Small molecule detection method

Abstract

The invention relates to a small molecule detection method. The method comprises the following steps: forming a multi-component compound containing'a first compound antibody-small molecules / small molecule binding partner compound-a second compound antibody ', and detecting the small molecules in a sample by detecting the multi-component compound, the micromolecule binding partner can be specifically bound with micromolecules, has at least two antigen binding sites, and can be bound with at least two micromolecules to obtain the micromolecule / micromolecule binding partner compound. And forming at least two new conformational epitopes different from the small molecule in the free state and the small molecule binding partner in the free state; each of the first complex antibody and the second complex antibody specifically binds to at least one new conformational epitope, and neither the first complex antibody nor the second complex antibody is an antibody of the small molecule antigen nor a specific antibody of the small molecule binding partner.

Description

Technical Field

[0001] The present invention generally relates to the field of small molecule detection technology, and in particular, to a method for detecting small molecules. Background Art

[0002] Due to their simple chemical structure and small molecular weight, small molecules have the problem of single antigen recognition epitope or multiple antigen recognition epitopes with overlapping or embedded epitopes. Currently, most of the rapid detection technologies for small molecules are competitive immunoassays (abbreviated as "competitive methods") based on the antigen-antibody specific recognition mechanism. However, the competitive method is only effective when the antibody affinity constant exceeds 10 11 Only when the concentration of the analyte is less than 1 L / mol can sensitivity reach the ng / L level, and ultra-high-affinity antibodies are difficult to prepare. Therefore, developing highly sensitive competitive assay kits is challenging, and it is difficult to distinguish negative samples during trace detection. Furthermore, competitive assays recognize the analyte at only one site, making cross-reactions prone to false positives.

[0003] Therefore, immunoassay methods for small molecules still need to be studied. Summary of the Invention

[0004] The present invention relates to a method for detecting small molecules, comprising: forming a multi-complex comprising a "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody", and detecting the small molecule in a sample by detecting the multi-complex;

[0005] Wherein: the molecular weight of the small molecule is less than 5000 Daltons;

[0006] The small molecule binding partner can specifically bind to the small molecule and has at least two antigen binding sites, and can bind to at least two small molecules to obtain the small molecule / small molecule binding partner complex, and form at least two new conformational epitopes that are different from the free small molecule and the free small molecule binding partner; the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope, and neither the first complex antibody nor the second complex antibody is an antibody to the small molecule antigen or a specific antibody to the small molecule binding partner.

[0007] According to another aspect of the present invention, it relates to a kit comprising the first complex antibody, a small molecule binding partner and a second complex antibody as defined above.

[0008] This new detection method can significantly improve the sensitivity and specificity of small molecule detection and solve the Hook effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0010] Figure 1 Schematic diagram of the multi-component complex structure of the present invention;

[0011] Figure 2 This is a correlation curve between the sandwich method and the competition method for cortisol detection provided by one embodiment of the present invention. DETAILED DESCRIPTION

[0012] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0013] Unless otherwise indicated, all terms (including technical and scientific terms) used to disclose the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0014] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0015] As used herein, the terms "comprising," "including," and "comprising" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0016] The recitation of numerical ranges herein by endpoints includes all numbers and fractions subsumed within the range, as well as the recited endpoints.

[0017] Concentration values used in this invention include fluctuations within a certain range. For example, fluctuations within a certain precision range are permitted. For example, for a 2% concentration, fluctuations within ±0.1% are permitted. For larger values or values that do not require overly precise control, greater fluctuations are also permitted. For example, for 100 mM, fluctuations within ±1%, ±2%, ±5%, etc. are permitted.

[0018] As used herein, the singular articles "a," "an," and "the" include plural referents unless otherwise indicated.

[0019] In the present invention, descriptions such as "plurality" and "multiple" refer to quantities greater than or equal to 2 unless otherwise specified.

[0020] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0021] In the present invention, "preferably", "better", "more preferably", and "suitably" are merely descriptions of preferred implementation methods or examples, and should be understood to not limit the scope of protection of the present invention. In the present invention, "optionally", "optional", and "optional" refer to being optional, that is, to being selected from either of the two parallel options of "with" or "without". If multiple "options" appear in a technical solution, unless otherwise specified and without contradiction or mutual restriction, each "optional" is independent.

[0022] In the present invention, the term "detection" refers to qualitative detection, quantitative detection or semi-quantitative detection of small molecules.

[0023] In the present invention, "sample" encompasses both biological and non-biological materials. Non-biological materials include environmental samples (e.g., water, for example, domestic water, river water, seawater, well water, etc.; soil; food; clothing). As used herein, the term "biological material" generally refers to biological materials suspected of containing small molecule analytes. Biological materials can be derived from any biological source, such as physiological fluids, including blood, plasma, serum, tissue fluid, saliva, lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstrual fluid, amniotic fluid, semen, and the like. Samples can be obtained from biological or non-biological sources and used directly, or after pretreatment to improve sample properties. For example, pretreatment may include preparing plasma from blood, diluting viscous fluids, and the like. Pretreatment methods also include filtration, precipitation, dilution, dissociation of small molecules from bound to free states, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, lysis, and the like. Furthermore, it may be advantageous to convert solid test samples into liquid media or release the analyte.

[0024] In the present invention, the term "small molecule" refers to a molecule with a molecular weight of less than 5000 Daltons. Such a small molecule may have a molecular weight of less than 3000 Daltons, and may have a molecular weight of less than 2000 Daltons, or may have a molecular weight of less than 1500 Daltons, or a molecular weight of less than 1200 Daltons, or a molecular weight of less than 1000 Daltons. In addition, if the complex is not particularly emphasized, then according to the context, "small molecule" generally refers to a state in which a complex is not formed with an anti-small molecule antibody. In addition, for certain small molecule types, such as hormones, etc., if not particularly emphasized, "small molecule" generally does not distinguish between free and bound states, and the "free state" and "bound state" here refer to the state in which the small molecule is non-bound / bound to other substances (such as plasma proteins, etc.) in the sample to be detected. If the bound state interferes with the detection of the small molecule, those skilled in the art can convert it into a free small molecule by known methods and then detect it.

