Improved assay compositions and methods

Abstract

The present disclosure provides improved assays and assay methods comprising compositions comprising an enzyme (e.g., ALP) conjugated to an antigen or antibody, as well as compositions comprising an enzyme substrate, where the compositions are optimized and suitable for providing improved assay performance. The present disclosure also describes compositions of matter for use in immunoassays and kits comprising the compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention relates to and claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 358,514 filed on July 5, 2022 and U.S. Provisional Patent Application Serial No. 63 / 389,136 filed on July 14, 2022, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Immunoassays are key tools in many areas of medicine and research and discovery, including disease diagnosis, disease prognosis, disease risk assessment, determining and monitoring therapeutic treatment and efficacy, and clinical pharmacokinetic and bioequivalence studies in drug discovery and the pharmaceutical industry. Continuing improvements in assay specificity, throughput, sensitivity, and increasing the number of validated target analytes will provide better clinical evaluation and therapeutic interventions.

[0004] Alkaline phosphatase (ALP) is an enzyme used in combination with a colorimetric reagent or a chemiluminescent reagent in an immunoassay. Generally speaking, after ALP catalyzes a substrate, a measurable and quantifiable chemiluminescent signal or a colored fluorescent signal is generated. The enzyme (e.g., ALP) and the related substrate molecules used in the assay and the composition comprising it can be the result effective variables of the overall assay performance. Therefore, the development provides improved assay performance (e.g., increased activity, stability, analyte signal and / or reduced background signal, lower detection limit and quantification limit, improved signal-to-noise ratio, and allows the use of lower concentrations and / or amounts of assay reagents) enzyme conjugates or derivatives, substrate compounds, and compositions comprising them have significant benefits. Summary of the invention

[0005] In one aspect, the present disclosure relates to a container comprising:

[0006] a. at least one agent comprising particles, wherein the mass of the particles is at least 0.1 μg and at most 100 μg; and

[0007] b. A chemiluminescent substrate preparation, wherein the chemiluminescent substrate preparation comprises a chemiluminescent compound of formula I or a salt thereof:

[0008]

[0009] in

[0010] A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent;

[0011] R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups;

[0012] R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring;

[0013] R 15 It is C 1-6 alkyl;

[0014] Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts;

[0015] Z is O or S; and

[0016] n is 0, 1 or 2.

[0017] In some embodiments of the present aspect, the container may be located in an immunoassay analyzer or instrument. In some additional embodiments, the immunoassay analyzer or instrument comprises a system for taking a sample aliquot from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide feedback on the performance of the immunoassay analyzer or instrument in real time, a dispenser that heats the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid, or a combination thereof. In yet other embodiments, the immunoassay system includes a photometer system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on the photons emitted from the assay over a period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube into a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sensing resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on the photons emitted from the assay over a period of time; and a photometer controller configured to: in response to the assay analog voltage being greater than a predetermined value, calculate a relative light unit value of the photons emitted from the assay over a period of time based on the assay analog voltage and an optimized linear function.

[0018] In some embodiments of this aspect, the container is suitable for use in an immunoassay method.

[0019] In another aspect, the present disclosure relates to an immunoassay analyzer or instrument comprising a container, wherein the immunoassay analyzer or instrument comprises a system for removing a sample aliquot from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide real-time feedback on the performance of the immunoassay analyzer or instrument, a dispenser that heats the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid, and / or wherein the immunoassay system comprises a photometer system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to generate a photon based on the photons emitted over a period of time. The invention relates to a photometer for providing an assay analog voltage based on photons emitted from the assay over a period of time, the analog circuit comprising: a current sensing resistor coupled to convert a current from the photomultiplier tube into a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sensing resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on photons emitted from the assay over a period of time; and a photometer controller configured to: in response to the assay analog voltage being greater than a predetermined value, calculate a relative light unit value of photons emitted from the assay over a period of time based on the assay analog voltage and an optimized linear function; and wherein the container contains up to 100 μg of paramagnetic microparticles.

[0020] In another aspect, the present disclosure relates to an immunoassay analyzer or instrument comprising a container, wherein the immunoassay analyzer or instrument comprises a system for removing a sample aliquot from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide feedback on the performance of the analyzer or instrument in real time, a dispenser that heats the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid, and / or wherein the immunoassay system comprises a photometer system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to generate a photon based on the photons emitted from the assay reaction over a period of time; The invention relates to a method for providing an assay analog voltage based on photons emitted from the assay, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube into a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sensing resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on photons emitted from the assay over a period of time; and a photometer controller configured to: in response to the assay analog voltage being greater than a predetermined value, calculate a relative light unit value of photons emitted from the assay over a period of time based on the assay analog voltage and an optimized linear function; and wherein the container comprises a sample aliquot having a volume of 2 μl to 50 μl.

[0021] In some related aspects and embodiments, the present disclosure relates to the use of an immunoassay instrument in an immunoassay method.

[0022] In another aspect, the disclosure relates to a method comprising: contacting a sample suspected of containing a target analyte with an antibody composition to form a mixture; incubating the mixture in a container or vessel; and adding a substrate composition to the container or vessel to form a reaction mixture; wherein the antibody composition comprises: an antibody to the target analyte conjugated to alkaline phosphatase (ALP) or an antigen conjugated to ALP; a buffer comprising a zinc salt and a magnesium salt; wherein when the pH of the composition is in the range of 5.5 to 6.4, the buffer frees Zn 2+ The concentration of free Mg was in the range of 5 μM to 1 mM. 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration of the substrate composition is within the range of 3500 times of that of the present invention; and wherein the substrate composition comprises: a chemiluminescent compound of formula I or a salt thereof:

[0023]

[0024] in

[0025] A is for C 1-6haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent;

[0026] R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups;

[0027] R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring;

[0028] R 15 It is C 1-6 alkyl;

[0029] Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts;

[0030] Z is O or S; and

[0031] n is 0, 1 or 2.

[0032] In another aspect, the disclosure relates to an immunoassay method comprising: contacting a sample suspected of containing an analyte with 0.1 μg to 100 μg of particles in a reaction vessel; and (a) subjecting the reaction vessel to further sample processing on an immunoassay instrument.

[0033] In another embodiment of this aspect, the present disclosure relates to an immunoassay method for detecting alpha-fetoprotein (AFP) in a sample, wherein the immunoassay method comprises: contacting a sample suspected of containing AFP and an anti-AFP antibody conjugated to an enzyme to form a mixture, wherein the sample is a blood sample, a plasma sample or an amniotic fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; incubating the reaction mixture; measuring the light generated by the reaction mixture; and detecting the concentration of AFP in the sample based on the light generated by the reaction mixture; wherein the limit of blank (LoB) of the immunoassay method is 0.08 to 0.16 ng / mL, the limit of detection (LoD) of the immunoassay method is 0.22 to 0.23 ng / mL, and / or the limit of quantitation (LoQ) of the immunoassay method is 0.13 to 0.20.

[0034] In another embodiment of this aspect, the present disclosure relates to detecting thyroid stimulating hormone (thyroid stimulating hormone) in a sample. The invention relates to an immunoassay method for detecting TSH in a sample suspected of containing TSH, wherein the immunoassay method comprises: contacting a sample suspected of containing TSH with an anti-TSH antibody conjugated to an enzyme to form a mixture, wherein the sample is a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample or a bronchoalveolar lavage fluid sample; incubating the mixture in a container or a vessel; adding a chemiluminescent substrate to the container or the vessel to form a reaction mixture; incubating the reaction mixture; measuring the light generated by the reaction mixture; and detecting the concentration of TSH in the sample based on the light generated by the reaction mixture; wherein the limit of blank (LoB) of the immunoassay method is 0.001 to 0.002 uIU / mL, the limit of detection (LoD) of the immunoassay method is 0.002 to 0.003 uIU / mL, and / or the limit of quantification (LoQ) of the immunoassay method is 0.0008 to 0.001 uIU / mL.

[0035] In another embodiment of this aspect, the present disclosure relates to detecting beta human chorionic gonadotropin in a sample. The invention relates to an immunoassay method for detecting βhCG in a sample suspected of containing βhCG, wherein the immunoassay method comprises: contacting a sample suspected of containing βhCG and an anti-βhCG antibody conjugated to an enzyme to form a mixture, wherein the sample is a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample or a bronchoalveolar lavage fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; incubating the reaction mixture; measuring the light generated by the reaction mixture; and detecting the concentration of βhCG in the sample based on the light generated by the reaction mixture; wherein the limit of blank (LoB) of the immunoassay method is 0.0 to 0.1 mIU / mL, the limit of detection (LoD) of the immunoassay method is 0.2 mIU / mL, and / or the limit of quantification (LoQ) of the immunoassay method is 0.1 to 0.2 mIU / mL.

[0036] In another embodiment of this aspect, the present disclosure relates to an immunoassay method for detecting free thyroxine 4 (T4) in a sample, wherein the immunoassay method comprises: contacting a sample suspected of containing T4 and an anti-T4 antibody conjugated to an enzyme to form a mixture, wherein the sample is a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample or a bronchoalveolar lavage fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; wherein the chemiluminescent substrate is a substrate for the enzyme; incubating the reaction mixture; measuring the light generated by the reaction mixture; and detecting the concentration of T4 in the sample based on the light generated by the reaction mixture; wherein the limit of blank (LoB) of the immunoassay method is less than 0.18 ng / mL.

[0037] These different aspects and embodiments are not intended to describe each disclosed embodiment or each implementation of the present disclosure, and the following description more specifically illustrates certain illustrative embodiments. In several places throughout the application, guidance is provided by example lists, which can be used in various combinations. In each case, the enumerated lists are only representative groups and should not be interpreted as exclusive lists. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A graphic representation of the Allowable Total Difference (ATD) region is shown. The image is adapted from the assay migration guidance document (see "Guidance for Industry and Staff: Assay Migration Studies for In Vitro Diagnostic Devices" published in 2013 and available online on the FDA website as a regulatory guidance document for assay migration studies for in vitro diagnostic devices.

[0039] Figure 2 The reproducibility of the Access AFP assay is shown, as evaluated on both the DxI 9000 and Access 2 immunoassay analyzers according to the Clinical & Laboratory Standard Institute (CLSI) guidelines in EP05-A3, including the between-site, between-batch, between-day, between-run, and within-run variance components. The reproducibility on the DxI 9000 immunoassay analyzer was significantly improved compared to the reproducibility on the Access2 immunoassay analyzer.

[0040] Figure 3 Results of a quantitative comparison study comparing the Access AFP assay on the DxI 9000 immunoassay analyzer to the Access AFP assay on the Access 2 immunoassay analyzer are shown. In addition to the comparison to the allowable total difference (ATD) area specified in the assay migration guidance (see 2013 guidance document above), a regression analysis per CLSI EP09C-ED3 was completed.

[0041] Figure 4 Results of an evaluation of Access AFP assay imprecision according to CLSI EP05-A3 on a DxI 9000 immunoassay analyzer for each of three reagent lots are shown. Representative reagent lots are shown for illustration; all lots produced acceptable performance.

[0042] Figure 5 Results of evaluation of the Access AFP assay limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) for each of three reagent lots on the DxI 9000 immunoassay analyzer according to CLSI EP17-A2 are shown. Representative precision curves at low concentrations are shown.

[0043] Figure 6Results of an evaluation of the linearity of the Access AFP assay across 3 reagent lots on a DxI 9000 immunoassay analyzer according to CLSI EP06-Ed 2 are shown. Acceptable non-linearity was observed for each individual evaluation.

[0044] Figure 7 The reproducibility of the Access hsTnI assay is shown, as evaluated according to CLSI EP05-A3 on both the DxI 9000 and Access 2 immunoassay analyzers, including between-site, between-batch, between-day, between-run, and within-run variance components. Independent studies were completed on both serum and lithium heparin plasma. The reproducibility on the DxI 9000 immunoassay analyzer was significantly improved compared to the reproducibility on the Access 2 immunoassay analyzer.

[0045] FIG. 8A to FIG. 8B Results of a quantitative comparative study comparing the Access hsTnI assay (lithium heparin plasma (8A); serum (8B)) on the DxI9000 immunoassay analyzer to the Access hsTnI assay on the Access 2 immunoassay analyzer are shown. In addition to the comparison to the allowable total difference (ATD) area specified in the assay migration guidelines, a regression analysis according to CLSI EP09C-ED3 was completed. The study design criteria were met.

[0046] Fig. 9 Results of an evaluation of the Access hsTnI assay imprecision according to CLSI EP05-A3 on a DxI 9000 immunoassay analyzer for each of three reagent lots are shown. Serum and lithium heparin plasma were evaluated separately. For illustration, representative reagent lots are shown; all lots produced acceptable performance.

[0047] Fig.10 Results of evaluation of the Access hsTnI assay LoB, LoD and LoQ for each of three reagent lots on the DxI 9000 immunoassay analyzer according to CLSI EP17-A2 are shown. Serum and lithium heparin plasma were evaluated separately. Representative precision curves at low concentrations are shown.

[0048] Fig.11 Results of an evaluation of the linearity of the Access hsTnI assay across 3 reagent lots on the DxI 9000 immunoassay analyzer according to CLSI EP06-Ed2 are shown. Independent studies on serum and lithium heparin plasma were completed. Acceptable nonlinearity was observed for each individual evaluation.

[0049] Fig.12An exemplary embodiment of APS-5 (acridinium phosphate) contained in a substrate LUMI-PHOS PRO (Lumigen, Inc., Southfield, MI) according to the present disclosure is shown.

[0050] Fig.13 Shown are relative light unit (RLU) responses when detected using LUMI-PHOS 530 (Lumigen, Inc., Southfield, MI) or an APS-5-containing substrate (LUMI-PHOS PRO) before (April curve) and after (October curve) storage of ALP-containing reagent packs at 4°C for 6 months.

[0051] FIG. 14A to FIG. 14C shows that at 37°C at pH 6 ( Fig.14A ), pH 7( Fig. 14B ) and pH 8( Fig. 14C ) on the percentage change of ALP activity after 3 days by varying the concentrations of zinc and magnesium.

[0052] Fig.15 Shown are Mg / Zn ratios plotted against relative light unit (RLU) loss as determined by cTnl, as further described in Example 3D. DETAILED DESCRIPTION

[0053] In a general sense, the present disclosure provides immunoassay methods and compositions, the compositions comprising assay substrates and preparations that can be used in those methods, and / or providing systems (including, for example, instruments or analyzers) for assays that have improved performance or other advantages relative to the prior art. In one aspect, the present disclosure provides improved assays comprising assay substrates (e.g., chemiluminescent substrates) and / or preparations comprising them according to the example embodiments described herein. In another aspect, the present disclosure provides improved assays comprising assay preparations (e.g., enzymes or conjugated enzymes in, for example, buffer preparations) according to the example embodiments described herein. In yet other aspects, the present disclosure provides improved assays comprising an assay substrate according to the example embodiments described herein and a combination of at least one assay preparation. In other aspects, the present disclosure provides kits and material compositions comprising an assay substrate according to the example embodiments described herein and a combination of at least one assay preparation. In another aspect, the disclosure relates to a method comprising: contacting a sample suspected of containing a target analyte with an antibody composition to form a mixture; incubating the mixture in a container or vessel; and adding a substrate composition to the container or vessel to form a reaction mixture; wherein the antibody composition comprises: an antibody to the target analyte conjugated to alkaline phosphatase (ALP) or an antigen conjugated to ALP; a buffer comprising a zinc salt and a magnesium salt; wherein when the pH of the composition is in the range of 5.5 to 6.4, the buffer frees Zn 2+ The concentration of free Mg was in the range of 5 μM to 1 mM. 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ within a range of 3500 times the concentration of; and wherein the substrate composition comprises a chemiluminescent compound as described herein. In some further aspects, the present disclosure provides one or more containers comprising: one or more buffers, one or more reagents comprising a solid substrate; antibodies; antibody-enzyme conjugates; and / or chemiluminescent compounds. In some further aspects, the present disclosure provides one or more containers comprising a chemiluminescent compound and an enzyme, wherein the ratio of the chemiluminescent compound to the enzyme is 1 μg of chemiluminescent compound to 1500 enzyme molecules. In other aspects, the present disclosure provides an immunoassay analyzer comprising: a distribution control system, an image capture device or camera unit, a dispenser or a mechanical device suitable for degassing a fluid, and a photometer system, and further comprising one or more containers according to the aspects and embodiments disclosed herein.

[0054] In some embodiments of the above aspects, the improvement of assay performance can refer to any result (i.e., one or more measurable parameters) of a specific assay, which shows an improvement in assay performance relative to one or more of the absence of an assay substrate, formulation, method, and / or system according to the present disclosure. In some other embodiments, the improvement of assay may include any one or more of the following: improved assay accuracy, improved assay sensitivity, improved assay reproducibility, improved assay signal-to-noise (S / N) ratio (e.g., improved signal, improved assay background / noise reduction, or both), improved assay blank limit (LoB), improved assay detection limit (LoD), improved assay quantitative limit (LoQ), and / or improved reagent consumption (e.g., increasing the number of tests per assay reagent pack / unit). The terms LoB, LoD, and LoQ used herein are consistent with the definitions provided in CLSI EP17-A2. (See CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline–Second Edition. CLSI document EP17-A2 Wayne, PA: Clinical and Laboratory Standards Institute; 2012.). In some other embodiments, the improvement of the assay relates to an increase in assay sensitivity (i.e., the assay provides detection of a lower amount of target analyte in a sample). In such embodiments, the assay may further provide such an increase in detection sensitivity without any loss of assay accuracy and / or reproducibility. Such an improvement in assay sensitivity may provide many advantages relative to prior art assays, including detecting a disorder or disease at an early stage (e.g., early onset, a high risk of disease, or an increased likelihood of disease), which may provide improved success and improvement associated with therapeutic interventions and patient outcomes. In some other embodiments, the improved assay sensitivity may allow detection of additional disease states associated with lower concentrations of target analyte concentrations, and the disease state is undetectable using prior art assay systems and methods. Thus, providing assays with increased sensitivity to one or more target analytes provides substantial improvements in the early identification of patient categories (e.g., patients at risk for developing a disease) and in the identification, diagnosis, and treatment of a variety of conditions, disorders, and diseases associated with one or more biomarkers (e.g., those described herein).

[0055] In some embodiments of the above aspects, the assay substrates, formulations, methods and / or systems can be used for methods comprising measuring the concentration of one or more target analytes in a liquid sample (e.g., a biological sample obtained from a subject (e.g., such as a human patient). Exemplary sample types include, but are not limited to, serum, plasma, urine, amniotic fluid, whole blood, synovial fluid, cerebrospinal fluid, saliva, semen, nasal fluid, mucus (e.g., swabs, sputum, etc.), or bronchoalveolar lavage fluid, etc.

[0056] definition

[0057] The terms "preferred" and "preferably" when used herein refer to embodiments of the present disclosure that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present disclosure.

[0058] The terms "comprise", "include" and "have" and similar variations thereof do not have a limiting meaning where these terms appear in the specification and claims. Such terms will be understood to imply the inclusion of the steps or elements, or groups of steps or elements, without excluding any other steps or elements, or groups of steps or elements. In contrast, the term "consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Substantially consisting of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activities or actions specified by the listed elements in the present disclosure. Thus, the phrase "substantially consisting of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not exist, depending on whether they substantially affect the activities or actions of the listed elements.

[0059] Unless otherwise specified, "a", "an", "the" and "at least one" are used interchangeably and mean one or more than one.

[0060] As used herein, the term "or" is generally used in its ordinary meaning, including "and / or", unless the content clearly dictates otherwise.

[0061] The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0062] As used herein, numerical ranges recited by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Additionally, reference within this disclosure to the phrase "up to" a stated value (e.g., up to 50) includes that value (e.g., 50).

[0063] When used herein, the terms "in the range" or "within a range" (and similar phrases) with respect to numerical values ​​are meant to include the endpoints of the numerical range.

[0064] For any methods disclosed herein that comprise discrete steps, the steps may be performed in any practicable order, and any combination of two or more steps may be performed simultaneously if appropriate and operationally possible.

[0065] Unless otherwise indicated, all headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading. In addition, all patent and non-patent references cited herein are incorporated herein by reference in their entirety.

[0066] References throughout this specification to "one embodiment," "embodiment," "certain embodiments," or "some embodiments" etc. mean that a particular feature, configuration, composition, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in multiple places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, in one or more embodiments, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner.

[0067] Unless otherwise stated, all numbers of the amount, measured value, molecular weight, etc. of the reflection component as used in the specification and claims should be understood to include the change of the measured amount, as those skilled in the art will expect when measuring and carrying out the level of care commensurate with the accuracy of the measurement purpose and the related measurement equipment. Therefore, unless otherwise indicated to the contrary, the numerical parameters listed in the specification and claims are approximate values, which can change according to the desired characteristics sought to be obtained by the present disclosure. At least each numerical parameter should be interpreted according to the number of reported significant figures and by applying common rounding techniques. For example, for the pH, temperature, amount or concentration listed, these values ​​can change with the amount without any significant effect on the resulting structure, stability, activity, or effective variable or parameter of the result.

[0068] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Those skilled in the art will understand that all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0069] The term "antibody" or its plural "antibodies" as used herein, also referred to as immunoglobulins, covers full-length antibody sequences, including, for example, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed by at least two different epitope binding fragments, bispecific antibodies, human antibodies, and humanized antibodies. Antibody "fragments" (or "antigen binding fragments", "binding fragments", "epitope binding fragments", etc.) described herein generally refer to any antibody sequence that is smaller than the full-length antibody sequence and still exhibits specific binding activity to the target antigen. In some exemplary embodiments, the antibody fragment generally comprises at least one combination of three CDR sequences (HCDR1, HCDR2, HCDR3) of the heavy chain variable domain and at least three CDR sequences (LCDR1, LCDR2, LCDR3) of the light chain variable domain. Some non-limiting examples of antibody fragments include single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, camelid antibodies, antibody fragments (e.g., antigen-binding portions) exhibiting desired biological activity, disulfide-linked Fv (dsFv) and anti-idiotypic (anti-Id) antibodies, intrabodies, and epitope-binding fragments of any of the above. In some embodiments, the disclosure provides antibodies comprising immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules comprising at least one antigen binding site. Antibodies and fragments thereof may also include peptide fusions with antibodies or portions thereof, such as proteins fused to Fc domains.

