Method for Reusing Test Probes and Reagents in Interferometry-Based Biochemical Assays

Abstract

The present invention relates to a biochemical assay method, which uses a hapten-fixed test probe and reagents repeatedly to quantify analytes in different samples or measure kinetic binding in different samples while maintaining acceptable assay performance, for any number of about 3 to 20 times. The method uses a conjugate solution comprising (i) an anti-hapten antibody and a capture antibody against the analyte or (ii) an anti-hapten antibody and streptavidin in each cycle, and after each reaction cycle, the hapten-coated test probe is regenerated by immersing the test probe in an acidic solution with a pH of about 1 to 4 and optionally further immersing it in a DMSO solution. The robustness of the hapten-fixed solid phase allows the use of denaturing reagents to efficiently elute immune complexes after each cycle without reducing the binding activity of the hapten on the solid phase.

Description

Technical Field

[0001] The present invention relates to a biochemical assay based on interferometry. The method uses a solution of a dual antibody conjugate in each cycle and regenerates the hapten-coated test probe by dipping the test probe in an acidic solution having a pH of about 1-4 after completion of each reaction cycle. The method repeatedly uses the hapten-fixed test probe and reagents to quantify analytes in different samples about 2 to 15 times. Background Art

[0002] Label-free detection methods such as biolayer interferometry (BLI) and surface plasmon resonance (SPR) have become standard methods in receptor / ligand binding studies in biomedical research and in therapeutic agent development. Across the healthcare industry, including research applications and drug development, cost control is a major issue. The cost of label-free sensors limits their use and thus the potential contribution of label-free sensors in research and development.

[0003] Typical methods for reducing the cost of immunoassays involve minimizing the manufacturing costs of materials, labor, and facility overhead.

[0004] Any method for recycling immunoassay reagents typically focuses on dissociating immune complexes with denaturants such as acidic / basic pH solutions, organic solvents, chaotropic agents, etc. However, the denaturation step typically alters the antibody charge, hydration, hydrogen bonding, and tertiary structure in a situation where the antibody no longer binds to the antigen. Reversing the exposure of the antibody to the initial binding conditions of near physiological pH and ionic strength is expected to restore the original binding activity. However, few antibodies can tolerate repeated exposure to denaturing conditions without adversely affecting their binding properties and thus some aspect of assay performance.

[0005] There is a need to reduce the cost of immunoassays while maintaining assay performance. Brief Description of the Drawings

[0006] Figure 1A Depicts a biosensor interferometer comprising a light source, a detector, a waveguide, and an optical assembly (also referred to as a "probe").

[0007] Figure 1B Depicts an example of a conventional probe.

[0008] Figure 2 Depicts another configuration of the probe.

[0009] Figures 3A - 3B Shows the detection principle in a thin film interferometer.

[0010] Figure 4Shows a first embodiment of the present invention for antigen quantification, wherein the anti-hapten antibody is covalently linked to the capture antibody.

[0011] Figure 5 Shows a second embodiment of the present invention for antigen-antibody binding kinetics, wherein the anti-hapten antibody is covalently linked to the capture antibody. (Ag = antigen, AB = antibody)

[0012] Figure 6 Shows a third embodiment of the present invention for quantifying biotinylated analytes, wherein the anti-hapten antibody is covalently linked to streptavidin.

[0013] Figure 7 Shows a fourth embodiment of the present invention for the binding kinetics of binding pairs, wherein the anti-hapten antibody is covalently linked to streptavidin. The binding pair is an antibody and a biotinylated antigen.

[0014] Figure 8 Also shows a fourth embodiment of the present invention for the binding kinetics of binding pairs, wherein the anti-hapten antibody is covalently linked to streptavidin. The binding pair in this figure is an antigen and a biotinylated antibody.

[0015] Figure 9 Shows data generated using the first embodiment of the present invention with multiple levels of murine IgG samples ranging from 1 μg / ml to 700 μg / ml under 10 regeneration cycles.

[0016] Figure 10 Shows the interference pattern of the binding kinetics of CRP and anti-CRP using an anti-mouse Fc-coated probe.

[0017] Figure 11 Shows the interference pattern of the binding kinetics of biotin-anti-His antibody and His-IL2r using a streptavidin-coated probe. Detailed Description

[0018] Definitions

[0019] The terms used in the claims and the specification shall be construed according to the ordinary meaning understood by those skilled in the art, except as defined below and as set forth hereinafter.

[0020] As used herein, "about" means within ±10% of the stated value.

[0021] As used herein, an "analyte-binding" molecule refers to any molecule that is capable of participating in a specific binding reaction with an analyte molecule. Examples include, but are not limited to, (i) an antigen molecule for detecting the presence of an antibody specific for said antigen; (ii) an antibody molecule for detecting the presence of an antigen; (iii) a protein molecule for detecting the presence of its protein binding partner; (iv) a ligand for detecting the presence of a binding partner; or (v) a single-stranded nucleic acid molecule for detecting the presence of a nucleic acid-binding molecule.

[0022] The "aspect ratio" of a shape refers to the ratio of its longer dimension to its shorter dimension.

[0023] A "binding molecule" refers to a molecule that is capable of binding to another molecule of interest.

[0024] As used herein, a "binding pair" refers to two molecules that attract each other and specifically bind to each other. Examples of binding pairs include, but are not limited to, an antigen and an antibody specific for said antigen, a ligand and its receptor, complementary strands of nucleic acid, biotin and avidin, biotin and streptavidin, a lectin and a carbohydrate. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti-fluorescein, digoxin / anti-digoxin.

[0025] A "bispecific antibody" is an antibody that can bind to two different types of antigens simultaneously.

[0026] As used herein, "immobilized" refers to fixing a reagent to a solid surface. When a reagent is immobilized on a solid surface, it is non-covalently or covalently bound to the surface.

[0027] As used herein, a "monolithic substrate" refers to a single piece of solid material, such as glass, quartz, or plastic having one refractive index.

[0028] As used herein, a "probe" refers to a monolithic substrate having an aspect ratio (length-to-width ratio) of at least 2:1, wherein a thin film layer is coated on the sensing side. The probe has a distal end and a proximal end. The proximal end (also referred to as the probe tip in this application) has a sensing surface coated with a thin layer of an analyte-binding molecule.

[0029] A "waveguide" refers to a device (e.g., a duct, a coaxial cable, or an optical fiber) that is designed to confine and guide the propagation of an electromagnetic wave (as light).

[0030] The present invention discloses a method for reusing biochemical assay test probes and reagents about 2 to 15 times in a biolayer interferometry (BLI) assay for quantification or kinetics while maintaining acceptable assay performance. The present invention reuses the test probes and reagents and saves costs on a per-test basis.