[0025] In the present invention, the term "small molecule binding partner" refers to a substance that can specifically bind to a small molecule and has at least two antigen-binding sites. It can bind to at least two small molecules to form a small molecule / small molecule binding partner complex and at least two new conformational epitopes. A typical structure is an anti-small molecule antibody. Alternatively, it can be a multimeric binding protein, where each monomer can be at least a portion of the variable region of an anti-small molecule antibody or a receptor / ligand specifically bound by the small molecule.

[0026] In the present invention, unless otherwise specified, the term "antibody" includes complete antibodies and antibody fragments. The term "antibody fragment" includes antigen compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv and antibody minimum recognition units, as well as single-chain derivatives of these antibodies and fragments, such as scFv-Fc. The types of antibodies can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In addition, the term "antibody" includes naturally occurring antibodies and non-naturally occurring antibodies, including, for example, chimeric, humanized antibodies and human antibodies, as well as related synthetic isoforms. The term "antibody" can be used interchangeably with "immunoglobulin". The species of antibodies include, but are not limited to, cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, etc. Specifically, the term "anti-small molecule antibody" refers to an antibody that specifically binds to a small molecule to be detected, including intact antibodies and antibody fragments containing at least two antigen-binding sites. The two antigen-binding sites refer to at least two variable regions on the anti-small molecule antibody that specifically bind to the small molecule to be detected. The amino acid sequences of the two variable regions can be identical or different, as long as they can specifically bind to the small molecule to be detected.

[0027] As used herein, the term "novel conformational epitope" refers to a novel conformational epitope resulting from the binding of a test small molecule to a small molecule binding partner to form a complex. This conformational epitope is selected from the region of the small molecule binding partner where the test small molecule is connected to the small molecule binding partner, or where the small molecule binding partner undergoes conformational change (for anti-small molecule antibodies, this region is typically located in the variable region, such as the CDR region, or the junction between the CDR region and the FR region). Since the small molecule binding partner possesses at least two antigen-binding sites, the binding of the two to form a small molecule / small molecule binding partner complex inevitably results in the presence of at least two novel conformational epitopes. The two novel conformational epitopes may be the same or different.

[0028] The term "anti-complex antibody" used in the present invention refers to an antibody that can specifically bind to the novel conformational epitope, and does not bind or weakly binds to a free small molecule binding partner or a small molecule to be detected.

[0029] As used herein, the term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope. In another specific embodiment, the CDR region or CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat.

[0030] All documents mentioned in the present invention are cited as references in the present invention, just as each document is cited as a reference separately. Unless they conflict with the purpose of the invention and / or technical solution of the present invention, the cited documents involved in the present invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into the present invention as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in the present invention, the present invention shall prevail or be modified adaptively based on the description of the present invention.

[0031] A first aspect of the present invention relates to a method for detecting a small molecule, comprising: forming a multi-complex comprising a "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody", and detecting the small molecule in a sample by detecting the multi-complex;

[0032] in:

[0033] The molecular weight of the small molecule is less than 5000 Daltons;

[0034] The small molecule binding partner can specifically bind to the small molecule and has at least two antigen binding sites, and can bind to at least two small molecules to obtain the small molecule / small molecule binding partner complex, and form at least two new conformational epitopes that are different from the free small molecule and the free small molecule binding partner; the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope, and neither the first complex antibody nor the second complex antibody is an antibody to the small molecule antigen or a specific antibody to the small molecule binding partner.

[0035] For a long time, the simple structure and small molecular weight of small molecules have hindered the development of non-competitive sandwich assays. This invention innovatively creates at least two new conformational epitopes by forming a small molecule / small molecule binding partner complex, enabling the construction of a double-complex antibody sandwich-based detection system, significantly improving the specificity and sensitivity of detection results.

[0036] As will be appreciated by those skilled in the art, a small molecule is typically a hapten, which can be an organic substance such as an organic compound or a short peptide.

[0037] Exemplary small molecules are any of drugs, biotoxins, environmental pollutants, hormones, metabolites.

[0038] The drugs may include pesticides, human drugs, and veterinary drugs. Drugs include those administered for therapeutic purposes and those administered for illicit purposes. The term "drug" also broadly encompasses drug intermediates or by-products. Exemplary drugs include immunosuppressants such as FK506, CSA, digoxin, everolimus, and sirolimus.

[0039] The biotoxins can be substances produced by various organisms (including bacteria, fungi, plants and animals) during their growth and metabolism that are toxic to other organisms. Exemplary biotoxins include aflatoxins, microcystins and T2 toxins.

[0040] The environmental pollutants may be, for example, endocrine disrupting chemicals (EDCs), chemical menaces (CMRs), persistent organic pollutants (POPs), and the like.

[0041] In some embodiments, the hormone is a thyroid hormone or a steroid hormone.

[0042] In some embodiments, the small molecule is selected from any one of triiodothyronine, tetraiodothyronine, trans-triiodothyronine, cortisol, estradiol, estriol, testosterone, progesterone, 17α-hydroxyprogesterone (17α-OHP), androstenedione, and aldosterone.

[0043] In some embodiments, the small molecule is selected from metabolites such as 25-hydroxyvitamin D.

[0044] In some embodiments, the small molecule is selected from vitamin B 12 , folic acid and other organic compounds, or short peptides selected from angiotensin I, angiotensin II and the like.

[0045] In some embodiments, the small molecule / small molecule binding partner complex is formed by contacting the small molecule with the free small molecule binding partner.