[0070] The immunoglobulin molecule can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subisotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or allotype (e.g., Gm, such as G1m (f, z, a, or x), G2m (n), G3m (g, b, or c), Am, Em, and Km (1, 2, or 3)). Antibodies and fragments thereof can be derived from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g., chickens).

[0071] In some aspects, the present disclosure provides assays, systems, kits and methods comprising one or more antibodies or compositions comprising the same. In some exemplary embodiments, the antibody is an IgG, scFv, Fab, monoclonal antibody (mAb) or single chain Fv-Fc (scFv-Fc) antibody. A typical or conventional mAb comprises two heavy chain subunits and two light chain subunits. Each mAb heavy chain comprises a variable domain (VH) that contributes to antigen binding and consists of three or four subregions (C H 1. C H 2. C H 3. C H 4) constitutes the constant domain (CH). VH contains three complementarity-determining regions (CDR): HCDR1, HCDR2 and HCDR3. Each mAb light chain contains a variable domain (VL) and a constant domain (CL). VL contains three CDRs, LCDR1, LCDR2 and LCDR3. Two isotypes of light chain constant domains, kappa (κ) and lambda (λ), exist in mammals. Disulfide bonds bind each C H 1 domain is connected to a CL domain and C H The 2 domains are connected to each other. Five types of heavy chains (α, δ, ε, γ, and μ) are present in different classes of antibodies (IgA, IgD, IgE, IgG, and IgM). mAb heavy chains have a hinge region that confers structural flexibility and mobility.

[0072] The "Fc" region encompasses domains from the constant region of an immunoglobulin heavy chain, including fragments, analogs, variants, mutants or derivatives thereof. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE and IgM. The Fc region may be a native sequence Fc region or an altered Fc region. The Fc region of an immunoglobulin generally comprises two constant domains, C H 2 domains and C H The "Fv" region encompasses the VH and VL domains of an immunoglobulin. As used herein, an "scFv-Fc" antibody refers to a fusion protein of a single VH and a single VL domain connected to a hinge region and a C domain of a single CH domain. H 2 domains and C H 3 domains.

[0073] The antibodies or antigen-binding fragments thereof generally described herein may comprise a binding domain that binds to an epitope of a target analyte. "Binding domain" or "binding sequence" may be used interchangeably with antibody fragment or "antigen-binding fragment" and as used herein refers to a portion of an antibody sequence that is sufficient and adequate to bind to or interact with a target structure, antigen or epitope. In some embodiments, antibodies and / or antibody compositions according to the present disclosure may comprise a commercially available antibody conjugated to an enzyme (e.g., ALP).

[0074] Exemplary Target Analytes

[0075] In some respects, the disclosure provides a determination substrate, preparation, method and / or system that can be used for the method for including measuring the concentration of one or more target analytes. Some exemplary analytes include troponin (e.g. cardiac troponin I), alpha-fetoprotein (AFP), thyroid stimulating hormone (TSH), free thyroxine (T4), creatinine kinase (CK-MB), sex hormone binding globulin (sex hormone binding globulin, SHBG) and cancer antigen 19-9 (CA 19-9, or "GI monitoring thing"). Many other analytes can also be detected by immunoassay, and the analytes listed herein are provided as examples, rather than being intended to be restrictive.

[0076] Troponin

[0077] In one aspect, the present disclosure provides a method comprising measuring the serum or plasma concentration of cardiac troponin I (cTnI) in a blood sample. cTnI is a recognized analyte associated with cardiac health, particularly as an indicator of cardiac damage caused by conditions such as heart attack. cTnI may also be associated with other diseases associated with troponin release, including, for example, acute pulmonary embolism, heart failure, myocarditis, and end-stage renal disease. The methods and assays disclosed herein may provide improved sensitivity, accuracy, or reproducibility, or a combination thereof, as well as improvements associated with therapeutic interventions.

[0078] In some embodiments, anti-cTnI antibodies can be used to detect troponin. In other embodiments, the anti-cTnI antibody is preferably a monoclonal anti-cTnI antibody.

[0079] In some embodiments, the method may include a two-site immunoenzymatic ("sandwich") assay. For example, a monoclonal anti-cTnI antibody conjugated to alkaline phosphatase may be added to a reaction vessel together with a buffer containing a surfactant and a sample. After a short incubation, paramagnetic particles coated with the monoclonal anti-cTnI antibody may be added. Human cTnI binds to the anti-cTnI antibody on the solid phase, while the anti-cTnI antibody-alkaline phosphatase conjugate reacts with different antigenic sites on the cTnI molecule. After incubation in the reaction vessel, the substance bound to the solid phase is maintained in a magnetic field, while the unbound substance can be washed away. A chemiluminescent substrate can then be added to the vessel, and the light produced by the reaction can be measured with a photometer. Light generation is generally proportional to the concentration of cTnI in the sample. The amount of analyte in the sample can be determined based on a stored (e.g., multi-point) calibration curve.

[0080] In some embodiments, the method comprises detecting cTnI using ACCESS hsTnI (available from Beckman Coulter, Inc., Brea, California).

[0081] In some embodiments, the method comprises comparing the cTnI concentration to the cTnI concentration in a normal population. In such embodiments, the difference between the cTnI concentration in a tested sample (e.g., a biological sample from a subject, such as a patient sample) and the normal population can be used to assess the cardiac health of the subject.

[0082] In some aspects, the method further comprises measuring the concentration of cTnI using a substrate preparation comprising a chemiluminescent compound. In one aspect, the substrate preparation can be a substrate preparation as described in U.S. Patent No. 10,703,971. In some preferred embodiments, the substrate preparation comprises a substrate and / or substrate preparation as described herein. In some preferred embodiments, the enzyme preparation comprises an enzyme preparation such as an ALP preparation, as described herein. In some further preferred embodiments, the method comprises both a substrate preparation and an enzyme preparation substantially as described herein.

[0083] As further described in some illustrative examples, the reproducibility (i.e., accuracy and / or sensitivity) of the cTnI assay performed on the DxI 9000 immunoassay analyzer was significantly improved compared to the reproducibility on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). According to the present disclosure, the improved assay may be attributable to one or a combination of the instrument platform (e.g., including improvements such as a distribution control system and an image capture device or camera unit that can be used to provide real-time feedback on the analyzer or instrument performance), the use of the substrate formulations disclosed herein, and / or the ALP formulation, and may also be associated with an improved signal-to-noise ratio, or both.

[0084] For example, as shown in the example embodiments described herein, for cTnI concentrations in plasma greater than 10 ng / L (which may also be reported as pg / mL), the measurement of cTnI exhibits a coefficient of variation of less than 4%. Additionally or alternatively, the example embodiments described herein show that for cTnI concentrations in plasma less than 10 ng / L, the measurement of cTnI may exhibit a standard deviation (SD) of less than 0.5 ng / L.

[0085] Also as shown in the exemplary embodiments described herein, for cTnI concentrations in serum greater than 15 ng / L, the measurement of cTnI can exhibit a coefficient of variation of less than 4%. Additionally or alternatively, some embodiments show that for cTnI concentrations in serum less than 15 ng / L, the measurement of cTnI exhibits a standard deviation (SD) of less than 0.5 ng / L.

[0086] The reproducibility conditions for the measurements for calculating the coefficient of variation or standard deviation (SD) or both include between-site and between-lot variance components. In some embodiments, the reproducibility conditions for the measurements also include between-day, between-run, and within-run variance components.

[0087] Alpha-fetoprotein (AFP)

[0088] In one aspect, the disclosure provides a method comprising measuring the concentration of alpha-fetoprotein (AFP) in a biological sample (e.g., a sample comprising serum or amniotic fluid). AFP is a recognized analyte associated with certain cancers and tumors and fetal development, particularly as an indicator of risk of congenital disabilities (e.g., birth defects or genetic disorders). The method can provide improved sensitivity, accuracy, or reproducibility, or a combination thereof.

[0089] In some embodiments, an anti-AFP antibody can be used to detect alpha-fetoprotein. In other embodiments, the anti-AFP antibody is preferably a monoclonal anti-AFP antibody.

[0090] In some embodiments, the method includes detecting AFP using an immunoassay analyzer or instrument. In some embodiments, the method includes detecting AFP in an apparatus comprising a distribution control system. An exemplary distribution control system according to the present disclosure includes, but is not limited to, a distribution control system described in U.S. Patent Publication No. 2020 / 0264207. In some embodiments, the method includes detecting AFP in an immunoassay analyzer or instrument comprising an image capture device or camera unit that can be used to provide feedback on the performance of the analyzer or instrument in real time. Exemplary immunoassay analyzers or instruments comprising an image capture device or camera unit according to the present disclosure include, but are not limited to, those described in U.S. Patent No. 11,263,433. In some preferred embodiments, the method includes detecting APF using a DxI 9000 immunoassay analyzer (available from Beckman Coulter, Inc., Brea, California).

[0091] In some embodiments, the method may include a two-site immunoenzymatic ("sandwich") assay. For example, a sample is added to a reaction vessel having an anti-AFP antibody conjugated to alkaline phosphatase (e.g., a mouse monoclonal anti-AFP-alkaline phosphatase conjugate) and particles (e.g., paramagnetic particles) coated with a second anti-AFP antibody (e.g., a second mouse monoclonal anti-AFP antibody). The AFP in the sample can bind to the monoclonal anti-AFP fixed on the particles, while the anti-AFP-alkaline phosphatase conjugate reacts with different antigenic sites on the sample AFP. After incubation in the reaction vessel, the substance bound to the solid phase is maintained in a magnetic field, while the unbound substance is washed away. Then, a chemiluminescent substrate can be added to the reaction vessel, and the light generated by the reaction can be measured with a photometer. Generally speaking, light generation is proportional to the concentration of AFP in the sample. The amount of analyte in the sample can be determined based on a stored (e.g., multi-point) calibration curve.

[0092] In some aspects, the method comprises detecting AFP using ACCESS AFP (available from Beckman Coulter, Inc., Brea, California).

[0093] In some aspects, the method includes comparing the AFP concentration to the AFP concentration in a normal population. In such embodiments, the difference between the AFP concentration in a tested sample (e.g., a biological sample from a subject, such as a patient sample) and the normal population can be used to assess the health status of the subject.

[0094] In some aspects, the method further comprises measuring the concentration of AFP using a substrate preparation comprising a chemiluminescent compound. In one aspect, the substrate preparation can be a substrate preparation as described in U.S. Patent No. 10,703,971. In some preferred embodiments, the substrate preparation comprises a substrate and / or substrate preparation as described herein. In some preferred embodiments, the enzyme preparation comprises, for example, an enzyme preparation such as an ALP preparation, as described herein. In some further preferred embodiments, the method comprises both a substrate preparation and an enzyme preparation substantially as described herein.

[0095] As further described in the illustrative examples, the reproducibility (i.e., accuracy and sensitivity) of the AFP assay performed on the DxI 9000 immunoassay analyzer is significantly improved compared to the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). According to the present disclosure, the improved assay may be attributable to one or a combination of the instrument platform (e.g., including improvements such as a distribution control system and an image capture device or camera unit that can be used to provide feedback about the analyzer or instrument performance in real time), the use of the substrate formulations disclosed herein, and / or the ALP formulation, and may also be associated with an improved signal-to-noise ratio, or both. Also as shown in the following examples, relative to the prior art, the assay formulation (e.g., a conjugate diluent) may allow for an improvement in assay results and / or maintain the same results achieved with a previous assay while consuming less reagent per assay, thereby providing an increased number of assays per unit of reagent (e.g., doubling the number of tests per reagent pack (e.g., 50 to 100 tests or 100 to 200 tests when the reagent packs are sold in units of two).

[0096] For example, as shown in the example embodiments described herein, for AFP concentrations greater than 10 ng / mL, the measurement of AFP exhibits a coefficient of variation (CV) of less than 7%. Additionally or alternatively, the example embodiments described herein show that for AFP concentrations less than 10 ng / mL, the measurement of AFP exhibits a standard deviation (SD) of less than 0.5 ng / mL.

[0097] The reproducibility conditions of the measurements used to calculate the coefficient of variation or standard deviation (SD) or both include between-site and between-batch variance components. In some aspects, the reproducibility conditions of the measurements also include between-day, between-run, and within-run variance components.

[0098] Thyroid stimulating hormone (TSH)

[0099] In one aspect, the disclosure provides such a method, which includes measuring the serum concentration or plasma concentration of thyroid stimulating hormone (TSH) in a blood sample. TSH is a recognized analyte associated with thyroid dysfunction or disease (e.g., hypothyroidism, hyperthyroidism, Hashimoto's disease, thyroid tumors, thyroid cancer and conditions related thereto), which can be manifested as a variety of clinical indications (e.g., autoimmune-related, body weight, temperature, strength, heart disease, mood, metabolism). The method can provide improved sensitivity, accuracy, or reproducibility, or a combination thereof.

[0100] In some embodiments, TSH can be detected using an anti-TSH antibody. In other embodiments, the anti-TSH antibody is preferably a monoclonal anti-TSH antibody. Generally speaking, the method for detecting TSH can be performed according to the method described in more detail above (for AFP and cTnI).

[0101] According to this illustrative embodiment, the reproducibility (i.e., accuracy and / or sensitivity) of the TSH assay performed on the DxI 9000 immunoassay analyzer is at least maintained when compared to the reproducibility achieved on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). In some embodiments, the reproducibility (i.e., accuracy and / or sensitivity) of the TSH assay performed on the DxI 9000 immunoassay analyzer when compared to the assay performed on the Access 2 immunoassay analyzer is expected to be significantly improved by one or more combinations of the features disclosed herein. According to the present disclosure, the improved assay may be attributed to one or a combination of the instrument platform (e.g., including improvements such as a system for removing aliquots of a sample from a sample using a disposable tip, a dispensing control system, an image capture device and / or camera unit that can be used to provide real-time feedback on the immunoassay analyzer or instrument performance, a dispenser that heats the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid), the use of the substrate formulation disclosed herein, and / or the ALP formulation, and may also be associated with an improved signal-to-noise ratio, or both. As also described in detail below, relative to the prior art, the assay formulation (e.g., conjugate diluent) is sufficient to enable assay results similar to those achieved with previous assays while consuming less reagent per assay and also providing an increased number of assays per unit of reagent (e.g., doubling the number of tests per reagent pack (e.g., 100 to 200 tests or 200 to 400 tests when the reagent pack (e.g., TSH reagent pack) is sold in units of two).

[0102] For example, a significant improvement in the number of assays per unit of reagent is achieved, i.e., an improvement associated with the same or similar assay performance relative to the prior art, exhibiting a coefficient of variation (CV) of 2% to 6%, a limit of blank (LoB) of 0.001 to 0.002 uIU / mL, a limit of detection (LoD) of 0.002 to 0.003 uIU / mL, and a limit of quantitation (LoQ) of 0.0008 to 0.001 uIU / mL at typical TSH concentrations.

[0103] The reproducibility conditions of the measurements used to calculate the coefficient of variation or standard deviation (SD) or both include the between-instrument variance components. In some aspects, the reproducibility conditions of the measurements may also include between-day, between-run, between-site, between-batch, and within-run variance components.

[0104] Beta human chorionic gonadotropin (βhCG)

[0105] In one aspect, the present disclosure provides methods comprising measurement of serum or plasma concentrations for quantification of beta human chorionic gonadotropin (βhCG) in a blood sample, also referred to as a βhCG test. βhCG is a well-recognized analyte associated with the pregnancy state and is sometimes referred to as the pregnancy hormone because it is produced by placental cells. Thus, the methods described herein can provide improved sensitivity, accuracy, or reproducibility, or a combination thereof, for tests indicating pregnancy.

[0106] In some embodiments, βhCG can be detected using an anti-βhCG antibody. In other embodiments, the anti-βhCG antibody is preferably a monoclonal anti-βhCG antibody. Generally speaking, methods for detecting βhCG can be performed according to the methods described in more detail above (for AFP and cTnI).

[0107] According to the present illustrative examples (e.g., Example 3), the reproducibility (i.e., accuracy and / or sensitivity) of the βhCG assay performed on the DxI 9000 immunoassay analyzer is at least maintained when compared to the reproducibility obtained on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). In some embodiments, the reproducibility (i.e., accuracy and / or sensitivity) of the βhCG assay performed on the DxI 9000 immunoassay analyzer when compared to the reproducibility of the assay performed on the Access 2 is expected to be significantly improved by one or more combinations of the features disclosed herein. According to the present disclosure, the improved assay may be attributable to one or a combination of the instrument platform (e.g., including improvements such as a distribution control system, a degassing module, and an image capture device or camera unit that can be used to provide feedback on the analyzer or instrument performance in real time), the use of the substrate formulations disclosed herein, and / or the ALP formulation, and may also be associated with an improved signal-to-noise ratio, or both. As also described in detail below, relative to the prior art, the assay formulation (e.g., conjugate diluent) is sufficient to enable assay results similar to those achieved with previous assays while consuming less reagent per assay and providing an increased number of assays per unit of reagent (e.g., doubling the number of tests per reagent pack (e.g., 50 to 100 tests or 100 to 200 tests when the reagent packs are sold in units of two).

[0108] For example, a significant improvement in the number of assays per unit of reagent is achieved relative to the prior art with the same or similar assay performance, exhibiting a coefficient of variation (CV) of 2.5% to 5%, a limit of blank (LoB) of 0.0 to 0.1 mIU / mL, a limit of detection (LoD) of 0.2 mIU / mL, and a limit of quantitation (LoQ) of 0.1 to 0.2 mIU / mL at typical hCG concentrations (i.e., above LoQ).

[0109] The reproducibility conditions of the measurements used to calculate the coefficient of variation or standard deviation (SD) or both include the between-instrument variance components. In some aspects, the reproducibility conditions of the measurements may also include between-day, between-run, between-site, between-batch, and within-run variance components.

[0110] Free thyroxine (T4)

[0111] In one aspect, the present disclosure provides a method comprising measuring the serum or plasma concentration of free thyroxine (T4) in a blood sample. T4 is very similar to TSH and is a recognized analyte associated with thyroid dysfunction or disease (e.g., hypothyroidism, hyperthyroidism, and conditions related thereto), which can be manifested as a variety of clinical signs (e.g., weight, temperature, physical strength, cardiac disease, mood, metabolism). The method can provide improved sensitivity, accuracy, or reproducibility, or a combination thereof.

[0112] In some embodiments, an anti-T4 antibody may be used to detect free T4. In other embodiments, the anti-T4 antibody is preferably a monoclonal anti-T4 antibody. Generally speaking, the method for detecting free T4 can be performed according to a conventional competitive assay method, and wherein the measured signal is inversely proportional to the concentration of free T4 in the sample. In some alternative embodiments, the method for detecting free T4 may include biotin as a specific binding agent.

[0113] According to the illustrative examples detailed below, the reproducibility (i.e., accuracy and sensitivity) of the free T4 assay performed on the DxI 9000 immunoassay analyzer is at least maintained when compared to the reproducibility obtained on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). In some embodiments, the reproducibility (i.e., accuracy and / or sensitivity) of the free T4 assay performed on the DxI 9000 immunoassay analyzer when compared to the assay performed on the Access 2 immunoassay analyzer is expected to be significantly improved by one or more combinations of the features disclosed herein. According to the present disclosure, the improved assay may be attributable to one or a combination of assay method changes (e.g., additional reaction mixing steps, improvements in dispense volume accuracy, etc.), instrument platform (e.g., including improvements such as a dispense control system and an image capture device or camera unit that can be used to provide feedback on analyzer or instrument performance in real time), use of the substrate formulations disclosed herein, and / or ALP formulations, and may also be associated with an improved signal-to-noise ratio, or both.

[0114] For example, improvements in precision were demonstrated with coefficients of variation (CV) of 2.7% to 5.2% and a limit of blank (LoB) of 0.18 ng / mL at typical free T4 concentrations. The updated assay also demonstrated improvements in limits of detection (LoD) and limits of quantitation (LoQ) for free T4 analyte measurements, features that have not been routinely measured on legacy assay platforms.

[0115] The reproducibility conditions of the measurements used to calculate the coefficient of variation or standard deviation (SD) or both include between-site and between-batch variance components. In some aspects, the reproducibility conditions of the measurements also include between-day, between-run, and within-run variance components.

[0116] Creatinine kinase (CK or CK-MB)

[0117] In one aspect, the disclosure provides a method comprising measuring the serum or plasma concentration of creatinine kinase (CK-MB) in a blood sample. Similar to cTnI, CK-MB is a recognized analyte associated with damage to cardiac tissue (and other organs and tissues that produce CK, such as the small intestine, brain, and uterus). The method can provide improved sensitivity, accuracy, or reproducibility, or a combination thereof.

[0118] In some embodiments, CK can be detected using anti-CK antibodies. In other embodiments, the anti-CK antibody is preferably a monoclonal anti-CK antibody. Generally speaking, the method for detecting CK can be performed according to the method described in more detail above (for AFP and cTnI).

[0119] According to this illustrative embodiment, the reproducibility (i.e., accuracy and sensitivity) of the CK-MB assay performed on the DxI 9000 immunoassay analyzer is expected to be significantly improved when compared to the reproducibility obtained on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). According to the present disclosure, the improved assay is expected to be due to one or a combination of assay method changes, instrument platform (e.g., including improvements such as distribution control systems and image capture devices or camera units that can be used to provide feedback on analyzer or instrument performance in real time), use of the substrate preparations disclosed herein, and / or ALP preparations, and may also be associated with improved signal-to-noise ratios, or both.

[0120] Sex hormone binding globulin (SHBG)

[0121] In one aspect, the disclosure provides such a method, which includes measuring the serum concentration or plasma concentration of sex hormone binding globulin (SHBG) in a blood sample. SHBG is a recognized analyte associated with testosterone levels. The method can provide improved sensitivity, accuracy, or reproducibility, or a combination thereof.

[0122] In some embodiments, SHBG can be detected using anti-SHBG antibodies. In other embodiments, the anti-SHBG antibody is preferably a monoclonal anti-SHBG antibody. Generally speaking, the method for detecting SHBG can be performed according to the method described in more detail above (for AFP and cTnI).