[0031] There are several key elements in implementing the present invention. The first feature of the present invention is that the solid phase (probe) is coated with a hapten. A hapten, typically defined as a small organic molecule with a molecular weight of less than about 1500 daltons, is antigenic but very poorly immunogenic. Therefore, a hapten must be conjugated to a larger polymer, typically a protein, to generate an anti-hapten antibody. The antibody binding of a hapten is based on its primary chemical structure, and there is no conformational dependence in its antibody binding. Thus, even after repeated steps that denature the anti-hapten antibody and the immune complex, the hapten antigen will still retain its antibody binding properties. The present invention uses a hapten-fixed test probe and an anti-hapten antibody that binds to the hapten-coated solid phase; the immune complex is then dissociated with a denaturing reagent. Subsequently, the hapten-coated solid phase is reimmersed in a reagent having an anti-hapten antibody to restore the original amount of the anti-hapten antibody bound to the solid phase. The hapten-coated solid phase can be subjected to multiple denaturation cycles and then to anti-hapten antibody binding. The robustness of the hapten-coated solid phase allows the use of a variety of denaturing reagents to efficiently elute the immune complex.

[0032] Suitable haptens of the present invention include, 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 Bodipy; polypeptides such as Myc, Flag, and polyhistidine; drugs 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 digoxin.

[0033] The second feature of the present invention requires the chemical conjugation of an anti-hapten to a member of a binding pair. Such a binding pair can be an antibody / antigen, receptor / ligand, or oligonucleotides with complementary sequences. For example, an anti-hapten antibody is conjugated to a capture antibody that binds to a specific analyte in a sample. The hapten probe / anti-hapten binding member format can be applied to a variety of binding assays.

[0034] The probe of the present invention is regenerated and recycled, while fresh anti-hapten / member of the binding pair is captured on the probe in each cycle, thereby maintaining assay performance.

[0035] A third feature of the present invention is the biolayer interferometry (BLI) detection of the binding of the second member of the binding pair to the probe surface. BLI detection is advantageous because no labeling that may alter the binding between the binding pairs is required. A denaturation step of eluting the complex from the probe surface is then performed, and the probe is cycled back to the anti-hapten / binding member conjugate. Since fresh coating of the anti-hapten / binding member is used in each cycle, assay performance can be maintained.

[0036] Other features of the present invention include small-diameter probes (≤5 mm). Due to their small surface area, the amount of anti-hapten / binding member reagent consumed by the probes is negligible, thus allowing the probes to be reused in subsequent cycles. The stability of the hapten-coated solid phase allows the use of a variety of denaturing reagents to efficiently elute the complex.

[0037] Biosensor interferometer system

[0038] The present invention is applicable to several biosensor interferometer systems. Figure 1A and Figure 1B An example of such a system is shown. Figure 1A A biosensor interferometer 100 (or simply "interferometer") is depicted that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a "probe"). The probe 108 can be connected to the waveguide 106 through a coupling medium.

[0039] The light source 102 can emit white light that is guided by the waveguide 106 towards the probe 108. For example, the light source 102 can be a light-emitting diode (LED) that is configured to produce light in a given spectral range (e.g., 400 nm or less to 700 nm or more) within at least a range of 50 nanometers (nm), 100 nm, or 150 nm. Alternatively, the interferometer 100 can employ multiple light sources having different characteristic wavelengths, such as LEDs designed to emit light at different wavelengths within the visible light range. The same effect can be achieved by a single light source with a suitable filter for directing light of different wavelengths onto the probe 108.

[0040] The detector 104 is preferably a spectrometer, such as an Ocean Optics USB4000, which is capable of recording the spectrum of the interfering light received from the probe 108. Alternatively, if the light source 102 operates to direct different wavelengths onto the probe 108, the detector 104 can be a simple photodetector capable of recording the intensity of each wavelength. In another embodiment, the detector 104 can include multiple filters that allow the detection of the intensity of each of multiple wavelengths.

[0041] The waveguide 106 can be configured to transmit light emitted by the light source 102 to the probe 108 and then transmit light reflected by the surface within the probe 108 to the detector 104. In some embodiments, the waveguide 106 is a bundle of optical fibers (e.g., a single-mode fiber optic cable), while in other embodiments, the waveguide 106 is a multimode fiber optic cable.

[0042] As Figure 1B shown, the probe 108 includes a monolithic substrate 114, a thin film layer (also referred to as an "interference layer"), and a biomolecular layer (also referred to as a "bio layer"), where the biomolecular layer includes analyte molecules 122 that have bound to analyte binding molecules 120. The monolithic substrate 114 includes a transparent material through which light can travel. The interference layer also includes a transparent material. When light impinges on the probe 108, the proximal surface of the interference layer can act as a first reflective surface, while the bio layer can act as a second reflective surface. As described further below, the light reflected by the first and second reflective surfaces can form an interference pattern that can be monitored by the interferometer 100.

[0043] The interference layer generally includes multiple layers combined in a manner that enhances the detectability of the interference pattern. Here, for example, the interference layer includes a tantalum pentoxide (Ta 2 O 5 ) layer 116 and a silica (SiO2) layer 118. The tantalum pentoxide layer 116 can be thin (e.g., approximately 10 nm - 40 nm) because its main purpose is to increase the reflectivity of the proximal surface of the interference layer. Meanwhile, the silica layer 118 can be relatively thick (e.g., on the order of 650 nm - 900 nm) because its main purpose is to increase the distance between the first and second reflective surfaces.

[0044] For testing, the probe 108 can be suspended in a micro well 110 (or simply referred to as a "well") containing a sample 112. During a diagnostic test, the analyte molecules 122 will bind to the analyte binding molecules 120 along the distal end of the probe 108, and these binding events will result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the bio layer formed along the distal end of the probe 108 by detecting an offset in the phase characteristics of the interference pattern.

[0045] Figure 2Shows another biosensor interferometer probe. The probe includes: a monolithic substrate having first and second surfaces that are substantially parallel to each other at opposite ends of the monolithic substrate; an interference layer coated on the second surface of the monolithic substrate; and an analyte-binding molecule layer coated on the interference layer. The interference layer generally includes magnesium fluoride (MgF2). When light is incident on the interferometric sensor, the first interface between the monolithic substrate and the interference layer acts as the first reflective surface, and when light is incident on the probe, the second interface between the biolayer formed by the binding of analyte molecules in the sample to the analyte-binding molecules and the solution containing the sample acts as the second reflective surface. As described above, the thickness of the biolayer can be estimated based on the interference pattern of the light reflected by the first and second reflective surfaces.

