Method for reusing hapten-coated probes in immunoassays

Abstract

The present invention relates to immunoassay methods that reuse hapten-immobilized test probes and reagents anywhere from about 3 to 20 times for the quantitation of an analyte in different samples while maintaining acceptable clinical assay performance. The methods use a solution of dual antibody conjugate comprising anti-hapten antibody and capture antibody against the analyte in each cycle. After each reaction cycle is completed, the test probes are immersed in an acidic solution having a pH of about 1-4 to elute the immune complexes formed on the probes and regenerate the hapten-immobilized probes. The robustness of the hapten-coated solid phase allows for the use of a variety of denaturing reagents to efficiently elute the immune complexes after each cycle without compromising the binding activity of the hapten on the solid phase.

Description

[0001] This application is a divisional application of Chinese national-phase invention patent application No. 202080026563.1, filed on April 2, 2020, entitled "Method for reusing hapten-coated probes in immunoassay". Technical Field

[0002] This invention relates to an immunoassay method in which a hapten-immobilized test probe is repeatedly used to quantify an analyte in different samples approximately 3 to 10 times. The method uses a biantibody conjugate solution in each cycle and regenerates the hapten-coated test probe by immersing it in an acidic solution with a pH of approximately 1-4 after each reaction cycle. Background Technology

[0003] Cost control is a primary objective for healthcare providers worldwide. In vitro diagnostics (IVD) is no exception, where the clinical utility of biomarkers in diagnosis and prognosis has become standard in patient management. Immunoassay technology accounts for a significant portion of the IVD industry and is steadily growing, at approximately 3% per year in the United States and 15-20% per year in developing countries. In some cases, such as in the diagnosis of myocardial infarction, continuous measurement of cardiac markers may limit the appropriate number of tests.

[0004] Typical approaches to reducing the cost of immunoassays require minimizing the manufacturing costs of materials, labor, and facility expenses.

[0005] Any method for recirculating immunoassay reagents typically focuses on dissociating the immune complex with denaturing agents such as acidic / alkaline pH solutions, organic solvents, or dissociation agents. This denaturation step alters the antibody's charge, hydration, hydrogen bonding, and tertiary structure when the antibody is no longer bound to the antigen. Reversing the exposure of the antibody to its initial binding conditions, close to physiological pH and ionic strength, is expected to restore its original binding activity; however, few antibodies can tolerate repeated exposure to denaturing conditions without adversely affecting their binding properties and thus some aspect of their assay performance.

[0006] There is a need to reduce the cost of immunoassays while maintaining clinical performance. Attached Figure Description

[0007] Figure 1A and 1BA procalcitonin (PCT) assay configuration is described, comprising a fluorescein (F)-coated probe, anti-F / anti-PCT as the capture antibody (AB), Cy5-anti-PCT as the signal AB, and a pH 2 eluted conjugate. The initial step in the recycling protocol is a “pre-read,” which measures the intrinsic fluorescence of the glass probe and any residual fluorescence retained on the probe tip from the previous cycle. This signal is subtracted from the final read step at the end of the assay to obtain the immunospecific fluorescence signal. The pH 2 elution step strips the immune complex containing the capture AB from the probe surface. Each cycle has a fresh coating of the capture AB, which is crucial for maintaining assay performance across multiple cycles. The 1 mm diameter probe has such a small surface area that any binding of the capture AB or signal AB consumes only negligible amounts of those reagents, thus allowing for reuse across multiple cycles. The fluorescein-coated probe can withstand multiple cycles up to pH 2 elution and return to neutral pH, enabling its reuse.

[0008] Figure 2 This shows that following scheme A, v1 utilizes Figure 1B Data from PCT assays performed using the fluorophore probe format described in [the document / reference].

[0009] Figure 3 The probe format coated with fluorescein is shown. Figure 1B The data of PCT standards using scheme A, v1 are used in the study.

[0010] Figure 4 The correlation between recirculation measurements and commercial IVD instruments (Biomerieux Vidas) was shown (R). 2 Results of PCT clinical serum samples (=0.95).

[0011] Figure 5 Results of a recirculation assay (Protocol A, v2) for cardiac troponin I (TnI), another immunoassay, are presented. The format is as follows: Figure 1B The difference is that the capture AB is anti-F connected with anti-TnI, and the signal AB is Cy5-anti-TnI.

[0012] Figure 6 The alternative recycling assay is demonstrated. Format and... Figure 1B They are the same, except that signal AB is labeled with a different fluorescent dye, Alexa Fluor 647.

[0013] Figure 7 Data from up to 10 cycles of different PCT samples using the signal AB (scheme A, v3) labeled with Alexa Fluor 647.

[0014] Figure 8 The recycling assays using different hapten / anti-hapten formats are shown. Formats and... Figure 1B Similarly, the difference is that the probe is coated with digitoxin, and the capture AB is an anti-digitoxin-anti-PCT conjugate.

[0015] Figure 9 PCT levels were presented for up to 10 cycles using the digitoxin format (Protocol A, v4). Results were compared with... Figure 3 The forms of fluorescein probes are very similar.

[0016] Figure 10 A recycling format utilizing additional amplification reagents is shown. In this example, the signal antibody is biotin-labeled, and Cy5-antibiotin- is present. Reagents. The molecular weight of the copolymer of sucrose and epichlorohydrin is in the millions of Daltons, and it is used to carry anti-biotin antibodies labeled with various dyes. The amplification reagent binds to the biotin-containing immune complex on the probe surface and produces a stronger signal than the direct dye labeling of signal AB.

[0017] Figure 11 The method shown is to use scheme B, v1 with pH 2 for elution. Figure 10 The PCT assay was performed using the recirculation format in the [process / system].

[0018] Figure 12 Alternative amplification reagents were demonstrated. The recycling format and... Figure 10 Similarly, the difference lies in the amplification reagent being Cy5-streptavidin- Streptavidin molecules are smaller than antibodies, therefore more streptavidin molecules can bind to antibodies. Polymer linkage to generate antimicrobial linkage Compared to the abundance of fluorescent signals, another advantage of streptavidin-based amplification reagents is their extremely high binding affinity for biotin.

[0019] Figure 13 It shows the use of Figure 12 PCT results of streptavidin amplification reagent eluted at pH 2 for scheme B, v2.

[0020] Figure 14 It shows something similar to Figure 12 The recycle format differs in that, after the pH 2 step, the probe is further exposed to DMSO (100%) for a second elution.

[0021] Figure 15 Contains such as Figure 14The results of the three PCT levels were determined using protocol C, which employed dual pH 2 and DMSO elution.

[0022] Figure 16A and 16B A two-reading scheme is shown, in which the fluorescence signal is read twice. The fluorescence signal is read as Read 1 before pH 2 acid elution, and then read again as Read 2 after pH 2 acid elution. The Read 2 signal is subtracted from Read 1, and the subtracted value is quantified against a calibration curve. Figure 16B format and Figure 14 The format is similar, the difference being that the fluorescence signal is read twice. Figure 16A The measurement format and Figure 16B The determination methods are similar, the difference being in the first binding step. Figure 16A In the process, after pre-reading, the sample is mixed with a dual antibody (anti-F-anti-PCT) and biotinylated anti-PCT. The remaining... Combining steps and Figure 16A The two reading steps in the middle and Figure 16B The steps are the same as those in the previous section.

[0023] Figure 17 This illustrates a two-read / subtraction scheme ( Figure 16B The results of the recirculation assays of three PCT levels (0, 10 and 100 ng / mL) performed in protocol D).

[0024] Figure 18 This illustrates a scheme involving two reads and re-looping. Figure 16B Results of 15 PCT clinical serum samples from protocol D.

[0025] Figure 19 Results from 15 PCT clinical serum samples and a two-read recirculation protocol are shown. Figure 16B Option D) has a high correlation with commercial IVD instruments (Biomerieux Vidas) (R 2 =0.98).

[0026] Figure 20 Results of the assay using protocol D-1 with three PCT levels of recirculation: (i) low pH with denaturant guanidine chloride (6M guanidine HCl, pH 1.6) and (ii) denaturant DMSO.

[0027] Figure 21 It shows the use of Figure 16A Data on the determination format and protocol E for PCT standards.

[0028] Figure 22The assay format for serum amyloid A (SAA) is shown using (i) elution with the denaturing agent guanidine chloride (6M guanidine HCl, pH 1.6) at low pH and (ii) elution with the denaturing agent DMSO.

[0029] Figure 23 This demonstrates the use of double low pH plus denaturing agent guanidine chloride elution and denaturing agent DMSO elution. Figure 22 The results of three SAA levels were obtained from the recirculation assay using the assay format and protocol F.

[0030] Figure 24 The optical configuration for detecting fluorescence on the probe tip is shown. Detailed Implementation

[0031] definition

[0032] The terms used in the claims and specification shall be interpreted in accordance with their ordinary meaning as understood by those skilled in the art, except as defined below.

[0033] As used in this article, “about” means within ±10% of the value.

[0034] As used herein, an "analyte-binding" molecule means any molecule capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to, (i) antigen molecules for detecting the presence of antibodies specific to said antigen; (ii) antibody molecules for detecting the presence of the antigen; (iii) protein molecules for detecting the presence of their protein binding chaperones; (iv) ligands for detecting the presence of binding chaperones; or (v) single-stranded nucleic acid molecules for detecting the presence of nucleic acid-binding molecules.

[0035] The aspect ratio of a shape refers to the ratio of its longer dimension to its shorter dimension.

