of BHQ10-NHS. In another embodiment, anti-CysB-BHQ is obtained by reacting Ra355 polyclonal-anti-CysB antibody with 4 equivalents of BHQ10-NHS. In yet another embodiment, anti-CysB-BHQ is obtained by reacting 7C2-anti-CysB antibody with 4 equivalents of BHQ10-NHS.

[0374] In one embodiment, the binding partner is an anti-CysB antibody conjugated to one or more boron-dipyrometen (BODIPY) molecules, i.e., anti-CysB-BODIPY. Anti-CysB-BODIPY can be obtained by reacting an anti-CysB antibody with BODIPY-NHS (N-hydroxysuccinimide ester of BODIPY, commercially available from Thermo Scientific in Waltham, Massachusetts). Typically, anti-CysB-BODIPY is obtained by reacting CysB with between 1 and 4 equivalents of BODIPY-NHS. In one embodiment, anti-CysB-BODIPY is obtained by reacting a 3H4-anti-cystatin-B antibody with 2 equivalents of BODIPY-NHS. In another embodiment, anti-CysB-BODIPY is obtained by reacting a 3H4-anti-cystatin-B antibody with 4 equivalents of BODIPY-NHS. Preferably, anti-CysB-BODIPY is obtained by reacting 3H4-anti-CysB antibody with 4 equivalents of BODIPY-NHS.

[0375] In one embodiment, the analyte is CysB; the fluorescent tracer is a fluorescent portion attached to an anti-CysB antibody; and the binding partner is CysB conjugated to one or more quenchers.

[0376] In one embodiment, the fluorescent tracer is an anti-CysB antibody reacted with various equivalents of fluorescein-NHS to provide anti-CysB-FL. While we do not wish to be constrained by theory, it is thought that fluorescein-NHS reacts with lysine residues on the anti-CysB antibody to yield anti-CysB-FL. Typically, anti-CysB-FL is obtained by reacting an anti-CysB antibody with between 1 and 4 equivalents of fluorescein-NHS. In one embodiment, anti-CysB-FL is obtained by reacting a 3H4-anti-CysB antibody with fluorescein-NHS. Exemplary examples of CysB antibodies, but not limited to them, include 3H4-anti-cystatin-B antibody, Ra355 polyclonal-anti-CysB antibody, and 7C2-anti-CysB antibody. In one embodiment, anti-CysB-FL is obtained by reacting a 3H4-anti-CysB antibody with 2 equivalents of fluorescein-NHS. Preferably, anti-CysB-FL is obtained by reacting a 3H4-anti-CysB antibody with 2 equivalents of fluorescein-NHS. In one embodiment, anti-CysB-FL is obtained by reacting a 3H4-anti-CysB antibody with 4 equivalents of fluorescein-NHS. In one embodiment, anti-CysB-FL is obtained by reacting a Ra355 polyclonal-anti-CysB antibody with 2 equivalents of fluorescein-NHS. In one embodiment, anti-CysB-FL is obtained by reacting a 7C2-anti-CysB antibody with 2 equivalents of fluorescein-NHS.

[0377] In one embodiment, the fluorescent tracer is an anti-CysB antibody, i.e., anti-CysB-FL-Ald, which is reacted with various equivalents of fluorescein aldehyde, i.e., fluorescein functionalized at the 4' position with an aldehyde group, to provide anti-CysB-FL conjugated with fluorescein aldehyde. Although we do not wish to be constrained by theory, it is thought that fluorescein aldehyde reacts with a lysine residue on the anti-CysB antibody to yield anti-CysB-FL-Ald. In one embodiment, anti-CysB-FL-Ald is obtained by reacting a 3H4-anti-CysB antibody with two equivalents of fluorescein aldehyde. Preferably, anti-CysB-FL-Ald is obtained by reacting a 3H4-anti-CysB antibody with two equivalents of fluorescein aldehyde.

[0378] In one embodiment, the binding partner is CysB conjugated to one or more BHQ10 molecules, i.e., CysB-BHQ. CysB-BHQ can be obtained by reacting CysB with BHQ10-NHS. Although we do not wish to be constrained by theory, it is thought that BHQ10-NHS reacts with lysine residues on CysB to yield CysB-BHQ. Typically, CysB-BHQ is obtained by reacting CysB with between 1 and 16 equivalents of BHQ10-NHS. In one embodiment, CysB-BHQ is obtained by reacting CysB with 4 equivalents of BHQ10-NHS. In another embodiment, CysB-BHQ is obtained by reacting CysB with 8 equivalents of BHQ10-NHS. Preferably, CysB-BHQ is obtained by reacting CysB with 8 equivalents of BHQ10-NHS. In one embodiment, CysB-BHQ is obtained by reacting CysB with 12 equivalents of BHQ10-NHS.

[0379] In one embodiment, the analyte is a polypeptide, and the fluorescent tracer is a fluorescent molecule linked to a peptide chain having an amino acid length shorter than the amino acid length of the polypeptide. In one embodiment, the peptide chain has an amino acid length of less than 20 amino acids. In one embodiment, the peptide chain has an amino acid length of less than 15 amino acids. In one embodiment, the peptide chain has an amino acid length of less than 10 amino acids. In one embodiment, the peptide chain has an amino acid length of less than 7 amino acids. In one embodiment, the polypeptide is a macromolecule (e.g., a protein). In one embodiment, the amino acid sequence of the peptide chain corresponds to the amino acid sequence of the epitope portion on the polypeptide.

[0380] In one embodiment, the analyte is a macromolecule such as a protein; the fluorescent tracer is a fluorescent portion attached to an amino acid chain having an amino acid length shorter than the amino acid length of the protein. The amino acid sequence of the amino acid chain corresponds to the amino acid sequence of an epitope on the protein responsible for complexation with the binding partner. In one embodiment, the binding partner is conjugated to a quencher.

[0381] The advantage of linking the fluorescent tracer to an amino acid chain with an amino acid length shorter than the amino acid length of the protein, rather than to the complete protein, is that it ensures that the distance between the fluorescent molecule and the quencher is close enough for effective quenching to occur. When the fluorescent tracer is attached to the complete protein, only the epitope site on the complete protein interacts with the binding partner. Therefore, if the fluorescent site on the protein is not close to the epitope site, the separation between the fluorescent site on the protein and the quencher on the binding partner when forming a complex may become too large for effective quenching to occur. This problem is minimized by linking the fluorescent site to an amino acid chain with an amino acid length shorter than the amino acid length of the protein.

[0382] In one embodiment, an amino acid sequence (i.e., a polypeptide having an amino acid sequence) is attached to the 5-position of fluorescein. In one embodiment, an amino acid sequence is attached to the 5-position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-.

[0383] In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In another embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In another embodiment, the linker is -CH2-.

[0384] For example, the analyte is canine CysB; the fluorescent tracer is a fluorescent moiety attached to an amino acid sequence of 5 to 15 amino acids, preferably 5 to 10 amino acids; and the binding partner is an anti-cystatin-B antibody (i.e., anti-Cys-B or anti-Cys-B antibody) conjugated to one or more quenchers.

[0385] In one embodiment, the fluorescent portion is attached to the amino acid sequence QTNKAKHDELAYF(P9)[SEQ ID NO:2] (i.e., attached to a polypeptide having an amino acid sequence). In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0386] In one embodiment, the fluorescent portion is attached to the amino acid sequence: YQTNKAKHDELAYF(P14) [SEQ ID NO: 3]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0387] In one embodiment, the fluorescent portion is attached to the amino acid sequence: GHDELAYF(P7) [SEQ ID NO: 4]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0388] In one embodiment, the fluorescent portion is attached to the amino acid sequence: GDELAYF(P6) [SEQ ID NO: 5]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0389] In one embodiment, the fluorescent portion is attached to the amino acid sequence: GELAYF(P5) [SEQ ID NO: 6]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0390] In one embodiment, the fluorescent portion is attached to the amino acid sequence: GLAYF(P4) [SEQ ID NO: 7]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0391] In one embodiment, the fluorescent portion is attached to the amino acid sequence:MMCGAPSASQPATADTQAIADQVKAQLEERENKKYTTFKAVTFRSQVVAGTPYFIKVQVDDDEFVHLRVFQSLPHENKPLALSSYQTNKAKHDELAYF[Sequence ID: 1]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0392] Another example is when the analyte is canine NT-proBNP (NT-pro-B type natriuretic protein); the fluorescent tracer is a fluorescent moiety attached to an amino acid sequence of 5 to 15 amino acids, preferably 5 to 10 amino acids; and the binding partner is an anti-NT-proBNP mab conjugated to one or more quenchers.

[0393] In one embodiment, the fluorescent portion is attached to the amino acid sequence: AEQLALEPLHRS(P1) [SEQ ID NO: 8]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0394] In one embodiment, the fluorescent portion is attached to the amino acid sequence: AEQLAL(P2) [SEQ ID NO: 9]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0395] In one embodiment, the fluorescent portion is attached to the amino acid sequence: EPLHRS(P3) [SEQ ID NO: 10]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0396] In one embodiment, the fluorescent portion is attached to the amino acid sequence: LALEPL(P4) [SEQ ID NO: 11]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0397] In one embodiment, the fluorescent portion is attached to the amino acid sequence: AEQLALE(P5) [SEQ ID NO: 12]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0398] In one embodiment, the fluorescent portion is attached to the amino acid sequence:LEPLHRS(P6)[SEQ ID NO:13]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0399] In one embodiment, the fluorescent portion is attached to the amino acid sequence:GRSPASEASEASEASGLWAVQ[SEQ ID NO:15]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0400] In one embodiment, the fluorescent portion is attached to the amino acid sequence: SHSPAEAPEAGGTPRGVLAPHDSVLQ [Sequence ID: 16]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0401] In one embodiment, the fluorescent portion is attached to the amino acid sequence:HPLGGRSPASEASEASEASGLWAVQELLGRLKDAVSELQAEQLALEPLHRSHSPAEAPEAGGTPRGVLAPHDSVLQALR[Sequence ID: 14]. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein. In one embodiment, the amino acid sequence is attached to the 5th position of fluorescein by a linker. In one embodiment, the linker is -NH-C(O)-. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein. In one embodiment, the amino acid sequence is attached to the 4' position of fluorescein by a linker. In one embodiment, the linker is -CH2-.

[0402] The fluorescent portion may be attached to any of the polypeptides disclosed in U.S. Patents 8,628,973; 8,778,699; 9605,068; 9005,984; and 1,072,5052, which are incorporated herein by reference in their entirety. Each obtained polypeptide tracer may be used in the method of the present invention in combination with a binding partner selected from any of the corresponding antibodies disclosed in U.S. Patents 8,628,973; 8,778,699; 9605,068; 9005,984; and 1,072,5052.

[0403] Examples The present invention is not limited in scope by the specific embodiments disclosed in the examples intended as illustrative of several aspects of the invention, and any functionally equivalent embodiment falls within the scope of the invention. In fact, various modifications of the invention to those shown and described herein will be obvious to those skilled in the art and are intended to fall within the scope of the appended claims. Such variations of the invention, including the substitution of all currently known or subsequently developed equivalents, and changes or minor modifications in formulation in experimental designs, which are in the eye of those skilled in the art, are considered to fall within the scope of the invention as incorporated herein. [Examples]

[0404] Preparation of fluorescein aldehyde Fluorescein aldehyde was prepared as shown in the reaction scheme provided below:

[0405] [ka]

[0406] Fluorescein (4 g, 12 mmol, commercially available from Sigma Aldrich in St. Louis, Missouri) was dissolved in 40 mL of 1 M sodium hydroxide solution and then heated to 70°C. Chloroform (8 mL, 45 mmol) was added dropwise to the heated solution, and the resulting mixture was maintained at the same temperature for 3 hours. After cooling to room temperature, the reaction mixture was poured into 250 mL of 1 M HCl solution to obtain a precipitate. The precipitate was collected by filtration, washed several times with water, dried, and protected from light.

[0407] Next, the crude product was purified by column chromatography using a silica gel column (2.5 cm × 30 cm) eluted with dichloromethane / acetonitrile (15:1). The fraction containing fluorescein aldehyde was identified using LC / MS equipped with a C18 reversed-phase column eluted with a gradient of 40% to 80% acetonitrile in water containing 1% acetic acid. The fractions containing fluorescein aldehyde were combined, the solvent was removed under reduced pressure, and the resulting residue was dried under high vacuum to obtain 0.28 g of pale yellow crystals. Mass spectrum m / z = 361.1 (M + H) + . [Examples]

[0408] Preparation of melamine-fluorescein (Mel-F) Mel-F was prepared as shown in the reaction scheme provided below:

[0409] [ka]

[0410] 2-Chloro-4,6-diamino-1,3,5-triazine (4.5 mg, 0.03 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) and 4'-aminomethylfluorescein (7.9 mg, 0.02 mmol, commercially available from AAT Bioquest, Sunnyvale, California) were dissolved in anhydrous dimethyl sulfoxide (DMSO) (1 mL). Anhydrous potassium carbonate (5.5 mg, 0.04 mmol) was added to the resulting solution, and the solution was heated to 95°C and stirred at this temperature overnight. The solvent was then removed under reduced pressure, and the resulting residue was dissolved in a 30% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid (TFA). The resulting solution was purified by column chromatography using a C18 reversed-phase column (10 g) eluted with an acetonitrile aqueous solution containing 0.1% TFA. A gradient from 30% acetonitrile to 60% acetonitrile was used as the gradient. The fractions containing pure Mel-F were combined and freeze-dried to obtain Mel-F as a yellow powder. Mass spectrum m / z = 471.7(M+H) + . [Examples]

[0411] Preparation of melamine-succinate-fluorescein (Mel-Su-F) Mel-Su-F was prepared as shown in the reaction scheme provided below:

[0412] [ka]

[0413] Melamine (416 mg, 3.3 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) and succinic anhydride (0.5 g, 5.0 mmol) were dissolved in dimethylformamide anhydride (DMF) (8 mL). The resulting solution was heated to 100°C and stirred at this temperature overnight. The solvent was then removed under reduced pressure to obtain the melamine-succinic acid derivative (Mel-Su).

[0414] Mel-Su (270 mg, 1.2 mmol), N',N'-dicyclohexylcarbodiimide (268 mg, 1.3 mmol), and N-hydroxysuccinimide (0.15 g, 1.30 mmol) were dissolved in anhydrous dimethyl sulfoxide (DMSO) (5 mL), and the resulting solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure to obtain (Mel-Su-NHS), which was used without further purification.

