A method, kit, and application for detecting IgM antibodies.

Abstract

This application relates to a method, kit, and application for detecting IgM antibodies. The method includes the following steps: mixing a hydrolysis reagent containing a proteolytic enzyme and an immunoassay reagent capable of specifically binding to the target IgM antibody in the test sample with the test sample to obtain a test reaction solution; detecting the signal value of the reaction solution; wherein the proteolytic enzyme can destroy the structure of interfering proteins in the test sample. The solution provided in this application can reduce or eliminate the interference of matrix effects produced by interfering proteins, thereby improving the detection accuracy of IgM antibodies.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a method, kit, and application for detecting IgM antibodies. Background Technology

[0002] Immunoglobulins (Ig) are globulins that possess antibody activity or have a chemical structure similar to antibodies, and are widely found in the serum, lymph, tissue fluid, and exocrine fluid of mammals. Based on their structure, immunoglobulins can be classified into five classes: IgG, IgA, IgM, IgD, and IgE.

[0003] When pathogens invade the human body, the body first produces specific antibodies IgM. Therefore, IgM can be used as a marker for acute, recurrent, or latent infections. As pathogens continue to stimulate and the immune response develops, the specific antibodies produced by the body gradually change from IgM to IgG. Therefore, IgG can be used as an indicator for assessing past infections.

[0004] In the detection of specific antibody IgM, there are two detection methods: homogeneous and heterogeneous. Compared with heterogeneous detection, homogeneous detection methods have the advantages of shorter reaction time, no need for separation and washing, simpler operation, higher detection precision, and higher sensitivity, and are used in many infectious disease immunoassays. However, because the sample components in the homogeneous IgM immunoassay method are not removed by the washing step, it is susceptible to interference from proteins such as rheumatoid factor, specific IgG antibodies, and heterophilic antibodies in the blood sample, which can lead to false negatives or other erroneous results. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a detection method, kit, and application for IgM antibodies, which can reduce or eliminate the interference of matrix effects generated by interfering proteins and improve the detection accuracy of specific IgM antibodies.

[0006] The first aspect of this application provides a method, kit, and application for detecting IgM antibodies, comprising the following steps: S1. Mix the hydrolysis reagent containing proteolytic enzymes and the immunoassay reagent that can specifically bind to the IgM antibody in the sample to be tested with the sample to obtain the test reaction solution; S2, the signal value of the detection reaction solution; The proteolytic enzyme can destroy the structure of interfering proteins in the test sample.

[0007] In some embodiments of this application, the immunoassay reagent includes a luminescent reagent and a labeling reagent; the steps specifically include: S1-1. Mix the sample to be tested with the luminescent reagent, labeling reagent and hydrolysis reagent, and incubate for the first time to obtain the first reaction solution; S1-2. Add the donor reagent to the first reaction solution, and after a second incubation, obtain the second reaction solution; S2. The second reaction liquid is irradiated with excitation light, and the light signal is detected. The luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles can react with reactive oxygen species to generate a detectable light signal; the labeling reagent includes a second biomolecule labeled with a tag; the donor reagent can generate reactive oxygen species under laser irradiation of a certain wavelength; both the first biomolecule and the second biomolecule can specifically bind to the IgM antibody to be tested in the sample.

[0008] In some embodiments of this application, step S1 specifically includes: first adding a hydrolysis reagent to the sample to be tested, and then adding an immunoassay reagent.

[0009] In some embodiments of this application, the incubation temperature is 25~45°C.

[0010] In some embodiments of this application, the first incubation time is 5 to 25 minutes; preferably 15 to 25 minutes.

[0011] In some embodiments of this application, the second incubation time is 5 to 20 minutes; preferably 5 to 15 minutes.

[0012] In some embodiments of this application, the proteolytic enzyme includes at least one of papain, pepsin, trypsin, bromelain, and figase; preferably papain and pepsin.

[0013] In some embodiments of this application, the hydrolysis reagent further includes a diluent and an enzymatic reaction promoter corresponding to the proteolytic enzyme.

[0014] In some preferred embodiments of this application, the enzyme reaction promoter includes at least one of acid, base, metal salt, and oligopeptide.

[0015] In some preferred embodiments of this application, the diluent includes at least one of physiological saline and buffer solution; more preferably, the buffer solution is selected from at least one of phosphate, citrate, Tris-HCl, and HEPES.

[0016] In some embodiments of this application, the hydrolysis reagent includes papain and glutathione; preferably, the papain content is 0.05~5 mg / mL and the reduced glutathione content is 1~50 mM; more preferably, the papain content is 0.1~2 mg / mL and the reduced glutathione content is 5~20 mM; even more preferably, the pH of the hydrolysis reagent is 5~7.

[0017] In some embodiments of this application, the hydrolysis reagent includes pepsin and hydrochloric acid; preferably, the pepsin content is 0.01~1 mg / mL and the hydrochloric acid content is 0.1~100 mM; more preferably, the pepsin content is 0.05~0.5 mg / mL and the hydrochloric acid content is 10~50 mM; even more preferably, the pH of the hydrolysis reagent is 1~4.