[0036] "Binding molecule" refers to a molecule that can bind to another molecule of interest.

[0037] As used herein, a “binding pair” refers to two molecules that attract each other and bind specifically to each other. Examples of binding pairs include, but are not limited to, antigens and antibodies against said antigens, ligands and their receptors, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectins and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and antifluorescein, and digitoxin / antidigitoxin. Biotin and avidin, including biotin derivatives and avidin derivatives such as streptavidin, can be used as intermediate binding substances in assays employing complex binding sequences. For example, an antibody can be biotinylated (“biotinylated”) and used to bind to a target substance previously immobilized on a solid surface. A fluorescent label can then be introduced using a fluorescent composition employing avidin or streptavidin according to the invention.

[0038] "Bispecific antibodies" are artificial proteins that can bind to two different types of antigens simultaneously.

[0039] As used in this article, "immobilization" refers to fixing a reagent to a solid surface. When a reagent is immobilized on a solid surface, it is either non-covalently or covalently bound to the surface.

[0040] As used in this article, "monolithic substrate" refers to a single solid material, such as glass, quartz, or plastic with a single refractive index.

[0041] As used herein, a "probe" refers to a substrate on which a thin film layer of analyte-binding molecules is coated on the sensing surface. A probe has a distal end and a proximal end. The proximal end (also referring to the probe tip in this application) has a sensing surface coated with a thin film of analyte-binding molecules.

[0042] This invention discloses a method for reusing immunoassay probes and reagents approximately 3 to 10 times while maintaining acceptable clinical assay performance. The immunoassay probes and reagents can be contained in a single test strip or a kit. This invention allows for the reuse of test probes and reagents, resulting in cost savings on a per-test basis.

[0043] Several key elements are involved in implementing this invention. A first feature of this invention is that the solid-phase capture reagent is coated with a hapten. Haptens, generally defined as small organic molecules smaller than about 1500 Daltons, are antigenic but have very poor immunogenicity. Therefore, haptens must be linked to larger polymers, typically proteins, to generate anti-hapten antibodies. Antibody binding of haptens is based on their primary chemical structure, and there is no conformational dependence in their antibody binding. Therefore, even after repeated steps of denaturing the anti-hapten antibody and the immune complex, the hapten antigen will retain its antibody-binding properties. This invention uses a hapten-immobilized test probe and an anti-hapten antibody to bind to a hapten-coated solid; the immune complex is then dissociated from the denaturing reagent. The hapten-coated solid phase is then re-immersed in a reagent containing the anti-hapten antibody to restore the original amount of anti-hapten antibody bound to the solid phase. The hapten-coated solid phase can be subjected to multiple denaturation cycles, followed by anti-hapten antibody binding. The robustness of the hapten-coated solid phase allows for efficient elution of the immune complex using a variety of denaturing reagents.

[0044] Suitable haptens of the present invention comprise, for example, small organic molecules such as nitrotyrosine, dinitrophenol, trinitrophenol, nitrophenol, and aminobenzoic acid; dyes such as Alexa Fluor, cyanine dyes, TRITC, Lucifer Yellow, Texas Red, and Bodipyrrole; polypeptides such as Myc, Flag, and multiple histidines; pharmaceuticals such as theophylline, phenytoin, phenobarbital, valproic acid, penicillin, and gentamicin; steroids such as progesterone, testosterone, and estradiol; and vitamins such as biotin and vitamin D. Preferred haptens are fluorescein, biotin, and digitalisin.

[0045] A second feature of the invention is that the assay uses a biantibody conjugate, wherein the anti-hapten antibody is chemically and covalently conjugated to a capture antibody that binds to a specific analyte in the sample. The capture antibody varies depending on the analyte being measured; this allows the assay format to be applied to many different immunoassays. The invention features solid-phase regeneration and recycling, with fresh biantibody conjugate reagents captured on the probe in each cycle, thereby maintaining assay performance.

[0046] A third feature of the invention is that the probe tip surface is small (diameter ≤ 5 mm), making the consumption of signal reagents and amplification reagents negligible, and eliminating the need to replenish those reagents during assay cycles.

[0047] Fluorescence detection system

[0048] This invention uses a fluorescence detection system for measuring fluorescence signals on a probe tip, as described in U.S. Patent No. 8,492,139, which is incorporated herein by reference. The system includes: (a) a probe having an aspect ratio of at least 5:1, the probe having a first end and a second end, the second end having a sensing surface associated with a fluorescent marker; (b) a light source for emitting excitation light directly onto the sensing surface of the probe; (c) a condenser lens directed toward the sensing surface; and (d) an optical detector for detecting emitted fluorescence light; wherein the condenser lens collects the emitted fluorescence light and directs the emitted fluorescence light to the optical detector.

[0049] The probe can be a monolithic substrate or an optical fiber. The probe can be of any shape, such as strip, cylinder, circle, square, triangle, etc., with a length-to-width ratio of at least 5:1, preferably 10:1. Because the probe is immersed in the sample solution and one or more reagent solutions during the immunoassay, a long probe with an aspect ratio of at least 5:1 is desirable to allow the probe tip to be submerged in the solution. Heterogeneous assays can be performed by transferring the long probe to different reaction chambers. This avoids the need for dispensing and aspirating reagents and samples during the assay. The sensing surface of the probe is coated with analyte-binding molecules and bound to a fluorescent label.

[0050] Any light source capable of emitting appropriate excitation light to the fluorescent marker is suitable for this invention. A preferred light source is a laser capable of emitting light with a wavelength suitable for the fluorescent marker. For example, for Cy5 fluorescent dye, the center wavelength of the laser is preferably 649 nm. Suitable optical detectors for detecting the emitted light are photomultiplier tubes (PMTs), charge-coupled devices (CCDs), or photodiodes.

[0051] The light source and the optical detector, which includes a condenser lens, are mounted on the same side of the probe tip surface (sensing surface). If the sensing surface is facing down, they are both mounted below the tip surface. If the sensing surface is facing up, they are both mounted above the tip surface. The light source is closer to the sensing surface than the other end of the probe. The sensing surface is always within the numerical aperture of the condenser lens. The probe can, but does not necessarily, need to be centered with the condenser lens.

[0052] Figure 24 An example of a fluorescence detection system is shown.

[0053] I. Detection of analytes using a recycling scheme

[0054] This invention relates to a method for detecting analytes in multiple liquid samples by fluorescence immunoassay using the same half-antigen-coated test probe, amplification reagent, and the same signal reagent for different samples.

[0055] The method includes the following steps: (a) immersing a probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip, wherein the probe includes a hapten immobilized on the probe tip, and the diameter of the tip surface is ≤5 mm; (b) forming an immune complex on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody against the hapten, and the signal antibody is conjugated to a fluorescent label; (c) [The text abruptly ends here, so the translation stops.] (d) The probe tip is immersed in a washing container containing a washing solution; the concentration of the analyte in the sample is determined by measuring the fluorescence signal of the immune complex at the probe tip, subtracting the pre-read fluorescence signal from (a), and quantifying the subtracted signal against a calibration curve; (e) The probe tip is immersed in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (f) Steps (a)-(e) are repeated, but the analyte from a new sample is used in step (b), thereby detecting the analyte in each of the plurality of liquid samples.

[0056] In step (e), after immersing the probe tip in an acidic solution with a pH of about 1.0-4.0, the probe is optionally further immersed in a denaturing agent or dissociating agent for further eluting the immune complex, such as dimethyl sulfoxide (DMSO), n-butanol, ethanol, guanidine hydrochloride (guanidine chloride), lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. DMSO is a preferred dissociating agent.

[0057] In one embodiment, the immune complex of step (b) is formed by: (b1) mixing a sample solution with a biantibody solution and a reagent solution to form a mixture, wherein the sample solution comprises an analyte, the biantibody solution comprises the anti-hapten antibody covalently linked to the capture antibody, and the reagent solution comprises a signal antibody conjugated to a fluorescent label; and (b2) immersing the probe tip in the mixture to form the immune complex on the probe tip. The probe can be immediately immersed in the mixture when the components are added together. Alternatively, the probe can be immersed in the mixture after incubating the mixture for a period of time, for example, 30 seconds to 15 minutes. This embodiment is shown in Figure 1A middle.

[0058] In one embodiment, the immune complex in step (b) is formed by: (b1) immersing the probe tip in a biantibody solution comprising the anti-hapten antibody covalently linked to the capture antibody; (b2) immersing the probe tip in a sample solution comprising the sample; and (b3) immersing the probe tip in a reagent solution comprising the signal antibody conjugated to a fluorescent label, to form the immune complex on the probe tip. This embodiment is shown in Figure 1B middle.

[0059] Example 1 shown

[0060] In another embodiment, the method for detecting the analyte includes the following steps: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) immersing the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip; (c) immersing the probe tip in a double antibody solution comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is a first antibody against the analyte; (d) immersing the probe tip in a sample solution comprising a liquid sample containing the analyte; and (e) immersing the probe tip in a reagent solution comprising a signal antibody conjugated to a fluorescent label to form a fluorescent label on the probe tip. An immune complex comprising an analyte, a capture antibody, and a signal antibody, wherein the signal antibody is a secondary antibody against the analyte; (f) immersing the probe tip in a washing container containing a washing solution; (g) determining the analyte concentration in the sample by measuring the fluorescence signal of the immune complex at the probe tip, subtracting the pre-read fluorescence signal from (b), and quantifying the subtracted signal against a calibration curve; (h) immersing the probe tip in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (i) repeating steps (b)-(h), but using a fresh sample solution comprising a new sample in step (d), thereby detecting the analyte in each of the plurality of liquid samples. This embodiment is shown in Figure 1B , 6 and 8.