[0046] Probe 200 includes an interference layer 204 fixed along the distal end of monolithic substrate 202. Analyte-binding molecules 206 can be deposited along the distal surface of interference layer 204. During a biochemical test, when analyte molecules 208 in the sample bind to analyte-binding molecules 206, a biolayer will be formed. As Figure 2 shown, monolithic substrate 202 has a proximal surface (also referred to as the "coupling side") that can be coupled to, for example, a waveguide of an interferometer and a distal surface (also referred to as the "sensing side") on which additional layers are deposited. Generally, the length of monolithic substrate 202 is at least 3 millimeters (mm), 5 mm, 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length-to-width ratio) of monolithic substrate 202 is at least 5:1. In such an embodiment, it can be said that monolithic substrate 202 has a columnar form. The cross-section of monolithic substrate 202 can be circular, oval, square, rectangular, triangular, pentagonal, etc. Monolithic substrate 202 preferably has a refractive index significantly higher than that of interference layer 204 such that the proximal surface of interference layer 204 effectively reflects the light guided to probe 200. The preferred refractive index of the monolithic substrate can be higher than 1.5, 1.8, or 2.0. Thus, monolithic substrate 202 can include a high refractive index material such as glass (refractive index of 2.0) rather than a low refractive index material such as quartz (refractive index of 1.46) or plastic (refractive index of 1.32 - 1.49).

[0047] The interference layer 204 comprises at least one transparent material coated on the distal surface of the monolithic substrate 202. These transparent materials are deposited on the distal surface of the monolithic substrate 202 in the form of thin films with a thickness ranging from a fraction of a nanometer (e.g., a single layer) to several micrometers. The thickness of the interference layer 204 can be at least 500 nm, 700 nm, or 900 nm. Exemplary thicknesses are between 500 nm and 5,000 nm (and preferably between 800 nm and 1,200 nm). Here, for example, the thickness of the interference layer 204 is approximately 900 nm - 1,000 nm or 940 nm.

[0048] Compared with conventional probes, the interference layer 204 has a refractive index substantially similar to that of the biological layer. This ensures that the reflection from the distal end of the probe 200 is mainly due to the analyte molecules 208 rather than the interface between the interference layer 204 and the analyte-binding molecules 206. In some embodiments, the interference layer 204 comprises magnesium fluoride (MgF 2 ), while in other embodiments, the interference layer 204 comprises potassium fluoride (KF), lithium fluoride (LiF), sodium fluoride (NaF), lithium calcium aluminum fluoride (LiCaAlF 6 ), sodium aluminum fluoride (Na3AlF 6 ), strontium fluoride (SrF 2 ), aluminum fluoride (AlF 3 ), sulfur hexafluoride (SF 6 ), etc. The refractive index of magnesium fluoride is 1.38, which is substantially the same as the refractive index of the biological layer formed along the distal end of the probe 200. For comparison, the interference layer of a conventional probe typically comprises silicon dioxide, and in the visible range, the refractive index of silicon dioxide is approximately 1.4 - 1.5. Since the interference layer 204 and the biological layer have similar refractive indices, light will experience minimal scattering when traveling from the interference layer 204 to the biological layer and then back from the biological layer to the interference layer 204.

[0049] In one embodiment, the probe 200 comprises an adhesion layer deposited along the distal surface of the interference layer 204 attached to the monolithic substrate 202. The adhesion layer can comprise a material that promotes the adhesion of the analyte-binding molecules 206. An example of such a material is silicon dioxide. Compared with the interference layer 204, the adhesion layer is typically very thin, so the effect of the adhesion layer on light traveling towards or back from the biological layer will be minimal. For example, the thickness of the adhesion layer 310 can be approximately 3 nm - 10 nm, while the thickness of the interference layer 304 can be approximately 800 nm - 1,000 nm. The thickness of the biological layer formed by the analyte-binding molecules 306 and the analyte molecules 308 is typically a few nanometers.

[0050] When light shines on the probe 200, the proximal surface of the interference layer 204 can act as a first reflective surface, and the distal surface of the biological layer can act as a second reflective surface. The presence, concentration, or binding rate of the analyte molecules 208 to the probe 200 can be estimated based on the interference of the light beams reflected by these two reflective surfaces. As the analyte molecules 208 attach to (or detach from) the analyte binding molecules 206, the distance between the first reflective surface and the second reflective surface will change. Since the dimensions of all other components in the probe 200 remain the same, the interference pattern formed by the light reflected from the first and second reflective surfaces undergoes a phase shift according to the change in the thickness of the biological layer due to the binding event.

[0051] In operation, the incident light signal 210 emitted by the light source is transmitted through the monolithic substrate 202 towards the biological layer. Inside the probe 200, the light will be reflected at the first reflective surface, generating a first reflected light signal 212. The light will also be reflected at the second reflective surface, generating a second reflected light signal 214. The second reflective surface initially corresponds to the interface between the analyte binding molecules 206 and the sample in which the probe 200 is immersed. Due to the binding occurring during the biochemical test, the second reflective surface becomes the interface between the analyte molecules 208 and the sample.

[0052] As Figure 3A shown, the first reflected light signal 212 and the second reflected light signal 214 form a spectral interference pattern. When the analyte molecules 208 bind to the analyte binding molecules 206 on the distal surface of the interference layer 204, the optical path of the second reflected light signal 214 will become longer. As a result, as Figure 3B shown, the spectral interference pattern shifts from T0 to T1. By continuously measuring the phase shift in real time, a kinetic binding curve can be plotted as a graph of the offset versus time. The association rate of the analyte molecules with the analyte binding molecules immobilized on the distal surface of the interference layer 204 can be used to calculate the analyte concentration in the sample. Therefore, the measure of the phase shift is the detection principle of the thin - film interferometer.

[0053] Recycling scheme for biochemical assays

[0054] The present invention relates to a biochemical assay that uses the same hapten - coated test probes and the same reagents for different samples. The test probes are regenerated by acid treatment and optionally by further DMSO treatment.

[0055] First Embodiment - Quantification

[0056] In a first embodiment, the method detects analytes in multiple samples. Figure 4 This embodiment is shown, where Fc is the analyte in the sample to be quantified.