[0415] Mel-Su-NHS (8 mg, 0.025 mmol) and 4'-aminomethylfluorescein (10 mg, 0.025 mmol, commercially available from AAT Bioquest, Sunnyvale, California) were dissolved in anhydrous DMF (0.5 mL). N'N'-diisopropylethylamine (DIPEA) (10 μL) was added to the resulting DMF solution, and the solution was stirred at room temperature for 2 hours. The solvent was removed under high vacuum to obtain the residue. The resulting residue was purified by column chromatography using a C18 reversed-phase column (10 g) eluting with a 40% to 60% acetonitrile gradient to obtain Mel-Su-F. Mass spectrum m / z = 570.2 (M+H) + . [Examples]

[0416] Preparation of biotin-fluorescein (biotin-F) Biotin-F was prepared as shown in the reaction scheme provided below:

[0417] [ka]

[0418] 4'-aminomethylfluorescein (3 mg, 7.54 μmol, commercially available from AAT Bioquest, Sunnyvale, California) and N-hydroxysuccinimidebiotin (2.5 mg, 7.54 μmol, commercially available from Thermo Scientific, Waltham, Massachusetts) were dissolved in anhydrous DMF (0.5 mL). N,N-diisopropylethylamine (4 μL) was added to the DMF solution, and the resulting solution was stirred at room temperature for 2 hours. The solution was then diluted with 40% acetonitrile in water containing 0.1% formic acid and purified by column chromatography using a C18 reversed-phase column (1 g) eluted with a mobile phase of 40% acetonitrile in water containing 0.1% formic acid. Biotin-F 3 mg was obtained as a yellow solid (yield: 68%). Mass spectrum m / z = 588.4 (M + H) + . [Examples]

[0419] Preparation of sulfadimethoxine succinate fluorescein (SDM-Su-F) SDM-Su-F was prepared as shown in the reaction scheme provided below:

[0420] [ka]

[0421] Sulfadimethoxine (SDM, 1.0 g, 3.2 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) and succinic anhydride (0.49 g, 4.9 mmol) were dissolved in anhydrous DMF (6 mL). The resulting solution was heated to 100°C and maintained at this temperature for 2 hours, after which the solvent was removed under reduced pressure to obtain the residue. The obtained residue was recrystallized from ethanol:water (1:1) to obtain 1 g of SDM-succinic acid (SDM-Su).

[0422] SDM-Su (0.51 g, 1.24 mmol), N',N'-dicyclohexylcarbodiimide (0.28 mL, 1.36 mmol), and N-hydroxysuccinimide (0.15 g, 1.30 mmol) were combined and dissolved in anhydrous DMF (5 mL) under an argon atmosphere. The resulting solution was stirred overnight at room temperature. The solvent was then removed under reduced pressure, and the resulting product (SDM-Su-NHS) was used without further purification.

[0423] SDM-Su-NHS (15.2 mg, 0.03 mmol) and 4'-aminomethylfluorescein (8 mg, 0.02 mmol, commercially available from AAT Bioquest, Sunnyvale, California) were dissolved in anhydrous DMF (0.5 mL). N'N'-diisopropylethylamine (7 μL, 0.04 mmol) was added to the DMF solution, and the solution was stirred at room temperature for 2 hours. The solvent was then removed under high vacuum to obtain the residue. The obtained residue was purified by column chromatography using a C18 reversed-phase column (10 g) eluted with 40% acetonitrile to obtain SDM-Su-F as a yellow powder. Mass spectrum m / z = 754.8 (M + H) + . [Examples]

[0424] Preparation of T3-ethyl fluorescein (T3-EF) T3-EF was prepared as shown in the reaction scheme provided below:

[0425] [ka]

[0426] Triiodothyronine (100 mg, 0.154 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) was dissolved in methanol (5 mL). Sodium hydroxide (45 μL, 10 M) and dimethoxyacetal aldehyde (230 mL, 60% aqueous solution, 1.5 mmol) were then added to the methanol solution, and the solution was maintained at room temperature for 30 minutes. Sodium borocyanohydride (50 mg, 0.77 mmol) was then added, and the resulting reaction mixture was stirred for 2 hours. Sodium hydroxide (0.1 mL, 1 M) was then added, and the resulting reaction mixture was stirred for a further 2 hours. Finally, 1 M HCl was added to the reaction mixture to obtain T3-ethyl acetal (100 mg), and the mass spectrum m / z = 740.3 (M+H) was obtained. + The precipitate was allowed to set, and this was used without further purification.

[0427] T3-Ethylaldehyde T3-ethyl acetal was hydrolyzed overnight in tetrahydrofuran and sulfuric acid (THF / H2SO4). The solvent was then removed under reduced pressure to obtain the residue. The obtained residue was dissolved in a 20% acetonitrile aqueous solution containing 1% acetic acid and purified by column chromatography using a C18 reversed-phase column (5g) with an elution gradient from 20% to 50% acetonitrile in water. The fractions containing T3-ethyl aldehyde were combined and freeze-dried to obtain a white powder.

[0428] T3-EF T3-ethylaldehyde (3 mg, 0.0042 mmol) and 4'-aminomethylfluorescein (0.9 mg, 0.002 mmol, commercially available from AAT Bioquest, Sunnyvale, California) were dissolved in THF (1 mL) and acetic acid (12 μL), and sodium triacetoxyborohydride (NaBH(OAc)3) (2 mg) was added. The resulting solution was stirred overnight at room temperature. The following day, the solvent was removed under reduced pressure, and the resulting residue was purified by column chromatography using a C18 reversed-phase column eluting with a gradient of 20% to 60% acetonitrile in water to obtain T3-EF. Mass spectrum m / z = 1039.4 (M + H) + . [Examples]

[0429] Preparation of sulfadimethoxine-fluorescein (SDM-F) SDM-F was prepared as shown in the reaction scheme provided below:

[0430] [ka]

[0431] Sulfadimethoxine (8.6 mg, 0.028 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) and fluorescein aldehyde (8 mg, 0.022 mmol) were dissolved in methanol (2 mL). Sodium triacetoxyborohydride (15 mg, 0.15 mmol) and acetic acid (4 μL) were added, and the resulting solution was stirred overnight at room temperature. The reaction solution was then diluted in 40% acetonitrile water containing 0.1% trifluoroacetic acid (TFA) and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 40% to 60% acetonitrile water containing 0.1% TFA. The fractions containing the SDM-F product were combined and lyophilized to obtain 1 mg of SDM-F as a yellow powder. Mass spectrum m / z = 623.7 (M + H) + . [Examples]

[0432] Preparation of T3-F T3-F was prepared as shown in the reaction scheme provided below:

[0433] [ka]

[0434] A 100 mL flask containing a mixture of 40 mL of sodium bicarbonate buffer (100 mM at pH 8.0) and 20 mL of dioxane was mixed with fluorescein aldehyde (180 mg, 0.5 mmol) and triiodothyroxine (390 mg, 0.6 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri). The resulting solution was stirred at room temperature for 1 hour. Then, sodium borocyanohydride (400 mg, 5 mmol) was added to the solution, and the resulting mixture was stirred overnight at room temperature in the dark. The pH of the resulting mixture was then adjusted to pH 5.0 using 1 M HCl, and the solvent was removed by lyophilization.

[0435] A flash column with a diameter of 38 mm and a length of 200 mm was prepared using 100 g of C18 reversed-phase silica. The column was equilibrated with 30% acetonitrile in water containing at least 3 column volumes of 0.1% acetic acid. The crude T3-F product was then dissolved in 30% acetonitrile in water containing 0.1% acetic acid and applied to the column. The column was eluted with 30% acetonitrile in water containing 0.1% acetic acid. The eluted fraction was monitored for the presence of T3-F using HPLC (4.6 mm × 100 mm XTerrao C18 reversed-phase column) equipped with a UV detector operated at 220 nm. The fractions containing T3-F were combined. The combined fraction showed a purity of more than 95% based on HPLC analysis using UV detection at 220 nm. 350 mg of the product was obtained by lyophilization of the combined fraction. Mass spectrum m / z = 996.6 (M + H) + The overall yield was approximately 50%.

[0436] T2-F and T4-F were prepared using a similar method. The products were characterized by their mass spectra. T2-F, m / z = 869.8 (M + H) + And T4-F, m / z=1122.1(M+H) + . [Examples]

[0437] Preparation of amoxicillin-fluorescein (AMO-F) AMO-F was prepared as shown in the reaction scheme provided below:

[0438] [ka]

[0439] To a 100 mL flask containing a mixture of 40 mL of sodium bicarbonate buffer (100 mM at pH 8.0) and 20 mL of dioxane, fluorescein aldehyde (360 mg, 1 mmol) and amoxicillin (547 mg, 1.5 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) were added. The resulting solution was cooled to 4°C using an ice bath and stirred for 40 minutes. Then, sodium borocyanohydride (314 mg, 5 mmol) was added, and the reaction mixture was stirred in the dark at 4°C. After about 2 hours, a further amount of sodium borocyanohydride (125 mg, 2 mmol) was added, and the reaction mixture was stirred in the dark at 4°C. The reaction mixture was monitored using HPLC. Once the reaction was complete (indicated by at least 90% product formation), the pH of the reaction mixture was adjusted to pH 5.5 with 1 M HCl, and the solvent was removed by lyophilization to obtain the crude product. One-third of the crude product was dissolved in 50 mL of 30% acetonitrile in water containing 0.1% acetic acid, yielding a clear yellow solution with a pH of 5.4. This solution was purified by column chromatography using a C18 reversed-phase column eluted with a mobile phase of 30% acetonitrile in water containing 0.1% acetic acid. The fractions containing AMO-F were combined. Analysis of the combined fractions by HPLC with a UV detector operating at 220 nm showed a purity of higher than 95% based on absorption at 220 nm. The combined fractions were then lyophilized to obtain 61 mg of the product. The overall yield of the product was approximately 42%. Mass spectrum m / z = 710.1795 (M+H) + .

[0440] Ampicillin-fluorescein (AMP-F) was prepared using a similar method. Mass spectrum m / z = 694.2 (M + H) + . [Examples]

[0441] Preparation of AMO-FITC AMO-FITC was prepared as shown in the reaction scheme provided below:

[0442] [ka]

[0443] Amoxicillin (9.4 mg, 0.026 mmol, commercially available from Sigma), fluorescein isothiocyanate (FITC) (10 mg, 0.026 mmol, commercially available from Thermo Scientific, Waltham, Massachusetts), and triethylamine (5.6 μL, 0.75 mmol) were dissolved in DMF (1 mL), and the resulting solution was stirred overnight at room temperature. The solution was then diluted with 30% acetonitrile in water containing 0.1% acetic acid and applied to a C18 reversed-phase column. The column was eluted with a gradient of 30% to 50% acetonitrile in water containing 0.1% acetic acid. The fractions containing AMO-FITC were combined, and the combined fraction was freeze-dried to obtain a yellow powder. Mass spectrum m / z = 755.2 (M + H) + . [Examples]

[0444] Preparation of cefotaxime-fluorescein (CEF-F) CEF-F was prepared as shown in the reaction scheme provided below:

[0445] [ka]

[0446] Cefotaximeic acid (63 mg, 0.138 mmol, commercially available from LKT Laboratories Inc., St. Paul, Minnesota), 4'-aminomethylfluorescein (50 mg, 0.126 mmol, commercially available from AAT Bioquest, Sunnyvale, California), and anhydrous potassium carbonate (52 mg, 0.378 mmol) were dissolved in anhydrous DMF (10 mL), and the resulting solution was stirred at room temperature for 10 minutes. 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HATU) (53 mg, 0.15 mmol) was added to the solution, and the resulting mixture was stirred at room temperature for 6 hours. The solvent was then removed under reduced pressure to obtain the residue. Next, the residue was dissolved in 30% acetonitrile in water containing 0.1% acetic acid. The resulting solution was applied to a C18 reversed-phase column (100 g) and eluted under a gradient of 40% to 50% acetonitrile in water containing 0.1% acetic acid. The fractions containing the CEF-F product were combined and freeze-dried to obtain 105 mg of the product. Mass spectrum m / z = 799.1477 (M + H) + . [Examples]

[0447] Preparation of CY3-, CY5-, IRDyeQC1, and BHQ1-labeled anti-T4-Mab A monoclonal anti-T4 antibody (anti-T4-Mab) (4 mg, commercially available from Meridian Life Sciences Inc. (Biodesign) in Memphis, Tennessee) was mixed with 0.5 mg of Cys3-NHS (i.e., N-hydroxysuccinimide ester of Cy3, commercially available from GE Healthcare in Chicago, Illinois), Cy5-NHS (N-hydroxysuccinimide ester of Cy5, commercially available from GE Healthcare in Chicago, Illinois), IRDyeQC1-NHS (N-hydroxysuccinimide ester of IRDyeQC1, commercially available from Li-Cor Biosciences in Lincoln, Nebraska), or BHQ1-NHS (N-hydroxysuccinimide ester of BHQ1, commercially available from LGC Biossearch Technologies in Petaluma, California) in 0.25 mL of DMSO to obtain a solution. The resulting solution was stirred overnight at 4°C. Next, the quencher-labeled antibody was purified by column chromatography using a Sephadex G-25 column eluted with PBS mobile phase. Protein concentrations were determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0448] Preparation of T3-FITC: T3-FITC was prepared as shown in the reaction scheme provided below:

[0449] [ka]

[0450] Triiodothyroxine (16.7 mg, 0.025 mmol), fluorescein isothiocyanate (FITC, 10 mg, 0.026 mmol, commercially available from Thermo Scientific, Waltham, Massachusetts), and triethylamine (5.6 μL, 0.75 mmol) were dissolved in DMF (1 mL), and the resulting solution was stirred overnight at room temperature. The solution was then diluted with 30% acetonitrile in water containing 0.1% acetic acid, and the resulting solution was purified by column chromatography using a C18 reversed-phase column eluting in water containing 0.1% acetic acid with a gradient from 30% to 70% acetonitrile. The fractions containing T3-FITC were combined and lyophilized to obtain a yellow powder. Mass spectrum m / z = 1040.8295 (M+H) + . [Examples]

[0451] Preparation of melamine-ED-F and melamine-OG:

[0452] [ka]

[0453] 2-Chloro-4,6-diamino-1,3,5-triazine (36 mg, 0.24 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri) and ethylenediamine (30 mg, 0.5 mmol) were dissolved in anhydrous DMSO (2 mL), anhydrous potassium carbonate (82 mg, 0.6 mmol) was added, and the resulting suspension was heated to 95°C and maintained overnight under an argon atmosphere. The solvent was then removed under reduced pressure to obtain the residue. The obtained residue was purified by column chromatography using a C18 reversed-phase column (10 g) eluted with 50% acetonitrile in water to obtain melamine-ethylamine.