[0061] The method uses the same probe and the same washing solution in all reaction cycles. Preferably, the method uses the same reagent solution in all reaction cycles. However, fresh reagent solutions can also be used in different cycles.

[0062] In step (a) of this method, a probe with a small tip for binding analytes is obtained. The tip has a small surface area, with a diameter ≤5 mm, preferably ≤2 mm or ≤1 mm. The small surface area of ​​the probe tip gives it several advantages. In solid-phase immunoassays, a small surface area is advantageous because it results in less nonspecific binding and thus a lower background signal. Furthermore, due to the small surface area of ​​the tip, the amount of reagent or sample carried on the probe tip is very small. This feature makes the probe tip easy to wash, and its contamination of the washing solution is negligible due to the larger volume of the washing solution. Another aspect of the small surface area of ​​the probe tip is its small binding capacity. Therefore, when the probe tip is immersed in the reagent solution, the binding of the reagent does not consume a large amount of reagent. The reagent concentration does not change practically. The negligible contamination of the washing solution and the low consumption of reagent allow the reagent and washing solution to be reused multiple times, for example, 3-10 times or 3-20 times.

[0063] Methods for immobilizing haptens onto a solid phase (the sensing surface of a probe tip) are common in immunochemistry and involve forming covalent, hydrophobic, or electrostatic bonds between the solid phase and the hapten. For example, haptens can be conjugated to carrier proteins by adsorption onto a solid surface or by covalent binding to aminopropylsilane coated on a solid surface, thus immobilizing the hapten protein.

[0064] In step (b), the fluorescence signal at the probe tip is pre-read by a fluorescence detection system in the read container (or read chamber or read orifice). The read container contains an aqueous solution, such as water or a buffer solution with a pH between 6.0 and 8.5. Preferably, the aqueous solution contains 1-10 mM or 1-100 mM phosphate buffer, Tris buffer, citrate buffer, or other buffer suitable for neutralizing the probe after low-pH regeneration with a pH between 6.0 and 8.5.

[0065] A preread is required before the first sample binding to establish a baseline for any potential background fluorescence in the first cycle. A preread is also required after probe tip regeneration and before the next sample binding to establish a baseline for subsequent cycles. Ideally, the preread signal should be the same after each cycle. If the preread signal is higher or lower than the preread signal of the previous cycle, subtracting the preread fluorescence signal from the measured fluorescence signal after each immunoassay in each cycle, and then quantifying the subtracted signal against a calibration curve, will still provide accurate quantification of the analyte.

[0066] In step (c) of the method, the probe tip is immersed in a dual-antibody container containing a dual-antibody solution. The dual-antibody solution contains an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is a first antibody against the analyte. Alternatively, the dual-antibody solution contains a bispecific antibody that binds to both the hapten and the analyte simultaneously.

[0067] In one embodiment, the anti-hapten antibody and the capture antibody are directly linked to each other without a connector.

[0068] In a preferred embodiment, both the anti-hapten antibody and the capture antibody are covalently linked to a polymer, which acts as a linker or spacer. The polymer typically has a molecular weight of 1,000 to 500,000 Daltons. The polymer can be a polysaccharide (e.g., dextran, amylose), a dendritic compound, or polyethylene glycol. In a preferred embodiment, the polymer is... (A copolymer of sucrose and epichlorohydrin).

[0069] In step (d) of the method, the probe tip is immersed in a sample container (or sample chamber or sample well) and incubated for 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 1 minute to bind the analyte to the first antibody on the probe tip.

[0070] Following step (d), the probe is optionally washed 1-5 times, preferably 1-3 times, in a washing container (or washing chamber or washing port) containing a washing solution. This additional washing step may not be necessary because the amount of residual solution is minimal due to the small binding surface area. The washing solution typically contains a buffer solution and a surfactant such as Tween 20.

[0071] In step (e) of the method, the probe tip is immersed in a reagent container (or reagent chamber or reagent well) for 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 1 minute to bind the reagent to the analyte on the probe tip. The reagent solution comprises a fluorescently labeled secondary antibody (signal antibody). Any suitable fluorescent label can be used in this method. Examples of fluorescent labels are arylsulfonate cyanine fluorescent dyes as described below: Mujumdar et al., (1993) *Bioconjugate Chemistry* 4:105-111; Southwick et al., (1990) *Cytometry* 11:418-430; and U.S. Patent No. 5,268,486. Cy5 is a preferred arylsulfonate cyanine fluorescent dye because of its high extinction coefficient and good quantum yield; it also has a fluorescence emission spectrum in the range of 500 nm to 750 nm, beyond the autofluorescence wavelengths of most biomaterials and plastics. In addition, Cy5 has good solubility in water and low non-specific binding properties.

[0072] Fluorescent labels can be covalently bound to signal antibodies using conventional conjugation chemistry as described in scientific and patent literature, through multiple motifs including disulfide bonds, hydroxyphenyl, amino, carboxyl, indole, or other functional groups. Exemplary techniques for binding arylsulfonate anthocyanin fluorescent dye labels to antibodies and other proteins are described in U.S. Patent Nos. 5,268,486 and 5,650,334, the contents of which are incorporated herein by reference. Techniques for linking preferred Cy5 fluorescent labels to antibodies are described in a technical publication, catalog number A25000, published by Biological Detection Systems, Inc., Pittsburgh, PA.

[0073] Following step (f), the probe is washed 1-5 times, preferably 1-3 times, in a washing container containing a washing solution. The washing solution typically contains a buffer solution and a surfactant such as Tween 20.

[0074] In step (g), the probe remains in the washing container or moves to the measurement container, and the fluorescence signal of the bound immune complex is detected by the fluorescence detection system as described above, wherein the light source and detector are mounted on the same side (proximal side) of the sensing surface of the probe. The measurement container may be a separate orifice or may be the same readout container.

[0075] Alternatively, the method of the present invention can be used for detection by other suitable fluorescence detection systems.

[0076] The concentration of the analyte in the sample is determined by measuring the fluorescence signal of the immune complex at the tip of the probe, subtracting the pre-read fluorescence signal from (b), and then quantifying against a calibration curve (standard curve).

[0077] The calibration curve is typically established in advance according to methods well known to those skilled in the art before measuring the sample. In a preferred embodiment, the fluorescence signal of the same sample (after subtracting the pre-read signal) is kept constant in each cycle, and the calibration curve is the same for each cycle.

[0078] In another embodiment, the fluorescence signal of the same sample (after subtracting the pre-read signal) increases or decreases in each cycle, and a cycle-specific calibration curve needs to be established for each cycle. In these cases where the fluorescence signal varies, the sample is quantified against the cycle-specific calibration curve, and the quantification results remain consistent despite the increase or decrease in fluorescence signal across different cycles.

[0079] In step (h), the probe is regenerated by employing denaturing conditions that dissociate the immune complex bound to the captured antibody on the solid phase. Typically, acids or acidic buffers with a pH of about 1 to about 4 are effective for regenerating the antibody probes of the present invention. For example, hydrochloric acid, sulfuric acid, nitric acid, or acetic acid can be used to regenerate the probe. The probe is first treated with acidic conditions and then neutralized with a neutral aqueous solution, such as a buffer with a pH between 6.0 and 8.5. In one embodiment, the probe treated with a low pH is conveniently neutralized in the reading container of step (b) prior to pre-reading. Alternatively, the probe treated with a low pH can be neutralized in a separate container with a buffer having a pH of 6.0 to 8.5. The regeneration process can be a single acidic treatment followed by neutralization. For example, a single exposure range of pH 1-3, or pH 1.5-2.5 (e.g., pH 2), for 10 seconds to 2 minutes is effective. The regeneration process can also be a “pulse” regeneration step, in which the probe is exposed to short pH treatments (e.g., 10-20 seconds) for 2-5 cycles (e.g., 3 cycles) and then neutralized at pH 6.5-8.0 (e.g., 10-20 seconds).

[0080] After probe regeneration, steps (b)-(h) are repeated 1-10, 1-20, 1-25, 3-20, 5-10, 5-20, 5-25, or 5-30 times with the same probe and reagents in subsequent cycles for different samples. In each cycle, the regenerated probe contains fresh double antibodies captured on it.

[0081] In one embodiment, the reaction is accelerated by stirring or mixing the solution in the container. For example, the solution can be induced to flow across the probe tip, such as lateral flow or orbital flow, in one or more reaction containers, including sample containers, reagent containers, washing containers, and regeneration containers, to accelerate binding and dissociation reactions. For example, the reaction container can be mounted on an orbital oscillator, and the orbital oscillator can rotate at a speed of at least 50 rpm, preferably at least 200 rpm or at least 500 rpm, such as 50-200 or 500-1,500 rpm. Additionally, the probe tip can move up and down at a speed of 0.01 to 10 mm / s and perpendicular to the plane of the orbital flow to induce additional mixing of the solution above and below the probe tip.