[0057] The method comprises 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) dipping the probe tip into a diabody container containing a diabody solution including an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is an anti-analyte antibody; (c) dipping the probe tip into a first washing container including a first aqueous solution for a first period of time to determine a baseline interferometry pattern of the probe tip; (d) dipping the probe tip into a sample container containing a liquid sample having the analyte for a second period of time to determine a second interferometry pattern of an immune complex formed at the probe tip; (e) determining the analyte concentration in the sample by measuring an interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern and quantifying the phase shift relative to a calibration curve; (f) dipping the probe tip into an acidic solution having a pH of about 1.0 to 4.0 to elute the immune complex from the probe tip; (g) dipping the probe tip into a second washing container including a second aqueous solution having a pH of 6.0 to 8.5; and (h) repeating steps (b) to (g) 2 to 15 times, except using a sample container including a new sample in step (d) of each cycle, thereby determining the analyte concentration of a plurality of samples. The sample container in the repeating cycle is preferably a new sample container.

[0058] The method uses the same probe, the same washing solution, and the same reagents in all reaction cycles. However, fresh reagent solutions can also be used in different cycles.

[0059] In step (a) of the present method, a probe having a small tip for binding an analyte is obtained. The tip has a small surface area with a diameter ≤ 5 mm, preferably ≤ 2 mm or ≤ 1 mm. The small surface of the probe tip confers several advantages. In solid-phase immunoassays, having a small surface area is advantageous because it has less non-specific binding and thus produces a lower background signal. Further, due to the small surface area of the tip, the reagent or sample carried on the probe tip is very small. This feature makes the probe tip easy to wash and the contamination of the washing solution negligible due to the large volume of the washing solution. Another aspect of the small surface area of the probe tip is its small binding capacity. Thus, when the probe tip is dipped into a reagent solution, the binding of the reagent does not consume a large amount of the reagent. The reagent concentration does not actually change. The negligible contamination of the washing solution and the small consumption of the reagent enable the reagent and the washing solution to be reused multiple times, such as 3 to 10 times, 3 to 15 times, or 3 to 20 times.

[0060] Methods for immobilizing haptens to a solid phase (the sensing surface of the probe tip) are common in immunochemistry and involve forming covalent, hydrophobic, or electrostatic bonds between the solid phase and the hapten. For example, the hapten can be conjugated to a carrier protein and the hapten-protein immobilized by adsorption onto a solid surface or by covalent attachment to aminopropylsilane coated on the solid surface.

[0061] In step (b) of the method, the probe tip is immersed in a diabody container containing a diabody solution. The diabody solution contains an anti-hapten antibody covalently linked to a capture antibody, where the capture antibody is a first antibody against the analyte. Alternatively, the diabody solution contains a bispecific antibody that binds to both the hapten and the analyte.

[0062] In one embodiment, the anti-hapten antibody and the capture antibody are directly linked to each other without a linker.

[0063] In a preferred embodiment, both the anti-hapten antibody and the capture antibody are covalently linked to a polymer that acts as a linker or spacer. The polymer typically has a molecular weight of 1,000 daltons to 500,000 daltons. The polymer can be a polysaccharide (e.g., dextran, amylose), a dendrimer, or polyethylene glycol. In a preferred embodiment, the polymer is (a copolymer of sucrose and epichlorohydrin).

[0064] In step (c), the probe is immersed in a first wash container (also acting as a baseline container for washing the probe) containing a first aqueous solution preferably having a pH of 6.0 to 8.5 for a first period of time (e.g., 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 1 minute) to determine the baseline interferometry pattern of the probe tip. The baseline container contains an aqueous solution such as water or a buffer 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 a pH between 6.0 and 8.5.

[0065] In step (d) of the method, the probe tip is immersed in a sample container (or sample chamber or sample well) containing a liquid sample having the analyte for a second period of time (e.g., 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 30 seconds to 1 minute) to determine the second interferometry pattern of the immune complex formed at the probe tip.

[0066] In step (e), the analyte concentration in the sample is quantified by measuring the interferometric phase shift between the second interferometric pattern and the baseline interferometric pattern and quantifying the wavelength shift relative to a calibration curve to determine the analyte concentration. The phase shift can be monitored kinetically or determined by the difference between a starting time point (T0) and an ending time point (T1) (see Figure 3B ).

[0067] In step (f), the probe is regenerated by applying denaturing conditions that dissociate the immune complex bound to the capture antibody on the solid phase. Typically, an acid or acidic buffer with a pH of about 1 to about 4 is effective in regenerating the antibody probes of the present invention. For example, hydrochloric acid, sulfuric acid, nitric acid, acetic acid can be used to regenerate the probe. The regeneration process can be a single acidic treatment followed by neutralization. For example, a single pH 1 - 3, or pH 1.5 - 2.5 (e.g., pH 2) exposure ranging from 10 seconds to 2 minutes is effective. The regeneration process can also be a "pulse" regeneration step where the probe is exposed to 2 - 5 cycles (e.g., 3 cycles) of short pH treatments (e.g., 10 - 20 seconds) and then neutralized at pH 6.5 - 8.0 (e.g., 10 - 20 seconds).

[0068] In step (g), after regeneration, the probe tip is immersed in a second wash container containing a second aqueous solution with a pH of 6.0 to 8.5 to neutralize the probe.

[0069] After probe regeneration and washing, steps (b) to (g) are repeated 1 - 10 times, 1 - 20 times, 1 - 25 times, 3 - 20 times, 5 - 10 times, 5 - 20 times, 5 - 25 times, or 5 - 30 times with the same probe and the same reagents for different samples in subsequent cycles.

[0070] In one embodiment, the reaction is accelerated by stirring or mixing the solution in a container. For example, solution flow across the probe tip can be induced in one or more reaction containers, including the sample container, reagent container, wash container, and regeneration container, such as lateral flow or orbital flow, to accelerate the binding reaction and dissociation. For example, the reaction containers can be mounted on an orbital shaker, and the orbital shaker rotates 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 perpendicular to the plane of the orbital flow to induce additional mixing of the solution above and below the probe tip.

[0071] Second Embodiment - Kinetics

[0072] The second embodiment of the present invention measures the binding kinetics of a sample antibody to a sample antigen in a plurality of samples each including the sample antibody. Figure 5 This embodiment is shown.