[0046] In some embodiments, the complex antibody is defined as the first complex antibody or the second complex antibody; the affinity constant between the complex antibody and the bound new conformational epitope is K D1 , the affinity constant between the complex antibody and the small molecule binding partner is K D2 , then K D2 / K D1 ≥10 2 :1, or ≥10 3 :1, or ≥10 4 : 1. The above-mentioned difference in affinity is conducive to the first complex antibody and the second complex antibody binding to the new conformational epitope more specifically, thereby improving the detection sensitivity.

[0047] As described above, it is easy to understand that the K of the first complex antibody D1 , K D2 and the K of the second complex antibody D1 , K D2 can be the same or different values, but their K D2 / K D1 All satisfy the above proportional relationship.

[0048] In some embodiments, the affinity constant K between the small molecule binding partner and the small molecule is D3 ≤9×10 -9 ;

[0049] In some embodiments, K D1 ≤9×10 -9 .

[0050] In some embodiments, K D2 ≥1×10 -6 .

[0051] In some embodiments, 1×10 -6 ≤K D2 ≤9×10 -4 , or 1×10 -6 ≤K D2 ≤9×10 -5 .

[0052] In some embodiments, the complexed antibody does not bind or weakly binds to free small molecules.

[0053] The present invention uses the SPR method to detect affinity constants. The detection principle is based on optical principles: when biomolecules bind or dissociate on the sensor chip surface, they cause changes in the chip surface's refractive index, which in turn leads to changes in the SPR angle. By monitoring these changes, intermolecular interactions can be analyzed in real time.

[0054] The exemplary process mainly includes the following steps: first, the anti-small molecule antibody / complex antibody to be tested is fixed on the surface of the sensor chip; then, different concentration gradients of the test standard / anti-small molecule antibody are flowed through the chip in sequence as the mobile phase, and the dynamic changes of the SPR angle during the molecular binding and dissociation process are monitored in real time to generate the binding / dissociation curve; finally, the affinity constant (K) is calculated by fitting the curve data through the software. D ), thereby evaluating antibody affinity.

[0055] The free state of the anti-small molecule antibody means that it is not coupled to a solid phase or a signal substance, and of course is not bound to the small molecule.

[0056] In some embodiments, the method further comprises pre-treating the bound small molecule to release the small molecule into a free state. Pre-treatment is generally performed before obtaining the small molecule / small molecule binding partner complex. Because a certain proportion of certain small molecules, particularly hormones (typically T3 and / or T4 antigens), exist in a bound state bound to a binding protein, a pre-treatment step can be selectively added before detection based on different detection requirements. The pre-treatment step can include adding a pre-treatment agent to the sample before the sample reacts with the small molecule binding partner to dissociate the small molecule in the sample from the binding substance (typically the binding protein) in the sample. Based on this, the present invention can not only measure the total amount of analyte, but also measure the amount of free analyte, the amount of bound analyte, the ratio of bound analyte to free and / or total analyte, or the ratio of free to bound and / or total analyte.

[0057] In some embodiments, the method further comprises: diluting the sample. Dilution is generally performed before obtaining the small molecule / small molecule binding partner complex.

[0058] In some embodiments, the sample is directly reacted with the small molecule binding partner, the first complex antibody, and the second complex antibody without dilution.

[0059] The solution of the present invention can solve the Hook effect, so that dilution can be avoided when detecting some samples (such as high-value samples), thereby avoiding the interference of the diluent matrix effect on the detection and accelerating the detection speed.

[0060] In some embodiments, the first complex antibody and the second complex antibody have the same or different CDR sequences. In some embodiments, the first complex antibody and the second complex antibody are antibodies derived from the same monoclonal antibody.

[0061] In some embodiments, the small molecule binding partner is an anti-small molecule antibody.

[0062] Advantageously, the anti-small molecule antibody, the first complex antibody, and the second complex antibody are all monoclonal antibodies.

[0063] In some embodiments, the anti-small molecule antibody is any one of IgG, IgA, IgM, IgD, and IgE.

[0064] In some embodiments, the anti-small molecule antibody is a whole immunoglobulin or an antibody fragment;

[0065] In some embodiments, the anti-small molecule antibody comprises two or more antibody variable regions, which may further be F(ab') 2. In some preferred embodiments, the anti-small molecule antibody comprises two or more antibody variable regions, and the sequences of the two or more variable regions are identical or inconsistent.

[0066] In some embodiments, the first complex antibody or the second complex antibody is a whole immunoglobulin or an antibody fragment;

[0067] In some embodiments, the first complex antibody or the second complex antibody comprises one or more antibody variable regions, which may further be Fv, Fab, Fab' or F(ab')2 fragments.

[0068] In some embodiments, the first complex antibody is conjugated to a solid phase.

[0069] "Solid phase" refers to a solid phase material that interacts with a liquid phase reagent through a heterogeneous reaction. The use of solid phase is well known in the fields of chemistry, biochemistry, pharmacy and molecular biology. Many types of solid phases have been developed according to the technical problems to be solved. Any of these can be used in the context of the present invention. For example, the solid phase used in the method of the present invention may include the following components: silicon dioxide, cellulose acetate, cellulose nitrate, nylon, polyester, polyethersulfone, polyolefin or polyvinylidene fluoride or a combination thereof. Other suitable solid phases include, but are not limited to, controlled pore glass, glass plates or slides, magnetic spheres, polystyrene and activated dextran. In other aspects, synthetic organic polymers (such as polyacrylamide, polymethacrylate and polystyrene) are also illustrative support surfaces. In addition, polysaccharides (such as cellulose and dextran) are also other illustrative examples of support surfaces. Other support surfaces such as fibers are also feasible.