[0123] According to this illustrative embodiment, the reproducibility (i.e., accuracy and sensitivity) of the SHBG assay performed on the DxI 9000 immunoassay analyzer is expected to be significantly improved when compared to the reproducibility obtained on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). According to the present disclosure, the improved assay is expected to be due to one or a combination of assay method changes, instrument platform (e.g., including improvements such as distribution control systems and image capture devices or camera units that can be used to provide feedback on analyzer or instrument performance in real time), use of the substrate formulations disclosed herein, and / or ALP formulations, and may also be associated with improved signal-to-noise ratios, or both.

[0124] Cancer Antigen 19-9 (CA19-9 or "GI Monitor")

[0125] In one aspect, the present disclosure provides a method comprising measuring the serum or plasma concentration of cancer antigen 19-9 (CA19-9 or "GI monitor") in a blood sample. CA 19-9 is a well-recognized analyte associated with various types of cancer, particularly digestive cancers and liver disease. The method may provide improved sensitivity, accuracy, or reproducibility, or a combination thereof.

[0126] In some embodiments, CA 19-9 can be detected using an anti-CA 19-9 antibody. In other embodiments, the anti-CA 19-9 antibody is preferably a monoclonal anti-CA 19-9 antibody. Generally speaking, the method for detecting CA 19-9 can be performed according to the method described in more detail above (for free T4).

[0127] According to this illustrative embodiment, the reproducibility (i.e., accuracy and sensitivity) of the CA19-9 assay performed on the DxI 9000 immunoassay analyzer is expected to be significantly improved when compared to the reproducibility obtained on the Access 2 immunoassay analyzer (i.e., a clinically approved assay platform). According to the present disclosure, the improved assay is expected to be due to one or a combination of assay method changes, instrument platform (e.g., including improvements such as distribution control systems and image capture devices or camera units that can be used to provide real-time feedback on analyzer or instrument performance), use of the substrate formulations disclosed herein, and / or ALP formulations, and may also be associated with improved signal-to-noise ratios, or both.

[0128] Assay Reagents and Compositions

[0129] In one aspect, the present disclosure provides a composition comprising ALP conjugated to an antigen or an antibody, wherein the composition is formulated to optimize the stability of the ALP during storage of the composition.

[0130] Alkaline phosphatase (ALP)

[0131] ALP is a homodimer. In the active site of each monomer, there are three different metal binding sites (M1, M2, and M3 sites). The catalytic activity of ALP is regulated by the metal content within these metal binding sites. When two of these sites (M1 and M2) are occupied by Zn 2+ and the third site (M3) is occupied by Mg 2+ When occupied, peak enzymatic activity is achieved.

[0132] Alkaline phosphatase substrates and chemiluminescent compounds

[0133] Substrates that react with ALP include, for example, combinations of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate (PNPP), and Lumigen PPD (as found in LUMI-PHOS 530 and LUMI-PHOS Plus (Lumigen, Inc., Southfield, MI)).

[0134] In some embodiments, the chemiluminescent substrate comprises a chemiluminescent substrate and its preparation as described in U.S. Pat. No. 10,703,971. In some aspects, the chemiluminescent substrate may include LUMIGEN APS-5 (Lumigen, Inc., Southfield, MI). The structure of the active component of APS-5 is Fig.12 Shown in.

[0135] In some aspects, the chemiluminescent substrate comprises a compound of Formula I or a salt thereof:

[0136]

[0137] Where A is C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent; R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl group; R7-R 14 Independently H, C 1-6 Alkoxy, halogen, or C 1-4 Alkyl, or R7 or R8-R9 or R9-R10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 can be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring; R 15 It is C 1-6 Alkyl; each M is independently selected from H, alkali metal, alkaline earth metal, transition metal, ammonium, Organic amine salts and amino acid salts; Z is O or S; and n is 0, 1 or 2. An "alkyl" group refers to a fully saturated straight or branched chain hydrocarbon containing 1 to 12 carbon atoms, which is attached to the molecule by a single bond. Alkyl groups may include C1-C 12 Alkyl, C1-C 10 The alkyl groups may be optionally substituted.

[0138] "Alkoxy" refers to a group of formula -OR, wherein R is an alkyl, alkenyl or alkynyl group as defined herein attached to the parent molecular moiety via an oxygen atom. Some non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy and hexyloxy. According to some exemplary embodiments, the alkoxy group may be optionally substituted.

[0139] The term "aryl" refers to a stable monocyclic (i.e., phenyl), bicyclic, tricyclic, or tetracyclic ring system containing 6 to 18 carbon atoms and at least one aromatic ring in the ring system. Aryl groups may contain fused and / or bridged ring systems. Some non-limiting examples of aryl groups include aceanthrene, acenaphthylene, acephenanthrene, anthracene, azulene, benzene, Fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. According to some exemplary embodiments, the aryl group may be optionally substituted.

[0140] The term "cycloalkyl" refers to a fully saturated ring system of a stable monocyclic, bicyclic, polycyclic or spirocyclic ring generally containing 3 to 20 carbon atoms. A monocyclic ring system is a cyclic hydrocarbon group containing 3 to 10 carbon atoms in some embodiments. Some non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptyl and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic or fused bicyclic rings. Bridged monocyclic rings include monocyclic cycloalkyl rings in which two non-adjacent carbon atoms of the monocyclic ring are connected by an alkylene bridge between one to three additional carbon atoms (i.e., in the form of -(CH2) w - bridging group, wherein w is 1, 2 or 3). Some non-limiting examples of bicyclic and polycyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane and bicyclo[4.2.1]nonane, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, and the like. According to some exemplary embodiments, the cycloalkyl group may be optionally substituted.

[0141] The terms "heterocyclyl" and "heterocycle" refer to 3- to 20-membered monocyclic, bicyclic, polycyclic or spirocyclic ring systems that may be saturated, unsaturated or aromatic and contain 1 to 6 heteroatoms, N, O or S. Monocyclic heterocycles include 3-, 4-, 5-, 6- and 7-membered rings containing at least 1 heteroatom independently selected from O, N and S. The heterocycle is attached to the parent molecular moiety via any carbon atom or any nitrogen atom contained within the heterocycle. Some non-limiting examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridine, diazepanyl, 1,3-dihydro-1,4-dinitro ... alkyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, Oxazoline, In some embodiments, the present invention relates to oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholine sulfone), thiopyranyl and trithianyl. Some non-limiting examples of bicyclic heterocycles include 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothiophene-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl and octahydrobenzofuranyl.

[0142] The terms "haloalkyl", "haloalkenyl", "haloalkynyl" and "haloalkoxy" refer to alkyl, alkenyl, alkynyl or alkoxy groups as defined above substituted with one or more halogen atoms at any available position. According to some exemplary embodiments, any of these "halo" groups may be optionally substituted.

[0143] In some embodiments, a substrate formulation (e.g., comprising a substrate capable of producing a signal (e.g., such as chemiluminescence)) comprises in an aqueous solution:

[0144] a) 0.01 mM to 50 mM of a compound of formula I or a salt thereof:

[0145]

[0146] b) 0.01 to 200 μM of a cationic aromatic compound (CAC);

[0147] c) 1 μM to 10 mM background reducing agent; and

[0148] d) 0.05 to 20 g / L of an ether-linked nonionic surfactant not containing a carboxylate group; or a hydrophilic polymer.

[0149] In some preferred embodiments, the compound of Formula I or a salt thereof is:

[0150]

[0151] In some embodiments, a substrate preparation comprising a chemiluminescent substrate is used, and the substrate preparation may include a cationic aromatic compound (CAC), a background reducing agent, or a surfactant, a hydrophilic polymer, or a combination thereof. In some aspects, the surfactant may include an ether-linked nonionic surfactant that does not contain a carboxylate group. Other suitable surfactants are described in WO 2021 / 086977.

[0152] In some embodiments, the composition comprises a cationic aromatic compound (CAC) according to Formula II:

[0153]

[0154] in:

[0155] Q is selected from halogen, cyano, -COOR', -COSR', -CONR 1 R 2 、-CON(R)SO2R”、naphthyl、anthryl、NC 1-4 Alkyl acridinium and halogen substituted NC 1-4 Alkyl acridinyl;

[0156] R is for C 1-4 alkyl;

[0157] R', R" are independently selected from C 1-6 Alkyl, aryl and alkyl-substituted aryl;

[0158] R 1 , R 2 Independently selected from H, C 1-6 Alkyl, aryl and alkylaryl; and

[0159] Z - is a halide or a nitrate; and

[0160] Y is selected from H, halogen and C 1-4 alkyl.

[0161] In some additional embodiments, the compound according to Formula II comprises a structure wherein Q is N-methylacridinyl, halogen-substituted N-methylacridinyl, naphthyl or anthracenyl; R is C 1-4 Alkyl; Z- is selected from Cl - Br - ,I - and NO3 - , and Y is H or halogen. Some embodiments provide exemplary CAC compounds, such as the CAC compounds disclosed in US Pat. No. 10,703,971.

[0162] In some additional embodiments, the substrate formulation comprises a background reducing agent comprising one or more of lithium sulfite, sodium sulfite, potassium sulfite, lithium bisulfite, sodium bisulfite, potassium bisulfite, lithium metabisulfite, sodium metabisulfite, potassium metabisulfite, butylated hydroxytoluene (BHT; 2,6-bis(1,10 dimethylethyl)-4-methylphenol), butylated hydroxyl anisole (BHA), 3-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, and an aromatic boronic acid of formula Ar-B(OH)2, wherein Ar is phenyl, substituted phenyl, a fused aromatic ring system which may or may contain heteroatoms, a substituted fused aromatic ring system which may or may contain heteroatoms, wherein the substituted aryl group may have 1 to 3 substituents independently selected from the following: C 1-6 Alkyl, halogen, alkoxycarbonyl or hydroxyl groups. In some embodiments, the background reducing agent can be selected from phenylboronic acid, 4-tolylboronic acid, 4-chloroboric acid, 4-iodoboric acid and 3-methoxycarbonylphenylboronic acid and sodium sulfite.

[0163] In other embodiments, the substrate formulation comprises an ether-linked nonionic surfactant of formula (III):

[0164]

[0165] Where R is selected from C 6-22 Alkyl, cycloalkyl, C 6-22 Alkyl-substituted cycloalkyl and C 6-22 Alkyl monosubstituted or C 6-22 alkyl disubstituted phenyl; n is a number from 2 to 200; X is selected from O or S; and Y is selected from H or C 1-4 alkyl.

[0166] In some additional embodiments, the substrate formulation comprises an ether-linked nonionic surfactant selected from the group consisting of polyoxyethylene glycol alkyl ether (BRIJ), polyoxyethylene glycol octylphenol ether (TRITON), or polyoxyethylene nonylphenyl ether (IGEPAL).

[0167] In other embodiments, the substrate formulation may comprise a hydrophilic polymer according to formula (IV):

[0168]

[0169] Wherein X1 and X2 are independently selected from O, S, N or NH, or are absent; Y1 and Y2 are independently selected from H, H2 or C 1-4and n is a number from 20 to 12,000. In some additional embodiments, the hydrophilic polymer comprises poly(ethylene glycol) having an average Mw ranging from 1,000 to 511,000.

[0170] In some additional embodiments, the substrate preparation further comprises a 10-22 Alkyl sulfates and C 10-22 Alkyl Sulfonate Anionic Surfactants In some further embodiments, the anionic surfactant may be selected from sodium tetradecyl sulfate, sodium dodecyl sulfate (SDS) and sodium tridecyl sulfate (STS).

[0171] In some additional embodiments, the substrate formulation further comprises an amine buffer selected from the group consisting of: iris (tromethamine); AMPD (2-amino-2-methyl-1,3-propanediol); DEA (diethanolamine); AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid); 221-amine (2-amino-2-methylpropan-1-ol); CHES (2-(N-cyclohexylamino)ethanesulfonic acid); glycine; and TAPS (N-tris(hydroxymethyl)aminopropanesulfonic acid) buffer.

[0172] In some further embodiments, the substrate formulation is suitable for use in providing one or more assay characteristics, such as, for example:

[0173] a) can reach maximum intensity (I) within ≤5 minutes, ≤4 minutes, ≤2 minutes, ≤1 minute, ≤45 seconds, ≤30 seconds, ≤20 seconds, ≤10 seconds, ≤5 seconds, ≤4 seconds, ≤3 seconds, or ≤2 seconds after exposure to alkaline phosphatase max );

[0174] b) after storage at 4°C for 300 days, can exhibit ≤ 10% loss of original RLU after exposure to alkaline phosphatase;

[0175] c) exhibits ≥90% or ≥95% retained activity (RLU) compared to the original RLU when stored at 4°C for 300 days or longer;

[0176] d) exhibits ≥ 90% retained activity when stored at 4°C for 400 days or longer; and / or

[0177] e) After storage at 30° C. for 15 days, the signal change in % / day may be <-0.50% / day.

[0178] In any of the above embodiments, the substrate preparation produces chemiluminescence in the presence of a phosphatase (eg, an ALP preparation described herein).

[0179] Optimization of ALP-conjugation compositions

[0180] Newer ALP-reactive substrates, such as those described in U.S. Pat. No. 10,703,971 and APS-5 ( Fig.12 ), exhibits a faster turnaround time (TAT) compared to commercially available substrates comprising compounds such as LUMI-PHOS 530. This faster substrate TAT allows for faster determination of TAT, but it provides less time required for ALP to reach optimal function in the reaction solution before the assay is read.

[0181] If ALP activity is not maintained before ALP is added to the substrate, dosage deviations may be observed. Fig.13 As shown in , in assays containing LUMI-PHOS 530 (LP530), little or no dosage deviation was observed. In assays containing LUMI-PHOS PRO (the ALP substrate described in U.S. Pat. No. 10,703,971), dosage deviation was observed due to loss of ALP activity during enzyme storage.

[0182] Dosage deviations can be particularly detrimental when ALP is used to detect the presence or absence or concentration of an analyte in the context of an immunoassay. During an immunoassay, the relationship between the measured signal and the measured analyte concentration must generally be established during a process known as assay calibration. Assay calibration involves the generation of a standard curve by using known concentrations of the analyte to be assayed. However, if the ALP activity in the sample used for the calibration curve is different from the ALP activity in the sample to be tested, inconsistent assay readings can result.

[0183] In some embodiments, in the presence of excess Zn 2+ Storing ALP in solution at a concentration of Mg 2- It displaces from the active site of the enzyme and inhibits enzymatic activity.

[0184] In some additional embodiments, ALP inhibition can be eliminated by optimizing the long-term ALP storage solution. Specifically, by ensuring storage conditions (i.e., Zn 2+ and Mg 2+ concentration) is optimized to make free Zn 2+ and Mg 2+The concentrations are consistent with the apparent dissociation constants between these ions and the M3 site of ALP, which can reduce the inhibition of ALP activity to an acceptable level or even avoid the inhibition of ALP activity. And, as further described in Example 2, the pH is the pH of these metal ions (Zn 2+ and Mg 2+ ) and the M3 site of the enzyme. Therefore, as shown in Example 2 and Figure 14, the Zn required to achieve long-term stability of ALP-conjugated antibodies or antigens during storage is 2+ and Mg 2+ The concentration is pH dependent.

[0185] Because immunoassays are performed at a specific pH and because antibodies and antigens conjugated to ALP need to be stored at a specific pH to maintain protein conformation, ALP conjugated to antibodies and antigens must be stored in a Zn free environment available to maintain enzymatic activity. 2+ and Mg 2+ Storage at pH within a very narrow concentration range.

[0186] Composition

[0187] In embodiments of this aspect, the disclosure describes a composition comprising an antibody conjugated to ALP or an antigen conjugated to ALP, a buffer, a zinc salt, and a magnesium salt (also referred to herein as an "antibody composition"). In some aspects, the composition may further comprise an albumin, including, for example, bovine serum albumin (BSA), human serum albumin, casein, hydrolyzed casein, or ovalbumin.

[0188] In some aspects, when the pH of the composition is in the range of 5.5 to 6.4, free Zn 2+ The concentration of Mg was in the range of 5 μM to 1 mM, and free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ In some aspects, free Zn 2+ The concentration of free Mg may be in the range of 5 μM to 100 μM, in the range of 5 μM to 500 μM, in the range of 50 μM to 150 μM, or in the range of 100 μM to 500 μM. 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+ The concentration of free Mg is 3500 times higher than that of 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+The concentration of free Mg is 1500 times higher than that of 2+ The concentration of free Zn 2+ The concentration is 500 times higher than that of free Zn 2+ The concentration of free Mg is 1500 times higher than that of 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration of free Mg is 1500 times higher than that of 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration of free Mg is within 1000 times of 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration range is 500 times.

[0189] In some aspects, when the pH of the composition is in the range of 6.5 to 7.4, free Zn 2+ The concentration of Mg was in the range of 1 μM to 5 mM, and free Mg 2+ The concentration of free Zn 2+ The concentration is 200 times higher than that of free Zn 2+ The concentration range is 3500 times.

[0190] In some aspects, when the pH of the composition is in the range of 7.5 to 8.4, free Zn 2+ The concentration of Mg was in the range of 100 nM to 300 μM, and free Mg 2+ The concentration of free Zn 2+ The concentration of free Zn is 100 times 2+ The concentration range is 1000 times.

[0191] In some aspects, free magnesium and / or free zinc can be assessed by first removing larger molecules from a storage solution by size exclusion chromatography, dialysis or other means. In some aspects, size exclusion chromatography is preferred. Once the separation is complete, quantitative analysis can be performed by, for example, mass spectrometry or colorimetry. For example, 5-bromo-paps can be used as a colorimetric indicator for zinc quantification. In some aspects, mass spectrometry is preferred.

[0192] In some aspects, the enzymatic activity of ALP changes by less than 10% from peak activity when the composition is stored at 37° C. for up to 3 days. It has been shown that incubating ALP (coated to PMP or conjugated to antibody or antigen) at 37° C. for 2 to 3 days gives similar results to incubating ALP (similarly coated or conjugated) at 2° C. to 8° C. for 3 to 6 months.

[0193] In some aspects, the enzymatic activity of ALP changes by less than 10% from peak activity when the composition is stored at a temperature in the range of 2°C to 6°C for up to 6 months, up to 9 months, or up to 1 year.

[0194] The ALP may include any suitable ALP. In some embodiments, the ALP is preferably an ALP suitable for use in immunoassays. In some aspects, the ALP may include bovine intestinal ALP.

[0195] The buffer may include any suitable buffer. Some exemplary buffers include ACES, HEPES, MES, phosphate, TRIS, ADA, PIPES, MOPSO, BTP, BES or Bis-TRIS, or a combination thereof. In some aspects, the buffer may include ACES, HEPES, MES, phosphate or TRIS. However, in some aspects, phosphate buffers may be excluded because phosphates may alter the interaction between the metal binding site in the ALP and one or more metal ions.

[0196] The zinc salt can include any suitable zinc salt. Some exemplary zinc salts include ZnSO4 and ZnCl2. In an exemplary aspect, the zinc salt includes ZnCl2.

[0197] The magnesium salt can include any suitable magnesium salt. Some exemplary magnesium salts include MgSO4, MgCl2, and magnesium pidolate. In an exemplary aspect, the magnesium salt includes MgCl2.

[0198] The antibody conjugated to ALP may include any antibody suitable for use in immunoassays. The term "antibody" as used herein refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a specific antigen target of interest. Thus, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof including antigen-binding fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimics that mimic the structure and / or function of antibodies or specific fragments or portions thereof, including single-chain antibodies and fragments thereof. Thus, antibodies of the present disclosure encompass antibody fragments, including antibody fragments capable of binding to biomolecules or portions thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAb), variable fragments (Fv), single chain variable fragments (scFv) or disulfide-linked Fv (sdFv), diabodies or bivalent diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Antibodies can be from any source, including, for example, humans, rodents, rabbits, cattle, sheep, pigs, dogs, other mammals, chickens, other birds, and the like.

[0199] In some exemplary embodiments, the antibody conjugated to ALP comprises an anti-troponin antibody, an anti-thyroid stimulating hormone (TSH) antibody, an anti-βhCG antibody, an anti-alpha-fetoprotein (AFP) antibody, an anti-sex hormone binding globulin (SHBG) antibody, an anti-CA19-9 antibody (ACCESS GI monitor assay), or an anti-creatine kinase-MB (CKMB) antibody, as well as other target antigens described herein (e.g., T4, etc.).

[0200] In some aspects, the antibody conjugated to ALP may be an antibody that needs to be maintained at a pH of less than 8, a pH of less than 7.5, a pH of less than 7, or a pH of less than 6.5 to maintain the integrity and / or protein conformation of the antibody.

[0201] The antigen conjugated to ALP may include any antigen suitable for use in an immunoassay. In some exemplary aspects, the antigen conjugated to ALP includes a thyroxine (T4) antigen or a triiodothyronine (T3) antigen.

[0202] In some aspects, the antigen conjugated to ALP may be an antigen that needs to be maintained at a pH of less than 8, a pH of less than 7.5, a pH of less than 7, or a pH of less than 6.5 to maintain the integrity and / or protein conformation of the antigen.

[0203] In some aspects, including, for example, when the antibody conjugated to ALP comprises an anti-troponin antibody, the pH of the composition can be in the range of 5.5 to 6.4; free Zn 2+ The concentration of free Mg may be in the range of 100 μM to 500 μM; and 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ In an exemplary embodiment further described in Example 3D, the pH of the composition is 6, the composition comprises 35 mM MgCl2, 250 μM ZnCl2, and free [Mg 2+ ] / free [Zn 2+ ]The ratio is 200.

[0204] In some aspects, including, for example, when the antibody conjugated to ALP comprises an anti-TSH antibody, the pH of the composition can be in the range of 5.5 to 6.4; free Zn 2+ The concentration of free Mg may range from 5 μM to 100 μM; and 2+ The concentration of free Zn 2+ The concentration is 500 times higher than that of free Zn 2+ In an exemplary embodiment further described in Example 3E, the pH of the composition is 6, the composition comprises 8 mM MgCl2, 10 μM ZnCl2, and free [Mg 2+ ] / free [Zn 2+ ]The ratio is 800.