[0073] The method can be used to measure the association rate and dissociation rate of an antibody to an antigen, and to determine the affinity of the antibody for the antigen. The method comprises the steps of: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface ≤ 5 mm; (b) dipping the probe tip into a diabody container containing a diabody solution including an anti-hapten antibody covalently linked to a first antibody, wherein the first antibody is an antibody against the Fc portion of the sample antibody; (c) dipping the probe tip into an antibody sample container containing the sample antibody to bind the sample antibody to the probe, wherein the sample antibody specifically binds to a sample antigen; (d) dipping the probe tip into a first wash container including a first aqueous solution for a first period of time to determine the baseline interferometry pattern of the probe tip; (e) dipping the probe tip into an antigen sample container including the sample antigen for a second period of time to bind the sample antigen to the probe and determine a second interferometry pattern; (f) calculating the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern to determine the binding kinetics of the sample antibody and the sample antigen; (g) dipping the probe tip into a second wash container including a second aqueous solution to measure a third interferometry pattern; (h) calculating the interferometry phase shift between the third interferometry pattern and the second interferometry pattern to determine the dissociation kinetics of the sample antibody and the sample antigen; (i) dipping the probe tip into p an acidic solution with H of about 1.0 to 4.0 to elute the remaining immune complexes on the probe tip; (j) dipping the probe tip into a third wash container including a third aqueous solution with a pH of 6.0 to 8.5; and (k) repeating steps (b) to (i) 3 to 15 times, except using an antibody sample container including a new sample antibody in step (c) of each cycle, thereby determining the binding kinetics of a plurality of sample antibodies.

[0074] The details of each step are similar to the details of the corresponding similar steps (if any) described above in the first embodiment.

[0075] An example of the first antibody in step (b) is an anti-mouse IgG antibody, and the sample antibody is a mouse antibody against the sample antigen.

[0076] Step (e) binds the sample antigen to the sample antibody on the probe. Step (f) calculates the association rate (binding) of the sample antibody with the sample antigen. Step (g) dissociates the sample antigen from the sample antibody on the probe by immersing the probe in a second wash solution that does not contain any sample antigen. Step (h) calculates the dissociation rate of the sample antibody from the sample antigen.

[0077] Third Embodiment - Streptavidin - Quantification

[0078] In the third and fourth embodiments, an anti - hapten and streptavidin conjugate is used instead of the double - antibody used in the first two embodiments.

[0079] In one embodiment, the anti - hapten antibody and streptavidin are directly linked to each other without a linker.

[0080] In a preferred embodiment, both the anti - hapten antibody and streptavidin are covalently linked to a polymer that acts as a linker or spacer. The molecular weight of the polymer is typically from 1,000 daltons to 500,000 daltons. The polymer can be a polysaccharide (e.g., dextran, amylose), a dendrimer, or polyethylene glycol. In one preferred embodiment, the polymer is (a copolymer of sucrose and epichlorohydrin).

[0081] There are several applications for using streptavidin - coated probes to detect biotin. The initial step involves incorporating biotin into biomolecules. There are many commercially available biotin analogs designed to couple with the primary and secondary amines, thiols, carboxyl groups, and carbohydrate moieties of biomolecules. Most coupling procedures are now considered standard. The most common analog is biotin - NHS ester for labeling primary amines on proteins. Recently, recombinant labeling methods have been developed. AviTagTM is a fusion peptide incorporated into recombinant proteins during expression. Then biotin ligase inserts biotin onto the lysine residue in the fusion peptide.

[0082] One application of streptavidin probes is the quantification of biotin - labeled proteins, commonly used to monitor the production and purification of recombinant proteins.

[0083] In the third embodiment, the method detects biotinylated analytes in multiple samples. Figure 6This embodiment is shown in which the biotinylated analyte in the sample is quantified. The method comprises the steps of: (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 solution comprising an anti-hapten antibody covalently linked to streptavidin; (c) immersing the probe tip in a first wash container comprising a first aqueous solution for a first period of time to determine the baseline interferometry pattern of the probe tip; (d) immersing the probe tip into a sample container containing a liquid sample having a biotinylated analyte for a second period of time to determine the second interferometry pattern of the complex formed at the probe tip; (e) determining the concentration of the biotinylated analyte in the sample by measuring the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern and quantifying the phase shift relative to a calibration curve;

[0084] (f) immersing the probe tip in an acidic solution having a pH of about 1.0 to 4.0 and then in a DMSO solution to elute the complex from the probe tip; (g) immersing the probe tip in a second wash container comprising a second aqueous solution having a pH of 6.0 to 8.5; (h) repeating steps (b) to (g) 3 to 15 times, except using a sample container comprising a new sample in step (d) of each cycle, thereby detecting the biotinylated analyte in a plurality of liquid samples.

[0085] The details of each step are similar to the details of the corresponding similar steps (if any) described above in the first embodiment.

[0086] The use of streptavidin conjugates requires more stringent elution conditions, and after acidic elution, a dimethyl sulfoxide (DMSO) solution is used as a second eluent. Generally, an aqueous solution of DMSO (water or buffer such as PBS) is used with DMSO in an amount of 20 wt% - 85 wt%, 30 wt% - 85 wt% or 40 wt% - 80 wt%.

[0087] Fourth Embodiment - Streptavidin - Kinetics

[0088] Another application of streptavidin is to obtain the affinity constant of a binding pair. Most commonly, it is used to optimize biotherapeutic antibodies. The affinity constant is typically determined by first binding a biotinylated antigen to a streptavidin probe, and then immersing the probe in a sample containing a known concentration of antibody to measure the association rate. The probe is then transferred to a buffer sample without antibody to measure the dissociation rate of the immune complex. A streptavidin probe with a regeneration protocol is most useful in an affinity maturation program to further enhance antibody affinity by generating random mutations in the CDR regions. Many antibody variants are generated, which require a rapid and cost-effective way to characterize the affinity of the antibodies.

[0089] In a fourth embodiment of the present invention, the method measures the binding kinetics of a binding pair. Examples of binding pairs include, but are not limited to, an antigen and an antibody against said antigen, a ligand and its receptor, complementary strands of nucleic acid, biotin and avidin, biotin and streptavidin, lectin and carbohydrate. Figure 7 and 8 shows the fourth embodiment. In Figure 7 , the first member of the binding pair is an antigen, and the second member of the binding pair is an antibody. In Figure 8 , the first member of the binding pair is an antibody, and the second member of the binding pair is an antigen.

[0090] The method comprises the steps of: (a) obtaining a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface ≤ 5 mm; (b) immersing the probe tip in a solution comprising an anti-hapten antibody covalently linked to streptavidin; (c) immersing the probe tip into a first sample container comprising a biotinylated first member of a binding pair;

[0091] (d) Immerse the probe tip in a first wash container containing a first aqueous solution for a first period of time to determine the baseline interferometry pattern of the probe tip; (e) Immerse the probe tip into a second sample container containing the second member of the binding pair for a second period of time to allow the second member of the binding pair to bind to the probe and determine a second interferometry pattern; (f) Calculate the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern to determine the binding kinetics of the first and second members of the binding pair; (g) Immerse the probe tip into a second wash container containing a second aqueous solution to measure a third interferometry pattern; (h) Calculate the interferometry phase shift between the third interferometry pattern and the second interferometry pattern to determine the dissociation kinetics of the first and second members of the binding pair; (i) Immerse the probe tip in an elution container containing an acidic solution with a pH of about 1.0 to 4.0 and then immerse the probe tip in a DMSO solution to elute the remaining immune complexes on the probe tip; (i) Immerse the probe tip in a third wash container containing a third aqueous solution with a pH of 6.0 to 8.5; and (k) Repeat steps (b) to (i) 3 to 15 times, except using a sample container containing a new second member of the binding pair in step (e) of each cycle, thereby determining the binding kinetics of the first and second members of the binding pair in multiple samples.