[0070] Common resin-based solid phases used, for example, in combinatorial chemistry or protein chemistry include: polystyrene resins (e.g., cross-linked with divinylbenzene); hydroxymethyl polystyrene; aminomethyl polystyrene; TentaGel resins (TG) and ArgoGel (AG): polystyrene / DVB poly(ethylene glycol) graft copolymer (PS-PEG)-Bayer; crown / pin (CP) (radiation-grafted polyethylene / polypropylene support); diatomaceous earth / polyacrylamide-based resins (KPA); controlled pore glass; PEGA-poly(ethylene glycol) / dimethylacrylamide copolymer.

[0071] Immobilization on a solid phase can be accomplished using a solid phase that has been modified or activated to contain functional groups that allow the entity or support to be covalently coupled to the first complex antibody. The functional groups are selected from one or more of carboxyl, amino, sulfhydryl, and tosyl groups. The first complex antibody can also be non-covalently attached to the surface by, for example, ionic or hydrophobic mechanisms and separated by a release agent that locally inhibits these mechanisms.

[0072] In one embodiment, the solid phase can be fibrous or granular, usually allowing for proper contact. The size of the solid phase suitable for the method of the present invention can vary according to the selected method. The capture molecule can only be bound to one solid phase (e.g., a porous plate), or can be bound to many solid phases (e.g., beads). In one embodiment, the solid phase can be fibrous or granular to allow for optimal contact. The size of the solid phase can vary and can be selected according to the method to be performed.

[0073] In some embodiments, the solid phase is a sheet, a preformed disk, a cylinder, a single fiber, or a solid support composed of particles.

[0074] In the present invention, the shape of the term "particles" (sometimes referred to as "beads" or "microbeads" or "microspheres") can generally vary. For example, in a particular embodiment, the particles are spherical. However, it should be understood that the present invention also contemplates other shapes, such as plates, rods, disks, strips, tubes, irregular shapes, etc. In addition, the size of the particles can also vary. For example, the average size (e.g., diameter) of the particles can range from about 0.1 nm to 1 mm, such as 1 nm, 10 nm, 100 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 500 μm.

[0075] In some embodiments, the particles are magnetic spheres, such as ferroferric oxide magnetic nanospheres.

[0076] In some embodiments, the second complex antibody is conjugated to a signaling substance.

[0077] The signal substance refers to any substance that can produce the signal for direct or indirect detection. Therefore, the signal substance can be detected directly or indirectly. For direct detection, the signal substance applicable to the present invention can be selected from any known detectable label group. In some embodiments, the signal substance is selected from chromogen, fluorescent group, chemiluminescent group (such as isoluminol and derivatives thereof, acridinium ester or dioxetane), electrochemiluminescent compound, catalyst, enzyme, enzyme substrate, dye, fluorescent dye (such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and derivatives thereof). Other examples of the signal substance have: luminescent metal complex such as ruthenium or europium complex (such as for ECLIA), enzyme (such as for ELISA) and radioactive isotope (such as for RIA). In some embodiments, the signal substance is selected from ABEI and derivatives thereof, acridinium ester, alkaline phosphatase and horseradish peroxidase.

[0078] Indirect detection systems include, for example, labeling the second complex antibody with the first partner of a bioaffinity binding pair. Examples of suitable binding pairs include: biotin or a biotin analog such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analog / complementary nucleic acid.

[0079] In some embodiments, the method includes: contacting the sample with the free small molecule binding partner, and then simultaneously or stepwise contacting the first complex antibody conjugated to the solid phase and the second complex antibody coupled to the signal substance for sufficient reaction to form a multi-complex comprising "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody", and detecting the small molecule in the sample by detecting the multi-complex.

[0080] In some embodiments, the method comprises:

[0081] a) contacting the small molecule with the free small molecule binding partner to form a small molecule / small molecule binding partner complex;

[0082] b) contacting the small molecule / small molecule binding partner complex, the first complex antibody conjugated to the solid phase, and the second complex antibody coupled to the signal substance simultaneously or stepwise, and incubating the system until the reaction is sufficient to obtain the multi-complex;

[0083] c) detecting the small molecule via the signal substance.

[0084] The assays of the present invention are typically performed under conventional conditions for such assays, for example, at a temperature of 4° C. to 45° C. More typically, 25° C. to 45° C., for example, 30° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C. The assays are typically performed in aqueous solution; and at a pH of 5 to 9, for example, pH 6, pH 7, or pH 8.

[0085] In some embodiments, step b) further comprises: performing at least one washing step to separate unbound substances from the solid phase.

[0086] The unbound substances mainly include impurities in the sample that are not bound to the solid phase, free small molecule binding partners that are not bound to small molecules in the system, and second complex antibodies coupled with signal substances that do not participate in forming a multi-complex.

[0087] In some embodiments, it can be a two-step washing process. The first washing step is performed after the small molecule / small molecule binding partner complex is contacted with the first complex antibody conjugated to the solid phase and incubated until the two are fully reacted, so that the first unbound substance is separated from the solid phase. The first unbound substance mainly includes impurities in the sample that are not bound to the solid phase and free small molecule binding partners that are not bound to small molecules in the system. The second washing step is performed after the second complex antibody coupled with the signal substance is contacted with the solid phase obtained in the first washing step and incubated until the two are fully bound to form a multi-complex, so that the second unbound substance is separated from the multi-complex. The second unbound substance mainly includes the second complex antibody coupled with the signal substance that does not participate in the formation of the multi-complex.

[0088] In some embodiments, a one-step wash is performed only after the small molecule / small molecule binding partner complex contacts the first complex antibody conjugated to the solid phase and the second complex antibody coupled to the signal substance and the system is incubated until the system is fully combined to form a multi-complex, so that the unbound substances mentioned above are separated from the system at one time in this wash.

[0089] In other embodiments, the method is a lateral flow assay. When a lateral flow assay is used for detection, advantageously, the solid phase is a strip-shaped solid phase, and is typically provided with a "test line" and a "control line".

[0090] According to the second aspect of the present invention, it also relates to a kit, which comprises the first complex antibody, a small molecule binding partner and a second complex antibody as defined in the first aspect above.