[0018] In some embodiments of this application, the biomolecule is selected from specific antigens or anti-human IgM antibodies capable of specifically binding to the IgM antibody to be tested.

[0019] A second aspect of this application provides a detection kit for IgM antibodies, comprising the above-mentioned hydrolysis reagent containing proteolytic enzyme, and an immunoassay reagent for detecting the IgM antibody to be tested.

[0020] The third aspect of this application provides the application of the above-mentioned detection method or kit in the homogeneous detection of the presence and / or content of specific IgM antibodies in blood samples.

[0021] It should be noted that the above-mentioned application of homogeneous detection of IgM antibodies in blood samples is for non-diagnostic purposes.

[0022] The technical solution provided in this application may include the following beneficial effects: Proteolytic enzymes can disrupt the structure of interfering proteins in the test sample, such as heterophilic antibodies, specific IgG antibodies, and rheumatoid factor. This reduces or eliminates the signal or shielding signal caused by the non-specific binding of interfering proteins to the effective components in the immunoassay reagent, reduces the interference of the sample matrix effect, reduces the probability of false positives or false negatives, and improves the detection accuracy of specific IgM antibodies.

[0023] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0024] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0025] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0026] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0027] I. Terminology The term "sample to be tested" as used herein refers to a mixture that may contain the analyte. Typical samples to be tested that can be used in this application include bodily fluids, including blood, plasma, serum, urine, semen, saliva, etc. The analyte may be pathogens (antigens) such as bacteria and viruses, antibodies, tumor markers, hormones, drugs, etc.

[0028] The term "biomolecule" as used herein refers to any type of molecule that can elicit an immune response in the body or that can specifically bind to immune products within the body. The biomolecule can be an antigen or an antibody.

[0029] The terms "antigen" and "specific antigen" used in this article refer to substances that can stimulate the body to produce an immune response and can bind to the immune response products, antibodies and sensitized lymphocytes, both in vivo and in vitro, to produce an immune effect. Antigens can be complete pathogens (such as bacteria and viruses), cellular components, proteins, polypeptides, polysaccharides, and many other substances. According to their properties, antigens can be classified as complete antigens and incomplete antigens (haptens). According to their preparation methods, antigens can be classified as natural antigens, recombinant antigens, synthetic antigens, etc. Where necessary, antigens can be further conjugated to other parts, such as specific binding pairs, for example, biotin or avidin.

[0030] The term "antibody" as used herein is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins containing the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin.

[0031] The terms “bonding”, “coupling”, or “coupling” as used herein refer to the direct connection between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0032] The term "specific binding" used in this article refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the reactants in the response.

[0033] The term "interfering protein" as used herein refers to proteins that, in addition to the target molecule itself, can interfere with the immunoassay results of the target molecule. These proteins can interact with antibodies, antigens, or other key components in the test reagent, thereby affecting the accuracy of the immunoassay and causing the results to deviate from the true value. Interfering proteins can be divided into endogenous and exogenous proteins. Endogenous proteins originate from the organism itself, such as autoantibody-associated interfering proteins, hygroscopic factors, and heterophilic antibodies. For example, in the detection of target-specific IgM, IgG is an antibody-associated interfering protein that can undergo complex interactions with components in the test reagent, leading to false positives. Exogenous proteins originate from the external environment, such as interfering proteins introduced during sample processing or impurity proteins present in the test reagent.

[0034] The term "rheumatoid factors" or RF (rheumatoid factors) used in this article refers to autoantibodies, primarily of the IgM type, but also of IgG, IgA, etc. They bind to the Fc fragment of immunoglobulins. In immunoassays, especially when antibody detection is involved, RF can act as interfering proteins. For example, when detecting serum IgM, if rheumatoid factor is present in the sample, it can non-specifically bind to the test reagent, increasing background signal and leading to false positive results.

[0035] The term "heterophilic antibody" or "HA (heterophilic antibody)" as used herein refers to a class of multispecific immunoglobulins produced by the human body in response to stimulation by known or unknown antigens. These antibodies can bind relatively weakly to immunoglobulins from multiple species. They can recognize certain similar antigenic determinants on immunoglobulins from different species, resulting in cross-reactivity. For example, in IgM detection, when animal-derived detection reagents (such as murine monoclonal antibodies) are used, heterophilic antibodies may bind to the antibodies in these reagents, mimicking an antigen-antibody reaction. These nonspecific bindings increase background signal, leading to false positive results.

[0036] The term "proteolytic enzyme" as used in this article is a general term for a class of enzymes that catalyze the hydrolysis of proteins. Their function is to break peptide bonds in protein molecules, breaking them down into smaller peptides or amino acids. Based on their site of action, they can be divided into endopeptides and exopeptides. Endopeptides cleave peptide bonds within the protein molecule, while exopeptides hydrolyze peptide bonds starting from the ends of the protein molecule. Based on the chemical properties of their active sites, they can be classified into serine proteases, cysteine ​​proteases, aspartic proteases, and metalloproteinases, etc. Serine proteases have serine residues in their active sites, such as trypsin. Cysteine ​​proteases have cysteine ​​residues in their active sites, such as papain, bromelain, and figase. Aspartic proteases have aspartic residues in their active sites, such as pepsin.