[0092] The details of each step are similar to the details of the corresponding similar steps (if any) described above in the first or third embodiment.

[0093] In all of the above methods, the reagent container is optionally covered with a layer of mineral oil to prevent or reduce evaporation of the solution in the container, which can increase the concentration of the signal antibody conjugate or amplification conjugate. Generally, 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 generally 20 - 80 μL or 30 - 50 μL. Mineral oil is typically used to minimize evaporation and subsequent condensation in PCR sample tubes. The inventors have demonstrated that the probe and immune complexes at the probe tip are not affected by passing through the mineral oil layer.

[0094] Probe including immobilized hapten

[0095] The present invention utilizes a probe, the probe comprising a hapten immobilized on the tip of the probe, wherein the aspect ratio of the length to the width of the probe is at least 5:1, the diameter of the surface of the probe tip is ≤5 mm, and the hapten does not dissociate from the probe after acid treatment; that is, after 1 to 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 generally carried out 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. DMSO treatment is generally carried out by immersing the probe in a DMSO solution for 10 seconds to 2 minutes.

[0096] The present 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 therein.

[0097] Examples

[0098] Example 1: Preparation of hapten-coated probes

[0099] Various hapten / anti-hapten pairs can be used in the present invention. Fluorescein and digoxigenin are useful haptens for the present invention because their analogs for labeling and their antibodies are readily available. The first step is to link the hapten to a polymer, usually a protein, such as BSA (bovine serum albumin). The protein is used for subsequent immobilization on the probe surface. For example, fluorescein-NHS ester (Invitrogen) or digoxigenin-NHS ester (ATT Bioquest) is reacted with BSA at a molar coupling ratio of 15:1 in PBS at pH 7.4 for 1 hour, followed by purification on a PD10 column (GE Healthcare).

[0100] The probes shown below are prepared as follows. The ends of glass (refractive index 2.0) rods with a length of 20 mm and a diameter of 1 mm are polished into mirror surfaces using an optical polishing machine. After cleaning and purifying the rods in pure water, they are arranged in a fixture and then loaded into an ion beam-assisted physical vapor deposition (PVD) machine. In the PVD machine, an electron beam is used to bombard and vaporize the target material to be coated onto the surface; then an ion beam is applied to deposit the vapor onto the surface to form a thin film layer. The glass rods are first coated with a 940 nm MgF Figure 2 layer, followed by a 5 nm SiO 2 layer. After coating the surface with the MgF 2 / SiO 2 / SiO 2 layer, the rods are placed in a chemical vapor deposition (CVD) machine to coat a thin layer of aminopropylsilane (APS) with a thickness generally of 1-2 nm.

[0101] Then immerse the probe tip in a solution of fluorescein-BSA or digoxin-BSA (30 μg / ml) in PBS at pH 7.4. After allowing BSA to adsorb to the probe for 10 minutes, wash the probe tip with PBS.

[0102] Example 2: Bispecific anti-hapten and anti-Fc preparations

[0103] Prepare a bispecific conjugate for IgG detection assays. Prepare the anti-hapten and anti-Fc conjugate as follows. React 2 mg of amino- (Skold Technolgy) / PBS, pH 7.4 with SPDP (ThermoFisher) at a molar conjugation ratio of 10:1 for 1 hour, followed by overnight dialysis. Deprotection of the thiol occurs by adding 30 μl of DTT (ThermoFisher). After 1 hour, purify the material on a PD10 column.

[0104] Mix 2 mg of anti-hapten-anti-fluorescein (Jackson Immunoresearch) or anti-digoxin (ThermoFisher) with 2 mg of anti-Fc (Jackson Immunoresearch) and react with SMCC (ThermoFisher) at a molar conjugation ratio of 15:1 for 1 hour, followed by purification on a PD10 column.

[0105] React the SMMC-labeled antibody mixture with the thiolated overnight and then purify on an agarose CL-6B column (GE Healthcare) to prepare the conjugate linked to anti-fluorescein and anti-Fc

[0106] Example 3: Preparation of anti-hapten conjugated to streptavidin

[0107] React 2 mg of amino- (Skold Technolgy) / PBS, pH 7.4 with SPDP (ThermoFisher) at a molar conjugation ratio of 10:1 for 1 hour, followed by overnight dialysis. Deprotection of the thiol occurs by adding 30 μl of DTT (ThermoFisher). After 1 hour, purify the material on PD10.

[0108] React 2 mg of anti-fluorescein with SMCC at a molar conjugation ratio of 15:1 for 1 hour, followed by purification on a PD10 column. In a parallel reaction, react 4 mg of streptavidin (Prozyme) with SMCC at a molar conjugation ratio of 15:1 and then purify on a PD 10 column.

[0109] SMCC-anti-fluorescein, SMCC-streptavidin and thiolated- The mixture was mixed together and after reacting overnight, it was purified on a Sepharose CL-6B column.

[0110] Example 4: Determination of IgG (Fc)

[0111] Figure 4 The IgG(Fc) assay protocol and the sequence of probe transfer through microwells containing IgG(Fc) samples and various reagents are shown. Initially, the probe with fluorescein (hapten)-BSA coated on the tip is immersed in a well containing In the reagent of the double antibody conjugate of anti-fluorescein connected with anti-mouse IgG (Fc) (Example 2). After the binding step and the probe washing step, the probe is immersed in the sample of mouse IgG. The binding of mouse IgG to the probe is monitored by the change of interference offset relative to time. After the binding step, the probe is immersed in a denaturing reagent, in this case 10 mM glycine / HCl buffer with a pH of 2.0. Low pH dissociates the immune complex from the probe surface, while leaving free fluorescein-BSA on the probe for subsequent binding. The probe is then cycled back to the anti-F-anti-mouse IgG (Fc) reagent to obtain a fresh antibody coating, and then another IgG sample is used to determine the sequence.

[0112] Details of the assay protocol, volumes, incubation times, etc. are depicted in Table 1. K buffer is 0.02% BSA, 0.002% Tween 20, PBS, pH 7.4. After regeneration, the probe cycles to step 1.