[0082] Examples shown 2

[0083] In a second embodiment for detecting the analyte, the method is similar to that of the first embodiment, except that steps (b) and (c) are in the reverse order. The method of the second embodiment includes the following steps: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) immersing the probe tip in a biantibody solution comprising biantibodies, the biantibody comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is a first antibody against the analyte;

[0084] (c) Immersing the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip; (d) Immersing the probe tip in a sample solution comprising a liquid sample containing the analyte; (e) Immersing the probe tip in a reagent solution comprising a signal antibody conjugated with a fluorescent label to form an immune complex comprising the analyte, a capture antibody, and a signal antibody at the probe tip, wherein the signal antibody is a secondary antibody against the analyte; (f) Immersing the probe tip in a washing solution; (g) Determining the concentration of the analyte in the sample by measuring the fluorescence signal of the immune complex at the probe tip, subtracting the pre-read fluorescence signal from (c), and quantifying the subtracted signal against a calibration curve; (h) Immersing the probe tip in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (i) Repeating steps (b)-(h), but using a fresh sample solution containing the fresh sample in step (d), thereby detecting the analyte in each of the plurality of liquid samples.

[0085] The details of each step are similar to those described above in the first embodiment.

[0086] In a second embodiment, the biantibody conjugate optionally includes a second fluorescent label that differs from the signal fluorescent label in terms of excitation and emission wavelengths. The pre-read in step (b) determines the signal of the second fluorescent label, indicating whether, after multiple sample reactions, the recycled probe still provides acceptable binding of the biantibody conjugate to the hapten-immobilized probe tip. If the probe is masked by interfering material after multiple sample reactions, the pre-read of the second fluorescent signal in step (b) will show a significant decrease in the signal of the second fluorescent label, and the assay should be repeated using a fresh probe.

[0087] II. Amplification using the second hapten and anti-second hapten antibody or streptavidin

[0088] The method described below adds an amplification step to Recycling Protocol I by amplifying the fluorescent signal using a second hapten and an anti-second hapten antibody or streptavidin. The second hapten is different from the hapten immobilized on the probe tip. The details of each step are the same as or similar to those of the corresponding steps described in Recycling Protocol I above.

[0089] The method includes the following steps: (a) immersing a probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip, wherein the probe includes a first hapten immobilized on the probe tip, and the diameter of the tip surface is ≤5 mm; (b) forming a first immune complex on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-first hapten antibody against the first hapten, and the signal antibody is conjugated to a second hapten, the first hapten and the second hapten being different; (c) immersing the probe tip in a washing solution; (d) immersing the probe tip in an aqueous solution containing a fluorescently labeled... (i) an amplification solution of anti-second hapten antibody or (ii) streptavidin to form a second immune complex comprising analyte, capture antibody, signal antibody, second hapten, and anti-second hapten antibody or streptavidin at the probe tip; (e) determining the concentration of analyte in the sample by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (a), and quantifying the subtracted signal against a calibration curve; (f) immersing the probe tip in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (g) repeating the same steps (a)-(f), but using the analyte from a new sample in step (b), thereby detecting the analyte in each of the plurality of liquid samples.

[0090] In one embodiment, the first immune complex in step (b) is formed by: (b1) mixing a sample solution with a dual antibody solution and a reagent solution to form a mixture, wherein the sample solution includes an analyte, the dual antibody solution includes the anti-first hapten antibody covalently linked to the capture antibody, and the reagent solution includes a signal antibody conjugated to the second hapten; and (b2) immersing the probe tip in the mixture to form the first immune complex on the probe tip.

[0091] In another embodiment, the first immune complex in step (b) is formed by: (b1) immersing the probe tip in a biantibody solution comprising the anti-first hapten antibody covalently linked to the capture antibody; (b2) immersing the probe tip in a sample solution comprising the sample; and (b3) immersing the probe tip in a reagent solution comprising a signal antibody conjugated to the second hapten to form the first immune complex on the probe tip.

[0092] IIA. Amplification using the second hapten and anti-second hapten antibody.

[0093] In this protocol, the fluorescence signal is amplified using the second hapten and anti-second hapten antibody.

[0094] The method includes the following steps: (a) immersing a probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip, wherein the probe includes a first hapten immobilized on the probe tip, and the diameter of the tip surface is ≤5 mm; (b) forming a first immune complex on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-first hapten antibody against the first hapten, and the signal antibody is conjugated to a second hapten, the first hapten and the second hapten being different; (c) immersing the probe tip in a washing solution; (d) immersing the probe tip in an aqueous solution containing a fluorescent label. (e) A second immune complex comprising the analyte, the capture antibody, the signal antibody, the second hapten, and the anti-second hapten antibody is formed on the probe tip in an amplification solution of the conjugated anti-second hapten antibody; (e) the concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (a), and quantifying the subtracted signal against a calibration curve; (f) the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (g) the same steps (a)-(f) are repeated, but the analyte from a new sample is used in step (b), thereby detecting the analyte in each of the plurality of liquid samples.

[0095] In step (f), the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0. Optionally, the acidic solution may include a denaturing agent or a dissociating agent. Optionally, the probe may be further immersed in a denaturing agent or a dissociating agent. Denaturing agents or dissociating agents may include, for example, dimethyl sulfoxide (DMSO), n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea for further elution of immune complexes. DMSO and guanidine hydrochloride are preferred denaturing agents or dissociating agents.

[0096] In one embodiment, the first immune complex in step (b) is formed by: (b1) mixing a sample solution with a dual antibody solution and a reagent solution to form a mixture, wherein the sample solution includes an analyte, the dual antibody solution includes the anti-first hapten antibody covalently linked to the capture antibody, and the reagent solution includes a signal antibody conjugated to the second hapten; and (b2) immersing the probe tip in the mixture to form the first immune complex on the probe tip.

[0097] In another embodiment, the first immune complex in step (b) is formed by: (b1) immersing the probe tip in a biantibody solution comprising the anti-first hapten antibody covalently linked to the capture antibody; (b2) immersing the probe tip in a sample solution comprising the sample; and (b3) immersing the probe tip in a reagent solution comprising a signal antibody conjugated to the second hapten to form the first immune complex on the probe tip.

[0098] Example 1 shown

[0099] For example, the method includes the following steps: (a) obtaining a probe having a first hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) immersing the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal of the probe tip; (c) immersing the probe tip in a double antibody solution comprising an anti-first hapten antibody covalently linked to a capture antibody, wherein the capture antibody is a first antibody against the analyte; (d) immersing the probe tip in a sample solution comprising a liquid sample containing the analyte; and (e) immersing the probe tip in a reagent solution comprising a signal antibody conjugated to a second hapten to form a first immune complex on the probe tip comprising the analyte, the capture antibody, and the signal antibody, wherein the signal antibody is a second antibody against the analyte, and the first hapten and the... The second hapten is different; (f) the probe tip is immersed in a washing solution; (g) the probe tip is immersed in an amplification solution comprising an anti-second hapten antibody conjugated with a fluorescent label to form a second immune complex on the probe tip comprising the analyte, the capture antibody, the signal antibody, the second hapten, and the anti-second hapten antibody; (h) the concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (b), and quantifying the subtracted signal against a calibration curve; (i) the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (j) the same steps (b)-(i) are repeated, but in step (d) a new sample solution containing the new sample is used, thereby detecting the analyte in each of the plurality of liquid samples. This embodiment is shown in Figure 10 In this context, biotin is the second half antigen.

[0100] Example 2 shown

[0101] In this embodiment, the method includes the following steps: (a) obtaining a probe having a first hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) immersing the probe tip in a biantibody solution comprising biantibodies, the biantibody comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is a first antibody against the analyte; (c) immersing the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal of the probe tip; (d) immersing the probe tip in a sample solution comprising a liquid sample containing the analyte; and (e) immersing the probe tip in a reagent solution comprising a signal antibody conjugated to a second hapten to form a first immune complex comprising the analyte, the capture antibody, and the signal antibody on the probe tip, wherein the signal antibody is a second antibody against the analyte. The first hapten and the second hapten are different; (f) the probe tip is immersed in a washing solution; (g) the probe tip is immersed in an amplification solution comprising an anti-second hapten antibody conjugated with a fluorescent label to form a second immune complex comprising the analyte, the capture antibody, the signal antibody, the second hapten, and the anti-second hapten antibody on the probe tip; (h) the concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (c), and quantifying the subtracted signal against a calibration curve; (i) the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (j) the same steps (b)-(i) are repeated, but in step (d) a new sample solution containing the new sample is used, thereby detecting the analyte in each of the plurality of liquid samples.

[0102] In both amplification methods, an anti-second hapten antibody and a fluorescently labeled conjugate can be combined with... Further conjugation was performed to increase the loading of anti-second hapten antibody and fluorescent label.

[0103] IIB. Amplification using biotin and streptavidin

[0104] The methods described below add an amplification step to recycling protocol I by amplifying the fluorescent signal using biotin and streptavidin. In these methods, the hapten on the probe tip is a non-biotinylated hapten. Using streptavidin amplification reagent requires more stringent elution conditions, and dimethyl sulfoxide (DMSO) is used as a second eluent after acidic elution.

[0105] The method includes the following steps: (a) immersing a probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip, wherein the probe comprises a non-biotinylated hapten immobilized on the probe tip, and the diameter of the tip surface is ≤5 mm; (b) forming a first immune complex on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody, and the signal antibody is conjugated to a biotin; (c) immersing the probe tip in a washing solution; (d) immersing the probe tip in a solution comprising streptavidin conjugated to a fluorescent label. (e) A second immune complex comprising the analyte, the capture antibody, the signal antibody, the biotin, and streptavidin is formed on the probe tip in the amplification solution; (e) the concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (a), and quantifying the subtracted signal against a calibration curve; (f) the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (g) the same steps (a)-(f) are repeated, but the analyte from a new sample is used in step (b), thereby detecting the analyte in each of the plurality of liquid samples.