[0205] In some aspects, including, for example, when the antibody conjugated to ALP comprises an anti-AFP antibody, the pH of the composition can be in the range of 5.5 to 6.4; free Zn 2+ The concentration of free Mg may range from 50 μM to 150 μM; and 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ In an exemplary embodiment further described in Example 3A, the pH of the composition is 6, the composition comprises 15 mM MgCl2, 85 μM ZnCl2, and free [Mg 2 + ] / free [Zn 2+ ]The ratio is 176.

[0206] In some aspects, including, for example, when the antibody conjugated to ALP comprises an anti-βhCG antibody, the pH of the composition is in the range of 5.5 to 6.4; free Zn 2+ The concentration of free Mg may range from 5 μM to 100 μM; and 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+ In an exemplary embodiment further described in Example 3B, the pH of the composition is 6, the composition comprises 32 mM MgCl2, 35 μM ZnCl2, and free [Mg 2+ ] / free [Zn 2+ ] has a ratio of 914.

[0207] In some aspects, including, for example, when the antigen conjugated to ALP comprises a triiodothyronine (T3) antigen, the pH of the composition can be in the range of 5.5 to 6.4; free Zn 2+ The concentration of free Mg may range from 5 μM to 100 μM; and 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ In an exemplary embodiment further described in Example 3C, the pH of the composition is 6, the composition comprises 2 mM MgCl2, 10 μM ZnCl2, and free [Mg 2+ ] / free [Zn 2+ ]The ratio is 200.

[0208] Methods of using the composition

[0209] According to other aspects and embodiments discussed herein, the present disclosure provides methods of using the compositions described herein in immunoassays, the compositions comprising an antibody conjugated to ALP or an antigen conjugated to ALP.

[0210] Any suitable immunoassay method can be used. Some non-limiting immunoassay methods include two-site immunoenzyme ("sandwich") assays and two-step enzyme immunoassays. Competitive assays are also foreseen and illustrated herein. In some embodiments, the immunoassay may preferably be performed on an immunoassay analyzer or instrument.

[0211] In some embodiments, the method of using the composition may include contacting the analyte with at least one composition to form a mixture in a container, the composition comprising at least one reagent; incubating the mixture; adding a substrate composition comprising a chemiluminescent compound to the container; and after adding the substrate composition to the container, measuring the light produced by the reaction. In some aspects, the method also includes a washing step before adding the substrate composition to the container. The light can be measured by placing the container in a photometer. In some embodiments of the method, one or more compositions and / or reagents and / or samples can be combined, contacted and / or mixed with one or more combinations of compositions, reagents and / or samples. In some embodiments of the method, a "reaction mixture" includes a combination of elements provided for the reaction under conditions that allow such a reaction. In some additional embodiments, the reaction mixture may produce light. In yet other embodiments, the reaction mixture may produce chemiluminescence. In such embodiments, the reaction mixture may include a sample, an antibody comprising an antibody to a target antigen conjugated to an enzyme (e.g., ALP) or an antigen conjugated to an enzyme (e.g., ALP), and a substrate composition comprising a chemiluminescent compound. In some embodiments of the methods described herein, one or more other mixtures can be formed prior to any reaction mixture, such as a first or second mixture comprising a sample and a composition comprising an antibody, or a sample and a substrate composition and an assay reagent, or a sample and a sample diluent, etc.

[0212] The container can be any suitable container. In some aspects, the container can be a tube including, for example, a reaction vessel (RV). In some aspects, the tube comprises polyethylene.

[0213] The at least one reagent may comprise one or more reagents. In some aspects, the at least one reagent comprises a buffer, a particle (including, for example, a paramagnetic particle), a capture antibody, or an antibody-enzyme conjugate (e.g., an antibody-ALP conjugate) or an antigen-enzyme conjugate (e.g., an antigen-ALP conjugate), or any combination thereof.

[0214] In some aspects, the agent comprises particles. In some aspects, the agent comprises at least 0.1 μg particles, at least 0.2 μg particles, at least 0.3 μg particles, at least 0.4 μg particles, at least 0.5 μg particles, at least 0.6 μg particles, at least 0.7 μg particles, at least 0.8 μg particles, at least 0.9 μg particles, at least 1.0 μg particles, at least 2.0 μg particles, at least 3.0 μg particles, at least 3.0 μg particles, at least 4.0 μg particles, at least 5.0 μg particles, at least 6.0 μg particles, at least 7.0 μg particles, at least 8.0 μg particles, at least 9.0 μg particles, at least 10 μg particles, at least 15 μg particles, at least 20 μg particles, at least 25 μg particles, at least 30 μg particles, at least 35 μg particles, at least 40 μg particles, at least 45 μg particles, at least 50 μg particles, at least 60 μg particles, or at least 70 μg particles, and / or a maximum of 10μg particles, a maximum of 11μg particles, a maximum of 12μg particles, a maximum of 13μg particles, a maximum of 14μg particles, a maximum of 15μg particles, a maximum of 16μg particles, a maximum of 17μg particles, a maximum of 18μg particles, a maximum of 19μg particles, a maximum of 20μg particles, a maximum of 25μg particles, a maximum of 30μg particles, a maximum of 35μg particles, a maximum of 40μg particles, a maximum of 45μg particles, a maximum of 50μg particles, a maximum of 55μg particles, a maximum of 60μg particles, a maximum of 65μg particles, a maximum of 70μg particles, a maximum of 75μg particles, a maximum of 80μg particles, a maximum of 85μg particles, a maximum of 90μg particles, a maximum of 100μg particles, a maximum of 110μg particles, a maximum of 120μg particles, a maximum of 130μg particles, a maximum of 140μg particles, or a maximum of 150μg particles. For example, exemplary ranges of particles include at least 0.1 μg and up to 100 μg, at least 0.1 μg and up to 150 μg, at least 5 μg and up to 100 μg, at least 10 μg and up to 100 μg, etc. In some cases, the agent comprises about 0.1 μg to about 150 μg, about 0.2 μg to about 140 μg, about 0.3 μg to about 130 μg, about 0.4 μg to about 120 μg, about 0.5 μg to about 110 μg, about 1.0 μg to about 100 μg, about 1.5 μg to about 90 μg, about 2.0 μg to about 80 μg, about 2.5 μg to about 70 μg, about 3 μg to about 60 μg, about 4 μg to about 50 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 50 μg, or about 0.5 μg to about 50 μg of particles. In some embodiments, the particle is a microparticle, a coated particle, a coated microparticle, a paramagnetic particle, or a paramagnetic microparticle. In some embodiments, the particle is coated with an antibody, a monoclonal antibody, or a target-specific antibody. When the particle comprises a paramagnetic particle, the particle can be separated from other components of the composition by subjecting it to a magnetic field.

[0215] In some aspects, the method comprises detecting AFP or βHCG using a reagent comprising particles. In some aspects, the agent comprises at least 0.1 μg of particles, at least 0.2 μg of particles, at least 0.3 μg of particles, at least 0.4 μg of particles, at least 0.5 μg of particles, at least 0.6 μg of particles, at least 0.7 μg of particles, at least 0.8 μg of particles, at least 0.9 μg of particles, at least 1.0 μg of particles, at least 2.0 μg of particles, at least 3.0 μg of particles, at least 3.0 μg of particles, at least 4.0 μg of particles, at least 5.0 μg of particles, at least 6.0 μg of particles, at least 7.0 μg of particles, at least 8.0 μg of particles, at least 9.0 μg of particles, at least 10 μg of particles, at least 15 μg of particles, at least 20 μg of particles, at least 25 μg of particles, at least 30 μg of particles, at least 35 μg of particles, at least 40 μg of particles, at least 45 μg of particles, or at least 50 μg of particles, and / or or up to 10 μg of particles, up to 11 μg of particles, up to 12 μg of particles, up to 13 μg of particles, up to 14 μg of particles, up to 15 μg of particles, up to 16 μg of particles, up to 17 μg of particles, up to 18 μg of particles, up to 19 μg of particles, up to 20 μg of particles, up to 25 μg of particles, up to 30 μg of particles, up to 35 μg of particles, up to 40 μg of particles, up to 45 μg of particles, up to 50 μg of particles, up to 55 μg of particles, up to 60 μg of particles, up to 65 μg of particles, up to 70 μg of particles, up to 75 μg of particles, up to 80 μg of particles, up to 85 μg of particles, up to 90 μg of particles, up to 100 μg of particles, up to 110 μg of particles, up to 120 μg of particles, up to 130 μg of particles, up to 140 μg of particles, or up to 150 μg of particles. For example, exemplary ranges of particles include at least 0.1 μg and up to 100 μg, at least 0.1 μg and up to 150 μg, at least 5 μg and up to 100 μg, at least 10 μg and up to 100 μg, etc. In some cases, the agent comprises about 0.1 μg to about 150 μg, about 0.2 μg to about 140 μg, about 0.3 μg to about 130 μg, about 0.4 μg to about 120 μg, about 0.5 μg to about 110 μg, about 1.0 μg to about 100 μg, about 1.5 μg to about 90 μg, about 2.0 μg to about 80 μg, about 2.5 μg to about 70 μg, about 3 μg to about 60 μg, about 4 μg to about 50 μg, about 0.1 μg to about 60 μg, about 0.1 μg to about 50 μg, or about 0.5 μg to about 50 μg of particles.

[0216] In some aspects, the method comprises detecting TSH using a reagent comprising particles. In some aspects, the reagent comprises at least 0.1 μg particles, at least 0.2 μg particles, at least 0.3 μg particles, at least 0.4 μg particles, at least 0.5 μg particles, at least 0.6 μg particles, at least 0.7 μg particles, at least 0.8 μg particles, at least 0.9 μg particles, at least 1.0 μg particles, at least 2.0 μg particles, at least 3.0 μg particles, at least 3.0 μg particles, at least 4.0 μg particles, at least 5.0 μg particles, at least 6.0 μg particles, at least 7.0 μg particles, at least 8.0 μg particles, at least 9.0 μg particles, at least 10.0 μg particles, at least 11.0 μg particles, at least 12.0 μg particles, at least 13.0 μg particles, at least 14.0 μg particles, at least 15.0 μg particles, at least 16.0 μg particles, at least 17.0 μg particles, at least 18.0 μg particles, at least 19.0 μg particles, at least 20.0 μg particles, at least 21.0 μg particles, at least 22.0 μg particles, at least 23.0 μg particles, at least 24.0 μg particles, at least 25.0 μg particles, at least 26.0 μg particles, at least 27.0 μg particles, at least 28.0 μg particles, at least 29.0 μg particles, at least 30.0 μg particles, at least 31.0 μg particles, at least 32.0 μg particles, at least 33.0 μg particles, at least 34.0 μg particles At least 10 μg particles, at least 15 μg particles, at least 20 μg particles, at least 25 μg particles, at least 30 μg particles, at least 35 μg particles, at least 40 μg particles, at least 45 μg particles, or at least 50 μg particles, and / or at most 10 μg particles, at most 11 μg particles, at most 12 μg particles, at most 13 μg particles, at most 14 μg particles, at most 15 μg particles, at most 16 μg particles, at most 17 μg particles, at most 18 μg particles, at most 19 μg particles, at most 20 μg particles, at most 25 μg particles, at most 30 μg particles. For example, exemplary ranges of particles include at least 0.1 μg and at most 20 μg, at least 0.1 μg and at most 20 μg, at least 5 μg and at most 20 μg, at least 10 μg and at most 20 μg, etc.

[0217] In some embodiments, the microparticles comprise paramagnetic or superparamagnetic materials, such as, for example, ferromagnetic iron oxides, Fe3O4 or Fe2O3. The terms "paramagnetism" and "superparamagnetism" refer to materials that undergo forces in a magnetic field gradient without becoming permanently magnetized. In a specific embodiment, the support comprises iron in the form of hematite or Fe2O3. In multiple embodiments, the average diameter of the microparticles is in the range of 100nm to 22,900nm. In a specific embodiment, the average diameter of the microparticles is in the range of about 750nm to about 3,000nm. In another specific embodiment, the average diameter of the microparticles is in the range of about 950nm to about 1,150nm.

[0218] In some aspects, the substrate composition may include a buffer. In some aspects, the volume of the buffer in the substrate composition is at least 50 μL, at least 100 μL, at least 150 μL, at least 200 μL. In some aspects, the volume of the buffer in the substrate composition is at most 100 μL, at most 150 μL, at most 200 μL, at most 250 μL or at most 300 μL. Therefore, in some exemplary embodiments, when the chemiluminescent compound is added to the reagent (comprising particles, capture antibodies, antibody-enzyme conjugates and / or antigen-enzyme conjugates), the volume of the buffer in the container can be at most 100 μL, or at most 150 μL, or at most 200 μL. In another exemplary embodiment, when the chemiluminescent compound is added to the reagent, the volume of the buffer in the container can be in the range of 150 μL to 300 μL.

[0219] In some embodiments, when a chemiluminescent compound is added to the reagent, the buffer may also contain at least 10,000 analyte molecules, at least 15,000 analyte molecules, at least 20,000 analyte molecules, or at most 20,000 analyte molecules, at most 25,000 analyte molecules, at most 35,000 analyte molecules, or at most 50,000 analyte molecules. For example, the buffer may contain 10,000 to 20,000 analyte molecules or 10,000 to 50,000 analyte molecules.

[0220] In some embodiments, when the chemiluminescent compound is added to the reagent, the mass of the chemiluminescent compound is at least 2 times the mass of the reagent, at least 5 times the mass of the reagent, at least 10 times the mass of the reagent, at least 20 times the mass of the reagent, at least 30 times the mass of the reagent, at least 40 times the mass of the reagent, at least 50 times the mass of the reagent, at least 60 times the mass of the reagent, at least 70 times the mass of the reagent, at least 80 times the mass of the reagent, or at least 90 times the mass of the reagent. In some embodiments, when the chemiluminescent compound is added to the reagent, the mass of the chemiluminescent compound is at most 50 times the mass of the reagent, at most 70 times the mass of the reagent, at most 80 times the mass of the reagent, at most 90 times the mass of the reagent, at most 100 times the mass of the reagent, or at most 150 times the mass of the reagent.

[0221] In some embodiments, when the chemiluminescent compound is added to the reagent, the ratio of chemiluminescent compound to enzyme is at least 1 μg of chemiluminescent compound to 1500 enzyme molecules.

[0222] In some embodiments, when a chemiluminescent compound is added to a reagent (e.g., to a particle, microparticle, paramagnetic particle, paramagnetic microparticle, etc.), the chemiluminescent compound is present at a concentration of at least 0.01 g / L, at least 0.02 g / L, at least 0.03 g / L, at least 0.04 g / L, at least 0.05 g / L, at least 0.1 g / L, at least 0.15 g / L, at least 0.2 g / L, at least 0.3 g / L, at least 0.4 g / L, at least 0.5 g / L, In some embodiments, the chemiluminescent compound is present in a concentration of at most 0.15 g / L, at most 0.2 g / L, at most 0.5 g / L, at most 1 g / L, at most 2 g / L, at most 3 g / L, at most 4 g / L, at most 5 g / L, at most 7 g / L, at most 9 g / L, at most 10 g / L, at most 12 g / L, at most 15 g / L, at most 20 g / L, at most 25 g / L, at most 35 g / L and at most 50 g / L. For example, when the chemiluminescent compound is added to the reagent, the chemiluminescent compound can be present in a concentration in the range of 0.01 g / L to 50 g / L or in the range of 0.1 g / L to 10 g / L. In some cases, the chemiluminescent compound is present at a concentration in the range of about 0.005 to about 20 g / L, about 0.01 to about 15 g / L, about 0.01 to about 10 g / L, about 0.01 to about 9 g / L, about 0.01 to about 8 g / L, about 0.01 to about 7 g / L, about 0.01 to about 6 g / L, about 0.01 to about 5 g / L, about 0.05 to about 10 g / L, about 0.05 to about 8 g / L, about 0.05 to about 6 g / L, about 0.05 to about 4 g / L, about 0.1 to about 10 g / L, about 0.1 to about 7 g / L, about 0.1 to about 8 g / L, about 0.1 to about 7 g / L, about 0.1 to about 6 g / L, or about 0.5 to about 10 g / L. In some cases, the chemiluminescent compound is present at a concentration of 0.1 g / L to 0.2 g / L, 0.1 to 0.5 g / L, 0.1 to 1 g / L, 0.1 to 2 g / L, 0.1 to 3 g / L, 0.1 to 4 g / L, 0.1 to 5 g / L, 0.1 to 6 g / L, 0.1 to 7 g / L, 0.1 to 8 g / L, 0.1 to 9 g / L, 0.1 to 10 g / L, 0.1 to 11 g / L, 0.1 to 12 g / L, or 0.1 to 15 g / L. In some cases, the chemiluminescent compound is present at a concentration of 0.05 to 0.5 g / L, 0.1 to 0.5 g / L, 0.2 to 0.5 g / L, 0.3 to 0.5 g / L, 0.4 to 0.5 g / L, 0.1 to 0.4 g / L, 0.1 to 0.3 g / L, or 0.1 to 0.2 g / L. The chemiluminescent compound can be any compound known in the art. In some cases, the chemiluminescent compound is a chemiluminescent substrate of Formula I or a salt thereof:

[0223]

[0224] in

[0225] A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent;

[0226] R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups;

[0227] R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring;

[0228] R 15 It is C 1-6 alkyl;

[0229] Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts;

[0230] Z is O or S; and

[0231] n is 0, 1 or 2.

[0232] In some aspects, the mass of the chemiluminescent compound used in the methods provided herein is at least 0.1 times, at least 0.5 times, at least 0.8 times, at least the same, at least 1.5 times, at least two times, at least three times, at least five times, at least ten times, at least twenty-five times, at least fifty times, at least one hundred times, or at least one hundred and fifty times the mass of the at least one reagent. In some embodiments, the at least one reagent is a particle, a microparticle, a paramagnetic particle, a paramagnetic microparticle, a coated particle, a coated paramagnetic particle, a coated microparticle, or a coated paramagnetic microparticle.

[0233] In some aspects, the at least one reagent comprises at least 0.01 μg and at most 1.0 μg, at least 0.01 μg and at most 0.7 μg, or at least 0.01 μg and at most 0.5 μg of capture antibody. The at least one reagent may comprise at least 0.001 μg, at least 0.005 μg, at least 0.01 μg, at least 0.02 μg, at least 0.03 μg, at least 0.04 μg, at least 0.05 μg, at least 0.06 μg, at least 0.07 μg, at least 0.08 μg, at least 0.09 μg, at least 0.1 μg, at least 0.2 μg, or at least 0.3 μg, and / or at most 0.05 μg, at most 0.06 μg, at most 0.07 μg, at most 0.08 μg, at most 0.09 μg, at most 0.1 μg, at most 0.2 μg, at most 0.3 μg, at most 0.4 μg, at most 0.5 μg, at most 0.6 μg, at most 0.7 μg, at most 0.8 μg, at most 0.9 μg, or at most 1.0 μg of capture antibody. Additional amounts of capture antibody are also envisioned to include, for example, at least 0.01 μg and up to 1.0 μg, at least 0.01 μg and up to 0.5 μg, 0.01 μg and up to 0.3 μg, and the like.

[0234] In some embodiments, light is measured at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes, at most 1 minute, at most 56 seconds, at most 55 seconds, at most 50 seconds, or at most 48 seconds after the substrate composition is added to the container. The use of newer ALP-reactive substrates that exhibit a faster turnaround time (TAT) allows for faster measurement of light compared to older chemiluminescent substrates that require waiting longer (e.g., more than 5 minutes or more than 6 minutes) before measuring the emitted light after the chemiluminescent substrate is added to the container. As further discussed herein, the compositions of the present disclosure reduce the inhibition of ALP activity when using chemiluminescent substrates with faster TATs, allowing for more accurate measurements. In some embodiments, including, for example, when the substrate comprises APS-5, light is preferably measured less than 1 minute after the chemiluminescent substrate is added to the container.

[0235] In other embodiments, substrate and / or substrate preparation provide the mensuration operation number / reagent unit of raising.The signal-to-noise ratio of the light produced by described substrate and / or the improvement of brightness (or amount / intensity) are obviously brighter than other substrates forming prior art.Therefore, the background (that is, the substrate signal under the situation of not having analyte) relevant with method and substrate / substrate composition described herein is usually much lower than known substrate / substrate composition.Therefore, with respect to background, the signal raising provides the opportunity that reduces the amount of the reagent used in reaction and keeps the measurement performance simultaneously.

[0236] In some embodiments, the sample may be present in an immunoassay analysis or instrument as an aliquot, for example, in a vessel or tube as an aliquot. In some embodiments, the volume of the sample aliquot is at most 2 μl, at most 5 μl, at most 8 μl, at most 10 μl, at most 15 μl, at most 20 μl, at most 30 μl, at most 40 μl, or at most 50 μl. In some cases, the volume of the sample aliquot is in the range of 1 μl to 2 μl, 1 μl to 5 μl, 1 μl to 8 μl, 2 μl to 10 μl, or 2 μl to 20 μl, or 2 μl to 50 μl. In some embodiments, the volume of the sample aliquot is at least 1 μl, at least 2 μl, or at least 3 μl.

[0237] In some embodiments, the method further comprises determining the amount of the analyte in the sample. For example, the amount of the analyte can be determined based on a stored multi-point calibration curve.

[0238] In an exemplary embodiment, the method may include contacting a sample comprising an analyte with a composition described herein, wherein the composition comprises an antibody that binds to ALP to form an ALP-antibody-analyte complex. The method may also include contacting the ALP-antibody-analyte complex with an anti-analyte antibody bound to a solid phase, and washing away sample that is not bound to the solid phase. Some exemplary solid phases include polymeric beads and magnetic beads, including, for example, paramagnetic particles.

[0239] In another exemplary embodiment, the method may include contacting a sample comprising an analyte with an antibody specific for the analyte to form a complex comprising the analyte, washing the complex, and contacting the complex with a composition described herein, wherein the composition comprises an antigen that binds to ALP.