[0091] In some embodiments, the small molecule binding partner is present in a free form in the detection kit, the first complex antibody is conjugated to a solid phase, and the second complex antibody is coupled to a signal substance.

[0092] The kit may further comprise one or more containers, which may contain a dilution buffer, a reaction solution, a color development reagent, etc. The kit may also comprise instructions for use.

[0093] The kit can be used for the diagnosis or prognosis of a disease or condition. For example, a biomarker (e.g., hormone) indicating a particular disease, condition, or prognosis can be determined in the context of the present invention. In particular, the kit and method of the present invention can be used for individual diagnosis, prognosis, risk assessment, risk stratification, monitoring, and / or therapeutic control. The term "individual" herein refers to a living human or non-human animal, such as a mammal, particularly a human. The individual is most suitably a patient. As used herein, the term "patient" refers to a living human or non-human animal (typically a human) who is receiving medical care or who should receive medical care due to a disease or medical condition. This includes people who are undergoing pathological examinations without clear disease. Therefore, the methods and assays described herein are applicable to both human and animal diseases.

[0094] In some embodiments, the kit further comprises a solid phase. In some embodiments, the solid phase is the solid phase defined in the first aspect above.

[0095] In some embodiments, the first complex antibody is conjugated to a solid phase.

[0096] In some embodiments, the second complex antibody is coupled to a signal substance. In some embodiments, the signal substance is the signal substance defined in the first aspect above.

[0097] In some embodiments, the small molecule binding partner is in a free state, that is, it does not form a complex with the small molecule.

[0098] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made to experimental manuals or conventional conditions in the art, other experimental methods known in the art, or conditions recommended by the manufacturer.

[0099] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0100] Example 1 Antibody Preparation

[0101] 1. Sources of Anti-Small Molecule Antibodies

[0102] Source of cortisol antibody: Snibe cortisol competition kit (product number: 130270002M, batch number: 468240111)

[0103] Source of 17α-OHP antibody: Snibe 17α-OHP competition kit (product number: 130201002M, batch number: 026240112).

[0104] 2. Preparation process of complex antibodies

[0105] 1) Preparation of Cortisol / Cortisol Antibody Complex Antibodies

[0106] Immunization of mice

[0107] Cortisol natural standard (Sigma) and the aforementioned cortisol antibody were dialyzed into 0.01 M PBS and incubated at a molar ratio of 20:1 at 37°C for 15 min to form a mixture containing a cortisol / cortisol antibody complex as the immunogen.

[0108] An equal volume of Freund's complete adjuvant (Sigma) was used to emulsify the antigen into water-in-oil droplets. 6-8 week old SPF grade Balb / c mice were injected subcutaneously at multiple points with 200 μg / mouse. Three weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple points with 100 μg / mouse. Boost the immunization twice and perform a shock injection into the peritoneum three days before fusion.

[0109] b) Fusion and cloning

[0110] Spleens from immunized mice were harvested and ground to isolate dispersed single splenocytes. These splenocytes were then fused with myeloma cells using polyethylene glycol using standard procedures. Following fusion, the cells were cultured for one week, followed by a change of medium. The following day, the supernatant was collected for indirect enzyme immunoassay analysis.

[0111] c) The detection method is as follows:

[0112] The cortisol antibody was treated with pepsin to obtain the F(ab')2 fragment as the coating antigen. After blocking, PBS or natural cortisol standard (Sigma) was added, and then the cell supernatant was added. The color developing agent was goat anti-mouse IgG Fcγ-HRP. Positive wells with no or low reaction with the cortisol antibody F(ab')2 fragment and strong reaction with the complex were selected. The positive wells were further subjected to competitive detection: cortisol-bovine IgG was used as the coating source. After blocking, PBS or natural cortisol standard was added, and then the cell supernatant was added. The color developing agent was goat anti-mouse IgG Fcγ-HRP. Positive wells with no competitive inhibition of cortisol were selected and further subcloned by limiting dilution. After one week of culture, enzyme immunoassay was continued. The detection method was the same as above. Positive wells were selected for limiting dilution and repeated 3-4 times until all the test wells were positive and the cells in the wells were single colonies. The cells in the best positive wells were selected for expansion culture to obtain specific hybridoma cell lines, which were anti-complex antibodies.

[0113] d) Preservation of positive cell lines

[0114] Hybridoma cell lines were frozen at -80°C in freezing medium (50% 1640, 40% FCS, 10% DMSO) using a cell cooling box (cooling rate -1°C / min) (Corning) and transferred to liquid nitrogen for storage after 24 hours.

[0115] e) Preparation of ascites from positive cell lines

[0116] The positive cell lines were expanded and cultured, and injected into mice that had been pre-immunized with IFA to prepare ascites. The ascites was collected to obtain anti-complex antibodies, which were affinity purified using the SPA method.

[0117] 2) Preparation of 17α-OHP / 17α-OHP Antibody Complex Antibody

[0118] The preparation method is similar to the preparation of the above-mentioned cortisol / cortisol antibody complex antibody.

[0119] 3. Binding characteristics of complex antibodies with complexes, anti-small molecule antibodies, and free antigens, as well as binding affinity assessment

[0120] The binding affinity assessment assay for the cortisol complex antibody is performed as follows:

[0121] a. Affinity detection of complex antibodies and small molecules or anti-small molecule antibodies

[0122] (1) Immobilization of complex antibodies: The ligand was immobilized by direct amino coupling. First, EDC / NHS (freshly prepared and used, 1:1 mixture) was injected for 5-10 minutes to activate the carboxyl group. Then, the complex antibody was diluted to 10 μg / ml with sodium acetate buffer at pH 4.5 and injected into the detection channel to the target coupling level (small molecule analytes, high coupling, 20,000 RU; large analytes, low coupling, 100-500 RU). Then, ethanolamine (pH 8.5) was injected for 5-10 minutes to block the unreacted sites. Finally, the equilibration buffer was used to rinse until the baseline was stable.