[0106] In step (f), after immersing the probe tip in an acidic solution with a pH of about 1.0-4.0, the probe is optionally further immersed in a denaturing agent or dissociating agent for further eluting the immune complex, such as dimethyl sulfoxide (DMSO), n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. DMSO is a preferred dissociating agent.

[0107] In one embodiment, the first immune complex in step (b) is formed by: (b1) mixing a sample solution with a biantibody solution and a reagent solution to form a mixture, wherein the sample solution includes an analyte, the biantibody solution includes the anti-hapten antibody covalently linked to the capture antibody, and the reagent solution includes a biotin-conjugated signal antibody; and (b2) immersing the probe tip in the mixture to form the first immune complex on the probe tip.

[0108] In one embodiment, the immune complex of step (b) is formed by: (b1) immersing the probe tip in a biantibody solution comprising the anti-hapten antibody covalently linked to the capture antibody; (b2) immersing the probe tip in a sample solution comprising the sample; and (b3) immersing the probe tip in a reagent solution comprising the signal antibody conjugated to the biotin to form the first immune complex on the probe tip.

[0109] Example 1 shown

[0110] In one embodiment, the method includes the following steps: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm and the hapten is not biotin; (b) immersing the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal of the probe tip; (c) immersing the probe tip in a double antibody solution comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is an antibody against an analyte; (d) immersing the probe tip in a sample solution containing a liquid sample having the analyte; (e) immersing the probe tip in a reagent solution comprising a signal antibody conjugated with biotin to form a first immune complex on the probe tip comprising the analyte, the capture antibody, and the signal antibody, wherein the signal antibody is a second antibody against the analyte; (f) immersing the probe tip in a reagent solution comprising a signal antibody conjugated with biotin to form a first immune complex comprising the analyte, the capture antibody, and the signal antibody, wherein the signal antibody is a second antibody against the analyte; The probe tip is immersed in a washing solution; (g) the probe tip is immersed in an amplification solution comprising streptavidin conjugated with a fluorescent label to form a second immune complex comprising the analyte, the capture antibody, the signal antibody, the biotin, and the streptavidin on the probe tip; (h) the concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (b), and quantifying the subtracted signal against a calibration curve; (i) the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0, optionally further immersed in a dissociative agent, such as dimethyl sulfoxide, for eluting the immune complex from the probe tip; and (j) the same steps (b)-(i) are repeated, but in step (d) a fresh sample solution comprising a new sample is used, thereby detecting the analyte in each of the plurality of liquid samples. This embodiment is shown in Figure 12 and 14 middle.

[0111] Example 2 shown

[0112] In another embodiment, the method includes the following steps: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm and the hapten is not biotin; (b) immersing the probe tip in a dual antibody container containing a dual antibody solution comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is a first antibody against the analyte; (c) immersing the probe in a prereading container comprising an aqueous solution with a pH of 6.0-8.5 to preread the fluorescence signal of the probe tip; (d) immersing the probe tip in a sample solution containing a liquid sample having the analyte; and (e) immersing the probe tip in a reagent solution comprising a biotin-conjugated signaling antibody to form a first immune complex on the probe tip comprising the analyte, the capture antibody, and the signaling antibody, wherein the signaling antibody is against the analyte. (b) immersing the probe tip in a washing solution; (c) immersing the probe tip in an amplification solution comprising streptavidin conjugated with a fluorescent label to form a second immune complex comprising the analyte, the capture antibody, the signal antibody, the biotin, and the streptavidin on the probe tip; (d) determining the concentration of the analyte in the sample by measuring the fluorescence signal of the second immune complex at the probe tip, subtracting the pre-read fluorescence signal from (b), and quantifying the subtracted signal against a calibration curve; (i) first immersing the probe tip in an acidic solution with a pH of about 1.0-4.0, optionally further immersing it in a dissociative agent, such as dimethyl sulfoxide, for eluting the immune complex from the probe tip; and (j) repeating the same steps (b)-(i), but using a fresh sample solution comprising a new sample in step (d), thereby detecting the analyte in each of the plurality of liquid samples.

[0113] In the amplification of the second embodiment, the biantibody conjugate optionally includes a second fluorescent label that differs from the signal fluorescent label in terms of excitation and emission wavelengths.

[0114] In both amplification methods, streptavidin and fluorescently labeled conjugates can be combined with... Further conjugation was performed to increase the loading of streptavidin and fluorescent markers.

[0115] III. Two-read scheme

[0116] The two-read protocol reads the first fluorescence signal from the second immune complex at the probe tip, then reads the second fluorescence signal from the acid-treated probe tip, and then subtracts the second fluorescence signal from the first fluorescence signal to quantify the subtracted signal against a calibration curve. Using this two-read and subtraction method, the background fluorescence signal remains low for more than 10 cycles. Low background improves the detection of low-concentration samples and increases the sensitivity of the assay.

[0117] The details of each step are the same as or similar to those of the corresponding steps described in Recycling Scheme I above.

[0118] The method includes the following steps: (a) obtaining a probe having a non-biotinylated hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) forming a first immune complex on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody, and the signal antibody is conjugated to a biotin; (c) immersing the probe tip in a washing solution; and (d) immersing the probe tip in an amplification solution comprising streptavidin conjugated to a fluorescent label to form on the probe tip the analyte, the capture antibody, and the signal antibody. (e) A signal antibody, a second immune complex of biotin and streptavidin; (f) Reading a first fluorescence signal of the second immune complex at the tip of the probe; (g) Immersing the probe tip in an acidic solution with a pH of about 1.0-4.0; (h) Reading a second fluorescence signal of the acid-treated probe tip; (i) Subtracting the second fluorescence signal from the first fluorescence signal and quantifying the subtracted signal against a calibration curve; (j) Immersing the probe tip in a plasma such as dimethyl sulfoxide; and (b) Repeating the same steps (i) but using a new sample solution including a new sample in step (b), thereby detecting the analyte in each of the plurality of liquid samples.

[0119] In one embodiment, the first immune complex in step (b) is formed by: (b1) mixing a sample solution with a biantibody solution and a reagent solution to form a mixture, wherein the sample solution comprises an analyte, the biantibody solution comprises the anti-hapten antibody covalently linked to the capture antibody, and the reagent solution comprises a biotin-conjugated signal antibody; and (b2) immersing the probe tip in the mixture to form the first immune complex on the probe tip. This embodiment is shown in Figure 16A middle.

[0120] In one embodiment, the immune complex of step (b) is formed by: (b1) immersing the probe tip in a biantibody solution comprising the anti-hapten antibody covalently linked to the capture antibody; (b2) immersing the probe tip in a sample solution comprising the sample; and (b3) immersing the probe tip in a reagent solution comprising the signal antibody conjugated to the biotin, to form the first immune complex on the probe tip. This embodiment is shown in Figure 16B middle.

[0121] For example, the method includes the following steps: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm, and the hapten is not biotin; (b) immersing the probe tip in a biantibody solution comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is an antibody against an analyte; (c) immersing the probe tip in a sample solution comprising a liquid sample containing an analyte; (d) immersing the probe tip in a reagent solution comprising a biotin-conjugated signal antibody to form a first immune complex on the probe tip comprising the analyte, the capture antibody, and the signal antibody, wherein the signal antibody is a second antibody against the analyte; (e) immersing the probe tip in a washing solution; (f) immersing the probe tip in a solution comprising... In an amplification solution of streptavidin conjugated with a fluorescent label, a second immune complex comprising the analyte, the capture antibody, the signal antibody, the biotin, and the streptavidin is formed on the probe tip; (g) a first fluorescence signal of the second immune complex at the probe tip is read; (h) the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0; (i) a second fluorescence signal of the acid-treated probe tip is read; (j) the second fluorescence signal is subtracted from the first fluorescence signal, and the subtracted signal is quantified against a calibration curve; (k) the probe tip is immersed in a plasma such as dimethyl sulfoxide; and (j) steps (b)-(k) are repeated, but in step (c) a fresh sample solution comprising a new sample is used, thereby detecting the analyte in each of the plurality of liquid samples. This embodiment is shown in Figure 16B middle.

[0122] In step (h), the acidic solution may further include a denaturing agent or dissociating agent, such as guanidine chloride, with a concentration of 2M-8M or 5-7M, for example 6M.

[0123] In the two-read protocol, it is optional to preread the probe's fluorescence signal in each cycle before immersing the probe in the sample solution. The probe can be preread before step (b) or after step (b) but before step (c).

[0124] In the two-read scheme, streptavidin and fluorescently labeled conjugates can be combined with... Further conjugation was performed to increase the loading of streptavidin and fluorescent markers.

[0125] IV. Dual-denaturant regimen

[0126] For some proteins that are sticky or prone to aggregation, using two different denaturing agents helps remove immune complexes formed on the probe after each immune response cycle.

[0127] The inventors have found that elution with guanidine chloride (guanidine hydrochloride) at an acidic pH (pH 1-4 or 1.5 to 2.5), followed by elution with DMSO, is effective for removing the immune complexes bound after each cycle and providing regenerated probes with good performance.