[0037] The term "enzyme reaction promoter" as used in this article refers to a class of substances that can increase the rate of enzyme-catalyzed reactions. They enhance the catalytic activity of enzymes by interacting with enzymes, substrates, or enzyme-substrate complexes, thereby accelerating the reaction process and achieving higher reaction rates even at lower substrate concentrations. Common enzyme reaction promoters include, but are not limited to, acids, bases, metal salts, coenzymes, and oligopeptides.

[0038] The term "reactive oxygen species" as used in this article refers to a general term for oxygen-containing and reactive substances in the body or natural environment. It mainly refers to excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH),) as well as nitric oxide and reactive oxygen species (NOS). 1 O2), etc.

[0039] As used herein, "luminescent microparticles" refers to polymeric microparticles filled with a luminescent composition capable of reacting with reactive oxygen species to generate a detectable light signal. Luminescent microparticles may also be called acceptor microspheres or luminescent microspheres. The luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. The luminescent composition may comprise a chemiluminescent compound and a metal chelate. The chemiluminescent compound is selected from olefin compounds, specifically, for example, dimethylthiophene, dibutyl dione compounds, dioxane, enol ethers, enamines, 9-alkylene-xanthane, 9-alkylene-N-9,10-dihydroacrylidine, arylethyl ethers, aryl imidazoles, and luster compounds, as well as their derivatives. The metal in the metal chelate is selected from rare earth metals or Group VIII metals, specifically, for example, europium, terbium, dysprosium, samarium, osmium, and ruthenium. The organic complexes of the metal chelates are selected from NHA (nickel azidohydrazine), BHHT (butylated hydroxytoluene), DPP (2,6-diphenylphenol), TTA (methylbenzotriazole), NTA (nitrotriacetic acid), TOPO (tri-n-octylphosphine oxide), TPPO (triphenylphosphonic acid), PFTU (2,2-dimethyl-4-perfluorobutyryl-3-butanone), BFTA, NPPTA, BHHCT, 2,2'-bipyridine, bipyridylcarboxylic acid, azacrown ethers, azacavitary ligands, and their derivatives.

[0040] As used herein, "photosensitive microparticles" refers to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microspheres, and solutions containing such photosensitive microparticles can be called photosensitive solutions or universal solutions. The photosensitizers can be, but are not limited to, those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll. Photosensitive microspheres can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0041] The term "microparticle" as used herein can refer to any size and shape, and can be expandable or non-expandable, porous or non-porous, having any density, but preferably close to that of water, preferably capable of floating in water, and composed of transparent, partially transparent, or opaque materials. Microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), or vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). The surface of the microparticles can be modified with active groups, such as carboxyl, amino, aldehyde, and thiol groups, through chemical coupling. A non-limiting example of microparticles suitable for use in this invention is aldehyde-based polystyrene latex microspheres.

[0042] The term "protein stabilizer" as used in this article refers to a class of substances that can maintain the structural stability of proteins and prevent protein denaturation and aggregation. They enhance the stability of the protein's native conformation by interacting with the protein, thereby protecting the protein's activity and function. Common protein stabilizers include, but are not limited to, polysaccharides, polyols, low-concentration salts, amino acids and their derivatives, proteins, and surfactants.

[0043] The term "photoinduced chemiluminescence detection method" used in this article is a highly sensitive detection technique primarily used for the detection of biomolecules (such as antigen-antibody pairs, nucleic acids, etc.). It is based on two special types of microparticles: photosensitive microparticles and luminescent microparticles. When a target molecule simultaneously binds to both photosensitive and luminescent microparticles, the photosensitive microparticles, when irradiated with light of a specific wavelength (such as a 680nm laser), generate reactive oxygen species. These reactive oxygen species can diffuse to the luminescent microparticles, triggering a chemical reaction in the luminescent composition on the microparticles to produce high-energy light. The number of emitted photons is then converted into a relative optical signal value using a single-photon counter and mathematical fitting.

[0044] II. Specific Implementation Plan This application will now be described in more detail.

[0045] The present application discloses a method for detecting IgM antibodies, which includes the following steps: S1. Mix the hydrolysis reagent containing proteolytic enzymes and the immunoassay reagent that can specifically bind to the IgM antibody in the sample to be tested with the sample to obtain the test reaction solution; S2, the signal value of the detection reaction solution; The proteolytic enzyme can destroy the structure of interfering proteins in the test sample.

[0046] In this embodiment of the application, by adding a hydrolysis reagent containing proteolytic enzymes during the detection process of IgM antibodies, the peptide bonds of proteins such as rheumatoid factor, specific IgG antibodies, and heterophilic antibodies present in the test sample can be hydrolyzed, thereby destroying the structure of these interfering proteins, achieving the effect of weakening or eliminating the interference of interfering proteins, reducing the probability of false positive results, and improving the accuracy of specific IgM antibody detection.