[0454] Melamine-ethylamine (0.5 mg, 0.003 mmol) and Oregon Green-NHS ester (1 mg, 0.002 mmol, commercially available from Invitrogen Life Technologies, Carlsbad, California) or 5-carboxyfluorescein succinimidyl ester (1 mg, 0.002 mmol, commercially available from Thermo Scientific, Waltham, Massachusetts) were dissolved in DMF (0.5 mL) containing triethylamine (8 μL) and incubated at room temperature for 6 hours. The resulting solution was diluted in 30% acetonitrile water containing 1% acetic acid and purified by column chromatography using a C18 reversed-phase column to obtain melamine-ED-F or melamine-OG. Melamine-ED-F, m / z = 528.2 (M + H) + Melamine-OG, m / z = 564.1(M+H) + . [Examples]

[0455] Fluorescence of mixtures of various fluorescent tracers and anti-melamine antibodies Various substituted fluorescent tracers in which melamine is conjugated to fluorescein were dissolved in DMSO (1 mM) and diluted in phosphate-buffered saline (PBS) (pH=7.3) to obtain melamine tracer solutions (1.2 μM). The following melamine tracers were used:

[0456] [ka]

[0457] Polyclonal anti-melamine antibody (anti-Mel-Ab, 4.65 mg / ml, commercially available from Meridian Life Sciences Inc. (Biodesign) in Memphis, Tennessee) was diluted in PBS and serially diluted to obtain anti-Mel-Ab solutions with anti-Mel-Ab concentrations ranging from 0 to 300 nM. Melamine tracer solution (5 μL) was mixed with each anti-Mel-Ab solution (195 μL) in a 96-well black assay plate with an unbound surface (commercially available from Corning Inc. in Corning, New York) to obtain solutions with different molar ratios of anti-Mel-Ab and melamine tracer. The concentration of the melamine tracer was 30 nM. The plates were gently shaken at room temperature for 30 minutes. Next, the fluorescence intensity of each solution was measured using a fluorescence plate reader (Synergy 4 Microplate Reader, commercially available from BioTek Instruments, Inc., Winooski, Vermont) with an excitation wavelength of 490 nm and emission readings at 520 nm. The results are shown in Figure 3.

[0458] Figure 3 shows that different melamine-fluorescein conjugates quench fluorescence to different degrees, and that the amount of quenching is a function of the molar ratio of anti-Mel-Ab to melamine tracer. Higher ratios of anti-Mel-Ab to melamine tracer result in greater quenching. These results also demonstrate that the 4'-substituted tracer (i.e., MelF) exhibited the highest quenching compared to other melamine tracers, reaching a saturation point when the molar ratio of anti-Mel-Ab to melamine tracer was 6:1. These results indicate that quenching efficiency (i.e., the percentage reduction in fluorescence at a 1:1 fluorescent antibody:antigen ratio compared to fluorescence in the absence of antibody) depends on the position where the ligand is attached to the fluorescein molecule, and that the 4'-substituted tracer exhibits superior quenching efficiency compared to the 5'-substituted tracer. These results also demonstrate that shorter linkers result in higher quenching efficiency.

[0459] Unless otherwise specified, an excitation wavelength of 490 nm and emission readings at 520 nm were used throughout the examples. Unless otherwise specified, all results are the average of three consecutive measurements. [Examples]

[0460] Fluorescence of Mel-F and anti-Mel-Ab solutions as a function of melamine concentration Aliquots of Mel-F (1.2 μM, 5 μL) and anti-Mel-Ab solution (6 μM, 5 μL) in PBS were added to a 96-well black assay plate and incubated at room temperature for 30 minutes with gentle shaking. Melamine standard solutions were prepared in PBS at concentrations ranging from 0 to 20 ng / ml. Each melamine standard solution was then added to the MelF and anti-Mel-Ab solutions in the 96-well assay plate. The plate was then incubated at room temperature for 1 hour, and the fluorescence intensity was measured. The results are shown in Figure 4, illustrating fluorescence recovery as a function of melamine concentration. In Figure 4, the concentration of Mel-F is 30 nM, and the concentration of anti-Mel-Ab is 150 nM. The results indicate that the dynamic range of melamine in PBS solution is approximately 0 to 8 ng / mL. [Examples]

[0461] Fluorescence of serum solutions of Mel-F and anti-Mel-Ab in the presence and absence of melamine. Untreated dog serum was adjusted to pH 7.3 using phosphate buffer and diluted 10-fold with PBS. To a PBS solution of Mel-F (1 μM, 6 μL) and a mixture of Mel-F (1 μM, 6 μL) and anti-Mel-Ab (1 μM, 6 μL) in PBS, PBS, serum, or diluted serum (i.e., serum diluted 1 / 10 with PBS) was added in the absence and presence of melamine (2 μM, 6 μL) to a final volume of 200 μL. The resulting solutions were incubated at room temperature for 1 hour, and then the fluorescence intensity was measured. The results are shown in Figure 5.

[0462] These results demonstrate that, for each solution, the fluorescence of Mel-F is quenched to about half its original value by anti-Mel-Ab, and that the fluorescence is restored by the addition of melamine. [Examples]

[0463] Fluorescence of anti-Mel-Ab and Mel-F solutions in milk in the presence of melamine Raw milk samples were diluted 10-fold with PBS. Melamine standard solutions from raw milk, diluted 10-fold with PBS, with melamine concentrations ranging from 0 to 250 μM, were prepared by serial dilution. Mel-F (2 μM, 5 μL) and anti-Mel-Ab (10 μM, 5 μL) in PBS were added to the wells of a 96-well black assay plate. Then, 190 μL of melamine standard solution was added to each well, and the plate was incubated at room temperature for 1 hour, and the fluorescence intensity was recorded. The results are shown in Figure 6.

[0464] Figure 6 shows the fluorescence intensity of a solution of Mel-F (50 nM) in the presence of anti-Mel-Ab (250 nM) in various concentrations of melamine (10% raw milk in PBS). The results indicate a dynamic range of approximately 0–30 μg / L for melamine detection. [Examples]

[0465] Fluorescence of the solution in the presence of biotin-F or biotin-ED and streptavidin. Biotin-F or biotin-ED (commercially available from Thermo Scientific, Waltham, Massachusetts) was dissolved in DMSO to obtain a DMSO solution (8.5 μM). The structure of biotin-ED is:

[0466] [ka] That is the case.

[0467] Next, the DMSO solution was diluted with PBS to obtain a solution with a biotin-F or biotin-ED concentration of 100 nM. Then, 20 μL aliquots of the obtained PBS-infused biotin-F or biotin-ED solution were added to the wells of a 96-well black assay plate. A PBS-infused streptavidin solution with streptavidin concentrations ranging from 0 to 100 nM was prepared and added to the biotin-F or biotin-ED solution in the plate (180 μL). The plate was incubated at room temperature for 30 minutes, and then the fluorescence intensity was recorded. The results are shown in Figure 7, which displays the percentage change in fluorescence intensity as a function of the molar ratio of streptavidin to tracer.

[0468] The results in Figure 7 show that the fluorescence of biotin F is quenched more efficiently than that of biotin ED. At the saturation point (1:1 molar ratio), the quenching efficiency of biotin-F is 70% higher than that of biotin-ED. The results indicate that the change in fluorescence for the 4'-substituted fluorescent tracer is greater than that for the 5-substituted fluorescent tracer. [Examples]

[0469] Fluorescence of T3-F and anti-T4-Mab solutions in the presence of T4 A mixture of T3-F (1 μM, 6 μL) and an anti-monoclonal anti-thyroxine antibody (anti-T4-Mab (commercially available from Biodesign Inc., Denver, Colorado)) (1 μM, 6 μL) in PBS was added to PBS in the absence and presence of L-thyroxine (T4, 2 μM, 6 μL, commercially available from Sigma Aldrich, St. Louis, Missouri) to a final volume of 200 μL. The resulting solution was incubated at room temperature for 1 hour, and then the fluorescence intensity was measured.

[0470] The solutions were prepared similarly, except that T3-F was replaced with T3-5-F, T3-EF, and T3-FITC, and the fluorescence was measured.

[0471] T3-5-F was synthesized using the following procedure: Preparation of T3-5-Fluorescein (T3-5-F)

[0472] [ka]

[0473] Triiodothyroxine (8.2 mg, 0.013 mmol), 5-carboxylfluorescein N-hydroxysuccinimide ("NHS") (5.9 mg, 0.013 mmol, commercially available from Thermo Scientific, Waltham, Massachusetts), and N'N-diisopropylethylamine (6.5 μL, 0.04 mmol) were dissolved in DMF (1 mL), and the resulting solution was stirred overnight at room temperature. The solution was purified by column chromatography using a C18 reversed-phase column (5 g) eluted with 40% acetonitrile in water containing 0.1% acetic acid to obtain T3-5-F.

[0474] The structure of each tracer is shown below:

[0475] [ka]

[0476] The results are shown in Figure 8. Figure 8 shows that fluorescence was not quenched by anti-T4-Mab for both T3-FITC and T3-5-F. For T3-EF and T3-F, fluorescence was quenched by anti-T4-Mab. This indicates that quenching is more efficient for 4'-substituted fluorescent tracers than for 5-substituted fluorescent tracers.

[0477] Figure 9 shows the amount of quenching and the recovery of quenching after the addition of T4 as a function of time when T3-F is added to anti-T4-Mab. The results show that a stable fluorescence signal is obtained within 10 minutes. [Examples]

[0478] T4 dose response in PBS A mixture of T3-F in PBS (1.0 μM, 8 μL) and anti-T4-Mab in PBS (1 μM, 8 μL) was incubated in the wells of a 96-well black assay plate at room temperature for 30 minutes. Then, a series of T4 standard solutions (184 μL) with T4 concentrations ranging from 0 to 32 μg / dL were added to the mixture of T3-F in PBS and anti-T4-Mab. The plate was then incubated at room temperature for 30 minutes, and the fluorescence intensity was recorded. The results are shown in Figure 10.

[0479] Figure 10 shows that fluorescence is recorded when T4 is added to solutions of T3-F (50 nM) and anti-T4-Mab (50 nM). The results indicate that the dynamic range for T4 detection in PBS is approximately 0–15 μg / dL. [Examples]

[0480] Fluorescence of T3-F and anti-T4-Mab solutions in the presence of T4 after lyophilization. T3-F (1.7 mg) was dissolved in 0.85 ml of DMSO to obtain a stock solution (2 mM). The T3-F stock solution was then diluted in PBS to obtain a 1 μM working solution, which was separated into two vials (1 mL each). PBS (9 mL) was added to the first vial, and PBS (8.9 mL) + anti-T4 Mab (15 μM, 0.1 mL) was added to the second vial. The vials were thoroughly mixed and incubated at room temperature for 30 minutes. The fluorescence intensity of each solution (200 μL) was determined. Aliquots of each solution (200 μL) were then placed in the wells of a 96-well black assay plate and frozen to dryness. To the dried residue, 200 μL of PBS, charcoal-treated horse serum (i.e., serum was "charcoal-treated" by dialyzing it into charcoal-containing PBS buffer over at least three buffer changes to remove small molecules), or horse serum treated with charcoal-containing T4 (10 μg / dL) were added, and the plate was incubated at room temperature for 30 minutes. The fluorescence intensity was then recorded again. The results are shown in Figure 11.

[0481] Figure 11 shows that the assay is valid even when T3-F and / or anti-T4-Mab are frozen. [Examples]

[0482] Fluorescence of a mixture of T4-F and Ab-Cy5 in the presence of T4 T4-F was dissolved in DMSO (1 mM) and diluted with PBS to obtain a stock solution (4 μM). A series of PBS solutions of Cy5-conjugated antibody (Ab-Cy5, prepared as in Example 12) with Ab-Cy5 concentrations ranging from 0 to 400 nM were prepared by serial dilution. T4-F (5 μL) in PBS and serially diluted Ab-Cy5 solutions (95 μL) were combined in the wells of a 96-well black assay plate, and the plate was shaken at room temperature for 30 minutes. Fluorescence intensity was measured using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Figures 12A and 12B show that the fluorescence of T4-F is quenched in the presence of Ab-Cy5 (quenching efficiency of approximately 70% with a T4-F:Ab-Cy5 ratio of approximately 1:1). Cy5 alone (i.e., not conjugated with anti-T4-Mab) did not quench the fluorescence.

[0483] To demonstrate that fluorescence can be restored in the presence of T4, aliquots of a mixture of T4-F (5 μL, 4 μM) in PBS and Ab-Cy5 solution (5 μL, 8 μM) were added to the wells of a 96-well black assay plate. Then, 90 μL of T4 solution in PBS (0, 1, 2, 4, 6, 8, 10, 12 μg / dL) were added to each well. The resulting 100 μL solution was incubated for 30 minutes, and fluorescence was measured. The results are shown in Figure 13.

[0484] Figure 13A shows that the fluorescence increases when T4 is added to a solution of T4-F and Ab-Cy5 (Ab-Cy5:T4-F approximately 2:1), and that the increase in fluorescence is proportional to the amount of T4 added. The dynamic range is approximately 0 to 12 μg / dL. Figure 13B shows the fluorescence recovery percentage as a function of T4-F concentration. [Examples]

[0485] Fluorescence of a mixture of T3-F or T4-F and Ab-Cy3 in the presence of T4 Charcoal-treated horse serum (50 μL) was mixed with 8-amino-naphthalene sulfonic acid (ANS) to obtain an ANS concentration of 0.5 mM, and the pH was adjusted to 7.3 with sodium hydroxide. A series of PBS or serum T4 standard solutions with concentrations ranging from 0 to 64 μg / dL were prepared by serial dilution. In a 96-well black assay plate, each T4 standard (PBS or serum, 80 μL) was added to a mixture of T3-F (or T4-F) in PBS (4 μM, 5 μL) and anti-T4-Mab conjugated to Cy3 in PBS (Ab-Cy3, prepared as described in Example 12) (4 μM, 5 μL). The resulting solutions were incubated at room temperature for 30 minutes, and then the fluorescence intensity was measured. The results are shown in Figure 14.

[0486] Figure 14A shows the fluorescence intensity as a function of T4 concentration when T4 was added to PBS and serum solutions of T3-F (200 nM) and Ab-Cy3 (200 nM). The results indicate a dynamic range of approximately 0–64 μg / L for T4 detection in PBS and serum.

[0487] Figure 14B shows the fluorescence intensity as a function of T4 concentration when T4 was added to PBS and serum solutions of T4-F (200 nM) and Ab-Cy3 (200 nM). The results indicate a dynamic range of approximately 0–64 μg / L for T4 detection in PBS and serum.