[0128] In one embodiment, the method includes the following steps: (a) immersing a probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip, wherein the probe includes a hapten immobilized on the probe tip, and the diameter of the tip surface is ≤5 mm; (b) forming an immune complex on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody against the hapten, and the signal antibody is conjugated to a fluorescent label; (c) immersing the probe tip in a washing solution. In a container; (d) reading a first fluorescence signal of the immune complex at the tip of the probe; (e) immersing the probe tip in an acidic solution containing guanidine chloride at a pH of about 1.0-4.0; (f) reading a second fluorescence signal of the acid-treated probe tip; (g) subtracting the second fluorescence signal from the first fluorescence signal and quantifying the subtracted signal against a calibration curve; (h) immersing the probe tip in a plasma such as dimethyl sulfoxide; and (i) repeating the same steps (b)-(h), but using a new sample solution including a new sample in step (b), thereby detecting the analyte in each of the plurality of liquid samples.

[0129] In one embodiment, the immune complex of step (b) is formed by: (b1) mixing a sample solution with a biantibody solution and a reagent solution to form a mixture, wherein the sample solution comprises an analyte, the biantibody solution comprises the anti-hapten antibody covalently linked to the capture antibody, and the reagent solution comprises a signal antibody conjugated to a fluorescent label; and (b2) immersing the probe tip in the mixture to form the immune complex on the probe tip. The probe may be immersed in the mixture immediately upon addition of the components together. Alternatively, the probe may be immersed in the mixture after incubating the mixture for a period of time, for example, 30 seconds to 15 minutes.

[0130] In one embodiment, the immune complex in step (b) is formed by: (b1) immersing the probe tip in a biantibody solution comprising the anti-hapten antibody covalently linked to the capture antibody; (b2) immersing the probe tip in a sample solution comprising the sample; and (b3) immersing the probe tip in a reagent solution comprising a signal antibody conjugated to a fluorescent label to form the immune complex on the probe tip. This embodiment is shown in Figure 22 middle.

[0131] In step (e), the concentration of guanidine chloride is typically 2-8M, or 3-7M, or 4-6M, for example 6M.

[0132] The details of each step are the same as or similar to those of the corresponding steps described in the above recycling schemes I-III.

[0133] In all the methods described above, the reagent containers and amplification containers are optionally coated with a layer of mineral oil to prevent or reduce evaporation of the solution within the container, which can increase the concentration of the signal antibody conjugate or amplification conjugate. Typically, the volume of the solution in the container is about 50-300 μL, preferably 100-200 μL. The volume of the mineral oil layer is typically 20-80 μL or 30-50 μL. Mineral oil is generally used to minimize evaporation and subsequent condensation in the PCR sample tube. The inventors have demonstrated that the probe and immune complex at the probe tip are unaffected by penetration through the mineral oil layer.

[0134] Probes including immobilized haptens

[0135] This invention utilizes a probe comprising a hapten fixed to its tip, wherein the aspect ratio of the probe's length to width is at least 5:1, the diameter of the probe tip surface is ≤5 mm, and the hapten substantially does not dissociate from the probe after acid treatment; that is, after 1-20 cycles of acid treatment, no more than 15%, preferably no more than 10% or 5%, of the hapten dissociates from the probe. Acid treatment is typically performed by immersing the probe in a low-pH buffer (pH 1-4, or 1-3, or 1.5-2.5) for 10 seconds to 2 minutes.

[0136] The invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention to the specific procedures described herein.

[0137] Example

[0138] Example 1: Hapten and fluorescent dye labeling

[0139] BSA was labeled with fluorescein (F) using standard methods. Biotin (B) was linked to anticalcitonin procalcitonin (PCT) and antitroponin I (TnI), both from Hytest Ltd., using standard methods. Digitoxadixyl (Dig) NHS (Sigma-Aldrich) was reacted with BSA at room temperature (RT) at a 10-molar coupling ratio (MCR) for 1 hour, followed by purification on a Sephadex G25 (GE Healthcare) column. Cy5 tm NHS (GE Healthcare) reacted anti-PCT antibodies, anti-TnI antibodies, anti-B antibodies (Jackson Immunoresearch), or streptavidin (Prozyme) with PBS at pH 7.4 at 10 MCR for 1 hour at RT, followed by G25 column purification. Anti-PCT-labeled Alexa Fluor was then reacted with PBS at pH 7.4 at 10 MCR at RT. TM The sample was purified on a 647 NHS (Thermo Fisher Scientific) column for 1 hour, followed by G-24 column purification.

[0140] Example 2A: Antibody-antibody conjugation

[0141] The conjugation of anti-F (Jackson Immunological Research, Inc.) with anti-PCT followed the established SMCC (4-[N-maleimidemethyl]cyclohexane-1-carboxylic acid succinimide) conjugation chemistry (Hermanson, GT (2008), Bioconjugate Techniques, 2nd ed., Academic Press, New York). SMCC (Sigma-Aldrich, Inc.) was reacted with anti-F in PBS, pH 7.4, at MCR 0f 15. After 1 hour at room temperature, the antibody was purified using a G-25 column. Anti-PCT was reacted with 6-[3-[2-pyridyldithio]propamido]hexanoic acid succinimide (SPDP, Sigma-Aldrich, Inc.) in PBS, pH 7.4, at RT for 1 hour at MCR 15, followed by purification using a G-25 column. The reaction of anti-PCT-SPDP with dithioether alcohol (DTT, Sigma-Aldrich) deprotected the thiol (SH) group, and after removing DTT by G-25 chromatography, anti-PCT-SH was mixed with anti-F-SMCC and allowed to react overnight at RT. The resulting anti-F-anti-PCT conjugate was purified using a Sepharose 4B CL (GE Healthcare) column. Anti-F conjugated with anti-TnI and anti-Dig (Jackson Immunological Research) conjugated with anti-PCT followed the same procedure.

[0142] Example 2B: with Antibody-antibody conjugation

[0143] The alternative biantibody conjugates against hapten and anti-PCT were prepared as follows. 2 mg of amino... The material was reacted with PBS (Skold Technology) at pH 7.4 and SPDP (Thermo Fisher Scientific) at a molar coupling ratio of 10:1 for 1 hour, followed by overnight dialysis. Deprotection of the thiol was induced by the addition of 30 μl of DTT (Thermo Fisher Scientific). After 1 hour, the material was purified on a PD10 column.

[0144] 2 mg of anti-hapten - antifluorescein (Jackson Immunological Research) or anti-digitoxin (Thermo Fisher Scientific) was mixed with 2 mg of anti-PCT (Hytest) and reacted with SMCC (Thermo Fisher Scientific) at a molar coupling ratio of 15:1 for 1 hour, and then purified on a PD10 column.

[0145] Mix the SMMC-labeled antibody mixture with thiol-modified... The reaction was allowed to proceed overnight, followed by purification on an agarose CL-6B column (GE Healthcare) to prepare a compound containing antifluorescein and antiPCT. This method is also used to prepare other biantibody conjugates, such as antifluorescein and antiTnI.

[0146] Example 3: Cross-linked preparation

[0147] Cross-linked Cy5-anti-F crosslinking Cross-linked with Cy5-streptavidin The preparation followed the procedure described in U.S. Patent No. 8,492,139. ​​Amination was performed with 88 amines per 20 mg / ml PBS. 400kD (Schkold Technologies) 2ml 400 mg (Sigma / Aldrich) was added to 10 μL of SPDP (DMF) at a concentration of 50 mg / mL (N,N-dimethylformamide). SPDP with The MCR was 15. The mixture was reacted at room temperature for 1 hour and then dialyzed. The thiol incorporation was estimated according to standard methods to be approximately 1. 400kD 5.5.

[0148] In order to make SPDP marking Thiol deprotection was performed on a 400 column by adding 30 μL of DTT in 38 mg / ml PBS to 1 ml PBS and allowing the reaction to proceed at room temperature for two hours. The reaction was then carried out on a PD10 column. Purification is performed.

[0149] The following describes the interaction between SMCC and amination. 400 (88 amines / ) Ligation: Add 10 mg of amination solution to 1 ml of PBS. 400 was mixed with 25 μL of MCC (Pierce Chemical) at a concentration of 10 mg / ml DMF. The MCR was 30. The mixture was allowed to react at room temperature for 1 hour and then purified on a PD10 column (GE Healthcare).

[0150] In order to 400 and 400 crosslinking, add 10 mg of SH- in 1 ml of PBS. 400 mg in 1 ml PBS Mix at 400°C. Allow the mixture to react overnight at RT.

[0151] To provide for subsequent cross-linking of antibodies or streptavidin. 400 conjugated linker sites were identified, and then the residual amine was reacted with excess SPDP. 20 mg of cross-linked... Mix 400 mg / ml DMF with 75 μL of PDP. Allow the mixture to react at room temperature for 1 hour, then dialyze against PBS.

[0152] Then, the SPDP-labeled crosslinks were crosslinked on a Sepharose 4B CL column. The 400 formulation was purified.

[0153] Example 4: Cy5-antibiotic cross-linked Preparation

[0154] Mix 1.5 mg / ml Cy5-anti-B in 1 ml PBS with 1.9 μL SMCC containing 5 mg / ml DMF and react at room temperature for 1 hour, then purify on a PD 10 column.

[0155] By adding 30 μL of DTT at a concentration of 38 mg / ml in 1 ml of PBS to 0.7 mg of crosslinked... The crosslinked material was reacted in 400-SPDP at room temperature for 1 hour, and then crosslinked by a PD 10 column. Purification is performed to crosslink the Thiol deprotection on 400-SPDP.

[0156] Enabling Cy5-anti-B-SMCC to crosslink The mixture was prepared in 400-SH and reacted overnight at room temperature. Then, 10 μL of NEM (N-ethyl-maleimide, Aldrich) at 12.5 mg / mL was added and the mixture was reacted at room temperature for 1 / 2 h. The conjugate was then purified on a Sepharose 4B CL column.