[0047] This application also relates to a homogeneous detection method for IgM antibodies, which detects IgM antibodies in a photo-induced chemiluminescence detection platform without separation and washing steps. At the same time, it has high detection sensitivity and is simple and easy to operate, which can further improve the detection efficiency and accuracy of IgM antibodies.

[0048] Immunoassay reagents suitable for photo-induced chemiluminescence detection platforms include luminescent reagents and labeling reagents. The homogeneous detection method for IgM antibodies specifically includes the following steps: S1-1. Mix the sample to be tested with the luminescent reagent, labeling reagent and hydrolysis reagent, and incubate for the first time to obtain the first reaction solution; S1-2. Add the donor reagent to the first reaction solution, and after a second incubation, obtain the second reaction solution; S2. The second reaction liquid is irradiated with excitation light, and the light signal is detected. The luminescent reagent includes luminescent microparticles and a first biomolecule bound to them. The luminescent microparticles can react with reactive oxygen species to generate a detectable light signal. The labeling reagent includes a second biomolecule labeled with a tag. The donor reagent can generate reactive oxygen species under laser irradiation of a certain wavelength. Both the first and second biomolecules can specifically bind to the target IgM antibody in the sample to form a sandwich immune complex.

[0049] In some embodiments, the luminescent microparticles are polymeric microparticles coated or filled with a luminescent composition, which can react with reactive oxygen species to generate a detectable light signal. The luminescent composition is preferably an organic complex of dimethylthiophene and europium, such as MTTA-EU. 3+ Furthermore, the luminescent microparticles can have functional groups introduced onto their surface using chemical coupling technology. These functional groups include, for example, carboxyl (-COOH), amino (-NH2), thiol (-SH), and aldehyde (-CHO).

[0050] In some embodiments, the tagging of the second biomolecule can be biotin. The biotin molecule has two ring structures: an imidazoline ring and a thiophene ring. Biotin has an extremely small molecular weight, enabling it to bind to the second biomolecule through chemical coupling modification or gene recombination, thereby achieving biotin labeling of the second biomolecule. The imidazoline ring in biotin is the main binding site for avidin, allowing for specific binding; avidin is a protein secreted by Streptomyces with a molecular weight of 65 kDa, exhibiting a very strong affinity for biotin.

[0051] Furthermore, the biotin can be activated biotin, such as biotin activated by carboxyl (-COOH), amino (-NH2), thiol (-SH), NHS ester, etc.

[0052] In some embodiments, the donor reagent comprises photosensitive microparticles. These photosensitive microparticles are polymeric microparticles coated or filled with a photosensitizer, capable of releasing reactive oxygen species upon excitation by excitation light. The donor reagent containing photosensitive microparticles may also be referred to as a photosensitive solution or a universal solution.

[0053] Furthermore, avidin is coated on the surface of the photosensitive microparticles. Through the specific interaction between avidin and biotin, the photosensitive microparticles can indirectly bind to a second biomolecule, thereby bringing the photosensitive microparticles closer to the sandwich immune complex and the luminescent microparticles. When the photosensitive microparticles are irradiated with a laser of a certain wavelength, the reactive oxygen molecules generated can be captured by the luminescent composition on the luminescent microparticles, enabling the luminescent microparticles to generate a detectable light signal.

[0054] In some embodiments, both the first and second biomolecules can be specific antigens or specific antibodies capable of specifically binding to the IgM antibody to be tested. Further, the specific antigen can be a natural antigen, recombinant antigen, or synthetic antigen, and may even be an artificially synthesized polypeptide. The specific antibody can be an anti-human IgM antibody, specifically including mouse anti-human, rabbit anti-human, sheep anti-human, or other animal anti-human IgM antibodies.

[0055] In some embodiments, the second biomolecule is preferably a mouse anti-human, rabbit anti-human, sheep anti-human, or other animal anti-human IgM antibody.

[0056] In some embodiments, the sample to be tested is a blood sample, or more specifically, a serum or plasma sample.

[0057] In some embodiments, the proteolytic enzyme includes at least one of papain, pepsin, trypsin, bromelain, and figase; preferably papain and pepsin.

[0058] For example, papain can hydrolyze lysine residues, arginine residues, or the carboxyl terminus of proteins, and can be used to treat various interfering proteins. Similarly, pepsin can hydrolyze peptide bonds of phenylalanine, tryptophan, glutamic acid, tyrosine, or leucine, and can also be used to treat various interfering proteins.

[0059] It should be noted that, depending on the testing requirements, any one or more of the above-mentioned proteolytic enzymes can be selected as the hydrolysis reagent.

[0060] In some embodiments, the hydrolysis reagent further includes a diluent for diluting the proteolytic enzyme and / or an enzyme-catalyzing promoter corresponding to the proteolytic enzyme. Therefore, the hydrolysis reagent may include a proteolytic enzyme and a diluent; the hydrolysis reagent may also include a proteolytic enzyme and an enzyme-catalyzing promoter; the hydrolysis reagent may further include a proteolytic enzyme, a diluent, and an enzyme-catalyzing promoter.

[0061] The diluents include, but are not limited to, physiological saline, buffer solutions, etc., and the buffer solutions may be selected from at least one of phosphate, citrate, Tris-HCl, and HEPES; preferably, disodium hydrogen phosphate-citrate buffer solution.