[0488] Figure 14C shows the fluorescence intensity as a function of T4 concentration when T4 is added to PBS solutions of T4-F (200 nM) and Ab-Cy3 (200 nM), with the sample excited at 490 nm and the emission measured at 615 nm. This decrease in emission is due to the loss of fluorescence resonance energy transfer (FRET). FRET involves energy transfer from the excited donor dye to the acceptor dye through a non-radiative means. In this example, the excitation of the donor (T4-F) at 490 nm resulted in efficient energy transfer to the acceptor dye (Ab-Cy3), leading to excitation at 615 nm. The FRET process is highly distance-dependent and only occurs when Ab-Cy3 is bound to T4-F. Upon addition of T4, the dissociation of Ab-Cy3 bound to T4-F leads to a decrease in FRET efficiency due to the increasing distance between the donor (T4-F) and acceptor (Ab-Cy3). [Examples]

[0489] Fluorescence of a mixture of beta-lactam antibiotic conjugated to fluorescein and antibody in the presence of free antibiotic. Beta-lactam antibiotics conjugated to fluorescein in PBS (i.e., AMO-FITC, AMO-F, AMP-F, and CEF-F) (400 nM, 100 μL) were mixed in 96-well black assay plates with anti-penicillin or anti-cefotaxime polyclonal antibodies (400 nM, 100 μL, commercially available from Novus Biologicals, LLC, Centennial, Colorado) in the absence and presence of free beta-lactam antibiotics (ampicillin (400 nM) was added to AMP-F, and cefotaxime (400 nM) was added to CEF-F). The solutions had tracer and antibody concentrations of 200 nM. The solutions were incubated at room temperature for 5 minutes, and fluorescence intensity was measured. The results are shown in Figure 15.

[0490] These results indicate that the fluorescence of AMO-FITC was not quenched by the addition of the anti-penicillin antibody, but the fluorescence of AMO-F and AMP-F was quenched by the same antibody. The results also indicate that the addition of ampicillin to the solution of the anti-penicillin antibody and AMP-F resulted in an increase in fluorescence intensity (i.e., recovery). Similarly, the fluorescence of CEF-F was quenched by the anti-cefotaxime antibody, and the addition of cefotaxime to the solution of the anti-cefotaxime antibody and CEF-F resulted in an increase in fluorescence intensity (i.e., recovery). [Examples]

[0491] Fluorescence of a mixture of SDM-F or SDM-Su-F and an anti-sulfadimethoxine monoclonal antibody in the presence of sulfadimethoxine. SDM-F or SDM-Su-F (400 nM, 100 μL) in PBS was mixed with anti-sulfadimethoxine monoclonal antibody (400 nM, 100 μL, commercially available from Genway Biotech, San Diego, California) in the absence and presence of sulfadimethoxine (SDM) (400 nM). The resulting solutions were incubated at room temperature for 5 minutes, and then the fluorescence intensity was measured. The results are shown in Figure 16.

[0492] These results indicate that when SDM-F and SDM-Su-F are combined with an anti-sulfadimethoxine monoclonal antibody, their fluorescence is quenched. The greater quenching for SDM-F compared to SDM-Su-F suggests that linker length influences quenching. The results also show that adding SDM to a solution of anti-sulfadimethoxine antibody and SDM-F or SDM-Su-F results in an increase in fluorescence intensity. [Examples]

[0493] Fluorescence quenching of T3-F by anti-T4-Mab T3-F was dissolved in DMSO (1 mM) and diluted in PBS to obtain a stock solution (4 μM). Monoclonal anti-T4 antibody (anti-T4-Mab, commercially available from Meridian Life Sciences Inc. (Biodesign) in Memphis, Tennessee) (4.65 mg / mL) was serially diluted in PBS buffer to obtain solutions with anti-T4-Mab concentrations ranging from 0 to 1000 nM. T3-F solution (5 μL) was added to a 96-well black assay plate containing serially diluted anti-T4-Mab solution (195 μL), mixed thoroughly, incubated for 30 minutes, and fluorescence intensity was recorded. The results are shown in Figure 17.

[0494] Figure 17 shows that T3-F fluorescence is quenched in the presence of anti-T4-Mab. Maximum quenching is reached at a molar ratio of approximately 1:1 antibody to tracer. Increasing the anti-T4-Mab to tracer ratio to 20:1 did not result in any further increase in the amount of quenching.

[0495] In another experiment, tracers T2-F, T3-F, or T4-F were dissolved in DMSO to obtain DMSO solutions with a tracer concentration of 1 mM. The DMSO solutions were then diluted with phosphate-buffered saline ("PBS") to obtain stock solutions of each tracer at a concentration of 10 μM. Monoclonal anti-T4-Mab or anti-T4-Mab conjugated with a quencher (Cy3-Ab, IRDyeQC1-Ab, Cy5-Ab, BHQ1-Ab) were serially diluted in PBS to obtain solutions containing anti-T4-Mab or anti-T4-Mab conjugated with a quencher at concentrations ranging from 0 to 2 μM. To a 96-well black assay plate containing serially diluted anti-T4-Mab or anti-T4-Mab conjugated with a quencher (95 μL), tracer T2-F, T3-F, or T4-F solution (5 μL) was added, thoroughly mixed, incubated for 30 minutes, and then fluorescence intensity was recorded at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The results of the maximum fluorescence quenching percentage for different tracer and quencher-modified antibodies are provided in the table below: Percentage of fluorescence quenching of fluorescein-conjugates with or without quencher-labeled anti-T4 antibody

[0496] [Table 1]

[0497] The results show that Cy3-Ab quenched more than 50% of the fluorescence of all three fluorescein conjugates (T2-F, T3-F, and T4-F), and IRdyeQC1-Ab quenched more than 50% of the fluorescence of both T3-F and T4-F. These combinations of labeled antibodies and fluorescein conjugates make them useful reagents for T4 assays. The data also showed that T3-F is the optimal conjugate for T4 assays compared to T2-F and T4-F. [Examples]

[0498] Fluorescence quenching of cortisol-4-Fl and cortisol-5-Fl by cortisol antibodies By dissolving hydrocortisone-3-(carboxymethyl)oxime (30 mg, 0.069 mmol, commercially available from Sigma Aldrich in St. Louis, Missouri), 4'-aminomethylfluorescein (25 mg, 0.063 mmol, commercially available from AAT Bioquest in Sunnyvale, California), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexafluorophosphate (HATU (29 mg, 0.075 mmol, commercially available from EMD Millipore in Burlington, Massachusetts)), and N'N-diisopropylethylamine (24 mg, 0.189 mmol) in anhydrous dimethylformamide (DMF, 1.5 mL), cortisol-4-Fl is obtained, i.e.:

[0499] [ka] A solution was prepared. The resulting mixture was stirred at room temperature for longer than 18 hours, then diluted in 25 mL of 50% acetonitrile aqueous solution (containing 0.1% acetic acid), and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 60% to 70% acetonitrile in water. The fractions containing cortisol-4-Fl were combined and freeze-dried to obtain a yellow solid product, as confirmed by LC-MS (M+1:779.4).

[0500] By dissolving hydrocortisone-3-(carboxymethyl)oxime (12 mg, 0.028 mmol, commercially available from Sigma Aldrich in St. Louis, Missouri), 5-aminomethylfluorescein (10 mg, 0.025 mmol, commercially available from Thermo Scientific in Waltham, Massachusetts), HATU (11 mg, 0.030 mmol, commercially available from EMD Millipore in Burlington, Massachusetts), and N'N-diisopropylethylamine (10 mg, 0.075 mmol) in anhydrous DMF (1.0 mL), cortisol-5-Fl, i.e.:

[0501] [ka] A solution was prepared. The resulting mixture was stirred at room temperature for longer than 18 hours, then diluted in 15 mL of 50% acetonitrile (containing 0.1% acetic acid) in water, and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 60% to 70% acetonitrile in water. The fractions containing cortisol-5-Fl were combined and freeze-dried to obtain a yellow solid product, as confirmed by LC-MS (M+1:779.4).

[0502] Cortisol-4-FL was combined with various commercially available α-cortisol antibodies from HyTest (Turku, Finland), Abcam (Cambridge, UK), and Bio-Connect BV (Fitzgerald Industries) (Netherlands) in varying equivalent amounts. Fluorescence was measured as a function of the ratio of antibody (Ab) to cortisol-4-FL. The results are shown in Figure 21.

[0503] The results indicate that when cortisol-4-Fl forms a complex with the antibody, its fluorescence is quenched. Maximum quenching is observed at an antibody / cortisol-4-Fl ratio of approximately 1:1.

[0504] Figure 22 shows the fluorescence quenching of cortisol-4-Fl and cortisol-5-Fl when combined with various equivalents of commercially available α-cortisol antibodies from HyTest (Turku, Finland). The results show that the fluorescence change for the 4'-substituted fluorescein tracer is greater than that for the 5-substituted fluorescein tracer. [Examples]

[0505] Synthesis of 4'-aminomethyldifluorofluorescein (4-AMDFF) According to the following synthesis scheme, 4-AMDFF is produced, i.e.:

[0506] [ka] Prepared:

[0507] [ka]

[0508] 0.1 g of chloroacetamide methanol (CLAM, 0.82 mmol) in concentrated sulfuric acid (1.5 mL) was added to a solution of 2',7'-difluorofluorescein (commercially purchased from Chemodex Ltd., Switzerland, 0.3 g, 0.82 mmol) in sulfuric acid (4 mL). After stirring for 18 hours under protection from light, the reaction mixture was poured onto ice (25 mL) to form an orange-colored precipitate. The orange precipitate was collected from the melted ice via filtration, washed with water, and dried to obtain 0.3 g of the product. The product was characterized by LC-MS ([M+1]=474.5).

[0509] 0.25 g of the above product was refluxed at approximately 160°C for 20 hours with 11 mL of diglym (3 mL) of medium-concentration HCl. After removing the solvent under reduced pressure, the residue was dissolved in 2 mL of DMF and purified using a reverse-phase chromatography system with elution via a water / acetonitrile gradient. A suitable fraction was collected, evaporated to dryness under vacuum, and characterized by LC-MS ([M+1]=398.4). [Examples]

[0510] Synthesis of 4'-aminomethyldichlorofluorescein (4-AMDCF) According to the following synthesis scheme, 4-AMDCF is produced, i.e.:

[0511] [ka] Prepared:

[0512] [ka]

[0513] 0.31 g of chloroacetamide methanol (CLAM, 2.5 mmol) in concentrated sulfuric acid (4 mL) was added to a solution of 2',7'-dichlorofluorescein (commercially available from Sigma Aldrich, St. Louis, Missouri, 1 g, 2.5 mmol) in sulfuric acid (12 mL). After stirring for 18 hours under protection from light, the reaction mixture was poured onto ice (100 mL) to form an orange-colored precipitate. The orange precipitate was collected from the melted ice via filtration, washed with water, and dried to obtain 1 g of the product. The product was characterized by LC-MS (purity higher than 90%, [M+1] = 505.8).

[0514] 1.0 g of the above product was refluxed at approximately 160°C for 20 hours with 11 mL of diglym (medium concentration HCl, 3 mL). After removing the solvent under reduced pressure, the residue was dissolved in 2 mL of DMF and purified using a reverse-phase chromatography system with elution via a water / acetonitrile gradient. A suitable fraction was collected, evaporated to dryness under vacuum, and characterized by LC-MS ([M+1]=430.4). [Examples]

[0515] Synthesis of SDMA-DFF According to the following synthesis scheme, SDMA-DFF is produced, i.e.:

[0516] [ka] Prepared:

[0517] [ka] [ka]

[0518] Synthesis of Fmoc-SDMA(Boc)2-DFF Fmoc-SDMA(Boc)2-OH (9.6 mg, 0.0148 mmol), 4'-aminomethyl 2',7'-difluorofluorescein (5.6 mg, 0.0141 mmol), HATU (6.2 mg, 0.0162 mmol), DIPEA (8.56 μL, 0.045 mmol), and anhydrous DMF (1.0 mL) were added to a 2 mL glass vial. The resulting reaction mixture was thoroughly mixed and stirred at room temperature for 18 hours. The reaction mixture was loaded into an automated purification system equipped with a reversed-phase column and eluted using a water / acetonitrile gradient (containing 0.1% formic acid). A suitable fraction was collected, and the solvent was removed by evaporation to obtain approximately 10 mg of the product as a red powder. The product was characterized using LC-MS ([M+1]=1004.6).

[0519] Synthesis of SDMA-DFF Fmoc-SDMA(Boc)2-DFF (10 mg, 0.01 mmol) was mixed with 5 mL of 20% piperidine in DMF, and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was then loaded into an automated purification system equipped with a reversed-phase column and eluted using a water / acetonitrile gradient (containing 0.1% formic acid). A suitable fraction was collected, and the solvent was removed by evaporation to obtain a red solid. The red solid was combined with 1 mL of dioxane and 0.28 mL of 4M HCl in the dioxane, stirred overnight at room temperature, and the solvent was removed by evaporation to obtain the crude product. The crude product was dissolved in water and purified using an automated purification system equipped with a reversed-phase column, and eluted using a water / acetonitrile gradient (containing 0.1% formic acid). A suitable fraction was collected, and the solvent was removed by evaporation to obtain yellow colored crystals, which were characterized by LC-MS ([M+1]=582.2). [Examples]

[0520] Synthesis of SDMA-DCF According to the following synthesis scheme, SDMA-DCF is produced, i.e.:

[0521] [ka] Prepared:

[0522] [ka]

[0523] Synthesis of Fmoc-SDMA(Boc)2-DCF Fmoc-SDMA(Boc)2-OH (20 mg, 0.0308 mmol), 4'-aminomethyl 2',7'-dichlorofluorescein (12.6 mg, 0.0294 mmol), HATU (13 mg, 0.034 mmol), DIPEA (17.8 μL, 0.088 mmol), and anhydrous DMF (1.0 mL) were added to a 2 mL glass vial. The resulting reaction mixture was thoroughly mixed and stirred at room temperature for 18 hours. The reaction mixture was loaded into an automated purification system equipped with a reversed-phase column and eluted using a water / acetonitrile gradient (containing 0.1% formic acid). A suitable fraction was collected, and the solvent was removed by evaporation to obtain approximately 25 mg of the product as a red powder. The product was characterized using LC-MS ([M+1]=1036.5).