[0123] (2) Analyte detection: Small molecules were diluted to 100 μM, or anti-small molecule antibodies were diluted to 10 μM, and then diluted 2-fold to 6 gradients, plus 0 concentration for a total of 8. A multi-cycle kinetic detection method was used, with injection from low to high concentrations at a flow rate of 10 μL / min for 180 s, a dissociation time of 600 s, and regeneration with 10 mM Gly-HCl (pH 2.0) for 120 s.

[0124] b. Affinity detection of complex antibodies and small molecules / anti-small molecule antibodies

[0125] (1) Immobilization of anti-small molecule antibodies: Ligand immobilization was performed using the amino direct coupling method. First, EDC / NHS (freshly prepared and used, 1:1 mixture) was injected for 5-10 minutes to activate the carboxyl group. Then, the anti-small molecule antibody was diluted to 10 μg / ml with sodium acetate buffer at pH 4.5 and injected into the detection channel to the target coupling level (large analyte, low coupling, 100-500 RU). Then, ethanolamine (pH 8.5) was injected for 5-10 minutes to block unreacted sites. (4) The equilibration buffer was used to rinse until the baseline was stable.

[0126] (2) Small molecule injection: The small molecule solution was diluted to 100 μM and injected until the curve stabilized.

[0127] (3) Co-injection of small molecules and complex antibodies: The complex antibody was diluted to 10 μM (containing 100 μM of the small molecule), and then diluted 2-fold to 6 concentrations (the dilution solution contained 100 μM of the small molecule). A multi-cycle kinetic detection method was used, and the samples were injected in the order from low concentration to high concentration at a flow rate of 10 μL / min for 120 s. The dissociation phase was still carried out with a 100 μM small molecule solution, followed by regeneration and baseline equilibration.

[0128] c. Use analysis software to fit and analyze data and calculate affinity.

[0129] The binding affinity of the 17α complex antibody was assessed similarly to the above procedure.

[0130] The test results are as follows:

[0131]

[0132]

[0133] The data showed that the complex antibody did not bind to free small molecules, had a weak binding force to anti-small molecule antibodies that were not bound to small molecules, and had a higher affinity for the complex formed by anti-small molecule antibodies and small molecules.

[0134] Example 2 Preparation and Performance Verification of Cortisol Kit

[0135] 1. Components of the Cortisol Kit

[0136] 1) Components of the first-generation small molecule sandwich assay kit

[0137] Buffer: PBS buffer containing 0.25% ANS.

[0138] Solid phase complex antibody reagent: TRIS buffer containing 0.2% NaN3, complex antibody that specifically binds to cortisol / cortisol antibody complex attached to the solid phase.

[0139] Labeled anti-small molecule antibody reagent: PBS buffer containing 0.2% NaN3, cortisol antibody linked to the signal substance.

[0140] 2) Reagent components for second-generation small molecule sandwich assays

[0141] Pre-reaction solution 1: TRIS buffer containing 0.25% ANS, cortisol antibody.

[0142] Solid phase first complex antibody reagent: TRIS buffer containing 0.2% NaN3, complex antibody that specifically binds to the cortisol / cortisol antibody complex is linked to the solid phase.

[0143] Labeled secondary complex antibody reagent: PBS buffer containing 0.2% NaN3, complex antibody that specifically binds to the cortisol / cortisol antibody complex linked to the signal substance.

[0144] Specific antibody information is as follows:

[0145]

[0146] 2. Detection process:

[0147] 1) First-generation small molecule sandwich detection process

[0148] ① Take 10 μL of sample, 150 μL of labeled anti-small molecule antibody reagent and 20 μL of solid phase complex antibody reagent and add them to 80 μL of buffer, mix well and incubate at 37°C for 4 minutes, wash and remove the supernatant;

[0149] ②Add substrate solution and detect light signal.

[0150] Detection process of the cortisol test kit related embodiment (one-step cleaning method)

[0151] ① Add 10 μL of sample to 100 μL of pre-reaction solution, mix well, and incubate at 37°C for 4 minutes;

[0152] ② Add 150 μL of labeled second complex antibody reagent and 20 μL of solid phase first complex antibody reagent to step ①, mix well and incubate at 37°C for 4 minutes, wash and remove the supernatant;

[0153] ③Add substrate solution and detect light signal.

[0154] 3) Detection process of the cortisol test kit related embodiments (two-step cleaning method)

[0155] The testing steps include:

[0156] ① Add 10 μL of sample to 100 μL of pre-reaction solution, mix well, and incubate at 37°C for 4 minutes;

[0157] ② Add 20 μL of solid phase first complex antibody reagent to step ①, mix well, incubate at 37°C for 4 minutes, and remove the supernatant after washing;

[0158] ③ Add 150 μL of labeled second complex antibody reagent to step ②, mix well, incubate at 37°C for 4 minutes, and remove the supernatant after washing;

[0159] ④Add substrate solution and measure the light signal.

[0160] Sensitivity, discrimination, and Hook effect verification results

[0161] Testing instrument: MAGLUMI X8

[0162] Sample preparation: DMSO was used to prepare the stock solution of the cortisol (Sigma) standard. PBS was used to prepare the cortisol standard (Sigma) standard at a gradient working concentration of 0 ng / mL (point A), 10 ng / mL (point B), 16.683 ng / mL (point C), 27.832 ng / mL (point D), 46.431 ng / mL (point E), 77.46 ng / mL (point F), 129.224 ng / mL (point G), 215.583 ng / mL (point H), 359.625 ng / mL (point I), and 600 ng / mL (point J). The evaluation results are as follows.