[0062] In some embodiments, the enzyme-catalyzed reaction promoter includes at least one of an acid, a base, a metal salt, and an oligopeptide. Specific examples include hydrochloric acid, sodium hydroxide, calcium chloride, SDS (sodium dodecyl sulfate), copper sulfate, glutathione, etc.

[0063] For example, when the proteolytic enzyme is papain, the hydrolysis reagent can be prepared by diluting the proteolytic enzyme with physiological saline.

[0064] When the proteolytic enzyme is papain, the hydrolysis reagent can also be prepared using physiological saline and glutathione.

[0065] In some embodiments, the hydrolyzing agent includes papain and glutathione. The proteolytic enzyme is papain, and the enzymatic reaction promoter is glutathione. Glutathione acts as an activator for papain to disrupt the structure of interfering proteins during the enzymatic hydrolysis reaction, thereby enhancing the effect of papain in weakening or eliminating the interference of interfering proteins. In some preferred embodiments, the pH of the hydrolyzing agent is 5-7.

[0066] The diluent for the above hydrolysis reagent can be selected from physiological saline; or it can be selected from a mixture of physiological saline and buffer such as citrate phosphate buffer.

[0067] In some specific embodiments, the papain content is 0.05~5 mg / mL, and the reduced glutathione content is 1~50 mM; preferably, the papain content is 0.1~2 mg / mL, and the reduced glutathione content is 5~20 mM. For example, the papain content can be 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, etc. The reduced glutathione content can be 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, etc.

[0068] In other embodiments, the hydrolysis reagent includes pepsin and hydrochloric acid. The proteolytic enzyme is pepsin, and the enzymatic reaction promoter is hydrochloric acid. Hydrochloric acid not only acts as an activator for papain to disrupt the structure of interfering proteins, but also as a pH adjuster for the hydrolysis reagent, allowing the pepsin in reagent R3 to cleave peptide chains under suitable conditions, thus enhancing the detection reagent's elimination effect on interfering proteins. In some preferred embodiments, the pH of the hydrolysis reagent is 5-7.

[0069] In some specific embodiments, the pepsin content is 0.01~1 mg / mL, and the hydrochloric acid content is 0.1~100 mM; preferably, the pepsin content is 0.05~0.5 mg / mL, and the hydrochloric acid content is 10~50 mM. For example, the pepsin content can be 0.01 mg / mL, 0.03 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, etc. The hydrochloric acid content can be 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, 100 mM, etc.

[0070] The immunoassay reagents involved in the embodiments of this application require dilution of each component to a suitable concentration during the detection process before they are used to detect the sample to be tested.

[0071] The dilution buffer solution for the immunoassay reagent can be selected from a buffer solution containing 0.1 to 2 wt% protein stabilizer.

[0072] The buffer solution may be selected from at least one of phosphate, citrate, Tris-HCl, and HEPES; preferably, HEPES buffer. The protein stabilizer may be selected from polyols, polysaccharides, amino acids and their derivatives, proteins, surfactants, low-concentration salts, such as BSA (bovine serum albumin), sodium chloride, Tween, glucose, sucrose, glycine, glycerol, sorbitol, polyethylene glycol, etc.

[0073] In some implementations, the pH of the dilution buffer solution for the immunoassay reagent is 5 to 9.

[0074] The detection method described in this application, by controlling the order of addition of each reagent, the incubation time, and the incubation temperature, can improve detection efficiency while reducing matrix effects, thereby further improving the accuracy of detection results.

[0075] In some embodiments, in step S1, the sample to be tested can be mixed with the hydrolysis reagent first, and then the immunoassay reagent can be added for a first incubation. The proteolytic enzyme preferentially contacts the interfering proteins in the sample, which is beneficial for the proteolytic enzyme to exert its function of destroying the structure of the interfering proteins.

[0076] In some embodiments, the first incubation time is 5-25 minutes. Preferably, the first incubation time is 15-25 minutes. The first incubation can pretreat the interfering proteins present in the test sample by the proteolytic enzymes, thereby reducing or eliminating the content of interfering proteins in the sample and preventing them from binding with specific antigens / antibodies to generate interfering signals. Therefore, the time of the first incubation can be increased to allow the proteolytic enzymes to fully contact the interfering proteins.

[0077] In some embodiments, the second incubation time is 5-20 minutes. Preferably, the second incubation time is 5-15 minutes. During the second incubation, the photosensitive microparticles in the donor reagent bind to the sandwich immune complex, meeting the requirements for photo-induced chemiluminescence detection. The reaction between avidin and biotin in the photosensitive microparticles is rapid; by reducing the second incubation time, the overall reaction time can be shortened, enabling rapid homogeneous detection of IgM antibodies. In some preferred embodiments, the first incubation time is 15-25 min, and the second incubation time is 5-15 min.

[0078] In some embodiments, the incubation temperature is 25~45°C. Depending on the different proteolytic enzymes, the corresponding optimal incubation temperature can be adjusted. For example, when papain is used as the proteolytic enzyme, the higher the incubation temperature, the better, preferably 37~45°C; when pepsin is used as the proteolytic enzyme, the incubation temperature is based on human body temperature, preferably around 37°C.