[0524] Synthesis of SDMA-DCF Fmoc-SDMA(Boc)2-DCF (15 mg, 0.0145 mmol) was mixed with 5 mL of 20% piperidine in DMF, and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was loaded into an automated purification system equipped with a reversed-phase column and eluted with a water / acetonitrile gradient (containing 0.1% formic acid). A suitable fraction was collected, and the solvent was removed by evaporation to obtain a red solid. The red solid was combined with 1 mL of dioxane and 0.28 mL of 4M HCl in the dioxane, stirred overnight at room temperature, and the solvent was removed by evaporation to obtain the crude product. The crude product was dissolved in water and purified using an automated purification system equipped with a reversed-phase column, and eluted with a water / acetonitrile gradient (containing 0.1% formic acid). A suitable fraction was collected, and the solvent was removed by evaporation to obtain an orange solid, which was characterized by LC-MS ([M+1]=615.6). [Examples]

[0525] Absorption and emission spectra of DFF, SDMA-DFF, DCF, SDMA-DCF, Fl, and SDMA-FL The maximum absorption of fluorescein (Fl) in PBS buffer is 489 nm, and the maximum fluorescence emission is 515 nm. When a fluorescein molecule is conjugated with an SDMA molecule via a -CH2NH- linker (i.e., obtaining SDMA-Fl), the maximum absorption and emission shift to 495 nm and 525 nm, respectively. When a fluorescein molecule is substituted with fluorine at the 2' and 7' positions (i.e., obtaining DFF), there is no change in maximum absorption and emission compared to unsubstituted fluorescein. When a fluorescein molecule is substituted with chlorine at the 2' and 7' positions (i.e., obtaining DCF), the maximum absorption and emission shift to red, to 500 nm and 525 nm, respectively. Conjugating DFF or DCF to SDMA via a -CH2NH- linker (i.e., obtaining SDMA-DFF and SDMA-DCF, respectively) also shifts the maximum absorption and emission to red. The maximum absorption and emission wavelengths of fluorescein (FL), 2',7'-difluorofluorescein (DFF), 2',7'-dichlorofluorescein (DCF), and conjugates with SDMA via a -CH2-NH-linker (i.e., SDMA-Fl (i.e., structure A3), SDMA-DFF, and SDMA-DCF) are listed in the table provided below. The absorption spectra of DFF, SDMA-DFF, DCF, SDMA-DCF, Fl, and SDMA-FL are shown in Figure 25A, and the emission spectra of SDMA-DFF, SDMA-DCF, Fl, and SDMA-FL are shown in Figure 25B.

[0526] [Table 2] [Examples]

[0527] Fluorescence of a mixture of various SDMA fluorescent tracers and anti-SDMA antibodies Solutions of anti-SDMA antibody or anti-SDMA antibody conjugated to BHQ10 (4 equivalents) in phosphate-buffered saline (PBS) were prepared at concentrations of 0, 0.25, 0.5, 1, 2, 4, 8, and 16 μM (antibody solutions). Solutions of SDMA fluorescence tracers (i.e., SDMA-Fl, SDMA-DFF, or SDMA-DCF) in PBS were prepared at a concentration of 100 nM (tracer solutions). 50 μL of antibody solution and 50 μL of tracer solution were combined in a 96-well UV plate and incubated at room temperature for 30 minutes. The fluorescence intensity of each mixture was read using an excitation wavelength of 490 nm, and emission wavelengths of 525 nm for SDMA-DFF and SDMA-Fl conjugates, and 535 nm for SDMA-DCF. The results are shown in Figures 26 and 27.

[0528] Figure 26 shows the fluorescence quenching percentage for different SDMA tracers as a function of increasing antibody concentration. Figure 26 shows that approximately 50% of the fluorescence of the SDMA-DFF tracer is quenched by the addition of anti-SDMA antibody, and approximately 30% of the fluorescence of the SDMA-Fl tracer is quenched by the addition of anti-SDMA antibody. Figure 26 shows that in SDMA-DFF, the quenching efficiency is nearly saturated when the molar ratio of antibody to SDMA-DFF is greater than 2:1. The quenching efficiencies of anti-SDMA antibody (not conjugated to a quencher) and SDMA-DFF tracers indicate that SDMA-DFF tracers can be used as an assay reagent in homogeneous SDMA assays with anti-SDMA antibody (not conjugated to a quencher).

[0529] Figure 27 shows the fluorescence quenching percentage for different SDMA tracers as a function of increasing concentrations of anti-SDMA antibody conjugated to BHQ10 (4 equivalents). Figure 27 shows that the quenching efficiency is slightly greater for SDMA-DFF compared to SDMA-Fl.

[0530] Solutions of various SDMA tracers (SDMA-Fl, SDMA-DFF, and SDMA-DCF) in PBS (containing 0.1% Tween) were prepared at a concentration of 400 nM. Solutions of anti-SDMA antibody or anti-SDMA antibody conjugated to BHQ10 (4 equivalents) in PBS were prepared at concentrations of 400 nM and 800 nM, respectively. The SDMA tracer solutions were mixed with the solutions of anti-SDMA antibody or anti-SDMA antibody conjugated to BHQ10 in a 1:1 (v / v) ratio and incubated at room temperature for 60 minutes to obtain SDMA tracer / SDMA antibody solutions.

[0531] SDMA standard solutions were prepared by serially diluting SDMA in PBS (containing 0.1% Tween) to final SDMA concentrations of 100, 50, 25, 12.5, 6.26, and 0 μg / dL.

[0532] 50 μl of each SDMA standard solution was added to the wells of a 96-well UV transparent plate. Then, 50 μl of SDMA tracer / SDMA antibody solution was added to each well, and the resulting solutions were thoroughly mixed by shaking and incubated at room temperature for 30 minutes. The fluorescence of each solution was then read using a plate reader, with an excitation wavelength of 490 nm and emission wavelengths of 525 nm for SDMA-DFF and SDMA-Fl, and 535 nm for SDMA-DCF.

[0533] The results are shown in Figures 28 to 30. Figures 28 to 30 show that there is an increase in fluorescence intensity as a function of SDMA concentration, i.e., a dose-response is observed.

[0534] Figure 28 shows the increase in fluorescence intensity when the SDMA tracer is SDMA-DFF. Figure 28 shows that when the SDMA tracer / SDMA antibody solution is SDMA-DFF and antibody (i.e., antibody not conjugated to BHQ10), the increase in fluorescence intensity is greater when the antibody / SDMA-DFF ratio is 2:1 than when the ratio is 1:1. Figure 28 further shows that an increase in fluorescence intensity is observed when the SDMA tracer / SDMA antibody solution is SDMA-DFF and antibody conjugated to BHQ10.

[0535] Figure 29 shows the increase in fluorescence intensity when the SDMA tracer is SDMA-DCF, and Figure 30 shows the increase in fluorescence intensity when the SDMA tracer is SDMA-DFl. Figures 29 and 30 show that the increase in fluorescence intensity is greater when the SDMA antibody is conjugated to BHQ10 compared to when the SDMA antibody is not conjugated to BHQ10. [Examples]

[0536] Synthesis of SDMA-Fl According to the following synthesis scheme, SMDA-Fl, i.e.:

[0537] [ka] Prepared:

[0538] [ka]

[0539] Fmoc-SDMA(Boc)2-Fl Fmoc-SDMA(Boc)2-OH (47 mg, 0.075 mmol, commercially available from Novabiochem, a division of Sigma Aldrich in St. Louis, Missouri), 4-aminomethylfluorescein (25 mg, 0.063 mmol, commercially available from ATT Bioquest in Sunnyvale, California), HATU (28.7 mg, 0.075 mmol), and N'N-diisopropylethylamine (23.6 mg, 0.189 mmol) were dissolved in anhydrous DMF (1.5 mL). The resulting mixture was stirred at room temperature for 18 hours, then diluted in 15 mL of 40% acetonitrile in water (containing 0.1% acetic acid), and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 20% to 50% acetonitrile in water. When the fractions containing Fmoc-SDMA(Boc)2-Fl were combined and freeze-dried, a yellow solid product was obtained, as confirmed by LC-MS (M+1:968.3).

[0540] SDMA(Boc)2-Fl Fmoc-SDMA(Boc)2-Fl (45 mg, 0.0465 mmol) was deprotected in 20% piperidine (8 mL) in DMF for 1 hour. The solvent was then removed under reduced pressure to obtain the residue. The obtained residue was dissolved in 40% acetonitrile in water containing 0.1% acetic acid and purified by column chromatography using a C18 reversed-phase column (5 g) eluted with 40% acetonitrile in water containing 0.1% acetic acid. The fractions containing SDMA(Boc)2-Fl were combined and freeze-dried to obtain an orange powder.

[0541] SDMA-Fl SDMA(Boc)2-Fl (21 mg, 0.028 mmol) was dissolved in dioxane (2 mL), and 4 M HCl (0.28 mL) was added to the solution in the dioxane. The resulting mixture was stirred overnight. The solvent was removed, and the residue was dissolved in 20% acetonitrile in water and purified by column chromatography using a C18 reversed-phase column (5 g) that elutes with 20% acetonitrile in water. The fractions containing SDMA-Fl were combined and freeze-dried to obtain SDMA-Fl as a yellow powder, and its structure was confirmed by LC-MS (M+1:546.9). [Examples]

[0542] Quenching of riboflavin fluorescence in milk by riboflavin-binding protein The following examples used "in-house" riboflavin-binding protein purified from chicken egg white (i.e., "IDEXX in-house" riboflavin-binding protein) and commercially available (Sigma Aldrich, St. Louis, Missouri) riboflavin-binding protein.

[0543] A stock solution of riboflavin (1 mM) was added to milk to obtain a final riboflavin concentration of 4 μM. Stock solutions of riboflavin-binding protein ("RBP," 49.2 mg / mL in phosphate-buffered saline ("PBS")), either from IDEXX or commercially available (Sigma), were serially diluted in PBS to obtain solutions with RBP concentrations ranging from 0 to 1600 μM. 20 μL of the serially diluted RBP solution was added to 2 mL of riboflavin-added milk to obtain RBP concentrations ranging from 0 to 20 μM. The resulting solutions were incubated at room temperature for 1 hour, and the fluorescence intensity of a 90 μL volume of the obtained solution was measured at an excitation wavelength of 350 nm and an emission wavelength of 538 nm. Figure 31 shows the riboflavin fluorescence in milk as a function of riboflavin-binding protein concentration.

[0544] The results show that when riboflavin-binding protein was added to milk at a concentration of 5 μM, more than 95% of the riboflavin fluorescence was quenched, and the fluorescence background of the milk was significantly reduced. The results are shown in Figure 31. [Examples]

[0545] Preparation of dry slides for SDMA assay Dry slides for SDMA assay were prepared using the following method:

[0546] A Melinex sheet (10'' thick × 125 μm × 24'' long) (commercially available from HiFi Film, Stevenage, UK, part number 506) was coated with a solution containing approximately equal by weight volumes of D4 Hydrogel (high viscosity polymer) and D4 Hydrogel (low viscosity polymer) (both commercially available from AdvanSource Biomaterials Corp, Wilmington, Massachusetts) to obtain a wet primer layer with a thickness of approximately 40 μm. The solvent was evaporated to obtain a dry primer layer containing approximately equal by weight volumes of D4 Hydrogel (high viscosity polymer) and D4 Hydrogel (low viscosity polymer).

[0547] A wet indicator layer with a thickness of approximately 93 μm was coated onto the dry primer layer using a solution containing SDMA-fluorescein (prepared as described above in Example 35) and anti-SDMA antibody-BHQ10 conjugate (prepared as described below in Example 38) in HEPES buffer (pH 8), HEPES buffer (pH 8), Merpol A, pullulan, and cellulose. The solvent was evaporated to obtain approximately 1.4 × 10⁻⁶ -1 wt% anti-SDMA antibody-BHQ10 conjugate, approximately 1.1 × 10⁻⁶ -3 A dry indicator layer was obtained containing wt% SDMA-fluorescein, approximately 13 wt% HEPES buffer, approximately 1.2 wt% Merpol A, approximately 28.5 wt% pullulan, and approximately 57 wt% cellulose.

[0548] A solution containing approximately equal by weight amounts of D4 Hydrogel (high viscosity polymer) and D4 Hydrogel (low viscosity polymer) (both commercially available from AdvanSource Biomaterials Corp., Wilmington, Massachusetts), cellulose (commercially available from Sigma Aldrich, St. Louis, Missouri), and titania microparticles (commercially available from Chemours, Fayetteville, North Carolina, part number R-706) was coated onto a dry indicator layer to obtain a wet titanium oxide layer with a thickness of approximately 130 μm. The solvent was evaporated to obtain a dry titanium oxide layer containing approximately 37 wt% D4 Hydrogel (as approximately equal by weight amounts of low viscosity and high viscosity D4 Hydrogel), approximately 20 wt% cellulose, and approximately 43 wt% titanium dioxide.

[0549] A solution containing approximately equal amounts of D4 Hydrogel (high viscosity polymer) and D4 Hydrogel (low viscosity polymer) (both commercially available from AdvanSource Biomaterials Corp., Wilmington, Massachusetts), as well as carbon black and Lamp Black 101 powder (commercially available from Orion Specialty Carbon Blacks, Belpre, Ohio), was coated onto a dry titanium oxide layer to obtain a wet carbon black layer with a thickness of approximately 140 μm. The solvent was evaporated to obtain a dry carbon black layer containing approximately 95 wt% D4 Hydrogel (as approximately equal amounts by weight of low-viscosity and high-viscosity D4 Hydrogel) and approximately 5 wt% carbon black.

[0550] A solution containing cellulose, tetramethylammonium hydroxide (TMAH), polyacrylic acid (PAA), molecular weight approximately 1 million, and polyvinylpyrrolidone (PVP) was coated onto a dry carbon black layer to obtain a wet diffusion layer with a thickness of approximately 310 μm. The solvent was evaporated to obtain a dry diffusion layer containing approximately 83% by weight of cellulose, approximately 0.5% by weight of PAA, approximately 0.4% by weight of TMAH, and approximately 16% by weight of PVP.

[0551] Each wet layer was coated using a Slot Die Loop Coater with a drying tunnel and a heating pad.

[0552] The resulting Melinex sheets, coated with a primer layer, indicator layer, titanium oxide layer, carbon black layer, and diffusion layer, were formed into 6.5 mm discs using a perforator. The resulting discs were placed in the adhesive pads of a slide housing for use with an IDEXX Catalyst Instrument (commercially available from IDEXX Laboratories, Inc. in Westbrook, Maine) and bonded to it, and the resulting slides were then wrapped in packaging.

[0553] Panel assay of serum with various levels of SDMA using slides Using an IDEXX Catalyst pipette tip (commercially available from IDEXX Laboratories, Inc., Westbrook, Maine), an IDEXX Catalyst sample cup (commercially available from IDEXX Laboratories, Inc., Westbrook, Maine), and an IDEXX Catalyst instrument (commercially available from IDEXX Laboratories, Inc., Westbrook, Maine) consisting of the slides described above, charcoal-treated dog serum (0 μg / dL SDMA), charcoal-treated dog serum supplemented with SDMA (5 μg / dL), and dog serum supplemented with SDMA (15, 30, 60, 100 μg / dL) were analyzed. The following procedure was followed: Remove the SDMA slide from the packaging and place it in the IDEXX Catalyst instrument; Pipette 300 μl of the panel into the sample cup; Heat the slide to 37°C on the IDEXX Catalyst instrument; After heating, the fluorescence intensity of the dry slide was measured by the instrument (λex=470 nm , λ em Measure and record the wavelength (=525nm) at 15-second intervals for 1.5 minutes; Next, using an IDEXX Catalyst instrument, 8 μl of the sample is pipetteed onto the slide, and then the fluorescence intensity is measured and recorded for 400 seconds at 15-second intervals as described above; The recorded measurements are compared to the calibration curve used to generate the SDMA dose levels.