[0163]

[0164] Compared with the commercial competitive method, the sensitivity and discrimination of the first-generation sandwich method and each embodiment are significantly improved and have obvious advantages. In the specific embodiments, that is, when the double complex antibody sandwich method is used to detect cortisol, the B / A value is significantly higher than the commercial competitive method detection kit, that is, the sensitivity of the embodiment provided by the present invention is improved compared with the competitive method. Further, the J / A value is higher than the numerical value of the commercial competitive method kit and the first-generation sandwich method kit, indicating that the embodiment provided by the present invention has better discrimination in samples with a larger span concentration gradient; further, for high-value samples (I point sample, J point sample), the ratio of its light signal is close to the concentration ratio of the I point and J point samples, which proves that the problem of the Hook effect is effectively solved in high-value samples. It can be seen that in the various embodiments provided by the present invention, the complex antibodies can choose the same strain of complex antibodies or different strains of complex antibodies, and the detection process can choose a one-step washing method or a two-step washing method. The scheme has great advantages in the detection of cortisol.

[0165] (1) Detection capability verification of plasma samples

[0166] The kit of Example 2.1 and the one-step cleaning detection process were used to detect 20 plasma samples. The test results and the test results of the same blood samples using a commercial competitive kit were collected and a correlation curve was drawn. Figure 2 shown.

[0167] In the blood sample testing, the sandwich method showed correlation with the competition method, indicating that the scheme can be applied to actual sample testing and is reliable.

[0168] (2) Specific performance verification

[0169] Testing instrument: MAGLUMI X8

[0170] Sample preparation: Cortisol analogs (fludrocortisone, dexamethasone, prednisolone, and corticosterone) were prepared in DMSO. For testing, the four main analogs were diluted in PBS to working concentrations of 1 μg / mL, 0.1 μg / mL, 1 μg / mL, and 0.1 μg / mL, respectively. The following assay steps were used for evaluation.

[0171] The above samples containing analogs were tested using the kit of Example 2.1 and the one-step cleaning method. The test results are as follows:

[0172]

[0173] As can be seen from the data, the double complex antibody sandwich method also performs relatively well in terms of specificity when used to detect cortisol. Since the complex antibody itself has a high specificity for the complex formed by anti-small molecule antibodies and small molecules, the simultaneous use of two complex antibodies further improves the specificity of the entire detection system.

[0174] Example 3 Preparation and Performance Verification of 17α-OHP Kit

[0175] 1.17α-OHP Kit Components

[0176] 1) Components of the first-generation small molecule sandwich assay kit

[0177] Buffer: PBS buffer.

[0178] Solid phase anti-small molecule antibody reagent: PBS buffer containing 0.2% NaN3, 17α-OHP antibody linked to the solid phase.

[0179] Labeled complex antibody reagent: TRIS-HCl buffer containing 0.2% NaN3, complex antibody that specifically binds to 17α-OHP / 17α-OHP complex linked to a signal substance.

[0180] 2) Reagent components for second-generation small molecule sandwich assays

[0181] Pre-reaction solution 1: TRIS buffer, 17α-OHP antibody.

[0182] Solid phase first complex antibody reagent: PBS buffer containing 0.2% NaN3, and a complex antibody that specifically binds to the 17α-OHP / 17α-OHP antibody complex linked to the solid phase.

[0183] Labeling the second complex antibody reagent: TRIS-HCl buffer containing 0.2% NaN3, a complex antibody that specifically binds to the 17α-OHP / 17α-OHP antibody complex and is linked to a signal substance.

[0184] Specific antibody information is as follows:

[0185]

[0186] 2. Detection process:

[0187] 1) First-generation small molecule sandwich detection process

[0188] ① Take 10 μL of sample and 20 μL of solid-phase anti-small molecule antibody reagent and add them to 100 μL of buffer, mix well and incubate at 37°C for 5 minutes, then wash and remove the supernatant;

[0189] ②Add 100 μL of labeled complex antibody reagent to step ①, mix well, incubate at 37°C for 5 minutes, and remove the supernatant after washing;

[0190] ③Add substrate solution and detect light signal.

[0191] Detection process of the 17α-OHP kit related embodiments (one-step cleaning method)

[0192] ① Take 10 μL of sample and 20 μL of solid phase first complex antibody reagent and add them to 100 μL of pre-reaction solution, mix well and incubate at 37°C for 5 minutes;

[0193] ② Add 160 μL of the reagent for removing the labeled second complex antibody to step ①, mix well, incubate at 37°C for 5 minutes, and remove the supernatant after washing;

[0194] ③Add substrate solution and detect light signal.

[0195] (1) Sensitivity, discrimination, and Hook effect verification results

[0196] Testing instrument: MAGLUMI X8

[0197] Sample preparation: DMSO was used to prepare the stock solution of the 17α-OHP standard (B&K). PBS was used for the assay to prepare a gradient working concentration of 0 ng / mL (point A), 0.1 ng / mL (point B), 0.204 ng / mL (point C), 0.416 ng / mL (point D), 0.849 ng / mL (point E), 1.732 ng / mL (point F), 3.534 ng / mL (point G), 7.209 ng / mL (point H), 14.706 ng / mL (point I), and 30 ng / mL (point J). The evaluation results are as follows.

[0198]

[0199] From the verification results, it can be seen that in each embodiment provided by the present invention, when using the complex antibody to detect 17-αOHP, the same strain of complex antibody or different strains of complex antibodies can be selected, and the double complex antibody sandwich method also has a good performance improvement in detection sensitivity, discrimination and high-value sample Hook effect.

[0200] (2) Specificity verification results

[0201] Testing instrument: MAGLUMI X8

[0202] Sample preparation: DMSO was used to prepare the stock solutions of 17α-OHP analogs: progesterone, 17α-hydroxypregnenolone, 11-deoxycortisol, and 21-deoxycortisol. For testing, the four main analogs were diluted in PBS to working concentrations of 100 ng / mL, 100 ng / mL, 20 ng / mL, and 20 ng / mL, respectively. The following assay steps were used for evaluation.