[0079] This application also relates to a detection kit for IgM type antibodies, which includes the above-described immunoassay reagent and a hydrolysis reagent containing a proteolytic enzyme.

[0080] Specifically, the kit includes a luminescent reagent, a labeling reagent, and a hydrolysis reagent.

[0081] The luminescent reagent includes luminescent microparticles and a first biomolecule bound to them. The luminescent microparticles can react with reactive oxygen species to generate a detectable light signal, and the first biomolecule can specifically bind to the IgM antibody to be tested in the sample.

[0082] The labeling reagent includes a tag-labeled second biomolecule capable of specifically binding to the target IgM antibody in the test sample. In some embodiments, the tag for labeling the second biomolecule is selected from biotin, meaning the labeling reagent includes a biotinylated second biomolecule.

[0083] The hydrolysis reagent includes proteolytic enzymes, which can disrupt the structure of interfering proteins in the test sample. In some embodiments, the proteolytic enzyme may be selected from at least one of papain, pepsin, trypsin, bromelain, and figase.

[0084] In addition, the immunoassay reagent may also include a donor reagent comprising avidin-coated photosensitive microparticles that can generate reactive oxygen species, and avidin that can specifically bind to biotin in the labeling reagent.

[0085] The kit provided in this application embodiment may also include only luminescent reagent and labeling reagent. The hydrolysis reagent can be prepared by diluting the corresponding proteolytic enzyme when detecting IgM antibody. The donor reagent can be a universal solution.

[0086] The detection methods or kits described in this application can be used to detect the presence and / or content of IgM antibodies in blood samples, i.e., for qualitative / quantitative analysis of IgM antibodies. Furthermore, the detection methods or kits described above can be used for homogeneous detection of IgM antibodies. Specifically, they are applicable to the application of IgM antibodies in photo-induced chemiluminescence detection.

[0087] The qualitative / quantitative analysis method for IgM antibodies described in this application can use blood samples, such as serum or plasma samples. The blood samples can be pre-diluted with a diluent such as physiological saline before detection to further reduce interference from non-specific adsorption, improve detection sensitivity and specificity, and increase the accuracy of the detection results.

[0088] III. Specific Implementation Examples To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0089] The following uses HBc-IgM (hepatitis B core antibody) and Rubella IgM (rubella virus antibody) as examples. Papain and pepsin were used as hydrolysis reagents, respectively. The light signal value of the test samples was detected in a photo-induced chemiluminescence detection platform to determine the effect of proteolytic enzymes on the homogeneous detection performance of IgM antibodies.

[0090] Example 1: Preparation of HBc-IgM photochemiluminescence detection kit 1. Preparation of R1 reagent (luminescent reagent) Aldehyde-based luminescent microparticles were mixed with the first biomolecule (HBc-specific antigen HBc Ag) at a mass ratio of 10:0.5 in 0.05M CB buffer and reacted at 37℃ for 4 hours. Sodium borohydride was added for reduction, and the mixture was reacted at 2-8℃ for 1 hour. The microparticles were then washed twice with luminescent buffer solution (50mM HEPES buffer containing protein stabilizer, pH=8.0) and the volume was adjusted to a microparticle concentration of 10mg / mL.

[0091] The antigen-coated luminescent microparticles were diluted to 25 mg / L using a luminescent buffer solution to prepare reagent R1.

[0092] 2. Preparation of R2 reagent (labeling reagent) The second biomolecule (mouse anti-human IgM antibody) was dialyzed in 0.1M NaHCO3 solution at room temperature for 2-4 hours, with the medium changed 2 or more times. The dialyzed antibody was placed in a centrifuge tube, and biotin dissolved in DMSO was added at a molar ratio of 1:30. After reacting at 2-8℃ for 2 hours, the antibody was dialyzed in 0.02M PBS buffer at 2-8℃ for at least 6 hours, with the medium changed 4 or more times.

[0093] The biotinylated mouse anti-human IgM antibody was diluted to 2.5 mg / L using biotinylate buffer (0.1 M Tris-HCl buffer, pH 8.0, containing protein stabilizer) to prepare reagent R2.

[0094] 3. Preparation of R3 reagent (hydrolysis reagent) Mode 1: Use physiological saline as reagent R3; Mode 2: Dilute papain to 1 mg / mL with physiological saline to prepare R3 reagent; Mode 3: Dilute papain to 1 mg / mL with physiological saline and add 10 mM glutathione to prepare R3 reagent.

[0095] 4. Combine the donor reagent (universal solution) containing avidin-coated photosensitive microparticles as reagent R4, and assemble the reagents into a kit.

[0096] Example 2: Effects of proteolytic enzymes and enzymatic reaction accelerators on homogeneous detection of HBc-IgM 1. Experimental Procedure Blood samples were diluted 20 times with physiological saline, and negative, positive and false positive samples were detected on a photocatalytic chemiluminescence detection platform using the kit described in Example 1. The changes in signal values ​​were recorded.