[0157] Example 5: Cy5-streptavidin cross-linked Preparation

[0158] Mix 1.5 mg / ml Cy5-streptavidin (SA) in 1 ml PBS with 5 μL SMCC containing 5 mg / ml DMF and react at room temperature for 1 hour, then purify on a PD 10 column.

[0159] By adding 30 μL of DTT at a concentration of 38 mg / ml in 1 ml of PBS to 0.7 mg of crosslinked... The crosslinked material was reacted in 400-SPDP at room temperature for 1 hour, and then crosslinked by a PD 10 column. Purification is performed to crosslink the Thiol deprotection on 400-SPDP.

[0160] To make Cy5-SA-SMCC cross-linked with The mixture was prepared in 400-SH and reacted overnight at room temperature. Then, 10 μL of NEM (N-ethyl-maleimide, Aldrich) at 12.5 mg / mL was added and the mixture was reacted at room temperature for 1 / 2 h. The conjugate was then purified on a Sepharose 4B CL column.

[0161] Example 6: Preparation of hapten-coated probes

[0162] Following the manufacturer's instructions, a quartz probe with a diameter of 1 mm and a length of 2 cm was coated with aminopropylsilane using a chemical vapor deposition process (Yield Engineering Systems, 1224P). The probe tip was then immersed in a solution of 30 μg / ml F-bovine serum albumin (BSA) or Dig-BSA in PBS at pH 7.4. After allowing BSA to adhere to the probe for 10 minutes, the probe tip was washed with PBS.

[0163] Example 7: Recirculating Immunoassay

[0164] 120 μL of reagent and sample were measured using 5 mg / ml PHS pH 7.4, 0.05% Tween 20 as buffer with 40 μL of mineral oil added. Wash buffer was 200 μL PBS pH 7.4, 0.05% Tween 20. The probe was held immobilized with microwells fixed on a track-guided oscillator platform with a diameter stroke of 1 mm. Track-guided flow was used to accelerate binding kinetics. Protocols A, B, C, and D describe detailed information on the binding assay procedure, timing, flow rate, and reagent concentrations. Elution reagents were 10 mM glycine / HCl, 200 μL; anhydrous DMSO (Sigma-Aldrich), 200 μL; and anhydrous DMSO plus 0.5 mg / ml sodium borohydride (Sigma-Aldrich).

[0165] The probe recirculation measurement format and measurement data are shown in Figure 1-16. Figure 17 The optical configuration used to detect fluorescence at the probe tip is shown. Subtracting the preread from the readout generates an immune-specific signal.

[0166] Example 8: Measurement Protocol

[0167] Scheme A, v1 (Table 1) consists of an anti-hapten antibody and a capture antibody linked together without spacers.

[0168] All other biantibody conjugates in this example (Table 2-11) include those obtained through cross-linking. Anti-hapten antibody and capture antibody linked together by 400 spacers.

[0169] Washing solution = PBS with Tween-20

[0170] Table 1

[0171]

[0172] Table 2

[0173]

[0174] Table 3

[0175]

[0176] Table 4

[0177]

[0178] Table 5

[0179]

[0180] Table 6

[0181]

[0182] Table 7

[0183]

[0184] Table 8

[0185]

[0186] Table 9

[0187]

[0188] Table 10

[0189]

[0190] Table 11

[0191]

[0192] Example 9: PCT Measurement Results (Program A, v1)

[0193] Figure 2 This shows that following scheme A, v1 utilizes Figure 1BData from PCT assays performed using the fluorophore probe format described in [the original text]. PCT samples from negative, 10 ng / ml, and 100 ng / ml samples showed consistent fluorescence signals throughout 10 cycles. The 100 ng / ml sample represented the maximum signal in this PCT assay. Previous experiments showed a steady increase in signal with each cycle, attributed to the evaporation of the AB trapping and signal AB reagents. Since the reagent volume is only 120 μL, water evaporation could lead to an increase in AB concentration. By adding 40 μL of mineral oil to form a layer on top of the reagent to minimize aqueous evaporation, [the following results were obtained]. Figure 2 Consistent signals were observed. Notably, the probe tip containing the immune complex was not adversely affected by probe tip transfer through the mineral oil layer.

[0194] Figure 3 The probe format coated with fluorescein is shown. Figure 1B Data from PCT standards in scheme A, v1 were used. Results at each PCT level were highly consistent across 10 cycles, with a CV of <10%. The high reproducibility of the assays indicates that the method can be applied to quantitative applications and requires only a single calibration curve, rather than cycle-specific calibration.

[0195] Figure 4 The correlation between recirculation measurements and commercial IVD instruments (Biomerieux Vidas) was shown (R). 2 Results for PCT clinical serum samples (p=0.95). To measure 15 samples, Vidas used 15 test strips, while the recycling assay used a single probe and reagent set, with samples measured in random order. The recycling assay format is tolerable for biological samples and yields accurate results.

[0196] Example 10: TnI Measurement Results (Program A, v2)

[0197] Figure 5 Results of myocardial TnI assay using a recirculation assay (Protocol A, v2) based on another immunoassay are presented. The format is as follows: Figure 1B The difference is that the capture AB is anti-F connected with anti-TnI, and the signal AB is Cy5-anti-TnI.

[0198] Example 11: PCT Measurement Results (Protocol A, v3)

[0199] Figure 7Data from up to 10 cycles of different PCT samples containing signal AB (scheme A, v3) labeled with Alexa Fluor 647 were presented. The results demonstrate that the invention can be applied to a variety of signal-generating dyes (fluorescence and chemiluminescence). The principle of the invention focuses on the dissociation of the anti-hapten antibody from the hapten-coated probe surface. The dye used should not affect the hapten / anti-hapten dissociation, as the dye is labeled as signal AB.

[0200] Example 12: PCT Measurement Results (Protocol A, v4)

[0201] Figure 9 The results presented PCT levels after up to 10 cycles using the digitoxin format (regimen A, v4) were compared with those of other regimens. Figure 3 The fluorophore probes in the samples are very similar in format. The results indicate that this invention can employ multiple hapten / anti-hapten pairs in the probe coating.

[0202] Example 13: PCT Measurement Results (Program B)

[0203] Figure 11 The method of using scheme B, v1 with pH 2 elution is shown. Figure 10 The PCT assay was performed using the recirculation format in the [process / system]. Figure 11 Three PCT levels were shown over a maximum of 10 cycles, demonstrating the feasibility of using labeled antibodies linked to high molecular weight polymers.

[0204] Example 14: PCT Measurement Results (Protocol B, v2)

[0205] Figure 13 PCT results using streptavidin amplification reagent eluted at pH 2 with scheme B, v2 are shown. The data show a progressively increasing pre-reading for each cycle. The PCT-specific signal is obtained by subtracting the pre-reading signal from the final read signal. In this case, the PCT-specific signal remained constant throughout 10 cycles. The data indicate that additional fluorescent dyes, besides the PCT immune complex, bind to the probe surface. This is likely due to streptavidin, as it is known to be robust even when pH 2 treatment does not inhibit its biotin-binding activity. Any residual streptavidin remaining on the probe tip after pH 2 elution will remain active in subsequent cycles.

[0206] Example 15: PCT Measurement Results (Protocol C)

[0207] Figure 15 Contains such as Figure 14The results of the use protocol C in this study were obtained from a recycle assay of three PCT levels using dual pH 2 elution and DMSO. The PCT signal remained consistent throughout the 10 cycles, with a significant reduction in the signal during the pre-readout. Although many organic solvents can serve as suitable denaturants, DMSO is one of the preferred choices due to its relatively low toxicity. The consistent PCT cycling also indicates that the hapten-coated probe surface is resistant to repeated DMSO exposure.

[0208] Example 16A: PCT Measurement Results (Protocol D)

[0209] Figure 17 This illustrates a two-read / subtraction scheme ( Figure 16B The results of the recirculation assays at three PCT levels (0, 10, and 100 ng / mL) were performed in protocol D. During 12 cycles, the fluorescence signal at PCT = 0 ng / mL was negligible (approximately 20-30 fluorescence units), which is significantly lower than... Figure 15 The results shown are between 80 and 110 fluorescence units after subtracting the pre-read fluorescence signal.

[0210] Figure 18 Results of 15 PCT clinical serum samples are shown using a double-read recirculation protocol (Protocol D). The 15 samples were measured twice, first in order of low to high concentration (shown as the Y-axis) and then in order of high to low concentration (shown as the X-axis). The results from high to low and low to high were highly correlated (R0). 2 =0.9985), which indicates that there are no residue problems regardless of the sample concentration.

[0211] Figure 19 Results from 15 PCT clinical serum samples were shown to be highly correlated with the two-read recirculation protocol (Protocol D) for commercial IVD instruments (Biomerieux Vidas). 2 =0.98). To measure 15 samples, Vidas used 15 test strips, while the recycle assay used a single probe and reagent set, in which samples were measured in random order.

[0212] Example 16B: PCT Measurement Results (Protocol D-1)

[0213] Figure 20 The results were obtained using protocol D-1, which involved recirculation at three PCT levels with the denaturing agent guanidine chloride (6M guanidine HCl, pH 1.6) and DMSO elution. The protocol is consistent with... Figure 16BSimilar to those described in protocol D, the difference is that the first elution was performed in 6M guanidine hydrochloride (pH 1.6). As with protocol D, the PCT signal remained consistent throughout 11 cycles, with a significant reduction in the pre-read signal. The consistent PCT cycling also indicates that the hapten-coated probe surface is resistant to repeated exposure to the denaturing agents guanidine chloride and DMSO.