[0240] In some embodiments, the method includes a two-site immunoenzyme ("sandwich") assay. For example, a sample can be added to a reaction vessel having a relevant antibody conjugated to alkaline phosphatase and paramagnetic particles coated with a secondary antibody. The analyte in the sample binds to the fixed antibody on the paramagnetic particles, while at the same time, the antibody conjugated to alkaline phosphatase reacts with different antigenic sites on the analyte. After incubation in the reaction vessel, the substance bound to the solid phase is maintained in a magnetic field, and the unbound substance is washed away. Then, a chemiluminescent substrate is added to the vessel, and the light generated by the reaction is measured with a photometer. Light generation is proportional to the concentration of the analyte in the sample. The amount of the analyte in the sample is determined according to a stored multi-point calibration curve.

[0241] In some embodiments, the method includes a two-step enzyme immunoassay. For example, an antibody specific for an analyte can be added to a reaction vessel, and the analyte is coupled to a solid phase coated with biotin, a sample, a buffered protein solution, and a streptavidin-coated solid phase. During this first incubation period, the anti-analyte antibody coupled to biotin binds to the solid phase and the free analyte in the sample. After incubation in the reaction vessel, the substance bound to the solid phase is maintained in a magnetic field, and the unbound substance is washed away. Next, a buffered protein solution and an antigen-ALP conjugate are added to the reaction vessel. The antigen-ALP conjugate binds to the unoccupied anti-analyte antibody binding site. After incubation in the reaction vessel, the substance bound to the solid phase is maintained in a magnetic field, and the unbound substance is washed away. Then, a chemiluminescent substrate is added to the vessel, and the light generated by the reaction is measured with a photometer. Light generation is inversely proportional to the concentration of free analyte in the sample. The amount of analyte in the sample is determined according to a stored multi-point calibration curve.

[0242] The methods and assays provided by the present disclosure are widely applicable to many patient populations. For example, the methods and assays can be used to assess a patient population under one or more illnesses, disorders and / or disease risks associated with a target antigen (e.g., the exemplary target antigens described in detail herein). The methods and assays can be used to assess a patient population suffering from one or more illnesses, disorders and / or diseases associated with a target antigen. The methods and assays can be used to assess a patient population experiencing a preventive and / or therapeutic intervention for preventing and / or treating one or more illnesses, disorders and / or diseases associated with a target antigen, to assess the effectiveness of a preventive or therapeutic intervention. Some non-limiting examples of patient populations that may benefit from the methods and assays provided by the present disclosure include patients who have or are at risk of developing cancer or a neoplastic disease; patients who are pregnant or who think they may be pregnant; pregnant patients being evaluated for risk of a fetal congenital or genetic disorder; patients who have an autoimmune disorder or one or more clinical symptoms associated with an autoimmune disorder; patients who have or are at risk of one or more heart diseases (e.g., myocardial infarction, heart failure, myocarditis); patients who have or are at risk of developing kidney disease; patients who have or are at risk of developing endocrine and / or hormonal imbalances (e.g., thyroid imbalances); patients who have or are at risk of developing tissue and organ damage (e.g., heart, brain, intestines, and uterus); and patients who have or are at risk of developing sex hormone imbalances.

[0243] Kits and compositions of matter for assays

[0244] In yet another aspect, the disclosure relates to a kit for detecting a target analyte or antigen in a sample. In some embodiments of this aspect, the kit may include any one of a substrate, an enzyme, a preparation or a composition, the composition including substrates, enzymes and preparations as generally described herein; and instructions or labels for instructing appropriate use. Optionally, the kit may also include one or more containers, reagents, reactants and / or assay diluents, or other devices that are convenient for use. The disclosure contemplates all or any subgroups of components for conducting research assays and / or diagnostic assays that may be included in the kit. Similarly, the kit may include instructions for preparing one or more assay solutions under suitable conditions, the assay solution including one or more assay biological samples, substrates, enzymes, preparations, or compositions thereof. In some other exemplary embodiments, the kit may include a solution of one or more kit components, or a dry preparation or a lyophilized preparation, and instructions for preparing the solution, or a dry preparation or a lyophilized preparation for use (e.g., for reconstructing a lyophilized or dried product, distributing and / or diluting a solution, etc.).

[0245] The present disclosure also encompasses finished packaged and labeled products (e.g., as a kit, or as one or more parts of a kit). Such articles include appropriate unit forms (e.g., concentrated assay formulations, ready-to-use assay formulations, etc.) in suitable vessels or containers (e.g., glass bottles or other generally sterile and sealed containers). In some example embodiments, containers may include, but are not limited to, vials, bottles, and / or prefilled syringes, etc. Optionally, associated with such kits and / or containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of the kits and / or assay components, which notice may reflect approval by the manufacturing, use, or sales agency for human use / diagnosis.

[0246] As described above, the kit may include instructions for use or other informational materials advising physicians, clinicians, and / or technicians on how to properly interpret specific assay results in use. In some additional embodiments, kits that are not exclusively used for research use are contemplated. Such kits may, for example, be similar to those described above, but may also include a label indicating that the kit and its use are limited to research purposes.

[0247] The kit comprises one or more of the following: a chemiluminescent substrate, an enzyme, and / or an assay reagent, or a composition comprising one or more thereof, and according to some aspects and embodiments of the present disclosure. In some embodiments, the kit may also comprise additional assay reagents, such as assay buffers, assay diluents, pH regulators, or additional reporters (e.g., for indirect or detected reporter molecules of target analytes or antigens). The kit may also comprise instructions for implementing any one of the assays or methods disclosed herein.

[0248] Immunoassay analyzers or instruments

[0249] The methods, compositions and kits disclosed herein can be used in combination with integrated system platforms. For example, methods, compositions and kits can be used for a combination system of commercial sales (e.g., sold by Beckman Coulter), such as immunoassay analyzers, including Access 2+, UniCel DxI 600, UniCel, DxI 800 or DxI 9000 immunoassay analyzers. In some exemplary embodiments, the integrated system platform is a DxI 9000 immunoassay analyzer. The methods, compositions and kits disclosed herein can also be used with one or more sample preparation system platforms. Similarly, the methods, compositions and kits disclosed herein can be used with instant system platforms and other available assay platforms. Therefore, the methods, compositions and kits of the present disclosure can be easily applicable to any number of devices, assay platforms and instruments, such as handheld fluorescence detectors, microfluidic devices, enzymatic detection systems, immunochromatographic strips and lateral flow devices.

[0250] Thus, in some embodiments, the methods of using the compositions described herein include using an immunoassay analyzer or instrument as described herein or otherwise as known in the art. In some embodiments, the methods may preferably include using a DxI 9000 immunoassay analyzer (available from Beckman Coulter, Inc., Brea, California), which includes a number of advantageous features (e.g., hardware and / or software) that can provide additional improved assay results when used in combination with the enzyme compositions and substrate compositions described herein, and in some embodiments, such a combination provides a synergistic improvement in assay results.

[0251] Immunoassay analyzers or instruments may be used in combination with enzymes and / or enzyme compositions as described herein. Immunoassay instruments or one or more features thereof may be used in combination with substrates and / or substrate compositions as described herein. In addition or in lieu thereof, immunoassay analyzers or instruments or one or more features thereof may be used in combination with enzymes and / or enzyme compositions as described herein.

[0252] In an exemplary embodiment, an immunoassay analyzer or instrument may load a sample container into the instrument and onto a sample rack at the same time, such as described in WO2018232364. As part of loading the sample container into the immunoassay analyzer or instrument, the device may prepare a barcode or other identification mark on the sample. Once the sample container is in the immunoassay analyzer or instrument, a disposable tip may be used to divide a portion of the sample from the sample container. Exemplary disposable tips are described in U.S. Patent Nos. D841182, D887574, and D899623. The sample may be placed in a second vessel (e.g., a reaction vessel or RV). The RV containing the sample aliquot may be used immediately or stored at 4°C. In order to create an immunoassay, an immunoassay analyzer or instrument may use a pipette to obtain and distribute an appropriate amount of reagent in a container, which also contains all or a portion of the aliquoted sample. Reagents may be obtained from a reagent pack. Some exemplary reagents include buffers, particles (including, for example, paramagnetic particles), capture antibodies, antibody enzyme conjugates or antigen-enzyme conjugates, and combinations thereof. In some embodiments, the enzyme is preferably ALP. The RV containing reagents and samples can be incubated, for example, at 37° C. to allow the components of the immunoassay to bind to each other (e.g., to allow antibodies or antigens to bind to analytes in the sample). In order to separate the bound antibodies or antigens from the unbound antibodies or antigens, the immunoassay analyzer or instrument will perform one or more washing steps. Such washing steps can be performed by a washing station. An exemplary washing station is described in U.S. Patent Publication No. 2021 / 0025910A1. In some aspects, it may be preferred that the washing fluid can be heated before being added to the reaction vessel. The particles bound to the bound antibody or bound antigen can be separated from the washing fluid by any suitable means. When the particles are magnetic particles, it is preferred that the particles are aggregated by magnetizing the particles, the supernatant is aspirated, the washing (e.g., buffer) solution is distributed and repeated 1 to 3 times to separate (e.g., subject to a magnetic field) and wash the particles. When the particles have been washed and the unbound antibodies or antigens have been removed, a substrate can be added to the container to generate a signal. Before measuring the emitted light, a container (e.g., a reaction vessel) containing particles and substrates can be incubated on an immunoassay analyzer or instrument. Incubation can be performed at any suitable temperature, including, for example, at 37°C. An immunoassay analyzer or instrument can measure the emitted light using any suitable method. In some embodiments, a photometer may be preferred. Exemplary photometers that can be used for immunoassay analyzers or instruments include those described in WO2019060375, WO2020219869, and / or U.S. Patent No. 11,604,146. An immunoassay analyzer or instrument can also use a calibration curve to correlate the measured light with the amount of analyte.

[0253] For example, in some embodiments, the immunoassay analyzer or instrument includes a system that uses a disposable tip to remove a sample aliquot from a sample for use on the immunoassay analyzer or instrument. Exemplary disposable tips are described in U.S. Patent Nos. D841182, D887574, and D899623. Using a disposable tip to remove a sample aliquot from a sample in a sample container can minimize potential contamination of the sample remaining in the sample container. In addition, once a sample aliquot is removed from the sample in the sample container, the sample container can be unloaded from the immunoassay analyzer or instrument and used for other purposes, including for other types of clinical analysis.

[0254] In some embodiments, the immunoassay analyzer or instrument includes a distribution control system. Non-limiting distribution control systems include those described in U.S. Patent Publication No. 2020 / 0264207. The distribution control system can be used to prepare a method for evaluating a fluid substance. An exemplary method includes aspirating a first volume of fluid substance from a first vessel to a distribution tip, and distributing a second volume of fluid substance from the distribution tip to a second vessel. Distributing the second volume of fluid substance from the distribution tip to the second vessel may include lowering the distribution tip to the second vessel at a first height, configuring the first height so that the distal end of the distribution tip remains above the surface level in the second vessel after distribution; distributing the fluid substance at least partially from the distribution tip to the second vessel; and lowering the distribution tip to a second height, configuring the second height so that the distal end of the distribution tip contacts the surface level in the second vessel after distribution. The method may also include distributing the remaining fluid substance from the distribution tip to a second container at a second height.

[0255] In some embodiments, the immunoassay analyzer or instrument includes an image capture device or camera unit that can be used to provide feedback about the performance of the analyzer or instrument in real time. Non-limiting immunoassay analyzers or instruments including image capture devices or camera units are described in U.S. Patent No. 11,263,433. For example, an immunoassay analyzer or instrument may include a container holder device configured to support one or more containers, a sample pipetting device configured to dispense fluid material in at least one container on the container holder device, an image capture device configured to capture an image of at least one container on the container holder device, and at least one processing device, wherein the system is configured to: use the sample pipetting device to dispense at least one fluid material into the container; use the image capture device to capture the image of the container on the container holder device; use at least one processing device to analyze the image of the container to determine the volume of at least one fluid material dispensed in the container; and use at least one processing device to analyze the image of the container to determine the particle concentration of the target particle based on the determined volume of the fluid material in the container. An immunoassay analyzer or instrument may be capable of evaluating a fluid substance in a container using a method comprising: dispensing at least one fluid substance into a container using a sample pipetting device; capturing an image of at least a portion of a container disposed on a container holder device configured to support one or more containers using an image capture device; analyzing the image of the container using at least one computing device to determine a volume of at least one dispensed fluid substance in the container; and analyzing the image of the container using at least one computing device to determine a particle concentration of a particle of interest based on the determined volume of at least one dispensed fluid substance in the container.

[0256] In some embodiments, the immunoassay instrument or analyzer or one or more features thereof (e.g., hardware and / or software features) are generally described in international patent application PCT / US2021 / 0=65852 (WO2022 / 147370).

[0257] In some specific embodiments, the method includes a mechanical device suitable for degassing a fluid, such as a sample containing a fluid, a fluid assay reagent, and / or a mixture containing a sample and one or more assay reagents or diluents. For example, the method and / or mechanical device for degassing a fluid may include such a dispenser, which is suitable for containing a heater and one or more tubes composed of a material connected to the system fluid. One or more tubes may include a first end operably connected to one or more fluid / liquid sources, and a second end. One or more tubes may be connected to a heater or heating element (for example, via a conductive path that thermally connects the heater to one or more tubes. The material comprising one or more tubes may be permeable so that a portion of the gas dissolved in the fluid or liquid passes through the material to an external area after being degassed from a portion of the liquid in one or more tubes. For such embodiments including a degassing element or feature, factors known in the art may be used and / or changed to control the amount of degassing (for example, the pressure and temperature difference of the fluid in one or more tubes of the distribution system. In some embodiments, the pressure difference between the fluid and the ambient atmospheric pressure may be constant. In some embodiments , the actual gauge pressure difference measured from the inside of the distribution system to the outside of the distribution system (i.e., ambient atmospheric conditions) can be measured, and the actual gauge pressure difference is not equal to zero. In some embodiments, the amount of gas dissolved in the liquid is proportional to the concentration of the gas and the pressure (and other factors) at which it is located. In some embodiments, heating the fluid (e.g., liquid sample or liquid assay reagent) in the degasser allows the fluid to expand and helps to increase the pressure between the pump inlet valve, pump, degasser tube or loop and the distributor valve. In such embodiments, the pressure increase can affect the ability of the heater to degas the liquid, which can affect the relative light units (RLU) produced by the liquid (e.g., a mixture of sample and assay reagent).

[0258] In some embodiments, immunoassay analyzer or instrument comprises the structure that fluid is heated before adding to reaction vessel.This structure may comprise distributor.Fluid may comprise washing fluid or substrate composition.Washing fluid may be added to the particle that is combined with antibody or antigen.Washing fluid may be added to particle (for example, paramagnetic particles may be gathered by being subjected to magnetic field) before and / or after particle aggregation.Substrate composition is usually added to particle after particle is washed.Any suitable structure may be used to heat fluid.Exemplary distributor for heating fluid is described in for example U.S. Patent No. 10,562,021.In some respects, the structure of heating fluid may comprise distributor, and this distributor comprises fluid path, heat source that is thermally connected with fluid path, and comprises inlet and outlet and holds probe of fluid.In some embodiments, distributor preferably distributes fluid at desired fluid temperature, so that desired fluid temperature or desired fluid temperature range is maintained under distribution rate, frequency and distribution volume, and distribution fluid will not be withdrawn and removed.

[0259] In some embodiments, the immunoassay analyzer or instrument comprises a photometer as described in WO2019060375, WO2020219869, and / or U.S. Patent No. 11,604,146. For example, the immunoassay analyzer or instrument may comprise: a reaction vessel chamber; an optical channel intersecting the reaction vessel chamber; a cover that, when in a closed configuration, forms a dark chamber by blocking light emitted by an external light source from entering the reaction vessel chamber and entering the optical channel, and when in an open configuration, provides an entrance to the reaction vessel chamber; and a light detector optically coupled to the optical channel, the light detector comprising a sensing element for receiving light from the optical channel. In addition or alternatively, the immunoassay analyzer or instrument may include a photodetector configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an analog signal based on the photons emitted from the assay reaction over a period of time; a counter circuit configured to provide a photon count based on the photons emitted from the assay reaction over a period of time; and a photometer controller configured to: in response to an analog signal value of the analog signal greater than a predetermined value, determine and report a measurement of the photons emitted from the assay reaction over a period of time based on the analog signal value of the analog signal and a linear function, wherein the linear function is derived from the relationship established between the analog signal and the photon count. In addition or alternatively, the immunoassay analyzer or instrument may include a photometer system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on the photons emitted from the assay over a period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube into a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between an end of the current sensing resistor and an end of the amplifier; a counter circuit configured to provide an assay photon count based on the photons emitted from the assay over a period of time; and a photometer controller configured to: in response to the assay analog voltage being greater than a predetermined value, calculate a relative light unit value of the photons emitted from the assay over a period of time based on the assay analog voltage and an optimized linear function.

[0260] The following examples are provided solely in an effort to more fully describe and illustrate some aspects and embodiments described herein.It should be understood that the specific examples, materials, amounts, and operations should be interpreted broadly in accordance with the scope and spirit of the specification as set forth herein.

[0261] Illustrative Embodiments

[0262] All reagents, starting materials, and solvents used in the examples were purchased from commercial suppliers (eg, Sigma Aldrich, St. Louis, MO) and used without further purification unless otherwise noted.

[0263] Example 1 - Assay Migration Studies on the Beckman Coulter DxI 9000 Access Immunoassay Analyzer

[0264] The U.S. FDA's guidance for industry and staff titled "Assay Migration Studies for In Vitro Diagnostic Devices" provides the least burdensome approach to transferring previously approved assays from existing systems to new systems. (See Guidance for Industry and Staff: Assay Migration Studies for In Vitro Diagnostic Devices, issued in 2013, available on the FDA website). This approach enables the use of rigorous analytical performance data instead of complete clinical data to implement licensed products on new platforms. The Beckman Coulter DxI 9000 Access immunoassay analyzer includes many updates and new features designed to improve laboratory workflows and provide quality results to support patient management. Such elements include improved pipetting capabilities, updated process monitoring, increased throughput, reliability enhancements, and software features focused on operator needs. The analyzer also utilizes a new alkaline phosphatase substrate reagent that provides reduced time-to-result per test, as well as other benefits including improved signal-to-noise ratio and reduced sensitivity to endogenous alkaline phosphatase interference. The existing menu of BeckmanCoulter Access reagents is being transferred to the system.

[0265] The data presented here summarize results from analytical studies described in the assay migration guideline and obtained during validation testing of assays for high sensitivity cardiactroponin I (hsTnI) and alpha-fetoprotein (AFP) on the DxI 9000 Access immunoassay analyzer. Quantitative assay analytical studies were conducted according to the assay migration guideline to compare the performance of the Access hsTnI and Access AFP assays on the existing Access 2 and new DxI 9000 immunoassay analyzer systems.

[0266] method

[0267] Reproducibility

[0268] A multisite study was conducted on DxI 9000 and Access 2 immunoassay analyzers to evaluate the reproducibility of the Access hsTnI and Access AFP assays using a protocol based on CLSI EP05-A3 (See, CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition. CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.).

[0269] Reproducibility was evaluated on three DxI 9000 and three Access 2 immunoassay analyzers in three external clinical laboratories. hsTnI was measured for both serum and lithium heparin plasma samples across the assay range, while AFP was measured for serum samples. Samples were tested on each of the three immunoassay analyzers in the three test sites, running twice a day with three replicates per run for five days. Reproducibility was evaluated using three different reagent pack batches and one calibrator batch. QC was tested every day.

[0270] Comparative Study

[0271] For both serum and plasma sample types, a method comparison study comparing the Access hsTnI assay on the DxI 9000 immunoassay analyzer with the Access hsTnI assay on the Access 2 immunoassay analyzer was completed. For serum, a method comparison study comparing the Access AFP assay on the DxI 9000 immunoassay analyzer with the Access AFP assay on the Access 2 immunoassay analyzer was completed. Each study used a protocol based on CLSI EP09C-ED3 and assay migration guidelines. (See, CLSI. Measurement Procedure Comparison and Bias Estimation Using Patient Samples- Third Edition. CLSI Document EP09c, 3rd Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2018; and FDA Migration Guidelines cited above) A method comparison study was performed on three DxI9000 immunoassay analyzers and three Access 2 immunoassay analyzers in three external laboratories.

[0272] >240 serum samples and >180 lithium heparin plasma samples containing hsTnI concentrations across the analytical measurement range of the assay were tested. >200 serum samples containing AFP concentrations across the analytical measurement range of the assay were tested. All samples were tested in duplicate in three reagent lots at three external testing sites. Each reagent lot was tested on a different DxI 9000 / Access2 immunoassay analyzer pair at different sites. QC was tested daily.

[0273] The above-mentioned Assay Migration Guidance document provides instructions for calculating the "Total Allowable Variation" or ATD region within which 95% of the individual sample variations are expected to fall (see Figure 1 ).

[0274] Imprecision

[0275] A study was conducted using a CLSI EP05-A3 based protocol (See, CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline—3rd Edition. CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.) to evaluate the imprecision of the Access hsTnI assay and the Access AFP assay on the DxI 9000 immunoassay analyzer.

[0276] Each study was performed on three DxI 9000 immunoassay analyzers, with three reagent batches and three calibrator batches. hsTnI was measured for both serum and lithium heparin plasma samples across the assay range, while serum samples were tested for AFP. Each sample was tested with two replicates per run for hsTnI and three replicates per run for AFP. For each immunoassay analyzer and reagent batch combination, two runs per day were completed over 20 days. For each assay, three commercial quality controls were run in duplicate each day to verify that the system was in control.

[0277] Detection Capabilities

[0278] A study was conducted using a CLSI EP17-A2-based protocol (See, CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition. CLSI document EP17-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.) to determine the limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) for the Access hsTnI assay and the Access AFP assay.

[0279] To estimate the LoB, three DxI 9000 immunoassay analyzers with three reagent batches and one calibrator batch were used in the study design. Four S0 calibrator preparations for each corresponding assay were used for LoB determination. For each packaging batch, blank samples were tested in one run per day over three days with five replicates per run.

[0280] To estimate LoD and LoQ, three DxI 9000 immunoassay analyzers were used in the study design with three reagent batches and one calibrator batch. hsTnI was measured for both serum and lithium heparin plasma samples containing low levels of analytes, while AFP was measured for serum. For each packaging batch and immunoassay analyzer, nine replicates were run per run to test the samples, with one run per day and a total of five days. This design resulted in a minimum of 40 replicates per sample on each packaging batch tested.