[0097] Reaction system: Add 15 μL of the sample to be tested to the reaction well; add 25 μL each of reagents R1, R2, and R3, and incubate for 15 min at 37℃; add 175 μL of universal solution and incubate for 10 min at 37℃; read the values. The detection results are shown in the table below.

[0098] 2. Experimental Results Table 1

[0099] 3. Experimental Data Analysis Compared to Mode 1 without papain, Mode 2 showed a significant improvement in detection accuracy. False positive samples (1-5) showed a significant decrease in signal values, while positive samples showed an increase or a slight decrease in signal values, indicating that papain effectively reduced interference from irrelevant proteins.

[0100] Papain's active site contains a sulfhydryl group. Reduced glutathione can break disulfide bonds and maintain the sulfhydryl state, acting as both an activator and stabilizer for papain. In Mode 3, the addition of 10 mM glutathione further enhanced the activity of papain, increasing the signal difference between positive and negative samples.

[0101] Therefore, Mode 2 (containing 1 mg / mL papain) can improve the accuracy of IgM detection. Mode 3 (containing 1 mg / mL papain and 10 mM glutathione) can further improve the accuracy of IgM detection.

[0102] Example 3: Effects of the concentrations of proteolytic enzymes and enzyme-catalyzing promoters on homogeneous detection of HBc-IgM 1. Experimental Procedure Based on Mode 3 (containing 1 mg / mL papain and 10 mM glutathione) shown in Example 2, the concentrations of papain were adjusted to 1, 2, and 3 mg / mL, and the concentrations of glutathione were adjusted to 5, 10, and 20 mM. Samples were then tested to analyze the effects of papain and glutathione concentrations on the IgM detection signal value. The detection results are shown in the table below.

[0103] 2. Experimental Results Table 2

[0104] 3. Experimental Data Analysis After adjusting the concentrations of papain and glutathione, the signal values ​​changed significantly. Optimizing the concentrations of papain and glutathione can further improve the accuracy of the detection.

[0105] When the papain concentration was 2 mg / mL, the efficiency of papain in destroying interfering proteins reached saturation, and further increasing the papain concentration had no significant difference. As the glutathione concentration increased, the detection signal value gradually decreased, and its inhibitory effect on false positive and true positive samples became increasingly stronger.

[0106] Based on the above experimental results, papain at a concentration of 2 mg / mL and glutathione at a concentration of 10 mM can effectively remove interfering proteins and improve the detection accuracy of IgM.

[0107] Example 4: Effect of pH of hydrolysis reagent on homogeneous detection of HBc-IgM 1. Experimental Procedure Based on Example 3, the R3 reagent, consisting of 2 mg / mL papain and 10 mM glutathione, was selected. The pH of the R3 reagent was adjusted to 4, 6, and 8 using 0.02 M disodium hydrogen phosphate-citrate buffer, and samples were tested to analyze the effect of pH changes on the IgM detection signal value. The detection results are shown in the table below.

[0108] 2. Experimental Results Table 3

[0109] 3. Experimental Data Analysis After adjusting the pH of reagent R3, the detection signal value of the sample changed significantly. Optimizing the pH of reagent R3 can further improve the accuracy of detection.

[0110] The optimal pH for papain is 5-7; when the pH is 4 or 8, the enzyme activity is inhibited, the interference elimination effect is weakened, and the signal value difference between positive and negative samples is reduced.

[0111] Example 5: Effect of the order of addition of hydrolysis reagents on homogeneous detection of HBc-IgM 1. Experimental Procedure Based on Example 3, reagent R3 consisting of 2 mg / mL papain and 10 mM glutathione was selected. The order of addition of reagents R1, R2, and R3 was adjusted, and samples were tested to analyze the effect of the addition order of reagent R3 on the IgM detection signal value. The order of reagent addition during the detection process is shown in the table below.

[0112] Table 4

[0113] Add the test reagents to the sample in the order shown in the table above, and the test results are shown in the table below.

[0114] 2. Experimental Results Table 5

[0115] 3. Experimental Data Analysis Different reagent addition orders result in slightly different reactivity of the sample for detection, and optimizing the reagent addition order can further improve the accuracy of the detection.

[0116] The signal value difference between positive and negative samples was most pronounced when R3 reagent was added first.

[0117] Example 6: Effect of incubation temperature on homogeneous detection of HBc-IgM 1. Experimental Procedure Based on Example 3, the R3 reagent consisting of 2 mg / mL papain and 10 mM glutathione was selected. 37℃ and 42℃ were chosen as the two-step incubation conditions, and the samples were tested to analyze the effect of incubation temperature on the IgM detection signal value. The detection results are shown in the table below.

[0118] Table 6

[0119] 3. Experimental Data Analysis When papain is used as a hydrolytic reagent containing proteolytic enzymes, within a specific temperature range, the higher the temperature, the stronger the antigen-antibody reactivity and the stronger the papain activity. Increasing the incubation reaction temperature can further improve the accuracy of detection.

[0120] The reaction signal value was significantly enhanced at 42℃, and the difference between the signal values ​​of positive and negative samples increased.