[0554] Figure 32 shows the assay results. Figure 32 shows the fluorescence intensity on slides observed with a Catalyst instrument over time when different levels of SDMA were dispensed (legend). The y-axis is intensity (relative fluorescence units (unitless)) and the x-axis is time (seconds). First, the fluorescence intensity reading of the dry slide is taken, and then the fluorescence intensity reading of the wet slide is measured at t=0 (i.e., when the sample is dispensed). Fluorescence intensity is read every 15 seconds. Each trace is the average of n=10 replicates. [Examples]

[0555] Preparation of anti-SDMA antibody-BHQ10 conjugate A stock solution of anti-mAb SDMA (custom-made by Leinco Technologies, Fenton, Missouri) at a concentration of 5 mg / mL is prepared in 250 mM HEPES (pH 8) by diluting anti-mAb SDMA to 2.5 mg / mL in 250 mM HEPES and 40% sucrose at pH 8 to obtain an antibody solution in 250 mM HEPES and 20% sucrose. Lyophilized BHQ10-NHS ester (commercially available from LGC Biosearch Technologies, Middlesex, UK, no. BHQ10S) is solvated in anhydrous DMSO to a final concentration of 5 mg / mL. Then, 20 molar equivalents of BHQ10 in anhydrous DMSO are added to the antibody solution with gentle mixing (e.g., 52 μL of BHQ-10 solution to 1 mL of anti-mAb SDMA solution). The resulting solution was incubated unmixed at room temperature for 1 hour, and the reaction was quenched by adding 10 μL of 1 M Tris buffer (commercially available from Alfa Aesar, Tewkesbury, Massachusetts). The resulting solution was incubated at room temperature for 10 minutes, centrifuged to remove precipitate, and used without further adjustment. [Examples]

[0556] Determination of SDMA in the presence of various interfering substances To evaluate the effects of interfering substances (lipidemia, bilirubin, hemolysis, and whole blood) on dry slide performance, fresh sample solutions were prepared in pooled charcoal-treated hybrid serum with SDMA (commercially available from BioIVT, Westbury, NY, No. DOG42577) at a range of interfering substance levels. Each interfering substance level at each SDMA concentration was prepared through dilution. Samples with lipemia as the interfering substance were prepared by forming stock solutions in charcoal-treated serum with a desired SDMA concentration (e.g., 100 μg / dL) and the highest interfering substance level, 1000 mg / dL lipemia (Intralipid 20% emulsion, commercially available from Sigma Aldrich, St. Louis, Missouri, No. 100267553). Next, the obtained stock solution was serially diluted with a control solution of 100 μg / dL SDMA in charcoal-treated serum to prepare solutions with lipidemia concentrations of 0, 250, 500, 750, and 1000 mg / dL. In this way, solutions with different levels of interfering agent were prepared at SDMA concentrations of 0, 15, 30, and 100 μg / dL to evaluate the effect of the interfering agent over a range of SDMA concentrations. For each solution, the SDMA concentration was determined by LC-MS, and the lipidemia concentration was confirmed using a Beckman AU5812 Clinical Chemistry Analyzer (commercially available from Beckman Coulter, Blair, California).

[0557] Using a similar dilution protocol, bilirubin (bilirubin conjugate, commercially available from Scripps, La Jolla, California, number B011490604) solutions were prepared with SDMA concentrations of 0, 15, 30, and 100 μg / dL, and bilirubin concentrations of 0, 7.5, 15, 22.5, and 30 mg / dL. Bilirubin concentrations were confirmed using a Beckman AU5812 Clinical Chemistry Analyzer (commercially available from Beckman Coulter, Blair, California). SDMA concentrations were determined by LC-MS.

[0558] Hemolytic interferant solutions were prepared from freshly collected and hemolyzed blood samples (commercially available from Pet Food Solutions, Ward, South Carolina) and diluted as described above to hemolyzed blood concentrations of 0, 125, 250, 375, and 500 mg / dL. Hemolyzed blood concentrations were confirmed using a Beckman AU5812 Clinical Chemistry Analyzer (commercially available from Beckman Coulter, Blair, California). SDMA concentrations were determined by LC-MS.

[0559] To prepare whole blood samples, lithium-heparinized anticoagulant-treated whole blood (commercially available from Pet Food Solutions, Ward, South Carolina) was diluted with treated serum to SDMA concentrations of 0, 15, 60, and 100 μg / dL, as well as whole blood percentages of 1, 5, and 10%. For each solution, the whole blood percentage was determined by measuring the hematocrit, and the SDMA concentration was determined by LCMS.

[0560] Figure 24 shows the results of an assay (using a slide with a filtering layer) to determine the concentration of SDMA in a sample containing a fixed amount of SDMA. The results indicate that interfering substances do not interfere with the analysis of SDMA. [Examples]

[0561] Synthesis of cholic acid-fluorescein conjugate (CA-Fl)

[0562] [ka]

[0563] Cholic acid (28 mg, 0.069 mmol, commercially available from Sigma Aldrich, St. Louis, Missouri), 4'-aminomethylfluorescein (25 mg, 0.063 mmol, commercially available from AAT Bioquest, Sunnyvale, California), HATU (29 mg, 0.075 mmol), and N'N-diisopropylethylamine (29 mg, 0.189 mmol) were dissolved in anhydrous DMF (1.5 ml). The resulting mixture was stirred at room temperature for longer than 20 hours, and the crude product was purified using a Biotage Selekt System (commercially available from Biotage, Sweden) with a Teledyne Isco RediSep RF Gold C18AQ 5.5 gm column eluted with a mobile phase gradient of 50% to 100% acetonitrile in water containing 0.1% formic acid. The fraction containing CA-Fl was recovered and freeze-dried to obtain a yellow solid, which was then characterized using LCMS (M+1:752.4). [Examples]

[0564] Fluorescence quenching of CA-Fl by anticholic acid antibody in PBS A working solution (100 nM) of CA-Fl in PBS was prepared. The working solution was then combined with various equivalents of α-cholic acid antibody (1:1, v / v) (commercially available from Fitzgerald Industries, Netherlands). Fluorescence was measured as a function of the ratio of antibody (Ab) to CA-Fl. The results are shown in Figure 33. The results indicate that when CA-Fl forms a complex with the antibody, its fluorescence is quenched, and the amount of quenching is a function of the molar ratio of antibody to CA-Fl tracer. The amount of quenching increases with increasing antibody-to-CA-Fl tracer ratio. The results also demonstrate that quenching saturated when the molar ratio of antibody to CA-Fl tracer was approximately 8:1.

[0565] Aliquotes of CA-Fl (200 nM) in PBS (containing 0.1% tween 20) were mixed 1:1 (v / v) with antibody solutions of 200, 400, or 800 nM concentrations. The resulting mixtures were incubated at 4°C for 30 minutes to obtain three assay reagents: Reagent 1 (Ab 100 nM / CA-Fl 100 nM), Reagent 2 (Ab 200 nM / CA-Fl 100 nM), and Reagent 3 (Ab 400 nM / CA-Fl 100 nM).

[0566] Standard solutions of bile acids (cholic acid or taurocholic acid) at concentrations ranging from 0 to 80 μM were prepared in PBS (containing 0.1% tween 20). Each bile acid standard solution (5 μl) was then added to reagent 1, 2, or 3 (95 μl) in a 96-well assay plate, and the resulting solutions were incubated at room temperature for 30 minutes with gentle shaking. Fluorescence intensity was then measured using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The results are shown in Figure 34A (cholic acid) and Figure 34B (taurocholic acid). Figures 34A and 34B show fluorescence recovery as a function of bile acid concentration. The results indicate that reagent 3, with an antibody:CA-Fl ratio of 4:1, exhibits the widest dynamic range for bile acids in PBS at concentrations ranging from 0 to 80 μM. [Examples]

[0567] Fluorescence quenching of CA-Fl by anticholic acid antibody as a function of serum bile acid concentration Charcoal-treated dog serum was adjusted to pH 7.3 with phosphate buffer. Then, bile acids (cholic acid or taurocholic acid) were added to the serum to achieve a bile acid concentration in the range of 0 to 40 μM.

[0568] A CA-Fl solution (200 nM) in PBS was mixed with an α-cholic acid antibody solution (400 nM) in a 1:1 (v / v) ratio, and incubated at 4°C for 30 minutes to obtain the assay reagent.

[0569] Next, a solution of canine serum containing bile acids (5 μl) was added to the assay reagent (95 μl) in a 96-well assay plate, and the resulting mixture was incubated at room temperature for 30 minutes with gentle shaking.

[0570] Fluorescence intensity was measured using an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The results for cholic acid (Figure 35A) and taurocholic acid (Figure 35B) are shown in Figure 35. Figures 35A and 35B show fluorescence recovery as a function of serum bile acid concentration. The results indicate that the assays for cholic acid and taurocholic acid in serum have a dynamic range of 0–40 μM. [Examples]

[0571] Synthesis of BHQ10-labeled cystatin-B (CysB):CysB-BHQ Recombinant cystatin-B protein (4.0 mg) in PBS (1 mL) was mixed with 2.0 mg (8 equivalents, 204 μL of 10 mg / mL stock) of BHQ10-NHS (N-hydroxysuccinimide ester of BHQ10, commercially available from LGC Biosearch Technologies, Petaluma, California) dissolved in DMSO (0.25 mL). The resulting solution was swirled for 2 hours. The quencher-labeled protein was then purified by dialysis against 3 × 4 L of PBS for a minimum of 2 hours per exchange. A 10 kDa MWCO filter (G2 cassette, available from Thermo Scientific, Waltham, Massachusetts) was used during dialysis. The protein concentration of the resulting product was determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0572] Fluorescein-labeled anti-CysB(3H4)-mAb, NHS ester pathway: Synthesis of anti-CysB-FL Monoclonal anti-CysB antibody (anti-CysB(3H4)-mAb) (5 mg) in PBS (1 mL) was mixed with 0.025 mg (2 equivalents, 12.5 μL of 2 mg / mL stock) of fluorescein-NHS (fluorescein N-hydroxysuccinimide ester, commercially available from Sigma Aldrich, St. Louis, Missouri) dissolved in DMSO. The resulting solution was swirled for 2 hours. The fluorescein-labeled antibody was then purified by dialysis against 3 × 4 L of PBS for a minimum of 2 hours per exchange. A 10 kDa MWCO filter (G2 cassette, available from Thermo Scientific, Waltham, Massachusetts) was used during dialysis. The protein concentration of the resulting product was determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0573] Fluorescein-labeled anti-CysB(3H4)-mAb, fluorescein aldehyde pathway: synthesis of anti-CysB-FL-Ald A monoclonal anti-CysB antibody (anti-CysB(3H4)-mAb) (5 mg) was mixed with 0.024 mg (2 equivalents) of fluorescein aldehyde dissolved in DMSO in PBS (1 mL). To the resulting solution, 0.02 mg (10 equivalents) of sodium borohydride dissolved in 1 N NaOH was added. The mixture was swirled for 2 hours. The fluorescein-labeled antibody was then purified by dialysis against 3 × 4 L of PBS for a minimum of 2 hours per exchange. A 10 kDa MWCO filter (G2 cassette, available from Thermo Scientific, Waltham, Massachusetts) was used during dialysis. The protein concentration of the resulting product was determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0574] BODIPY-labeled anti-CysB(3H4)-mAb: Synthesis of anti-CysB-BODIPY 5 mg of monoclonal anti-CysB antibody (anti-CysB(3H4)-mAb) in 1 mL of PBS was mixed with 0.026 mg (2 equivalents) of BODIPY-NHS (commercially available from Thermo Scientific, Waltham, Massachusetts) dissolved in DMSO. The resulting solution was swirled for 2 hours. The fluorescein-labeled antibody was then purified by dialysis against 3 × 4 L of PBS for a minimum of 2 hours per exchange. A 10 kDa MWCO filter (G2 cassette, available from Thermo Scientific, Waltham, Massachusetts) was used during dialysis. The protein concentration of the resulting product was determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0575] 96-well plate quenching and dose-response assay for CysB The following examples use CysB-BHQ prepared as described in Example 41 and anti-CysB-FL prepared as described in Example 42. 1. A 10-fold dilution of the BHQ- and fluorescein-conjugate stock solution was obtained by diluting it 10-fold in PBS containing 1% BSA with 0.005% Tween 20, pH=7.4, to obtain an intermediate stock solution. 2. The intermediate stock solution was diluted four-fold to working concentration in PBS containing 1% BSA with 0.005% Tween20, pH=7.4. [Anti-CysB-FL]=400nM and [CysB-BHQ]=800nM. 3. 50 μL of CysB-BHQ solution and 50 μL of anti-CysB-FL solution were added to the wells of a 96-well black-bottom plate. The 96-well plate was gently shaken for 30 seconds, incubated at room temperature for 5 minutes, and read with a plate reader at 37°C. Final [anti-CysB-FL] = 200 nM, final [CysB-BHQ] = 200 nM; excitation wavelength: 470 nm, and emission wavelength: 520 nm. 4. 100 μL of 2x working solution of different concentrations of recombinant CysB standard (PBS, pH=7.4) was added to each well. The 96-well plate was then gently shaken for 30 seconds, incubated at 37°C for 30 minutes, and fluorescence was measured. Final [anti-CysB-FL] = 100 nM, [CysB-BHQ] = 200 nM, [CysB] = 0, 0.25, 0.5, 1, 2, 5 μg / mL. Excitation wavelength: 470 nm, emission wavelength: 520 nm.

[0576] Figure 36 shows the fluorescence intensity (λex=470 nm , λ ex This is a plot of fluorescence recovery (at 520 nm) versus CysB concentration (μg / mL). Figure 36 shows fluorescence recovery as a function of CysB concentration, demonstrating that the assay can be used to measure CysB concentration in a sample. The assay has sensitivity up to 125 ng / mL, covering a clinically relevant range. The maximum recovery of the fluorescence signal was 75% of unbound anti-CysB-FL fluorescence. [Examples]

[0577] Catalyst slide dose-response test The slides were prepared using the following procedure: A mixture of CysB-BHQ (1600 nM) prepared as described in Example 41 and anti-CysB-FL (800 nM) prepared as described in Example 42, or anti-CysB-BODIPY (800 nM) prepared as described in Example 44, in PBS containing 1% BSA with 1.005% Tween20, pH 7.4, was spotted (12 μL) onto a 7 mm diameter Fusion 5 film mounted on a plastic slide housing. 2. The spotted slide was protected from light and dried at 40°C for 30 minutes. 3. Next, the slide was cooled to room temperature and loaded into a Catalyst Dx instrument (commercially available from IDEXX Laboratories in Portland, Maine). 4. Serum samples were supplemented with recombinant CysB protein at concentrations of 0, 250, 500, 1000, and 2000 ng / mL and loaded into a Catalyst Dx instrument. 5. Next, the sample was dispensed into slides (10 μL) and the fluorescence intensity was measured over time.