[0203] The above samples containing analogs were tested using the kit of Example 3.1 and the one-step cleaning method. The test results are as follows:

[0204] The test results are as follows:

[0205]

[0206] As can be seen from the data, the double complex antibody sandwich method also performs relatively well in terms of specificity when used to detect 17α-OHP. Since the complex antibody itself has a high specificity for the complex formed by anti-small molecule antibodies and small molecules, the simultaneous use of two complex antibodies further improves the specificity of the entire detection system.

[0207] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the concept of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A method for detecting small molecules, comprising: forming a multi-complex comprising a "first complex antibody-small molecule / small molecule binding partner complex-second complex antibody", and detecting the small molecule in the sample by detecting the multi-complex; Wherein: the molecular weight of the small molecule is less than 5000 Daltons; The small molecule binding partner can specifically bind to the small molecule and has at least two antigen binding sites, and can bind to at least two small molecules to obtain the small molecule / small molecule binding partner complex, and form at least two new conformational epitopes that are different from the free small molecule and the free small molecule binding partner; the first complex antibody and the second complex antibody each specifically bind to at least one new conformational epitope, and neither the first complex antibody nor the second complex antibody is an antibody to the small molecule antigen or a specific antibody to the small molecule binding partner.

2. The method of claim 1, wherein the small molecule has a molecular weight of less than 3000 Daltons; alternatively, a molecular weight of less than 2000 Daltons; alternatively, a molecular weight of less than 1500 Daltons; alternatively, a molecular weight of less than 1000 Daltons; Optionally, the small molecule is any one of a drug, a biotoxin, an environmental pollutant, a hormone, and a metabolite; Optionally, the hormone is a thyroid hormone or a steroid hormone; Optionally, the small molecule is selected from any one of triiodothyronine, tetraiodothyronine, trans-triiodothyronine, cortisol, estradiol, estriol, testosterone, progesterone, 17α-hydroxyprogesterone, androstenedione, and aldosterone; Optionally, the small molecule / small molecule binding partner complex is formed by contact between the small molecule and the free small molecule binding partner; Optionally, the method further comprises: pre-treating the bound small molecules to release the small molecules into a free state; optionally, the method further comprises: diluting the sample.

3. The method according to claim 1, wherein the complex antibody is defined as the first complex antibody or the second complex antibody; the affinity constant between the complex antibody and the bound new conformational epitope is K D1 , the affinity constant between the small molecule and the binding partner is K D2 , then K D2 / K D1 ≥10 2 :1, or ≥10 3 :1, or ≥10 4 :1; Optionally, the affinity constant K between the small molecule binding partner and the small molecule D3 ≤9×10 -9 ; Optionally, K D1 ≤9×10 -9 ; Optionally, K D2 ≥1×10 -6 ; Optionally, the first complex antibody and the second complex antibody have the same or different CDR sequences.

4. The method according to claim 1, wherein the small molecule binding partner is an anti-small molecule antibody; Optionally, the anti-small molecule antibody is selected from any one of IgG, IgA, IgM, IgD, and IgE; Optionally, the anti-small molecule antibody is a complete immunoglobulin or an antibody fragment; Optionally, the anti-small molecule antibody comprises two or more antibody variable regions, which may further be F(ab')2. Optionally, the anti-small molecule antibody comprises two or more antibody variable regions, and the sequences of the two or more variable regions are identical or inconsistent.

5. The method according to claim 1, wherein the first complex antibody or the second complex antibody is a complete immunoglobulin or an antibody fragment; Optionally, the first complex antibody or the second complex antibody comprises one or more antibody variable regions, which may further be Fv, Fab, Fab' or F(ab')2 fragments.

6. The method according to any one of claims 1 to 5, wherein the first complex antibody is conjugated to a solid phase; Optionally, the solid phase is a sheet, a preformed disk, a cylinder, a single fiber, or a solid support composed of particles; preferably the particles are magnetic spheres.

7. The method according to claim 6, wherein the second complex antibody is coupled with a signal substance; Optionally, the signal substance is selected from chromogens, fluorescent groups, chemiluminescent groups (such as acridinium esters or dioxetanes), electrochemiluminescent compounds, catalysts, enzymes, enzyme substrates, dyes, fluorescent dyes (such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and derivatives thereof); Optionally, the signal substance is selected from ABEI and its derivatives, acridinium ester, alkaline phosphatase and horseradish peroxidase.

8. The method according to claim 7, comprising: a) contacting the small molecule with the free small molecule binding partner to form a small molecule / small molecule binding partner complex; b) contacting the small molecule / small molecule binding partner complex, the first complex antibody conjugated to the solid phase, and the second complex antibody coupled to the signal substance simultaneously or stepwise, and incubating until fully reacted to obtain the multi-complex; c) detecting the small molecule via the signal substance; Optionally, step b) further comprises at least one washing step to separate unbound substances from the solid phase.

9. A kit comprising the first complex antibody, a small molecule binding partner and a second complex antibody as defined in any one of claims 1 to 8.

10. The kit according to claim 9, further comprising a solid phase; Optionally, the first complex antibody is conjugated to a solid phase; Optionally, the solid phase is a sheet, a preformed disk, a cylinder, a single fiber, or a solid support composed of particles; Preferred particles are magnetic spheres; Optionally, the second complex antibody is coupled with a signal substance; Optionally, the signal substance is selected from chromogens, fluorescent groups, chemiluminescent groups, electrochemiluminescent compounds, catalysts, enzymes, enzyme substrates, dyes, fluorescent dyes (such as fluorescein, coumarin, rhodamine, oxazine, resorufin, cyanine and derivatives thereof); Optionally, the signal substance is selected from ABEI and its derivatives, acridinium ester, alkaline phosphatase and horseradish peroxidase; optionally, the small molecule binding partner is in a free state.

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