[0214] Example 17: PCT Measurement Results (Protocol E)

[0215] Figure 21 The fluorescently coated probe format using scheme E is shown. Figure 16A Data from PCT standards were used. Before contacting the probe, the sample was first mixed with a dual antibody anti-F-anti-PCT and a signaling antibody biotin-anti-PCT. Results at each PCT level were highly consistent across 12 cycles, with a CV < 10%.

[0216] Example 18: Serum amyloid A (SAA) assay results (Protocol F)

[0217] Figure 23 Contains such as Figure 22 The results of the assay using protocol F were obtained by recirculation of three SAA levels using (i) low pH with denaturing agent guanidine chloride (6M guanidine HCl, pH 1.6) and (ii) denaturing agent DMSO. Recombinant SAA, capture antibody monoclonal SAA antibody (6F9), and signal antibody monoclonal SAA antibody (8C7) were obtained from GenScript.

[0218] SAA tends to polymerize and adhere to the probe. In the absence of guanidine chloride, pH 2 acid elution and DMSO elution did not appear to substantially dissociate the immune complex from the probe. The readout signal progressively increased in each cycle, leading to a decrease in the specific SAA binding signal in each cycle (data not shown here).

[0219] However, the inventors discovered that by adding guanidine chloride during acid elution, specifically in both (i) low pH elution with denaturing agent guanidine chloride and (ii) elution with denaturing agent DMSO, the preread signal remained low, and the SAA signal remained consistent throughout 11 cycles. Figure 23 ).

[0220] The invention and the ways and means of making and using it are now described herein in the full, clear, concise and precise terminology to enable those skilled in the art to make and use the invention. It should be understood that preferred embodiments of the invention have been described above, and modifications may be made without departing from the scope of the invention as set forth in the claims. The following claims summarize this specification in particular and clearly claim protection for the subject matter considered to be inventive.

Claims

1. A biantibody-polymer conjugate comprising a first antibody, a second antibody, and a polymer, wherein the first antibody and the second antibody are covalently linked to the polymer, and the polymer is a sucrose and epichlorohydrin copolymer.

2. The biantibody-polymer conjugate according to claim 1, wherein the polymer is FICOLL.

3. The biantibody-polymer conjugate according to claim 2, wherein the FICOLL has a molecular weight of millions.

4. The biantibody-polymer conjugate according to claim 1, 2, or 3, wherein the first antibody is an anti-hapten antibody.

5. The biantibody-polymer conjugate according to claim 4, wherein the hapten is fluorescein, biotin, digitalisin, nitrotyrosine, dinitrophenol, trinitrophenol, nitrophenol, aminobenzoic acid, Alexa Fluor, anthocyanin dye, TRITC, fluorescent yellow, Texas red, fluoroborpyrrole, Myc, Flag, polyhistidine, theophylline, phenytoin, phenobarbital, valproic acid, penicillin, gentamicin, progesterone, testosterone, estradiol, or vitamin D.

6. A method for detecting an analyte in multiple liquid samples, the method comprising the following steps: (a) Immerse the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the probe tip, wherein the probe includes a first hapten fixed to the tip of the probe and the diameter of the tip surface is ≤5 mm. (b) A first immune complex is formed on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-first hapten antibody against the first hapten, and the signal antibody is conjugated to a second hapten, the first hapten and the second hapten being different. (c) Immerse the probe tip in a washing solution; (d) Immerse the probe tip in an amplification solution comprising an anti-second hapten antibody or streptavidin conjugated with a fluorescent label to form a second immune complex on the probe tip comprising the analyte, the capture antibody, the signal antibody, the second hapten, and the anti-second hapten antibody or streptavidin. (e) The concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the tip of the probe, subtracting the pre-read fluorescence signal from (a), and quantifying the subtracted signal against a calibration curve. (f) Immersing the probe tip in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; and (g) Repeat the same steps (a)-(f), but in step (b) use the analyte from the new sample, thereby detecting the analyte in each of the plurality of liquid samples.

7. The method of claim 6, wherein the first immune complex in step (b) is formed by: (b1) A sample solution is mixed with a dual antibody solution and a reagent solution to form a mixture, wherein the sample solution comprises the analyte, the dual antibody solution comprises the anti-first hapten antibody covalently linked to the capture antibody, and the reagent solution comprises the signal antibody conjugated to the second hapten; and (b2) Immerse the probe tip in the mixture to form the first immune complex on the probe tip.

8. The method of claim 6, wherein the first immune complex in step (b) is formed by: (b1) Immerse the probe tip in a double antibody solution, the double antibody solution comprising the anti-first hapten antibody covalently linked to the capture antibody; (b2) Immerse the probe tip in a sample solution containing the sample; (b3) Immerse the probe tip in a reagent solution comprising the signaling antibody conjugated with the second hapten to form the first immune complex on the probe tip.

9. A method for detecting an analyte in multiple liquid samples, the method comprising the following steps: (a) Immerse the probe tip in a biantibody solution comprising biantibodies, the biantibodies comprising an anti-first hapten antibody covalently linked to a capture antibody, wherein the probe has a first hapten immobilized on the tip of the probe and the diameter of the tip surface is ≤5 mm, and the capture antibody is a first antibody against the analyte; (b) Immerse the probe in an aqueous solution with a pH of 6.0-8.5 to pre-read the fluorescence signal at the tip of the probe; (c) Immerse the probe tip in a sample solution, the sample solution comprising a liquid sample containing the analyte; (d) Immerse the probe tip in a reagent solution comprising a signal antibody conjugated to a second hapten to form a first immune complex comprising the analyte, the capture antibody and the signal antibody on the probe tip, wherein the signal antibody is a second antibody against the analyte and the first hapten and the second hapten are different; (e) Immerse the probe tip in a washing solution; (f) Immerse the probe tip in an amplification solution comprising an anti-second hapten antibody conjugated with a fluorescent label to form a second immune complex comprising the analyte, the capture antibody, the signal antibody, the second hapten, and the anti-second hapten antibody on the probe tip; (g) The concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the tip of the probe, subtracting the pre-read fluorescence signal from (c), and quantifying the subtracted signal against a calibration curve. (h) Immerse the probe tip in an acidic solution with a pH of about 1.0-4.0 to elute the immune complex from the probe tip; as well as (i) Repeat the same steps (a)-(h), but in step (c) use a new sample solution containing the new sample, thereby detecting the analyte in each of the plurality of liquid samples.

10. A method for detecting an analyte in multiple liquid samples, the method comprising the following steps: (a) Immerse the probe tip in a dual antibody container containing a dual antibody solution comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the probe has a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface is ≤5 mm, and the hapten is not biotin, and the capture antibody is an antibody against the analyte; (b) Immerse the probe in a readout container containing an aqueous solution with a pH of 6.0-8.5 to read out the fluorescence signal at the tip of the probe; (c) Immerse the probe tip in a sample solution containing a liquid sample with the analyte; (d) Immersing the probe tip in a reagent solution comprising a biotin-conjugated signaling antibody to form a first immune complex comprising the analyte, the capture antibody and the signaling antibody on the probe tip, wherein the signaling antibody is a second antibody against the analyte; (e) Immerse the probe tip in a washing solution; (f) Immerse the probe tip in an amplification solution comprising streptavidin conjugated with a fluorescent label to form a second immune complex comprising the analyte, the capture antibody, the signal antibody, the biotin, and the streptavidin on the probe tip; (g) The concentration of the analyte in the sample is determined by measuring the fluorescence signal of the second immune complex at the tip of the probe, subtracting the pre-read fluorescence signal from (b), and quantifying the subtracted signal against a calibration curve. (h) First, the probe tip is immersed in an acidic solution with a pH of about 1.0-4.0, and then immersed in dimethyl sulfoxide to elute the immune complex from the probe tip; as well as (i) Repeat the same steps (a)-(h), but in step (c) use a new sample solution including the new sample, thereby detecting the analyte in each of the plurality of liquid samples.

11. A method for detecting an analyte in multiple liquid samples, the method comprising the following steps: (a) Obtaining a probe having a non-biotinylated hapten fixed to the tip of the probe, wherein the diameter of the tip surface is ≤5 mm; (b) A first immune complex is formed on the probe tip comprising the analyte from the sample, a capture antibody, and a signal antibody, wherein the capture antibody and the signal antibody are two different antibodies against the analyte, the capture antibody is covalently linked to an anti-hapten antibody, and the signal antibody is conjugated to biotin. (c) Immerse the probe tip in a washing solution; (d) Immerse the probe tip in an amplification solution comprising streptavidin conjugated with a fluorescent label to form a second immune complex comprising the analyte, the capture antibody, the signal antibody, the biotin, and streptavidin on the probe tip; (e) Read the first fluorescence signal of the second immune complex at the tip of the probe; (f) Immerse the probe tip in an acidic solution with a pH of about 1.0-4.0; (g) Read the second fluorescence signal from the acid-treated probe tip; (h) Subtract the second fluorescence signal from the first fluorescence signal, and quantify the subtracted signal against the calibration curve; (i) Immerse the probe tip in dimethyl sulfoxide; as well as (j) Repeat the same steps (b)-(i), but in step (b) use a new sample solution including the new sample, thereby detecting the analyte in each of the plurality of liquid samples.

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