[0578] Figure 37 plots the rate of increase in fluorescence intensity (measured over time intervals of 30–100 seconds) versus the concentration of CysB in a dry slide format. These results indicate that the anti-CysB-FL / CysB-BHQ immunocomplex remains intact after drying on a solid support, and that adding the sample to a dry slide can result in a clinically relevant dose-response range. [Examples]

[0579] Synthesis of fluorescein-labeled cystatin-B conjugated to FL via lysine residues Recombinant cystatin-B protein (4.0 mg) in PBS (1 mL) was mixed with 0.34 mg (2 equivalents, 17 μL of 20 mg / mL stock) of fluorescein-NHS (commercially available from Sigma-Aldrich) dissolved in DMSO. The resulting solution was swirled for 2 hours. Subsequently, fluorescein-labeled CysB (lysine-modified) was purified by dialysis against 3 × 4 L of PBS for a minimum of 2 hours per exchange. A 10 kDa MWCO filter (G2 cassette, available from Thermo Scientific, Waltham, Massachusetts) was used during dialysis. The protein concentration of the resulting product was determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0580] Synthesis of fluorescein-labeled cystatin-B conjugated to FLT via cysteine ​​residues Recombinant cystatin-B protein (4.0 mg) in PBS (1 mL) was mixed with 10 μL of 0.5 M EDTA (commercially available from Alfa Aesar, Hevalle, Massachusetts) to a final concentration of 5 mM EDTA. The resulting solution was added to 0.5 mL of TCEP-binding resin (commercially available from Thermo Scientific, Waltham, Massachusetts) in a centrifuge tube containing a 0.2 μm filter and centrifuged at room temperature for 2 hours. The reaction mixture was then briefly centrifuged at 8000 rpm for 1 minute. The flow-through was collected to obtain reduced CysB at a concentration of 4 mg / mL (containing one activated cysteine ​​residue). This activated CysB was mixed with 0.31 mg (2 equivalents, 15 μL of 20 mg / mL stock) of fluorescein-maleimide (commercially available from Sigma Aldrich, St. Louis, Missouri) dissolved in DMSO. The resulting solution was centrifuged for 2 hours. Next, fluorescein-labeled CysB (cysteine-modified) was purified by dialysis in 3 × 4 L of PBS for a minimum of 2 hours per exchange. A 10 kDa MWCO filter (G2 cassette, available from Thermo Scientific, Waltham, Massachusetts) was used during dialysis. The protein concentration of the resulting product was determined using a BCA kit (commercially available from Thermo Scientific, Waltham, Massachusetts). [Examples]

[0581] CysB peptide-fluorophore conjugate Peptide conjugated at the 5th position of fluorescein

[0582] [ka] Peptide conjugated at the 4' position of fluorescein

[0583] [ka]

[0584] Canine CysB Sequence Inucystatin-B, full length: MMCGAPSASQPATADTQAIADQVKAQLEERENKKYTTFKAVTFRSQVVAGTPYFIKVQVDDDEFVHLRVFQSLPHENKPLALSSYQTNKAKHDELAYF[Sequence ID:1] P9: QTNKAKHDELAYF[Sequence ID:2] P14: YQTNKAKHDELAYF[Sequence ID:3] P7: GHDELAYF[Sequence ID:4] P6: GDELAYF[Sequence ID: 5] P5: GELAYF[Sequence ID:6] P4: GLAYF[Sequence ID:7]

[0585] Preparation of canine CysB peptide-Fl conjugate conjugate in position 5 CysB peptides P7, P6, P5, and P4 were conjugated at the 5-position of fluorescein as follows: CysB peptide P6 (15 mg, 0.0184 mmol, custom synthesized by GenScript in Piscataway, New Jersey) having the amino acid sequence GDELAYF [SEQ ID NO: 6], 5-carboxyfluorescein succinimidyl ester (9.2 mg, 0.0194 mmol, commercially available from Thermo Fisher in Waltham, Massachusetts), and N'N-diisopropylethylamine (9.67 μl, 0.055 mmol) were dissolved in anhydrous DMF (0.5 ml). The resulting mixture was stirred overnight at room temperature and then diluted in 1 ml of 30% ACN in water (containing 0.1% formic acid) and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 30% to 100% acetonitrile in water in a Biotage Isolera® purification system. The fractions were automatically recovered by monitoring absorption at both 257 nm and 470 nm. Fractions with absorption at 470 nm were analyzed by LC-MS to identify fractions containing the target peptide P6-Fl conjugate. The desired fractions were combined and freeze-dried to obtain a final yellow solid product confirmed by LC-MS (M+1=1172).

[0586] Using substantially the same synthesis and purification procedures as described above for CysB peptide P6, CysB peptides P7, P5, and P4 conjugates with fluorescein at position 5 were synthesized. The final conjugates were characterized by LCMS as GHDELAYF, P7-Fl, LCMS(M+1=1309); GELAYF, P5-Fl, LCMS(M+1=1057); and GLAYF, P4-Fl, LCMS(M+1=928).

[0587] Preparation of canine CysB peptide-DFF conjugates conjugated at position 5. CysB peptides P7, P6, P5, and P4 were conjugated at the 5-position of DFF as follows: CysB peptide P6 (4.2 mg, 0.0052 mmol, custom synthesized by GenScript, Piscataway, NJ) having the amino acid sequence GDELAYF [SEQ ID NO: 6], 5-difluorocarboxyfluorescein succinimidyl ester (2.5 mg, 0.0049 mmol, commercially available from ATT Bioquest, Sunnyvale, California), and N'N-diisopropylethylamine (4.29 μl, 0.0245 mmol) were dissolved in anhydrous DMF (0.5 ml). The resulting mixture was stirred overnight at room temperature and then diluted in 1 ml of 30% ACN in water (containing 0.1% formic acid) and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 30% to 100% acetonitrile in water in a Biotage Isolera® purification system. The fractions were automatically recovered by monitoring absorption at both 257 nm and 470 nm. Fractions with absorption at 470 nm were analyzed by LC-MS to identify fractions containing the target peptide P6-DFF conjugate. The desired fractions were combined and lyophilized to obtain a final yellow solid product confirmed by LC-MS (M+1=1208). Using substantially the same synthesis and purification procedures as described above for CysB peptide P6, as well as appropriate starting materials, CysB peptides P7, P5, and conjugates with DFF at positions P4 and P5 were synthesized. The final conjugates were characterized by LC-MS as GHDELAYF, P7-DFF, LCMS (M+1=1345); GELAYF, P5-DFF, LCMS (M+1=1093); GLAYF, P4-DFF, LCMS (M+1=964).

[0588] Preparation of canine CysB peptide-4-Fl conjugate conjugate conjugated at the 4' position. CysB peptides P6, P5, and P4 were conjugated at the 4' position of fluorescein as follows: Fluorescein aldehyde (2.21 mg, 0.0061 mmol), CysB peptide P6 (5 mg, 0.0061 mmol) (custom synthesized by GenScript in Piscataway, New Jersey), and potassium carbonate (4.25 mg, 0.0307 mmol) were added to a 2 mL glass vial containing 1 mL of methanol, and the resulting solution was stirred at room temperature for 1 hour. Then, sodium borocyanohydride (0.77 mg, 0.0123 mmol) was added to the solution, and the resulting mixture was stirred overnight at room temperature in the dark. The resulting crude product was diluted in 1 mL of 30% ACN in water (containing 0.1% formic acid) and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 30% to 100% acetonitrile in water in a Biotage Isolera® purification system. The fractions were automatically recovered by monitoring absorption at both 257 nm and 470 nm. The fractions with absorption at 470 nm were analyzed by LC-MS, and the fractions containing the target peptide P6-4-Fl conjugate were combined and lyophilized to obtain a final yellow solid product confirmed by LC-MS (M+1=1158). Conjugates of CysB peptides P5 and P4 with fluorescein at the 4' position were synthesized using substantially the same synthesis and purification procedures as described above for CysB peptide P6. The final conjugates were characterized by LC-MS as GELAYF, P5-4-Fl, LC-MS (M+1=1043); GLAYF, P4-4-Fl, LC-MS (M+1=914).

[0589] Preparation of canine CysB peptide-4-DFF conjugate conjugate conjugated at the 4' position. CysB peptides P6, P5, and P4 were conjugated at the 4' position of DFF as follows: Difluorofluorescein aldehyde (2.43 mg, 0.0061 mmol), CysB peptide P6 (5 mg, 0.0061 mmol) (custom synthesized by GenScript in Piscataway, New Jersey), and potassium carbonate (4.25 mg, 0.0307 mmol) were added to a 2 mL glass vial containing 1 mL of methanol, and the resulting solution was stirred at room temperature for 1 hour. Then, sodium cyanoborohydride (0.77 mg, 0.0123 mmol) was added to the solution, and the resulting mixture was stirred overnight at room temperature in the dark. The obtained crude product was diluted in 1 ml of 30% ACN in water (containing 0.1% formic acid) and purified by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 30% to 100% acetonitrile in water in a Biotage Isolera® purification system. The fractions were automatically recovered by monitoring absorption at both 257 nm and 470 nm. The fractions with absorption at 470 nm were analyzed by LC-MS, and the fractions containing the target peptide P6-4-DFF conjugate were combined and lyophilized to obtain a final yellow solid product confirmed by LC-MS (M+1=1194). Using substantially the same synthesis and purification procedures as described above for CysB peptide P6, and appropriate starting materials, conjugates of CysB peptides P5 and P4 with DFF at the 4' position were synthesized. The final conjugates were characterized by LCMS as GELAYF, P5-4-DFF, LCMS(M+1=1079); GLAYF, P4-4-DFF, LCMS(M+1=950).

[0590] Fluorescence quenching of canine CysB P6-Fl and P6-DFF by anti-CysB Mab or anti-CysB Mab-BHQ10 CysB peptide-fluorescein (Fl) or difluorofluorescein (DFF) conjugate was dissolved in DMSO (0.5 mM) and diluted with Tris buffer (50 nM at pH 8.0) to obtain a stock solution (100 nM). Monoclonal anti-CysB antibody (produced against a peptide with the amino acid sequence of 7C2, SEQ ID NO: 14) (8 mg / ml) was serially diluted in PBS buffer to obtain solutions with anti-CysB-Mab concentrations ranging from 0 to 800 nM in Tris buffer (containing 0.5% sarcosyl (sodium lauroyl sarcosinate), 50 mM at pH 8.0). 50 μL of peptide-Fl or peptide-DFF solution was added to a 96-well black assay plate containing the serially diluted anti-CysB-Mab solution, mixed well, incubated for 30 minutes, and fluorescence intensity was recorded. Results are provided in Figures 38, 39, 40, and Table 1.

[0591] Figure 38 shows that the fluorescence of P6-Fl was quenched in the presence of anti-CysB-Mab, and weaker quenching was observed for P7-Fl. High quenching (approximately 30%) was observed at an antibody-to-P6-Fl molar ratio of approximately 8:1. Figure 39 shows that the fluorescence of P6-DFF was quenched in the presence of anti-CysB-Mab. Maximum quenching (approximately 45%) was observed at an antibody-to-P7-Fl molar ratio of approximately 4:1. Figure 40 shows that the fluorescence of P6-Fl was quenched in the presence of anti-CysB Mab-BHQ10. Quenching in the presence of anti-CysB Mab-BHQ10 was slightly higher than that observed with the unmodified mAb. Similar results for P6-DFF in the presence of Ab-BHQ10 are shown in Figures 41 and 42.

[0592] Tracers of CysB peptides P6, P5, and P4 were conjugated to the 4' position of fluorescein (Fl) or difluorofluorescein (DFF). Fluorescence quenching of other CysB peptide-fluorophore conjugates by anti-CysB Mab is summarized in Table 1.

[0593] As shown in Table 1, fluorescence quenching of peptides-Fl or DFF conjugated at the 4' position of the fluorophore was observed upon addition of anti-CysB mAb. Generally, quenching of peptides conjugated at the 4' position of the fluorophore was lower than that of peptides conjugated at the 5' position of the fluorophore, with the exception of P5-Fl, which showed higher quenching with 4'-conjugated fluorescein.

[0594] [Table 3]

[0595] Peptide / full-length canine CysB protein dose response in Tris buffer and serum. A mixture of CysB P6-Fl or P6-DFF (200 nM, 25 μL) in Tris buffer containing 0.1% Tween 20 and anti-Cys-Mab (400 nM, 25 μL) in Tris buffer containing 0.1% Tween 20 was incubated in the wells of a 96-well black assay plate at room temperature for 30 minutes. Then, a series of CysB peptide P6, P7, and P14 standard solutions (50 μL) with peptide concentrations ranging from 0 to 3200 nM were added to the mixture of P6-Fl or P6-DFF and anti-CysB Mab in Tris buffer. The plate was then incubated at room temperature for 30 minutes, and fluorescence intensity was recorded at excitation at 485 nm and emission at 520 nm. The results are shown in Figures 43, 44, 45, and 46.