[0281] Three quality controls were run in duplicate for each assay each day to verify that the system was in control.

[0282] In some cases, the analyte detected by the immunoassay comprises AFP. The LoB of the AFP immunoassay can be at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, or 0.14 ng / mL and / or at most 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.25, or 0.3 ng / mL. The LoB of the AFT immunoassay can be in the range of about 0.05 to about 0.3, about 0.06 to about 0.25, about 0.07 to about 0.2, or about 0.08 to about 0.15 ng / mL. The LoD of the AFP immunoassay can be at least 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, or 0.22 ng / mL, and / or at most 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.28, or 0.3 ng / mL. The LoD of the AFT immunoassay can be in the range of about 0.1 to about 0.3, about 0.12 to about 0.28, about 0.14 to about 0.26, about 0.16 to about 0.24, about 0.18 to about 0.24, about 0.2 to about 0.24, or about 0.22 to about 0.24 ng / mL. The LoQ of the AFP immunoassay may be at least 0.08, 0.09, 1.1, 0.11, 0.12, or 0.13 ng / mL, and / or at most 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.24, 0.26, 0.28, or 0.3 ng / mL. The LoQ of the AFP immunoassay may be in the range of about 0.08 to about 0.3, about 0.1 to about 0.25, about 0.12 to about 0.2, or about 0.14 to about 0.18 ng / mL. In some cases, the LoB of the AFP immunoassay is in the range of about 0.05 to about 0.3, about 0.06 to about 0.25, about 0.07 to about 0.2, or about 0.08 to about 0.15 ng / mL, the LoD is in the range of about 0.1 to about 0.3, about 0.12 to about 0.28, about 0.14 to about 0.26, about 0.16 to about 0.24, about 0.18 to about 0.24, about 0.2 to about 0.24, or about 0.22 to about 0.24 ng / mL, and the LoQ is in the range of about 0.08 to about 0.3, about 0.1 to about 0.25, about 0.12 to about 0.2, or about 0.14 to about 0.18 ng / mL.

[0283] In some cases, the analyte detected by the immunoassay comprises TSH. The LoB of the TSH immunoassay can be at least 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.0011, 0.0012, 0.0013, 0.0014, or 0.0015 uIU / mL, and / or at most 0.0016, 0.0017, 0.0018, 0.0019, 0.002, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, or 0.003 uIU / mL. The LoB of the TSH immunoassay may be in the range of about 0.0005 to about 0.003, about 0.0006 to about 0.0025, about 0.0008 to about 0.0022, about 0.001 to about 0.002, about 0.0012 to about 0.0018, or 0.0014 to about 0.0016 uIU / mL. The LoD of the TSH immunoassay may be at least 0.001, 0.0012, 0.0014, 0.0016, 0.0018, 0.002, 0.0022, or 0.0024 uIU / mL, and / or at most 0.0026, 0.0028, 0.003, 0.0032, 0.0034, 0.0036, 0.0038, or 0.004 uIU / mL. The LoD of the TSH immunoassay may be in the range of about 0.001 to about 0.004, about 0.0015 to about 0.0035, about 0.002 to about 0.003, or about 0.0022 to about 0.0028 uIU / mL. The LoQ of the TSH immunoassay may be at least 0.0003, 0.0004, 0.0005, 0.0006, or 0.0007 uIU / mL, and / or at most 0.0008, 0.0009, 0.001, 0.0011, 0.0012, 0.0013, 0.0014, or 0.0015 uIU / mL. The LoQ of the TSH immunoassay may be in the range of about 0.0003 to about 0.0015, about 0.0005 to about 0.0012, or about 0.0008 to about 0.001. In some instances, the LoB of the TSH immunoassay is in the range of about 0.0005 to about 0.003, about 0.0006 to about 0.0025, about 0.0008 to about 0.0022, about 0.001 to about 0.002, about 0.0012 to about 0.0018, or 0.0014 to about 0.0016 uIU / mL, the LoD is in the range of about 0.001 to about 0.004, about 0.0015 to about 0.0035, about 0.002 to about 0.003, or about 0.0022 to about 0.0028 uIU / mL, and the LoQ is in the range of about 0.0003 to about 0.0015, about 0.0005 to about 0.0012, or about 0.0008 to about 0.001.

[0284] In some cases, the analyte detected by the immunoassay comprises hCG. The LoB of the hCG immunoassay can be at least 0.0, 0.0005, 0.001, 0.01, or 0.05 mIU / mL, and / or at most 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, or 0.2 mIU / mL. The LoB of the hCG immunoassay can be in the range of about 0.0 to about 0.2, about 0.0005 to about 0.15, about 0.001 to about 0.15, about 0.01 to about 0.1, or about 0.05 to about 0.1 mIU / mL. The LoD of the hCG immunoassay may be at least 0.001, 0.005, 0.01, or 0.05 mIU / mL, and / or at most 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.25, or 0.3 mIU / mL. The LoD of the hCG immunoassay may be in the range of about 0.001 to about 0.3, about 0.005 to about 0.3, about 0.01 to about 0.25, about 0.1 to about 0.25, or about 0.15 to about 0.2 mIU / mL. The LoQ of the hCG immunoassay may be at least 0.001, 0.005, 0.01, 0.05, or 0.1 mIU / mL, and / or at most 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, or 0.3 mIU / mL. The LoQ of the hCG immunoassay may be in the range of about 0.001 to about 0.3, about 0.01 to about 0.25, about 0.05 to about 0.25, 0.1 to about 0.2, or 0.12 to about 0.18 mIU / mL. In some instances, the LoB of the hCG immunoassay is in the range of about 0.0 to about 0.2, about 0.0005 to about 0.15, about 0.001 to about 0.15, about 0.01 to about 0.1, or about 0.05 to about 0.1 mIU / mL, the LoD is in the range of about 0.001 to about 0.3, about 0.005 to about 0.3, about 0.01 to about 0.25, about 0.1 to about 0.25, or about 0.15 to about 0.2 mIU / mL, and the LoQ is in the range of about 0.001 to about 0.3, about 0.01 to about 0.25, about 0.05 to about 0.25, 0.1 to about 0.2, or 0.12 to about 0.18 mIU / mL.

[0285] In some cases, the analyte detected by the immunoassay comprises free T4. The LoB of the free T4 immunoassay can be at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.12, or 0.14 ng / mL, and / or at most 0.16, 0.18, 0.2, 0.22, or 0.24 ng / mL. The LoB of the free T4 immunoassay can be in the range of about 0.001 to about 0.3, about 0.01 to about 0.25, about 0.1 to about 0.2, or about 0.15 to about 0.2 ng / mL.

[0286] Linear

[0287] A study was conducted based on CLSI EP06-Ed2 (see, CLSI. Evaluation of Linearity of Quantitative Measurement Procedures. CLSI document EP06-Ed2. Wayne, PA: Clinical and Laboratory Standards Institute; 2020.) to evaluate the linearity of the Access hsTnI assay and the Access AFP assay on the DxI9000 immunoassay analyzer.

[0288] The samples covering the whole analytical measurement range of each determination are used for linear determination. The hsTnI of serum and lithium heparin plasma sample types are evaluated independently, and the AFP in serum is detected simultaneously. The natural samples with the low end value concentration of the measurement interval are obtained. The high sample is prepared by adding the antigen to the low sample until the concentration higher than the highest commercial calibrator is reached. In addition to the high concentration and low concentration samples, seven mixtures are tested in this study. These samples are independently prepared by using the high sample with the ratio of dilution with the low sample to reach the concentration covering the determination range. The low sample is run with eight replicates, and all other samples are run with four replicates. This study is run on a DxI 9000 immunoassay analyzer, using three reagent batches and one calibrator batch. Three quality controls are run with two replicates every day for each determination to verify that the system is controlled.

[0289] result

[0290] The results for Access AFP (alpha-fetoprotein) are Figures 2 to 6 and described in its description.

[0291] Results for Access hsTnI (cardiac troponin) Figures 7 to 11 and described in its description.

[0292] in conclusion

[0293] Individual assay data for the Access hsTnI and Access AFP assays generated on the DxI 9000Access immunoassay analyzer, including accuracy, imprecision, detectability, and linearity, met U.S. FDA assay migration guidance study design criteria and demonstrated acceptable assay performance.

[0294] Example 2 - Determination of performance using improved compositions

[0295] Alkaline phosphatase (ALP) was coated onto paramagnetic particles (PMPs) and subsequently stored at 37°C for 0, 1 and 3 days in various diluents (pH 6, 7 or 8). It has been previously shown that incubating ALP coated on PMPs for 3 days at 37°C achieved almost the same results as incubating ALP coated on PMPs for 6 months at 4°C.

[0296] The activity of the ALP-coated particles was then assessed on a DxI 800 immunoassay analyzer (Beckman Coulter, Inc., Brea, CA). The ALP-coated particles were loaded into a reagent pack and then loaded onto the DxI 800 immunoassay analyzer. The system washes the particles to remove storage solution and then assesses activity using a LUMI-PHOS PRO (Lumigen, Inc., Southfield, MI).

[0297] The results after 3 days at 37°C are shown in Figure 14. As shown in Figure 14, while a wide range of magnesium and zinc concentrations allowed ALP stability at pH 8 at 37°C for more than 3 days, a much narrower range of magnesium and zinc concentrations allowed ALP stability at pH 7 at 37°C for more than 3 days, and a very narrow range of magnesium and zinc concentrations allowed optimized ALP stability and activity at pH 6 at 37°C for more than 3 days.

[0298] Table 1 details the estimated apparent dissociation constants between metal species and the M3 site of bovine intestinal ALP as a function of pH.

[0299] Table 1.

[0300] pH <![CDATA[K 表观(Mg) ]]> <![CDATA[K 表观(Zn) ]]> 6 10mM 100uM 7 2mM 100uM 8 <<2mM >100uM

[0301] Example 3 - Targeted Antigen Assay Using Improved Compositions

[0302] This example describes the optimization of free magnesium and zinc ion concentrations in compositions where ALP is conjugated to an antibody or antigen for a particular immunoassay. The optimal ALP activity (as measured by relative light units (RLU)) is balanced with other features that affect assay performance, including antibody conjugate or antigen conjugate stability, pH, buffer, albumin (e.g., BSA) concentration, and dose recovery, to determine the final amounts of magnesium and zinc selected.

[0303] Example 3A-AFP

[0304] This example describes an immunoassay for detecting alpha-fetoprotein (AFP) in a sample. This example also describes an exemplary composition for use in the immunoassay, comprising a mouse monoclonal anti-AFP antibody conjugated to ALP.

[0305] The assay for detecting AFP is a two-site immunoenzymatic ("sandwich") assay. The sample is added to a reaction vessel with a mouse monoclonal anti-AFP-alkaline phosphatase conjugate and paramagnetic particles coated with a second mouse monoclonal anti-AFP antibody. The AFP in the sample binds to the immobilized monoclonal anti-AFP on the solid phase, while at the same time, the monoclonal anti-AFP-alkaline phosphatase conjugate reacts with a different antigenic site on the sample AFP.

[0306] Using a disposable pipette tip, 10 μL of serum sample (or alternatively, amniotic fluid sample) containing a range of AFP concentrations is loaded onto the DxI 9000 Immunoassay Analyzer. The Immunoassay Analyzer aliquots the diluted sample into individual reaction vessels (RVs).

[0307] To perform the immunoenzymatic assay, the immunoassay analyzer dispenses 52 μg of paramagnetic microparticles (PMPs) coated with anti-AFP antibodies to each RV. The immunoassay analyzer also dispenses anti-AFP antibodies conjugated to ALP to each RV. The contents of the RV tubes are mixed and incubated at 37°C for about 5 minutes. After incubation in the RV, the substances bound to the PMP are kept in the magnetic field, while the unbound substances are washed away. Then, 200 μL of LUMI-PHOS PRO is added to each RV tube. The light generated by the reaction is measured with a photometer. Light generation is proportional to the concentration of AFP in the sample. The amount of analyte in the sample is determined based on the stored multi-point calibration curve.

[0308] The pH of the composition containing mouse monoclonal anti-AFP antibody conjugated to ALP was 6. The composition was optimized to contain 15 mM MgCl2 and 100 μM ZnCl2, resulting in an estimated free Mg 2+ The concentration was 15 mM and the estimated free Zn 2+ The concentration of free [Mg 2+] / free [Zn 2+ ] was 176. Without wishing to be bound by theory, it is believed that additional ZnCl2 is required to achieve free Zn in the desired range. 2+ (See Example 1) because the composition comprising mouse monoclonal anti-AFP antibody conjugated to ALP contains albumin complexed with zinc ions. 2+ The concentrations are based on a 1:1 binding between BSA and Zn and do not include the weaker interaction with PO4.

[0309] Example 3B - βhCG

[0310] This example describes an immunoassay for detecting beta human chorionic gonadotropin (βhCG) in a sample. This example also describes an exemplary composition for use in the immunoassay, comprising a rabbit anti-βhCG antibody conjugated to ALP.

[0311] The assay for detecting βhCG is a two-site immunoenzymatic ("sandwich") assay. The sample is added to a reaction vessel along with a citrate buffer. After an initial incubation, a rabbit anti-βhCG alkaline phosphatase conjugate and paramagnetic particles coated with a goat anti-mouse IgG:mouse monoclonal anti-βhCG complex are added. hCG binds to the immobilized monoclonal anti-βhCG on the solid phase, while at the same time, the rabbit anti-βhCG alkaline phosphatase conjugate reacts with a different antigenic site on hCG.

[0312] 13 μL of serum sample (or alternatively, plasma sample) containing a range of βhCG concentrations is loaded onto the DxI 9000 immunoassay analyzer using a disposable pipette tip. The immunoassay analyzer aliquots the sample into individual reaction vessels (RVs).

[0313] To perform the immunoenzymatic assay, the immunoassay analyzer dispenses 50 μg of paramagnetic microparticles (PMPs) coated with anti-βhCG antibodies to each RV. The immunoassay analyzer also dispenses βhCG antibodies conjugated to ALP to each tube. The contents of the RV tubes are mixed and incubated at 37°C for approximately 6 minutes. After incubation in the RV, substances bound to the PMP are maintained in a magnetic field, while unbound substances are washed away. Then, 200 μL of LUMI-PHOS PRO is added to each RV tube. The light produced by the reaction is measured with a photometer. Light production is proportional to the concentration of total βhCG in the sample. The amount of analyte in the sample is determined based on a stored multi-point calibration curve.

[0314] The pH of the composition containing rabbit anti-βhCG antibody conjugated to ALP was 6. The composition was optimized to contain 32 mM MgCl2 and 50 μM ZnCl2, resulting in an estimated free Mg 2+ The concentration was 32 mM and the estimated free Zn2+ The concentration of free [Mg 2+ ] / free [Zn 2+ ] was 914. Without wishing to be bound by theory, it is believed that additional ZnCl2 is required because the composition comprising rabbit anti-βhCG antibody conjugated to ALP comprises bovine serum albumin (BSA) complexed with zinc ions. The estimated free Zn 2+ The concentration is based on the binding between albumin and Zn and does not include the weaker / more complex interaction with ACES.

[0315] Example 3C - Free T4

[0316] This example describes an immunoassay for detecting thyroxine (T4). This example also describes an exemplary composition for use in an immunoassay comprising a triiodothyronine (T3) antigen conjugated to ALP.

[0317] The assay for detecting (free) T4 is a two-step enzymatic immunoassay. A monoclonal anti-thyroxine (T4) antibody coupled to biotin, the sample, a buffered protein solution, and a streptavidin-coated solid phase are added to the reaction vessel. During this first incubation period, the anti-T4 antibody coupled to biotin binds to the solid phase and free T4 in the sample. After incubation in the reaction vessel, the material bound to the solid phase is maintained in a magnetic field, while the unbound material is washed away. Next, a buffered protein solution and a triiodothyronine (T3)-ALP conjugate are added to the reaction vessel. The T3-ALP conjugate binds to the unoccupied anti-T4 antibody binding sites.

[0318] 30 μL of serum sample (or alternatively, plasma sample) containing a range of concentrations of (free) T4 is loaded onto the DxI 9000 immunoassay analyzer using a disposable pipette tip. The immunoassay analyzer aliquots the sample into individual reaction vessels (RVs).

[0319] To perform the immunoassay, the immunoassay analyzer dispenses anti-T4 antibody coupled to biotin, 18 μg of paramagnetic microparticles (PMP) coated with streptavidin, and triiodothyronine (T3) conjugated to ALP to each RV. The contents of the RV tubes are mixed and incubated at 37°C for approximately 13 minutes. After incubation in the RV, the substances bound to the PMP are maintained in the magnetic field, while the unbound substances are washed away. Then, 200 μL of LUMI-PHOS PRO is added to each RV tube. The light generated by the reaction is measured with a photometer. Light generation is inversely proportional to the concentration of free T4 in the sample. The amount of analyte in the sample is determined based on a stored multi-point calibration curve.

[0320] The pH of the composition containing T3 antigen conjugated to ALP was 6. The composition was optimized to contain 2 mM MgCl2 and 10 μM ZnCl2, resulting in an estimated free Mg 2+ The concentration was 2 mM and the estimated free Zn 2+ The concentration of free [Mg 2+ ] / free [Zn 2+ ] is 200. Without wishing to be bound by theory, it is believed that the additional ZnCl2 is required because the composition comprising T3 antigen conjugated to ALP comprises ovalbumin complexed with zinc ions.

[0321] Example 3D-cTnI

[0322] This example describes an immunoassay for detecting cardiac troponin 1 (cTnI). This example also describes an exemplary composition for use in an immunoassay comprising a monoclonal anti-cTnI antibody conjugated to ALP.

[0323] The assay for detecting cTnI is a two-site immunoenzymatic ("sandwich") assay. A monoclonal anti-cTnI antibody conjugated to alkaline phosphatase is added to a reaction vessel along with a buffer containing a surfactant and the sample. After a short incubation, paramagnetic particles coated with the monoclonal anti-cTnI antibody are added. Human cTnI binds to the anti-cTnI antibody on the solid phase, while the anti-cTnI antibody-ALP conjugate reacts with a different antigenic site on the cTnI molecule.

[0324] 50 μL of serum sample (or alternatively, plasma sample) containing a range of cTnI concentrations was loaded onto the DxI 9000 immunoassay analyzer using a disposable pipette tip. The immunoassay analyzer aliquoted the sample into individual reaction vessels (RVs).

[0325] To perform the immunoenzymatic assay, the immunoassay analyzer dispenses 75 μg of a solution of paramagnetic microparticles (PMPs) coated with anti-cTnI antibodies to each RV. The immunoassay analyzer also dispenses anti-cTnI antibodies conjugated to ALP to each tube. The contents of the RV tubes are mixed and incubated at 37°C for approximately 5 minutes. After incubation in the RV, the substances bound to the PMP are maintained in a magnetic field, while the unbound substances are washed away. Then, 200 μL of LUMI-PHOS PRO is added to each RV tube. The light generated by the reaction is measured with a photometer. Light generation is proportional to the concentration of cTnI in the sample. The amount of analyte in the sample is determined based on a stored multi-point calibration curve.

[0326] The pH of the composition comprising the monoclonal anti-cTnI antibody conjugated to ALP was 6. The composition was optimized to contain 35 mM MgCl2 and 250 μM ZnCl2, resulting in an estimated free Mg2+ The concentration was 35 mM and the estimated free Zn 2+ The concentration of is 175 μM. Without wishing to be bound by theory, it is believed that the additional ZnCl2 is required because the composition comprising a monoclonal anti-cTnI antibody conjugated to ALP comprises bovine serum albumin (BSA) and ACES buffer, each of which complexes with zinc ions.

[0327] Free [Mg 2+ ] / free [Zn 2+ ] is 200. Fig.15 As shown in , the selected Mg / Zn ratio was not optimized for RLU loss alone, but still provided acceptable performance.

[0328] Example 3 E-TSH

[0329] This example describes an immunoassay for detecting human thyroid stimulating hormone (hTSH) in a sample. This example also describes an exemplary composition for use in an immunoassay comprising a mouse anti-hTSH antibody conjugated to ALP.

[0330] The assay for detecting hTSH is a two-site immunoenzymatic ("sandwich") assay. The sample is added to a reaction vessel with mouse anti-hTSH-alkaline phosphatase conjugate, a buffered protein solution, and paramagnetic particles coated with immobilized mouse monoclonal anti-hTSH antibodies. hTSH binds to the immobilized monoclonal anti-hTSH antibody on the solid phase, while the mouse anti-hTSH-alkaline phosphatase conjugate reacts with different antigenic sites on hTSH.

[0331] 25 μL of serum sample (or alternatively, plasma sample) containing a range of hTSH concentrations is loaded onto the DxI 9000 immunoassay analyzer using a disposable pipette tip. The immunoassay analyzer aliquots the sample into individual reaction vessels (RVs).

[0332] The immunoassay analyzer dispenses 13 μg of paramagnetic microparticles (PMPs) coated with anti-hTSH antibodies to each tube. The immunoassay analyzer also dispenses hTSH antibodies conjugated to ALP to each tube. The contents of the RV tubes are mixed and incubated at 37°C for approximately 14 minutes. After incubation in the RV, the substances bound to the PMPs are maintained in a magnetic field, while the unbound substances are washed away. Then, 200 μL of LUMI-PHOS PRO is added to each RV tube. The light generated by the reaction is measured with a photometer. Light generation is proportional to the concentration of hTSH in the sample. The amount of analyte in the sample is determined based on a stored multi-point calibration curve.

[0333] The pH of the composition containing mouse anti-hTSH antibody conjugated to ALP was 6. The composition was optimized to contain 8 mM MgCl2 and 25 μM ZnCl2, resulting in an estimated free Mg 2+ The concentration was 8 mM and the estimated free Zn 2+ The concentration of ZnCl2 was 10 μM. Without wishing to be bound by theory, it is believed that additional ZnCl2 is required because the composition comprising mouse anti-hTSH antibody conjugated to ALP comprises bovine serum albumin (BSA) and ACES buffer, each of which complexes with zinc ions. The estimated free Zn 2+ The concentrations are based on a 1:1 binding between BSA and Zn and do not include the weaker / more complex interaction with ACES. Free [Mg 2+ ] / free [Zn 2+ ]The ratio is 800.