[0113] Table 1: Assay protocol (Example 1)

[0114]

[0115] For these examples, the probe was kept stationary while the microwell (1 mm diameter) mounted on an orbital shaker was at 1000 rpm to generate flow to accelerate binding kinetics. Figure 9 Contains data generated with multiple levels of mouse IgG samples ranging from 1 to 700 μg / ml at 10 regeneration cycles. At each level, the same fluorescein-coated probe and the same anti-fluorescein-anti-mouse Fc reagent were used.

[0116] Table 2 shows the Figure 4 Data from the human IgG assay using an anti-fluorescein-anti-human IgG (Fc) conjugate with the protocol described in. A total of 23 regeneration cycles were performed with consistent nm shifts in the case of IgG samples ranging from 4 μg / ml to 333 μg / ml.

[0117] Table 2: Determination of human IgG

[0118]

[0119]

[0120] Example 5: Determination of biotinylated proteins

[0121] Figure 6 The transfer sequence is illustrated where a fluorescein-coated probe passes through a micro-well containing an anti-fluorescein-streptavidin conjugate reagent and is then immersed in a biotinylated IgG sample. Due to the high affinity of streptavidin, two denaturation steps are required to release the immune complex from the probe. The first denaturation is 10 mM glycine / HCl pH 2.0, followed by DMSO (usually 20 - 40% in PBS).

[0122] Table 3 shows details of the assay protocol, volumes, incubation times, etc.

[0123] Table 3: Protocol using streptavidin probe, biotin-mouse IgG sample

[0124]

[0125]

[0126] Table 4 shows the results of 15 regeneration cycles of a 10 μg / ml biotin IgG sample using the same fluorescein-coated probe and anti-fluorescein-streptavidin binding reagent. Three probes were tested repeatedly, showing consistent results between probes.

[0127] Table 4: Biotin-IgG sample

[0128]

[0129]

[0130] Example 6: Kinetics using an anti-mouse Fc probe

[0131] Figure 5 The protocol for this example is shown.

[0132] Materials:

[0133] Probe: F-BSA

[0134] Dual antibody: anti-F - anti-mouse Fc

[0135] Sample antibody: anti-CRP, mouse antibody C54.9 mg / ml, HyTes

[0136] Sample antigen: Recombinant human CRP, Sino Biological Inc.

[0137] Washing: PBS or K buffer (0.02% BSA, 0.002% Tween 20, PBS pH 7.4)

[0138] Regeneration buffer: 10 mM glycine pH 2.0

[0139] Table 5 shows the details of the assay protocol.

[0140] Table 5: Anti-MuFc probe, Mu anti-CRP / CRP kinetics

[0141]

[0142] Figure 10 Shows the nm shift of the interference pattern. The first 300 seconds show the association of anti-CRP and CRP. The second 300 seconds show the dissociation of anti-CRP and CRP in buffer.

[0143] Table 6 shows the association rate, dissociation rate, and K D results. K D = 0.36 ± 0.1 nM.

[0144] Table 6: Anti-MuFc probe, Mu anti-CRP / CRP kinetics

[0145] <![CDATA[k off > <![CDATA[k on > <![CDATA[K D > Measurement 1 1.56E-04 3.40E+05 0.46 nM Measurement 2 1.08E-04 3.26E+05 0.33 nM Measurement 3 1.36E-04 3.00E+05 0.46 nM Measurement 4 1.44E-04 3.19E+05 0.45 nM Measurement 5 6.60E-05 3.04E+05 0.22 nM Measurement 6 5.70E-05 2.98E+05 0.19 nM Measurement 7 1.36E-04 3.00E+05 0.46 nM Measurement 8 9.93E-05 2.68E+05 0.37 nM Measurement 9 8.90E-05 2.60E+05 0.34 nM

[0146] Example 7: Kinetics using streptavidin probe

[0147] Figure 8 Shows the protocol for this example.

[0148] Materials:

[0149] Probe: F-BSA

[0150] Antibody-streptavidin: Anti-F-streptavidin

[0151] Sample antibody: Biotinylated anti-His monoclonal antibody (Biospacific)

[0152] Sample antigen: His-IL2r, Sino Biological Inc.

[0153] Washing: PBS or K buffer (0.02% BSA, 0.002% Tween 20, PBS pH 7.4)

[0154] Regeneration buffer: 10 mM glycine pH 2.0 and PBS containing 75% DMSO

[0155] Table 7 shows the details of the assay protocol.

[0156] Table 7: Streptavidin probe: anti-His-His-IL2r kinetic protocol

[0157]

[0158]

[0159] Figure 11 Shows the nm shift of the interference pattern. The first 200 seconds show the association of biotin-anti-His with IL2r-His. The next 900 seconds show the dissociation of anti-biotin-anti-His and IL2r-His in buffer.

[0160] Table 8 shows the association rate, dissociation rate, and K D results.

[0161] Table 8: Streptavidin probe: anti-His-His-IL2r kinetic results

[0162] Run koff (1 / sec) kon (1 / msec) KD (M) Measurement 1 8.81E-04 1.63E+05 5.42E-09 Measurement 2 1.36E-03 1.90E+05 7.18E-09 Measurement 3 1.12E-03 2.13E+05 5.25E-09 Measurement 4 1.53E-03 2.28E+05 6.72E-09 Measurement 5 1.63E-03 2.21E+05 7.36E-09 Measurement 6 7.01E-04 2.47E+05 2.84E-09 Measurement 7 9.61E-04 2.59E+05 3.71E-09 Measurement 8 1.26E-03 2.61E+05 4.84E-09 Measurement 9 1.49E-03 2.44E+05 6.13E-09 Measurement 10 1.68E-03 2.58E+05 6.51E-09 Measurement 11 2.22E-03 2.50E+05 8.89E-09

[0163] The present invention and the manner and method of making and using the same are now described in terms that are complete, clear, concise, and exact so that those skilled in the art to which it pertains can make and use the invention. It should be understood that the foregoing describes preferred embodiments of the invention and that modifications may be made without departing from the scope of the invention as claimed. To particularly point out and clearly claim the subject matter regarded as the invention, the following claims conclude this specification.