[0596] Figure 43 shows an increase in fluorescence intensity as a function of increasing concentrations of CysB peptides P6, P7, or P14; i.e., a dose-response was observed when each of these peptides was added to a mixture of antibody and P6-Fl or P6-DFF. Figure 44 shows an increase in fluorescence intensity as a function of increasing concentrations of CysB full-length protein; i.e., a dose-response was observed when this protein was added to a mixture of antibody and P6-Fl (Figure 44A) or P1-DFF (Figure 44B) in the presence of various concentrations of sarcosyl. The magnitude of the dose-response increased with increasing sarcosyl concentration. A dose-response was also observed when serum CysB full-length protein was added to a mixture of antibody or antibody-BHQ10 and P6-Fl (Figure 45) or P6-DFF (Figure 46) in the presence of a washing agent sarcosyl (1%). [Examples]

[0597] Canine NT-proBNP peptide conjugate: Canine NT-proBNP sequence: P1: AEQLALEPLHRS[Sequence ID:8] P2: AEQLAL[Sequence ID:9] P3: EPLHRS[Sequence ID:10] P4: LALEPL[Sequence ID:11] P5: AEQLALE[Sequence ID:12] P6: LEPLHRS[Sequence ID:13] Canine NT-ProPNP, full length: HPLGGRSPASEASEASEASGLWAVQELLGRLKDAVSELQAEQLALEPLHRSHSPAEAPEAGGTPRGVLAPHDSVLQALR[Sequence ID:14] Canine NT-proPNP stable epitope: GRSPASEASEASEASGLWAVQ [SEQ ID NO: 15] Canine NT-proPNP, stable epitope 2: SHSPAEAPEAGGTPRGVLAPHDSVLQ [Sequence ID: 16]

[0598] Preparation of canine NT-proBNP peptide-Fl conjugate conjugate in position 5. NT-proBNP peptide P1 (3.02 mg, 0.0022 mmol, custom synthesized by GenScript in Piscataway, New Jersey) having the amino acid sequence AEQLALEPLHRS [SEQ ID NO: 8], 5-carboxyfluorescein succinimidyl ester (1 mg, 0.0021 mmol, commercially available from Thermo Fisher in Waltham, Massachusetts), and N'N-diisopropylethylamine (1.6 μL, 0.0089 mmol) were dissolved in anhydrous DMSO (0.5 ml). The resulting mixture was stirred overnight at room temperature and then diluted in 1 ml of 30% ACN in water (containing 0.1% formic acid). Purification was performed by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 30% to 100% acetonitrile in water in a Biotage Isolera® purification system. The fractions were automatically recovered by monitoring absorption at both 257 nm and 470 nm. The fraction with absorption at 470 nm was analyzed by LC-MS, and the fraction containing the target peptide P1-Fl conjugate was combined and freeze-dried to obtain a final yellow solid product confirmed by LC-MS (M2+=861.7). Using substantially the same synthesis and purification procedures and starting materials as described above for NT-proBNP peptide P1, conjugates of NT-proBNP peptides P2:AEQLAL [SEQ ID NO: 9]; P3:EPLHRS [SEQ ID NO: 10]; P4:LALEPL [SEQ ID NO: 11]; P5:AEQLALE [SEQ ID NO: 12]; and P6:LEPLHRS [SEQ ID NO: 13] with fluorescein at position 5 were synthesized. The final conjugate was characterized by LCMS as P2-Fl(M+1=1002.5); P3-Fl(M+1=1096.7); P4-Fl(M+1=1013.4): P5-Fl(M+1=1131.8); P6-Fl(M+1=1209.9).

[0599] Preparation of canine NT-proBNP peptide-DFF conjugate conjugate in position 5. NT-ProBNP peptide P1 (2.81 mg, 0.0021 mmol, custom synthesized by GenScript in Piscataway, New Jersey) having the amino acid sequence AEQLALEPLHRS, 5-difluorocarboxyfluorescein succinimidyl ester (1.0 mg, 0.0021 mmol, commercially available from ATT Bioquest in Sunnyvale, California), and N'N-diisopropylethylamine (1.5 μl, 0.0089 mmol) were dissolved in anhydrous DMSO (0.5 ml). The resulting mixture was stirred overnight at room temperature and then diluted in 1 ml of 30% ACN in water (containing 0.1% formic acid). Purification was performed by column chromatography using a C18 reversed-phase column (5 g) eluting with a gradient of 30% to 100% acetonitrile in water in a Biotage Isolera® purification system. The fractions were automatically recovered by monitoring absorption at both 257 nm and 470 nm. The fraction with absorption at 470 nm was analyzed by LC-MS, and the fraction containing the target peptide P1-DFF conjugate was combined and freeze-dried to obtain a final yellow solid product confirmed by LC-MS (M2+=879.8). Using substantially the same synthesis and purification procedures and starting materials as described above for NT-proBNP peptide P1, conjugates of NT-proBNP peptides P2:AEQLAL [SEQ ID NO: 9]; P3:EPLHRS [SEQ ID NO: 10]; P4:LALEPL [SEQ ID NO: 11]; P5:AEQLALE [SEQ ID NO: 12]; and P6:LEPLHRS [SEQ ID NO: 13] with DFF at position 5 were synthesized. The final conjugates were characterized by LCMS as P2-DFF(M+1=1039.1); P3-DFF(M+1=1132.2); P4-DFF(M+1=1049.5): P5-DFF(M+1=1167.9); P6-DFF(M+1=1245.9).

[0600] Fluorescence quenching of canine NT-proBNP P1-F1 and P1-DFF by anti-NT-proBNP Mab and Ab-BHQ10 NT-proBNP-peptide-fluorescein (Fl) or -difluorofluorescein (DFF) conjugate was dissolved in DMSO (0.4 mM) and diluted in PBS to obtain a stock solution (100 nM). Monoclonal anti-NT-proBNP antibody (ADX-15 Mab, IDEXX Laboratories Inc., Westbrook, Maine) (6.84 mg / ml) was serially diluted in PBS buffer to obtain solutions with antibody concentrations ranging from 0 to 800 nM in PBS. To a 96-well black assay plate containing serially diluted antibody solutions (50 μL), P1-Fl or P1-DFF, or other peptide-Fl or peptide-DFF solution (50 μL) was added, mixed well, incubated for 30 minutes, and fluorescence intensity was recorded (excitation at 485 nm and emission at 520 nm). Results are provided in Figures 47, 48, and Table 2.

[0601] Figure 47 shows that the fluorescence quenching of P1-Fl upon addition of anti-NT-pro-BNP antibody reached a maximum of 70%, and the quenching approached the saturation point when the molar ratio of anti-NT-pro-BNP Ab to P1-Fl was 2:1. The fluorescence quenching of P1-Fl upon addition of anti-NT-pro-BNP antibody-BHQ10 reached a maximum of 40%, and the quenching approached the saturation point when the molar ratio of anti-NT-pro-BNP Ab-BHQ10 to P1-Fl was 2:1. Figure 48 shows that the fluorescence of P1-DFF was quenched by up to approximately 70% in the presence of anti-NT-pro-BNP-Mab, and the quenching approached saturation with a molar ratio of antibody to P1-DFF of 1:1. The fluorescence of P1-DFF was quenched by up to approximately 65% ​​in the presence of anti-NT-pro-BNP Ab-BHQ10. All other NT-proBNP peptide-Fl or-DFF conjugates (P2, P3, P4, P5) did not show quenching upon addition of antibody or antibody-BHQ10 at any molar ratio from 1:1 to 8:1. NT-proBNP P6-Fl and P6-DFF showed slight fluorescence quenching (1.0–3.7%) at a 1:1 ratio, which increased to approximately 10% at an 8:1 ratio. The results at a 1:1 molar ratio are summarized in Table 2.

[0602] [Table 4]

[0603] Dose-response of canine NT-proBNP peptide P1 in PBS and serum A mixture of NT-proBNP P1-Fl or P1-DFF (100 nM, 25 μM) in PBS and anti-NT-proBNP-Mab (100 nM, 25 μL) in PBS was incubated in the wells of a 96-well black assay plate at room temperature for 30 minutes. Then, a series of PBS or charcoal-treated serum NT-proBNP peptide P1 standard solutions (50 μL) with peptide or protein concentrations ranging from 0 to 320 nM were added to the mixture of P1-Fl or P1-DFF in PBS and anti-NT-proBNP-Mab. The plates were then incubated at room temperature for 30 minutes, and fluorescence intensity was recorded at excitation at 485 nm and emission at 520 nm. The results are shown in Figures 49, 50, 51, and 52.

[0604] Figure 49 shows an increase in fluorescence intensity as a function of increasing NT-pro-BNP peptide P1 concentration, i.e., a dose-response was observed when the peptide was added to a mixture of P1-Fl and anti-NT-pro-BNP antibody (Ab), or antibody-BHQ10 (Ab-BHQ10). Figure 50 also shows an increase in fluorescence intensity as a function of increasing NT-pro-BNP peptide P1 concentration, i.e., a dose-response was observed when the peptide was added to a mixture of P1-DFF and anti-NT-pro-BNP antibody (Ab), or antibody-BHQ10 (Ab-BHQ10). The P1 peptide also yielded a dose-response in serum containing the antibody or antibody-BHQ10 and a mixture of P1-Fl (Figure 51) or P1-DFF (Figure 52).

[0605] Canine NT-ProBNP Full-Length Protein Dose-Response A mixture of NT-proBNP P1-Fl or P1-DFF in PBS (200 nM, 10 μL) and anti-NT-proBNP Mab in PBS (200 nM, 10 μL) was incubated in the wells of a 96-well black assay plate at room temperature for 30 minutes. Then, a series of NT-proBNP peptide P1 standard solutions (80 μL) containing NT-proBNP full-length protein (lyophilized powder synthesized by New England Biolabs, Ipswich, Massachusetts) in PBS at concentrations ranging from 0 to 320 nM was added to the mixture of P1-Fl or P1-DFF in PBS and anti-NT-proBNP Mab. The plate was then incubated at room temperature for 30 minutes, and fluorescence intensity was recorded at excitation at 485 nm and emission at 520 nm. The results are shown in Figures 53 and 54.

[0606] Figure 53 shows an increase in fluorescence intensity as a function of increasing concentration of full-length NT-proBNP protein, i.e., a dose-response was observed when the protein was added to a mixture of P1-Fl and anti-NT-proBNP antibody in a 1:1 P1-Fl / Ab ratio. Similarly, a dose-response was observed when the protein was added to a mixture of P1-DFF and antibody in a 1:1 ratio, as shown in Figure 54. A dose-response was also observed when full-length NT-proBNP was added to charcoal-treated serum and then to either a P1-Fl / Ab (Figure 55) or P1-DFF / Ab (Figure 56) mixture. Assay sensitivity is improved by reducing the relative amount of antibody in the mixture, as shown in Figure 55 for P1-Fl / Ab=2:1 and in Figure 56 for 2:1 P1-DFF / Ab.

[0607] The entire disclosure of all cited references is incorporated herein by reference.

Claims

1. A method for determining the presence or amount of an analyte in a sample, (i) the step of preparing a sample suspected to contain the analyte; (ii) A step of contacting the sample with a fluorescent tracer and a binding partner to obtain an assay composition, wherein the binding partner is specific to the analyte and the fluorescent tracer; (iii) The step of irradiating the assay composition with light of a first wavelength; (iv) A step of measuring the intensity of light emitted at a second wavelength and Includes, Here (a) When the fluorescent tracer is not bound to the binding partner, the intensity of light emitted at the second wavelength is greater than when the fluorescent tracer is bound to the binding partner. (b) The light of the first wavelength is not linearly polarized, and (c) Fluorescent tracers are as follows: 【Chemistry 1】 Here, T is a bond or linking group, and E is an epitope. Selected from the group consisting of; and A method wherein the intensity of light emitted at the second wavelength is directly proportional to the concentration of the analyte in the sample.

2. The method according to claim 1, wherein the analyte is a polymer.

3. The method according to claim 2, wherein the analyte is a protein.

4. (i) The epitope portion is an amino acid side chain, and the sequence of the amino acid side chain includes the amino acid sequence of the epitope on the protein responsible for complexing with the binding partner, (ii) The method according to claim 3, wherein the bonding partner is optionally conjugated to a quencher.

5. The aforementioned quencher is as follows: 【Chemistry 2】 【change】 The method according to claim 4, wherein the wavy line indicates the connection position to the coupling partner, selected from the group comprising:

6. The method according to claim 4, wherein the binding partner is an antibody.

7. The method according to claim 4, wherein the protein is selected from the group consisting of canine CysB protein and canine NT-proBNP.

8. The method according to claim 7, wherein the protein is canine CysB protein.

9. The method according to claim 8, wherein the binding partner is an anti-cystatin-B antibody, and the fluorescent tracer is fluorescein attached to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:

7.

10. The aforementioned amino acid sequence is -CH ２ - The method according to claim 9, wherein the linker is attached to the 4' position of the fluorescein.

11. The method according to claim 9, wherein the anti-cystatin-B antibody is conjugated to the quencher.

12. The method according to claim 1, wherein the analyte is an antigen and the binding partner is an antibody.

13. The method according to claim 12, wherein the antigen is SDMA and the antibody is an antibody against SDMA.

14. The aforementioned fluorescent tracer, 【Transformation 3】 In the equation, each X is -H, or each X is -F. The method according to claim 13, selected from the group consisting of the following.

15. The method according to claim 14, wherein the antibody against the SDMA is conjugated to the quencher.

16. The method according to claim 15, wherein the quencher is selected from the group consisting of CY3, CY5, IRDyeQC1, BHQ1, and BHQ10.

17. (A) In order, (i) A support layer selected from the group consisting of glass, polystyrene, polyester, polycarbonate, cellulose derivatives, polyethylene terephthalate, and mixtures thereof, A support layer that is optically transparent to the first and second wavelengths; (ii) A primer layer containing polyurethane; (iii) An indicator layer comprising the fluorescent tracer and the binding partner dispersed in a second polymer, wherein the second polymer is selected from the group consisting of cellulose, cellulose derivatives, polysaccharides, gelatin, gelatin derivatives, polyvinyl alcohol, polyvinylpyrrolidone, acrylamide polymer, polyurethane, alginate, xantham, and mixtures thereof; (iv) A filtering layer containing polyurethane and cellulose; and (v) A diffusion layer comprising a mixture of cellulose and a hydrophilic polymer, wherein the hydrophilic polymer is selected from the group consisting of polyacrylic acid, polyvinylpyrrolidone, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyacrylamide, and polyethyleneimine. The steps include: preparing slides that include; (B) The step of depositing the sample onto the diffusion layer of the slide; (C) The step of diffusing the sample from the diffusion layer to the indicator layer so that the sample can come into contact with the fluorescent tracer and the binding partner; (D) The step of irradiating the support layer of the slide with light that is not polarized at a first wavelength; (E) A step of measuring the intensity of light emitted from the support layer of the slide at the second wavelength. The method according to claim 1, including the method described in claim 1.

18. The method according to claim 17, wherein the analyte is SDMA and the binding partner is an antibody against SDMA.

19. A method for determining the presence or amount of an analyte in a sample, (i) the step of preparing a sample suspected to contain the analyte; (ii) A step of contacting the sample with a fluorescent tracer and a binding partner to obtain an assay composition, wherein the binding partner is specific to the analyte and the fluorescent tracer; (iii) The step of irradiating the assay composition with light of a first wavelength; (iv) A step of measuring the intensity of light emitted at a second wavelength and Includes, Here (a) When the fluorescent tracer is not bound to the binding partner, the intensity of light emitted at the second wavelength is greater than when the fluorescent tracer is bound to the binding partner. (b) The light of the first wavelength is not linearly polarized, (c) Fluorescent tracers are as follows: 【Chemistry 4】 Here, T is a bond or linking group, and E is an epitope moiety that can specifically bind to a binding partner. Selected from the group consisting of; (d) The intensity of the light emitted at the second wavelength is directly proportional to the concentration of the analyte in the sample, and (e) The analyte is selected from the group consisting of SDMA, melamine, T4, bile acids, biotin, thyroxine, cystatin-B, NT-proBNP, antibiotics, sulfadimethoxine, cortisol, and progesterone. method.

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