[0334] Example 4 - Dose Deviation Determination

[0335] To determine whether ALP activity is maintained before ALP is added to the substrate, a dose deviation assay was performed. The RLU response activity of LUMI-PHOS 530 (Lumigen, Southfield, MI) and LUMI-PHOS PRO (Lumigen, Southfield, MI) was measured for known concentrations of analytes using an immunoassay reagent composition that contained ALP but did not formulate the Mg / Zn ratio to optimize ALP activity under all conditions. The immunoassay reagent composition was then stored at 4°C for 6 months (from April to October). The RLU response activity of LUMI-PHOS 530 and LUMI-PHOS PRO was used again to detect the analyte.

[0336] like Fig.13 As shown in , little or no dosing deviation was observed in assays containing LUMI-PHOS 530. In assays containing LUMI-PHOS PRO, dosing deviation was observed due to loss of ALP activity of the enzyme in the immunoassay reagent composition during storage.

[0337] Exemplary Aspects

[0338] Some exemplary aspects relate to methods comprising:

[0339] measuring a serum concentration or a plasma concentration of a target analyte in a biological sample;

[0340] wherein the measuring comprises detecting the target analyte using an antibody against the target analyte or an antigen associated with the target analyte; and

[0341] The method further comprises measuring the concentration of the target analyte using a substrate formulation comprising a chemiluminescent compound.

[0342] Some exemplary aspects relate to methods comprising any one or more of the following A1 to A12 and / or B1 to B10:

[0343] A1. Includes the following methods:

[0344] measuring serum or plasma concentrations of cardiac troponin I (cTnI) in blood samples;

[0345] wherein the method comprises detecting cTnI using an anti-cTnI antibody, and

[0346] The method further comprises measuring the concentration of cTnI using a substrate preparation comprising a chemiluminescent compound.

[0347] A2. The method of A1, wherein the method comprises measuring cTnI in an apparatus comprising a distribution control system, an image capture device or a camera unit, or a combination thereof.

[0348] A3. The method of A1 or A2, wherein the method further comprises comparing the cTnI concentration with a calibration curve.

[0349] A4. The method of any one of A1 to A3, wherein the chemiluminescent compound comprises Formula I or a salt thereof:

[0350]

[0351] in

[0352] A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent;

[0353] R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups;

[0354] R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring;

[0355] R 15 It is C 1-6 alkyl;

[0356] Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts;

[0357] Z is O or S; and

[0358] n is 0, 1, or 2;

[0359] A5. The method of any one of A1 to A4, wherein the substrate preparation further comprises:

[0360] Cationic aromatic compounds (CAC);

[0361] Background reducing agent; or

[0362] an ether-linked nonionic surfactant not comprising a carboxylate group; or a hydrophilic polymer; or

[0363] Its combination.

[0364] A6. The method of any one of A1 to A5, wherein the substrate preparation comprises

[0365] a) 0.01 mM to 50 mM of a chemiluminescent compound of formula I or a salt thereof;

[0366] b) 0.01 to 200 μM of a cationic aromatic compound (CAC);

[0367] c) 1 μM to 10 mM background reducing agent; and

[0368] d) 0.05 to 20 g / L of an ether-linked nonionic surfactant not containing a carboxylate group; or a hydrophilic polymer.

[0369] A7. The method of any one of A1 to A6, wherein the measurement of cTnI exhibits a coefficient of variation of less than 4% for cTnI concentrations in plasma greater than 10 ng / mL.

[0370] A8. The method of any one of A1 to A7, wherein the measurement of cTnI exhibits a standard deviation (SD) of less than 0.5 ng / mL for cTnI concentrations in plasma of less than 10 ng / mL.

[0371] A9. The method of any one of A1 to A6, wherein the measurement of cTnI exhibits a coefficient of variation of less than 4% for cTnI concentrations in serum greater than 15 ng / mL.

[0372] A10. The method of any one of A1 to A6 or A9, wherein the measurement of cTnI exhibits a standard deviation (SD) of less than 0.5 ng / mL for cTnI concentrations in serum less than 15 ng / mL.

[0373] A11. A method as described in any of A7 to A10, wherein the reproducibility conditions of the measurements include at least between-site and between-batch variance components.

[0374] A12. A method as described in any of A7 to A10, wherein the reproducibility conditions of the measurement include variance components between sites, between batches, between days, between runs, and within runs.

[0375] B1. Methods including the following:

[0376] measuring the serum or plasma concentration of alpha-fetoprotein (AFP) in a blood sample;

[0377] wherein the method comprises detecting AFP using an anti-AFP antibody, and

[0378] The method further comprises measuring the concentration of AFP using a substrate preparation comprising a chemiluminescent compound.

[0379] B2. The method described in B1, wherein the method includes measuring AFP in a device comprising a distribution control system, an image capture device or camera unit, or a combination thereof.

[0380] B3. The method described in B1 or B2, wherein the method further comprises comparing the cTnI concentration with a calibration curve.

[0381] B4. The method of any one of B1 to B3, wherein the chemiluminescent compound comprises formula I or a salt thereof:

[0382]

[0383] in

[0384] A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent;

[0385] R1 is selected from C5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups;

[0386] R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring;

[0387] R 15 It is C 1-6 alkyl;

[0388] Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts;

[0389] Z is O or S; and

[0390] n is 0, 1, or 2;

[0391] B5. The method of any one of B1 to B4, wherein the substrate preparation further comprises:

[0392] Cationic aromatic compounds (CAC);

[0393] Background reducing agent; or

[0394] an ether-linked nonionic surfactant not comprising a carboxylate group; or a hydrophilic polymer; or

[0395] Its combination.

[0396] B6. The method of any one of B1 to B5, wherein the substrate preparation comprises

[0397] a) 0.01 mM to 50 mM of a chemiluminescent compound of formula I or a salt thereof;

[0398] b) 0.01 to 200 μM of a cationic aromatic compound (CAC);

[0399] c) 1 μM to 10 mM background reducing agent; and

[0400] d) 0.05 to 20 g / L of an ether-linked nonionic surfactant not containing a carboxylate group; or a hydrophilic polymer.

[0401] B7. The method described in any one of B1 to B6, wherein the measurement of AFP exhibits a coefficient of variation (CV) of less than 7% for AFP concentrations greater than 10 ng / mL.

[0402] B8. The method described in any one of B1 to B7, wherein the measurement of AFP exhibits a standard deviation (SD) of less than 0.5 ng / mL for AFP concentrations less than 10 ng / mL.

[0403] B9. A method as described in any of B7 or B8, wherein the reproducibility conditions of the measurement include at least between-site and between-batch variance components.

[0404] B10. A method as described in any of B7 or B8, wherein the reproducibility conditions of the measurement include variance components between locations, between batches, between days, between runs, and within runs.

[0405] Some exemplary aspects relate to the following compositions comprising any one or more of C1 to C20 and related methods comprising any one or more of C21 to C35:

[0406] C1. A composition comprising

[0407] an antibody conjugated to alkaline phosphatase (ALP) or an antigen conjugated to ALP;

[0408] Buffers;

[0409] Zinc salts; and

[0410] Magnesium salts;

[0411] in

[0412] When the pH of the composition is in the range of 5.5 to 6.4, free Zn 2+ The concentration of free Mg was in the range of 5 μM to 1 mM. 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration range is 3500 times.

[0413] C2. The composition described in C1, wherein the enzymatic activity of ALP changes by less than 25% of the peak activity when the composition is stored at 37°C for up to 3 days.

[0414] C3. The composition described in C1 or C2, wherein the enzymatic activity of ALP changes by less than 25% of the peak activity when the composition is stored at a temperature in the range of 2°C to 6°C for up to 6 months, up to 9 months or up to 1 year.

[0415] C4. A composition comprising

[0416] an antibody conjugated to alkaline phosphatase (ALP) or an antigen conjugated to ALP;

[0417] Buffers;

[0418] Zinc salts; and

[0419] Magnesium salts;

[0420] in

[0421] When the pH of the composition is in the range of 5.5 to 6.4, free Zn 2+ The concentration of free Mg was in the range of 5 μM to 1 mM. 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+ The concentration of 1% chlorine is within 3500 times of that of 1% chlorine;

[0422] When the pH of the composition is in the range of 6.5 to 7.4, free Zn 2+ The concentration of free Mg was in the range of 1 μM to 5 mM. 2+ The concentration of free Zn 2+ The concentration is 200 times higher than that of free Zn 2+ or

[0423] When the pH of the composition is in the range of 7.5 to 8.4, free Zn 2+ The concentration of Mg was in the range of 100 nM to 300 μM and free Mg 2+ The concentration of free Zn 2+ The concentration of free Zn is 100 times 2+ The concentration range is 1000 times.

[0424] C5. The composition described in C4, wherein the enzymatic activity of ALP changes by less than 10% of the peak activity when the composition is stored at 37°C for up to 3 days.

[0425] C6. The composition described in C4 or C5, wherein the enzymatic activity of ALP changes by less than 10% of the peak activity when the composition is stored at a temperature in the range of 2°C to 6°C for up to 6 months, up to 9 months or up to 1 year.

[0426] C7. A composition as described in any one of C1 to C6, wherein the ALP comprises bovine intestinal ALP.

[0427] C8. A composition as described in any one of C1 to C7, wherein the buffer comprises ACES, HEPES, MES, phosphate, TRIS, ADA, PIPES, MOPSO, BTP, BES or Bis-TRIS, or a combination thereof.

[0428] C9. A composition as described in any one of C1 to C8, wherein the zinc salt comprises ZnCl2.

[0429] C10. The composition of any one of C1 to C9, wherein the magnesium salt comprises MgCl2.

[0430] C11. The composition of any one of C1 to C10, wherein the composition further comprises albumin.

[0431] C12. The composition described in C11, wherein the albumin comprises bovine serum albumin, human serum albumin, casein, hydrolyzed casein, or ovalbumin.

[0432] C13. A composition described in any one of C1 to C12, wherein the antibody conjugated to ALP comprises an anti-troponin antibody, an anti-thyroid stimulating hormone (TSH) antibody, an anti-alpha-fetoprotein (AFP) antibody, an anti-β-human chorionic gonadotropin (βhCG) antibody, an anti-sex hormone binding globulin (SHBG) antibody, an anti-CA19-9 antibody, or an anti-creatine kinase-MB (CKMB) antibody.

[0433] C14. The composition described in C13, wherein

[0434] The antibody conjugated to ALP comprises an anti-troponin antibody;

[0435] The pH of the composition is in the range of 5.5 to 6.4; and

[0436] Free Zn 2+ The concentration of Mg was in the range of 100 μM to 500 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration range is 500 times.

[0437] C15. The composition described in C13, wherein

[0438] The antibody conjugated to ALP comprises an anti-TSH antibody;

[0439] The pH of the composition is in the range of 5.5 to 6.4; and

[0440] Free Zn 2+ The concentration of Mg was in the range of 5 μM to 100 μM and free Mg2+ The concentration of free Zn 2+ The concentration is 500 times higher than that of free Zn 2+ The concentration range is 1500 times.

[0441] C16. The composition described in C13, wherein

[0442] The antibody conjugated to ALP comprises an anti-AFP antibody;

[0443] The pH of the composition is in the range of 5.5 to 6.4; and

[0444] Free Zn 2+ The concentration of Mg was in the range of 50 μM to 150 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration range is 500 times.

[0445] C17. The composition described in C13, wherein

[0446] The antibody conjugated to ALP comprises an anti-βhCG antibody;

[0447] The pH of the composition is in the range of 5.5 to 6.4; and

[0448] Free Zn 2+ The concentration of Mg was in the range of 5 μM to 100 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+ The concentration range is 1500 times.

[0449] C18. The composition of any one of C1 to C18, wherein the antigen conjugated to ALP comprises a triiodothyronine (T3) antigen.

[0450] C19. The composition described in C18, wherein

[0451] The pH of the composition is in the range of 5.5 to 6.4; and

[0452] Free Zn 2+ The concentration of Mg was in the range of 5 μM to 100 μM, and free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ 500 times the concentration.

[0453] C20. The composition of any one of C1 to C19, wherein the concentration of free magnesium, free zinc, or a combination thereof is determined by removing larger molecules from a storage solution by size exclusion chromatography and then quantifying the free magnesium, free zinc, or a combination thereof by mass spectrometry.

[0454] C21. A method of using the composition of any one of C1 to C20 in an immunoassay.

[0455] C22. The method described in C21, wherein the immunoassay is performed on an immunoassay analyzer or instrument.

[0456] C23. The method described in C21 or C22, wherein the method comprises

[0457] contacting the analyte with the composition of any one of C1 to C20 in a vessel to form a mixture,

[0458] Incubate the mixture,

[0459] adding a chemiluminescent substrate to the vessel, and

[0460] After adding the chemiluminescent substrate to the vessel, light produced by the reaction is measured for up to 5 minutes, up to 4 minutes, up to 3 minutes, up to 2 minutes, up to 1 minute, up to 56 seconds, up to 55 seconds, up to 50 seconds, or up to 48 seconds.

[0461] C24. The method described in C23 also includes a washing step before adding the chemiluminescent substrate to the vessel.

[0462] C25. The method of C23 or C24, wherein the chemiluminescent substrate comprises formula I or a salt thereof:

[0463]

[0464] in

[0465] A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent;

[0466] R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups;

[0467] R7-R 14 Independently H, C 1-6 Alkoxy, halogen, or C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring;

[0468] R 15 It is C 1-6 alkyl;

[0469] Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts;

[0470] Z is O or S; and

[0471] n is 0, 1 or 2.

[0472] C26. The method described in C25, wherein the substrate preparation containing the chemiluminescent substrate further comprises:

[0473] Cationic aromatic compounds (CAC);

[0474] Background reducing agent; or

[0475] an ether-linked nonionic surfactant not comprising a carboxylate group; or a hydrophilic polymer; or

[0476] Its combination.

[0477] C27. The method of any one of C23 to C26, wherein the chemiluminescent substrate comprises APS-5.

[0478] C28. A method as described in any one of C23 to C27, wherein the method further comprises determining the amount of an analyte in the sample.

[0479] C29. The method described in C28, wherein the amount of the analyte is determined based on a stored multi-point calibration curve.

[0480] C30. The method of any one of C23 to C29, wherein the method comprises contacting a sample comprising the analyte with the composition, wherein the composition comprises an antibody that binds to ALP, and further wherein an ALP-antibody-analyte complex is formed.

[0481] C31. The method described in C30, wherein the method further comprises contacting the ALP-antibody-analyte complex with an anti-analyte antibody bound to a solid phase, and washing away sample that is not bound to the solid phase.

[0482] C32. The method described in C31, wherein the solid phase comprises paramagnetic particles.

[0483] C33. A method described in any one of C23 to C32, wherein the method comprises contacting a sample containing the analyte with an antibody specific for the analyte to form a complex containing the analyte, washing the complex, and contacting the complex with the composition, wherein the composition comprises an antigen that binds to ALP.

[0484] C34. The method described in any one of C23 to C33, wherein the method further comprises storing the composition described in any one of C1 to C20 for at least 1 month, at least 2 months, at least 3 months, at least 4 months, or at least 5 months before using the composition in an immunoassay.

[0485] C35. The method described in C34, wherein the method further comprises storing the composition of any one of C1 to C20 at a temperature in the range of 2°C to 6°C for up to 6 months or up to 1 year before using the composition in an immunoassay.

Claims

1. A container comprising: a. at least one agent comprising particles, wherein the mass of the particles is at least 0.1 μg and at most 100 μg; and b. A chemiluminescent substrate preparation, wherein the chemiluminescent substrate preparation comprises a chemiluminescent compound of formula I or a salt thereof: in: A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent; R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups; R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring; R 15 It is C 1-6 alkyl; Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts; Z is O or S; and n is 0, 1 or 2.

2. The container of claim 1, wherein the container further comprises: a. up to 25,000 analyte molecules, up to 35,000 analyte molecules, or up to 50,000 analyte molecules; b. up to 60,000, up to 70,000, up to 80,000, up to 90,000, up to 100,000, or up to 200,000 enzyme molecules, or c. combinations thereof.

3. The container of claim 1 or 2, wherein the container further comprises 10,000 to 15,000, 10,000 to 20,000, 10,000 to 25,000, 15,000 to 20,000, or 15,000 to 25,000 analyte molecules.

4. The container of any one of claims 1 to 3, wherein the container comprises a buffer.

5. The container of any one of claims 1 to 4, wherein the particles comprise paramagnetic particles or paramagnetic microparticles.

6. The container of any one of claims 1 to 5, wherein the particles comprise coated paramagnetic particles.

7. The container of any one of claims 1 to 6, wherein the particles are coated with antibodies or antigens.

8. The container of any one of claims 1 to 7, wherein the at least one reagent comprises At least 0.1 μg paramagnetic particles, at least 0.5 μg paramagnetic particles, at least 1.0 μg paramagnetic particles, at least 2.5 μg paramagnetic particles, at least 5 μg paramagnetic particles, at least 7.5 μg paramagnetic particles, at least 10 μg paramagnetic particles, at least 15 μg paramagnetic particles, or at least 20 μg paramagnetic particles and / or Up to 10 μg paramagnetic particles, up to 15 μg paramagnetic particles, up to 20 μg paramagnetic particles, up to 25 μg paramagnetic particles, up to 30 μg paramagnetic particles, up to 35 μg paramagnetic particles, up to 40 μg paramagnetic particles, up to 45 μg paramagnetic particles, up to 50 μg paramagnetic particles, up to 50 μg paramagnetic particles, up to 75 μg paramagnetic particles, up to 100 μg paramagnetic particles.

9. The container of any one of claims 1 to 8, wherein the mass of the chemiluminescent compound is at least 0.1 times, at least 0.5 times, at least the same, at least 1.5 times, at least two times, at least three times, at least five times, at least ten times, at least twenty-five times, at least fifty times, at least one hundred times, or at least one hundred and fifty times the mass of the at least one reagent.

10. The container of any one of claims 1 to 9, wherein the container further comprises an enzyme, an antibody, a capture antibody, an antibody-enzyme conjugate, or an antigen-enzyme conjugate.

11. The container of claim 10, wherein the enzyme is alkaline phosphatase; the antibody-enzyme conjugate comprises ALP; or the antigen-enzyme conjugate comprises ALP.

12. The container of any one of the preceding claims, wherein the container is a tube comprising polyethylene.

13. The container of any one of the preceding claims, wherein the container is in a photometer.

14. The container of any of the preceding claims, wherein the chemiluminescent compound is present at a concentration of 0.01 g / L to 50 g / L, 0.1 g / L to 10 g / L, 0.01 g / L to 10 g / L, 0.1 g / L to 30 g / L, or 0.1 g / L to 50 g / L.

15. The container of any of the preceding claims, wherein the chemiluminescent compound is present at a concentration of at least 0.01 g / L, at least 0.05 g / L, at least 0.1 g / L, at least 0.2 g / L, at least 0.5 g / L, at least 1 g / L; or at most 0.5 g / L, at most 1 g / L, at most 2 g / L, at most 5 g / L, at most 7 g / L, or at most 10 g / L.

16. The container of any of the preceding claims, wherein the chemiluminescent compound is present in a volume of 150 μL of buffer.

17. The container of any of the preceding claims, wherein the chemiluminescent compound is present in a volume of at least 150 μL and at most 300 μL of buffer.

18. The container of any of the preceding claims, wherein the chemiluminescent compound is present in a volume of at most 200 μL of buffer or at most 500 μL of buffer.

19. The container of any of the preceding claims, wherein the antibody or antigen conjugated to the enzyme comprises an anti-troponin antibody, an anti-thyroid stimulating hormone (TSH) antibody, an anti-alpha-fetoprotein (AFP) antibody, an anti-β-human chorionic gonadotropin (βhCG) antibody, an anti-sex hormone binding globulin (SHBG) antibody, an anti-CA19-9 antibody, or an anti-creatine kinase-MB (CKMB) antibody.

20. The container of any of the preceding claims, wherein the container is located in an immunoassay analyzer or instrument.

21. The container of claim 20, wherein the immunoassay analyzer or instrument comprises a system for removing sample aliquots from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide real-time feedback on the performance of the immunoassay analyzer or instrument, a dispenser for heating the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid, or a combination thereof.

22. The container of claim 20 or 21, wherein the immunoassay system comprises a photometric system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on photons emitted from the assay during the period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube to a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sense resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on photons emitted from the assay over the period of time; as well as A photometer controller is configured to calculate a relative light unit value of photons emitted from the assay during the period of time based on the assay analog voltage and an optimized linear function in response to the assay analog voltage being greater than a predetermined value.

23. Use of the container according to any one of claims 1 to 22 in an immunoassay method.

24. An immunoassay analyzer or apparatus comprising a container, wherein the immunoassay analyzer or instrument comprises a system for removing sample aliquots from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide real-time feedback on the performance of the immunoassay analyzer or instrument, a dispenser that heats the wash fluid before adding the wash fluid to the reaction vessel, or a mechanism suitable for degassing the fluid, and / or Wherein the immunoassay system comprises a photometric system, the photometric system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on photons emitted from the assay during the period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube to a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of said current sense resistor and terminals of said amplifier; a counter circuit configured to provide an assay photon count based on said photons emitted from said assay over said period of time; as well as a photometer controller configured to: in response to the assay analog voltage being greater than a predetermined value, calculate a relative light unit value of photons emitted from the assay during the period of time based on the assay analog voltage and an optimized linear function; and The container contains at most 100 μg of paramagnetic microparticles.

25. The immunoassay analyzer or instrument of claim 24, wherein the container further comprises an analyte to be detected by the immunoassay analyzer or instrument.

26. An immunoassay analyzer or apparatus comprising a container, wherein the immunoassay analyzer or instrument comprises a system for removing sample aliquots from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide real-time feedback on the performance of the analyzer or instrument, a dispenser that heats the wash fluid before adding the wash fluid to the reaction vessel, or a mechanism suitable for degassing the fluid, and / or Wherein the immunoassay system comprises a photometric system, the photometric system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on photons emitted from the assay during the period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube to a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sense resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on photons emitted from the assay over the period of time; as well as a photometer controller configured to: in response to the assay analog voltage being greater than a predetermined value, calculate a relative light unit value of photons emitted from the assay during the period of time based on the assay analog voltage and an optimized linear function; and wherein the container contains a sample aliquot having a volume of 2 μl to 50 μl.