Claims

1. A method for detecting an analyte in a plurality of liquid samples comprising the analyte, the method comprising the steps of: (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 dual antibody solution comprising an anti-hapten antibody covalently linked to a capture antibody, wherein the capture antibody is an antibody against the analyte; (c) immersing the probe tip in a first washing container comprising a first aqueous solution having a pH of 6.0 to 8.5 for a first period of time to determine a baseline interferometry pattern of the probe tip; (d) immersing the probe tip into a sample container containing a liquid sample having the analyte for a second period of time to determine a second interferometry pattern of the immune complex formed at the probe tip; (e) determining the analyte concentration in the sample by measuring the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern and quantifying the phase shift relative to a calibration curve; (f) immersing the probe tip in an elution container comprising an acidic solution having a pH of 1.0 to 4.0 to elute the immune complex from the probe tip; (g) immersing the probe tip in a second washing container comprising a second aqueous solution having a pH of 6.0 to 8.5; and (h) repeating steps (b) to (g) 3 to 15 times, except using a container comprising a new sample in step (d) of each cycle, thereby determining the analyte concentration of a plurality of samples.

2. The method according to claim 1, wherein both the anti-hapten antibody and the capture antibody are covalently linked to a polymer having a molecular weight of 1,000 daltons to 500,000 daltons.

3. A method for measuring the binding kinetics of a sample antibody to a sample antigen in a plurality of samples each comprising the sample antibody, the method comprising the steps of: (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 dual antibody solution comprising an anti-hapten antibody covalently linked to a first antibody, wherein the first antibody is an antibody against the Fc portion of the sample antibody; (c) immersing the probe tip into an antibody sample container containing the sample antibody to bind the sample antibody to the probe, wherein the sample antibody specifically binds to a sample antigen; (d) immersing the probe tip in a first washing container comprising a first aqueous solution for a first period of time to determine a baseline interferometry pattern of the probe tip; (e) immersing the probe tip into an antigen sample container comprising the sample antigen for a second period of time to bind the sample antigen to the probe and determine a second interferometry pattern; (f) calculating the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern to determine the binding kinetics of the sample antibody and the sample antigen; (g) Immerse the probe tip into a second washing container containing a second aqueous solution to measure a third interferometry pattern; (h) Calculate the interferometric phase shift between the third interferometry pattern and the second interferometry pattern to determine the dissociation kinetics of the sample antibody and the sample antigen; (i) Immerse the probe tip in an elution container containing an acidic solution with a pH of 1.0 to 4.0 to elute the remaining immune complexes on the probe tip; (i) Immerse the probe tip in a third washing container containing a third aqueous solution with a pH of 6.0 to 8.5; and (k) Repeat steps (b) to (i) 3 to 15 times, except using a container containing a new sample antibody in step (c) of each cycle, thereby determining the binding kinetics of multiple sample antibodies.

4. The method according to claim 3, wherein both the anti - hapten antibody and the first antibody are covalently linked to a polymer with a molecular weight of 1,000 daltons to 500,000 daltons.

5. The method according to claim 2 or 4, wherein the polymer is a polysaccharide, a dendrimer, or polyethylene glycol.

6. The method according to claim 4, wherein the polymer is a copolymer of sucrose and epichlorohydrin.

7. The method according to claim 1 or 3, wherein the pH of the acidic solution is 1.5 to 2.

5.

8. The method according to claim 1 or 3, wherein the probe tip is exposed to the acidic solution once for 10 seconds to 2 minutes.

9. The method according to claim 1 or 3, wherein the probe tip is exposed to a pulse treatment consisting of 2 to 5 cycles of acidic solution treatment followed by neutralization in the solution for 10 to 20 seconds.

10. A method for detecting biotinylated analytes in multiple liquid samples, the method comprising the following steps: (a) Obtain a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface ≤ 5 mm; (b) Immerse the probe tip in a solution containing an anti - hapten antibody covalently linked to streptavidin; (c) Immerse the probe tip in a first washing container containing a first aqueous solution for a first period of time to measure the baseline interferometry pattern of the probe tip; (d) Immerse the probe tip into a sample container containing a liquid sample with a biotinylated analyte for a second period of time to measure the second interferometry pattern of the complex formed at the probe tip; (e) Determine the concentration of the biotinylated analyte in the sample by measuring the interferometric phase shift between the second interferometry pattern and the baseline interferometry pattern and quantifying the phase shift relative to a calibration curve; (f) Immerse the probe tip in an acidic solution with a pH of 1.0 to 4.0 and then in a DMSO solution to elute the complex from the probe tip; (g) Immerse the probe tip in a second washing container containing a second aqueous solution with a pH of 6.0 to 8.5; and (h) Repeat steps (b) to (g) 3 to 15 times, except that in step (d) of each cycle, use a container including a new sample, thereby detecting biotinylated analytes in multiple liquid samples.

11. A method for measuring the binding kinetics of a binding pair, the method comprising the following steps: (a) Obtain a probe having a hapten immobilized on the tip of the probe, wherein the diameter of the tip surface ≤ 5 mm; (b) Immerse the probe tip in a solution including an anti-hapten antibody covalently linked to streptavidin; (c) Immerse the probe tip into a first sample container including a biotinylated first member of a binding pair; (d) Immerse the probe tip in a first washing container including a first aqueous solution for a first period of time to determine the baseline interferometry pattern of the probe tip; (e) Immerse the probe tip into a second sample container including a second member of the binding pair for a second period of time to bind the second member of the binding pair to the probe and determine a second interferometry pattern; (f) Calculate the interferometry phase shift between the second interferometry pattern and the baseline interferometry pattern to determine the binding kinetics of the first member and the second member of the binding pair; (g) Immerse the probe tip into a second washing container including a second aqueous solution to measure a third interferometry pattern; (h) Calculate the interferometry phase shift between the third interferometry pattern and the second interferometry pattern to determine the dissociation kinetics of the first member and the second member of the binding pair; (i) Immerse the probe tip in an elution container including an acidic solution with a pH of 1.0 to 4.0 and then immerse the probe tip in a DMSO solution to elute the remaining immune complexes on the probe tip; (j) Immerse the probe tip in a third washing container including a third aqueous solution with a pH of 6.0 to 8.5; and (k) Repeat steps (b) to (i) 3 to 15 times, except that in step (e) of each cycle, use a container including a new second member of the binding pair, thereby determining the binding kinetics of the first member and the second member of the binding pair in multiple samples.

12. The method according to claim 11, wherein the first member of the binding pair is an antigen, and the second member of the binding pair is an antibody.

13. The method according to claim 11, wherein the first member of the binding pair is an antibody, and the second member of the binding pair is an antigen.

14. The method according to claim 10 or 11, wherein both the streptavidin and the anti-hapten antibody are covalently linked to a polymer having a molecular weight of 1,000 daltons to 500,000 daltons.

15. The method according to claim 14, wherein the polymer is a polysaccharide, a dendrimer, or polyethylene glycol.

16. The method according to claim 14, wherein the polymer is a copolymer of sucrose and epichlorohydrin.

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