27. The immunoassay instrument of any one of claims 24 to 26, wherein the container contains a sample aliquot having a volume of at most 2 μl, at most 5 μl, at most 8 μl, at most 10 μl, at most 15 μl, at most 20 μl, at most 30 μl, at most 40 μl or at most 50 μl.

28. The immunoassay instrument of claim 26 or 27, wherein the container is a disposable pipette tip.

29. The immunoassay instrument of any one of claims 26 to 28, wherein the sample aliquot is a serum aliquot, a plasma aliquot, a urine aliquot, an amniotic fluid aliquot, a whole blood aliquot, a synovial fluid aliquot, a cerebrospinal fluid aliquot, a saliva aliquot, a semen aliquot, a nasal fluid aliquot, a mucus aliquot, or a bronchoalveolar lavage fluid aliquot.

30. The immunoassay instrument of any one of claims 26 to 29, wherein the container is a polyethylene tube.

31. Use of the immunoassay apparatus according to any one of claims 26 to 30 in an immunoassay method.

32. A method comprising: contacting a sample suspected of containing a target analyte with the antibody composition to form a mixture; incubating the mixture in a container or vessel; as well as adding a substrate composition to the container or vessel to form a reaction mixture; The antibody composition comprises: an antibody against the target analyte conjugated to alkaline phosphatase (ALP) or an antigen conjugated to ALP; a buffer comprising a zinc salt and a magnesium salt; in: When the pH of the composition is in the range of 5.5 to 6.4, free Zn 2+ The buffer concentration ranged from 5 μM to 1 mM and the free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ within 3500 times the concentration of The substrate composition comprises: The chemiluminescent compound of formula I or its salt: in: A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent; R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups; R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring; R 15 It is C 1-6 alkyl; Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts; Z is O or S; and n is 0, 1 or 2.

33. The method of claim 32, wherein the ALP enzymatic activity of the ALP-conjugate in the antibody composition changes by less than 25% from peak activity when the composition is stored at 37°C for up to 3 days.

34. The method of claim 32 or 33, wherein the ALP enzymatic activity of the ALP-conjugate in the antibody composition changes by less than 25% from peak activity when the composition is stored at a temperature in the range of 2°C to 6°C for up to 6 months, up to 9 months, or up to 1 year.

35. The method of any one of claims 32 to 34, Wherein in the antibody composition, (a) When the pH of the composition is in the range of 5.5 to 6.4, free Zn 2+ The buffer concentration ranged from 5 μM to 1 mM and the free Mg 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+ The concentration of 1% chlorine is within 3500 times of that of 1% chlorine; (b) When the pH of the composition is in the range of 6.5 to 7.4, free Zn 2+ The buffer concentration ranged from 1 μM to 5 mM and the free Mg 2+ The concentration of free Zn 2+ The concentration is 200 times higher than that of free Zn 2+ within 3500 times the concentration of (c) When the pH of the composition is in the range of 7.5 to 8.4, free Zn 2+ The buffer concentration ranged from 100 nM to 300 μM and the free Mg 2+ The concentration of free Zn 2+ The concentration of free Zn is 100 times 2+ The concentration range is 1000 times.

36. The method of claim 35, wherein the ALP enzymatic activity of the ALP-conjugate in the antibody composition changes by less than 10% from peak activity when the composition is stored at 37°C for up to 3 days.

37. The method of claim 35 or 36, wherein the ALP enzymatic activity of the antibody composition changes by less than 10% from peak activity when the composition is stored at a temperature in the range of 2°C to 6°C for up to 6 months, up to 9 months, or up to 1 year.

38. The method of any one of claims 32 to 37, wherein the ALP in the antibody composition comprises bovine intestinal ALP.

39. The method of any one of claims 32 to 38, wherein the buffer in the antibody composition comprises ACES, HEPES, MES, phosphate, TRIS, ADA, PIPES, MOPSO, BTP, BES or Bis-TRIS, or a combination thereof.

40. The method of any one of claims 32 to 39, wherein the zinc salt comprises ZnCl2.

41. The method of any one of claims 32 to 40, wherein the magnesium salt comprises MgCl2.

42. The method of any one of claims 32 to 41, wherein the antibody composition further comprises albumin.

43. The method of claim 42, wherein the albumin comprises bovine serum albumin, human serum albumin, casein, hydrolyzed casein, or ovalbumin.

44. The method of any one of claims 32 to 43, wherein the antibody or antigen conjugated to ALP comprises an anti-troponin antibody (cTnI), an anti-thyroid stimulating hormone (TSH) antibody, an anti-alpha-fetoprotein (AFP) antibody, a triiodothyronine (T3) antigen, an anti-beta human chorionic gonadotropin (βhCG) antibody, an anti-sex hormone binding globulin (SHBG) antibody, an anti-CA19-9 antibody, or an anti-creatine kinase-MB (CKMB) antibody.

45. The method of claim 44, wherein: The antibody conjugated to ALP comprises an anti-troponin I (cTnI) antibody; The pH of the antibody composition is in the range of 5.5 to 6.4; and Free Zn 2+ The concentration of Mg was in the range of 100 μM to 500 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration range is 500 times.

46. ​​The method of claim 44, wherein: The antibody conjugated to ALP comprises an anti-TSH antibody; The pH of the antibody composition is in the range of 5.5 to 6.4; and Free Zn 2+ The concentration of Mg was in the range of 5 μM to 100 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 500 times higher than that of free Zn 2+ The concentration range is 1500 times.

47. The method of claim 44, wherein: The antibody conjugated to ALP comprises an anti-AFP antibody; The pH of the antibody composition is in the range of 5.5 to 6.4; and Free Zn 2+ The concentration of Mg was in the range of 50 μM to 150 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 150 times that of free Zn 2+ The concentration range is 500 times.

48. The method of claim 44, wherein: The antibody conjugated to ALP comprises an anti-βhCG antibody; The pH of the antibody composition is in the range of 5.5 to 6.4; and Free Zn 2+ The concentration of Mg was in the range of 5 μM to 100 μM and free Mg 2+ The concentration of free Zn 2+ The concentration is 700 times higher than that of free Zn 2+ The concentration range is 1500 times.

49. The method of claim 44, wherein the antigen conjugated to ALP comprises a triiodothyronine (T3) antigen.

50. The method of any one of claims 32 to 49, wherein the concentration of free magnesium, free zinc, or a combination thereof in the antibody composition is determined by removing larger molecules from a storage solution by size exclusion chromatography followed by quantification of the free magnesium, free zinc, or a combination thereof by mass spectrometry.

51. The method of any one of claims 32 to 50, wherein the substrate composition further comprises Cationic aromatic compounds (CAC); Background reducing agent; or An ether-linked nonionic surfactant not containing a carboxylate group; or a hydrophilic polymer; or Its combination.

52. The method of claim 51, wherein the substrate composition comprises: a) 0.01 mM to 50 mM of the chemiluminescent compound of formula I or a salt thereof; as well as b) 0.01 to 200 μM of the cationic aromatic compound (CAC); c) 1 μM to 10 mM of the background reducing agent; or d) 0.05 to 20 g / L of the ether-linked nonionic surfactant not containing a carboxylate group; or a hydrophilic polymer; or A combination of a) and one or more of b), c) and / or d).

53. The method of any one of claims 32 to 52, wherein the substrate composition comprises the following compound:

54. The method of any one of claims 32 to 53, wherein the method is an immunoassay method.

55. The method of claim 54, wherein the immunoassay is performed on an immunoassay analyzer or instrument.

56. The method of any one of claims 32 to 55, wherein the method further comprises: After adding the substrate composition to the vessel, light generated by the reaction is measured for up to 5 minutes, up to 4 minutes, up to 3 minutes, up to 2 minutes, up to 1 minute, up to 56 seconds, up to 55 seconds, up to 50 seconds, or up to 48 seconds.

57. The method of any one of claims 32 to 56, wherein the method further comprises a washing step prior to adding the substrate composition to the vessel.

58. The method of any one of claims 32 to 57, wherein the method further comprises determining the amount of an analyte in the sample.

59. The method of claim 58, wherein the amount of the analyte is determined based on a stored multi-point calibration curve.

60. The method of any one of claims 56 to 59, wherein light produced by the reaction is measured using a photometer.

61. The method of any one of claims 32 to 56, wherein the method further comprises at least two washing steps prior to adding the substrate composition to the vessel.

62. The method of any one of claims 32 to 61, wherein the sample suspected of containing the target analyte comprises serum, plasma, urine, amniotic fluid, whole blood, synovial fluid, cerebrospinal fluid, saliva, semen, nasal fluid, mucus, or bronchoalveolar lavage fluid.

63. The method of any one of claims 32 to 62, wherein the sample suspected of containing the target analyte is a serum sample, a plasma sample, a urine sample, an amniotic fluid sample, or a whole blood sample.

64. The method of any one of claims 32 to 63, wherein the method comprises contacting a sample comprising an analyte with the antibody composition, wherein the antibody composition comprises an antibody that binds to ALP, and further wherein an ALP-antibody-analyte complex is formed.

65. The method of claim 64, wherein the method further comprises contacting the ALP-antibody-analyte complex with an anti-analyte antibody bound to a solid phase, and washing away sample that is not bound to the solid phase.

66. The method of claim 65, wherein the solid phase comprises paramagnetic particles, paramagnetic microparticles, or particles.

67. The method of any one of claims 57 to 66, wherein the method comprises contacting a sample comprising an analyte with an antibody specific for the analyte to form a complex comprising the analyte, washing the complex, and contacting the complex with the substrate composition, wherein the antibody composition comprises an antigen that binds to ALP.

68. The method of any one of claims 35 to 67, wherein the method further comprises storing the antibody composition for at least 1 month, at least 2 months, at least 3 months, at least 4 months, or at least 5 months prior to using the composition in an immunoassay.

69. The method of claim 68, wherein the method further comprises storing the antibody composition at a temperature in the range of 2°C to 6°C for up to 6 months or up to 1 year prior to using the composition in an immunoassay.

70. The method of any one of claims 35 to 69, further comprising a reproducibility condition of measurement, wherein the reproducibility condition of measurement comprises at least one of between-site, between-batch, between-day, between-instrument, between-run, and within-run variance components.

71. The method of any one of claims 35 to 70, wherein the target analyte comprises cardiac troponin I (cTnI), alpha-fetoprotein (AFP), thyroid stimulating hormone (TSH), beta human chorionic gonadotropin (βhCG), free thyroxine (T4), creatinine kinase (CK-MB), sex hormone binding globulin (SHBG), or cancer antigen 19-9 (CA19-9).

72. The method of any one of claims 35 to 71, wherein the target analyte comprises cardiac troponin I (cTnI), alpha-fetoprotein (AFP), thyroid stimulating hormone (TSH), beta human chorionic gonadotropin (βhCG), free thyroxine (T4).

73. The method of any one of claims 35 to 72, wherein the target analyte comprises cardiac troponin I (cTnI).

74. The method of any one of claims 35 to 72, wherein the target analyte comprises alpha-fetoprotein (AFP).

75. The method of any one of claims 35-72, wherein the target analyte comprises thyroid stimulating hormone (TSH).

76. The method of any one of claims 35-72, wherein the target analyte comprises beta human chorionic gonadotropin (βhCG).

77. The method of any one of claims 35-72, wherein the target analyte comprises free thyroxine (T4).

78. An immunoassay method comprising: a. contacting a sample suspected of containing an analyte with 0.1 μg to 100 μg of particles in a reaction vessel; as well as b. The reaction vessel is subjected to further sample processing on an immunoassay instrument.

79. The immunoassay method of claim 78, wherein the immunoassay analyzer or instrument comprises a system for removing sample aliquots from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide real-time feedback on the performance of the immunoassay analyzer or instrument, a dispenser for heating the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid, or a combination thereof.

80. The immunoassay method of claim 78 or 79, wherein the immunoassay system comprises a photometric system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on photons emitted from the assay during the period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube to a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sense resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on photons emitted from the assay over the period of time; as well as A photometer controller is configured to calculate a relative light unit value of photons emitted from the assay during the period of time based on the assay analog voltage and an optimized linear function in response to the assay analog voltage being greater than a predetermined value.

81. The immunoassay method of any one of claims 78 to 80, further comprising A chemiluminescent substrate is added to the reaction vessel, wherein the chemiluminescent substrate is a chemiluminescent compound of Formula I or a salt thereof: in: A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent; R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups; R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring; R 15 It is C 1-6 alkyl; Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts; Z is O or S; and n is 0, 1 or 2.

82. The immunoassay method of any one of claims 78 to 81, wherein the further sample processing comprises: Incubating the reaction vessel; washing the paramagnetic particles; applying a magnetic field to the paramagnetic particles; or Any combination thereof.

83. The immunoassay method of any one of claims 78 to 82, wherein the immunoassay method further comprises allowing the chemiluminescent compound to generate a signal and counting the signal generated by the chemiluminescent compound.

84. The immunoassay method of any one of claims 78 to 83, wherein the immunoassay method further comprises determining the amount of an analyte in the sample.

85. The immunoassay of any one of claims 78 to 84, wherein the amount of the analyte is determined according to a stored multi-point calibration curve.

86. The immunoassay method of any one of claims 78 to 85, wherein the analyte detected by the immunoassay comprises cardiac troponin I (cTnI), alpha-fetoprotein (AFP), thyroid stimulating hormone (TSH), beta human chorionic gonadotropin (βhCG), free thyroxine (T4), creatinine kinase (CK-MB), sex hormone binding globulin (SHBG), or cancer antigen 19-9 (CA 19-9).

87. The immunoassay method of any one of claims 78 to 85, wherein the analyte detected by the immunoassay comprises alpha-fetoprotein (AFP), thyroid stimulating hormone (TSH), beta human chorionic gonadotropin (βhCG), creatinine kinase (CK-MB), sex hormone binding globulin (SHBG), or cancer antigen 19-9 (CA19-9).

88. The immunoassay method of any one of claims 78 to 87, wherein the sample is a serum sample, a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample, or a bronchoalveolar lavage fluid sample.

89. The immunoassay method of any one of claims 78 to 88, wherein the sample is a serum sample, a plasma sample, or an amniotic fluid sample.

90. The immunoassay method of any one of claims 78 to 89, wherein the analyte detected by the immunoassay comprises AFP and has a limit of blank (LoB) of 0.08 to 0.16 ng / mL, a limit of detection (LoD) of 0.22 to 0.23 ng / mL, and / or a limit of quantitation (LoQ) of 0.13 to 0.

20.

91. The immunoassay method of any one of claims 78 to 89, wherein the analyte detected by the immunoassay comprises TSH and the limit of blank (LoB) is 0.001 to 0.002 uIU / mL, the limit of detection (LoD) is 0.002 to 0.003 uIU / mL, and / or the limit of quantification (LoQ) is 0.0008 to 0.001 uIU / mL.

92. The immunoassay method of any one of claims 78 to 89, wherein the analyte detected by the immunoassay comprises hCG and the limit of blank (LoB) is 0.0 to 0.1 mIU / mL, the limit of detection (LoD) is 0.2 mIU / mL, and / or the limit of quantitation (LoQ) is 0.1 to 0.2 mIU / mL.

93. The immunoassay method of any one of claims 78 to 89, wherein the analyte detected by the immunoassay comprises free T4 and the limit of blank (LoB) is 0.18 ng / mL.

94. The immunoassay method of any one of claims 78 to 93, wherein the particle is a paramagnetic particle, microparticle, or paramagnetic microparticle.

95. The immunoassay method of any one of claims 78 to 94, wherein the particles are coated particles.

96. The immunoassay of any one of claims 78 to 95, wherein the particles are coated with antibodies.

97. The immunoassay method of claim 96, wherein the particles are coated with a monoclonal antibody, a target-specific antibody, or a target-specific monoclonal antibody.

98. The immunoassay method of any one of claims 78 to 97, wherein the particles comprise at least 0.1 μg of paramagnetic particles, at least 0.5 μg of paramagnetic particles, at least 1.0 μg of paramagnetic particles, at least 2.5 μg of paramagnetic particles, at least 5 μg of paramagnetic particles, at least 7.5 μg of paramagnetic particles, at least 10 μg of paramagnetic particles, at least 15 μg of paramagnetic particles, or at least 20 μg of paramagnetic particles and / or Up to 10 μg paramagnetic particles, up to 15 μg paramagnetic particles, up to 20 μg paramagnetic particles, up to 25 μg paramagnetic particles, up to 30 μg paramagnetic particles, up to 35 μg paramagnetic particles, up to 40 μg paramagnetic particles, up to 45 μg paramagnetic particles, up to 50 μg paramagnetic particles, up to 50 μg paramagnetic particles, up to 75 μg paramagnetic particles, up to 100 μg paramagnetic particles.

99. An immunoassay for detecting alpha-fetoprotein (AFP) in a sample, wherein the immunoassay comprises: contacting a sample suspected of containing AFP and an anti-AFP antibody conjugated to an enzyme to form a mixture, wherein the sample is a blood sample, a plasma sample, or an amniotic fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; incubating the reaction mixture; measuring light generated by the reaction mixture; as well as detecting the concentration of AFP in the sample based on light generated by the reaction mixture; wherein the limit of blank (LoB) of the immunoassay method is 0.08 to 0.16 ng / mL, the limit of detection (LoD) of the immunoassay method is 0.22 to 0.23 ng / mL, and / or the limit of quantification (LoQ) of the immunoassay method is 0.13 to 0.

20.

100. An immunoassay for detecting thyroid stimulating hormone (TSH) in a sample, wherein the immunoassay comprises: contacting a sample suspected of containing TSH and an anti-TSH antibody conjugated to an enzyme to form a mixture, wherein the sample is a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample, or a bronchoalveolar lavage fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; incubating the reaction mixture; measuring light generated by the reaction mixture; as well as detecting the concentration of TSH in the sample based on light generated by the reaction mixture; The limit of blank (LoB) of the immunoassay method is 0.001 to 0.002 uIU / mL, the limit of detection (LoD) of the immunoassay method is 0.002 to 0.003 uIU / mL, and / or the limit of quantification (LoQ) of the immunoassay method is 0.0008 to 0.001 uIU / mL.

101. An immunoassay method for detecting beta human chorionic gonadotropin (βhCG) in a sample, wherein the immunoassay method comprises: contacting a sample suspected of containing βhCG and an anti-βhCG antibody conjugated to an enzyme to form a mixture, wherein the sample is a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample, or a bronchoalveolar lavage fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; incubating the reaction mixture; measuring light generated by the reaction mixture; as well as detecting the concentration of βhCG in the sample based on light generated by the reaction mixture; The limit of blank (LoB) of the immunoassay method is 0.0 to 0.1 mIU / mL, the limit of detection (LoD) of the immunoassay method is 0.2 mIU / mL, and / or the limit of quantification (LoQ) of the immunoassay method is 0.1 to 0.2 mIU / mL.

102. An immunoassay for detecting free thyroxine 4 (T4) in a sample, wherein the immunoassay comprises: contacting a sample suspected of containing T4 and an anti-T4 antibody conjugated to an enzyme to form a mixture, wherein the sample is a plasma sample, a urine sample, an amniotic fluid sample, a whole blood sample, a synovial fluid sample, a cerebrospinal fluid sample, a saliva sample, a semen sample, a nasal fluid sample, a mucus sample, or a bronchoalveolar lavage fluid sample; incubating the mixture in a container or vessel; adding a chemiluminescent substrate to the container or vessel to form a reaction mixture; wherein the chemiluminescent substrate is a substrate for the enzyme; incubating the reaction mixture; measuring light generated by the reaction mixture; as well as detecting the concentration of T4 in the sample based on the light generated by the reaction mixture; The limit of blank (LoB) of the immunoassay method is less than 0.18 ng / mL.

103. The immunoassay method of any one of claims 99 to 102, wherein the chemiluminescent substrate comprises a chemiluminescent compound of formula I or a salt thereof: in: A is for C 1-6 haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein the substituted phenyl contains 1 to 3 halogens, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C(O)R 15 , CN or NO2 substituent; R1 is selected from C 5-14 Aryl, C 1-6 Alkyl, C 1-6 Haloalkyl and C 5-14 Aralkyl groups; R7-R 14 Independently H, C 1-6 Alkoxy, halogen, C 1-4 Alkyl, or R7 or R8-R9 or R9-R 10 or R 11 -R 12 or R 12 -R 13 or R 13 -R 14 may be linked together as a carbocyclic or heterocyclic ring system containing at least one 5-membered or 6-membered ring; R 15 It is C 1-6 alkyl; Each M is independently selected from H, alkali metals, alkaline earth metals, transition metals, ammonium, Organic amine salts and amino acid salts; Z is O or S; and n is 0, 1 or 2.

104. The immunoassay method of claim 103, wherein the immunoassay method is performed using an immunoassay analyzer or instrument.

105. The immunoassay method of claim 104, wherein the immunoassay analyzer or instrument comprises a system for removing sample aliquots from a sample using a disposable tip, a dispensing control system, an image capture device and / or a camera unit that can be used to provide real-time feedback on the performance of the immunoassay analyzer or instrument, a dispenser for heating the wash fluid before adding the wash fluid to the reaction vessel, or a mechanical device suitable for degassing the fluid, or a combination thereof.

106. The immunoassay method of any one of claims 104-105, wherein the immunoassay method is performed using an immunoassay system comprising a photometric system, the photometric system comprising: a photomultiplier tube configured to sense photons emitted from an assay reaction over a period of time; an analog circuit configured to provide an assay analog voltage based on photons emitted from the assay during the period of time, the analog circuit comprising: a current sensing resistor coupled to convert current from the photomultiplier tube to a voltage; an amplifier configured to amplify the voltage; and a dedicated electrical connection between terminals of the current sense resistor and terminals of the amplifier; a counter circuit configured to provide an assay photon count based on photons emitted from the assay over the period of time; as well as A photometer controller is configured to calculate a relative light unit value of photons emitted from the assay during the period of time based on the assay analog voltage and an optimized linear function in response to the assay analog voltage being greater than a predetermined value.

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