[0121] Example 7: Effect of incubation time on homogeneous detection of HBc-IgM 1. Experimental Procedure (1) Based on Example 3, R3 reagent with 2 mg / mL papain and 10 mM glutathione was selected. The incubation time for the first step was 5 min, 10 min, 15 min and 20 min respectively; the incubation time for the second step was 10 min. The samples were tested and the effect of the first incubation time on the IgM detection signal value was analyzed.

[0122] (2) Based on Example 3, R3 reagent with 2 mg / mL papain and 10 mM glutathione was selected. The first step incubation time was 15 min; the second step incubation time was 5 min, 10 min, 15 min and 20 min respectively. The samples were tested and the effect of the second incubation time on the IgM detection signal value was analyzed.

[0123] The test results are shown in the table below.

[0124] 2. Experimental Results (1) Optimize the detection results of the first step of incubation time.

[0125] Table 7

[0126] (2) Optimize the detection results of the second step incubation time.

[0127] Table 8

[0128] 3. Experimental Data Analysis As shown in Table 7, the longer the incubation time in the first step, the longer the antigen-antibody reaction time and the papain action time, which can significantly enhance the reaction signal value and increase the difference between the signal values ​​of positive and negative samples.

[0129] As shown in Table 8, the second incubation time had no significant effect on the antigen-antibody reaction and the effect of papain, and the second incubation time had little impact on the detection results.

[0130] Therefore, the detection speed and accuracy can be further improved by increasing the first step reaction time and shortening the second step reaction time.

[0131] Example 8: Preparation of Rubella IgM Photochemiluminescence Detection Kit 1. Preparation of R1 reagent (luminescent reagent) Aldehyde-based luminescent microparticles were mixed with first biomolecule (Rubella Ab, monoclonal antibody) at a mass ratio of 10:0.5 in 0.05M CB buffer and reacted at 37℃ for 4 h. Sodium borohydride was added for reduction, and the mixture was reacted at 2-8℃ for 1 h. The microparticles were then washed twice with luminescent buffer solution (50mM HEPES buffer containing protein stabilizer, pH=8.0) and the volume was adjusted to a microparticle concentration of 10 mg / mL.

[0132] The antigen-coated luminescent microparticles were diluted to 25 mg / L using a luminescent buffer solution to prepare reagent R1.

[0133] 2. Preparation of R2 reagent (labeling reagent) Dialyze the second biomolecule (Rubella Ag) in 0.1M NaHCO3 solution at room temperature for 2-4 hours, changing the medium 2 or more times. Place the dialyzed antibody in a centrifuge tube, add biotin dissolved in DMSO at a molar ratio of 1:30, react at 2-8℃ for 2 hours, and then dialyze with 0.02M PBS buffer at 2-8℃ for at least 6 hours, changing the medium 4 or more times.

[0134] The biotinylated mouse anti-human IgM antibody Rubella Ag was diluted to 4 mg / L using biotin buffer (0.1 M Tris-HCl buffer, pH 8.0, containing protein stabilizer) to prepare reagent R2.

[0135] 3. Preparation of R3 reagent (hydrolysis reagent) Mode 1: Use physiological saline as reagent R3; Mode 2: Use 50 mM hydrochloric acid as reagent R3; Mode 3: Dilute pepsin to 0.05 mg / mL with 50 mM hydrochloric acid to prepare R3 reagent.

[0136] 4. Combine the donor reagent (universal solution) containing avidin-coated photosensitive microparticles as reagent R4, and assemble the reagents into a kit.

[0137] Example 9: Effect of proteolytic enzymes on homogeneous detection of Rubella IgM 1. Experimental Procedure Blood samples were diluted 20 times with physiological saline, and negative, positive and false positive samples were detected on a photo-induced chemiluminescence detection platform using the kit described in Example 8. The changes in signal values ​​were recorded.

[0138] Reaction system: Add 15 μL of the sample to be tested to the reaction well; add 25 μL each of reagents R1, R2, and R3, and incubate for 15 min at 37℃; add 175 μL of universal solution and incubate for 10 min at 37℃; read the values. The detection results are shown in the table below.

[0139] 2. Experimental Results Table 9

[0140] 3. Experimental Data Analysis Compared with Mode 1 and Mode 2 without pepsin, although the overall signal value of Mode 3 decreased, the decrease was greater in false positive samples than in positive samples. Therefore, it can improve the signal value difference between positive and negative samples, indicating that although pepsin has a certain impact on antigen-antibody response, it can significantly reduce the interference of irrelevant proteins.

[0141] Mode 3 (containing 0.05 mg / mL pepsin) can significantly improve the accuracy of Rubella IgM detection.

[0142] Example 10: Effect of proteolytic enzyme concentration on homogeneous detection of Rubella IgM 1. Experimental Procedure Based on Mode 3 (containing 0.05 mg / mL pepsin) shown in Example 9, the concentration of pepsin was adjusted to 0.025, 0.05, 0.1, and 0.2 mg / mL, and samples were tested to analyze the effect of pepsin concentration on the IgM detection signal value. The detection results are shown in the table below.

[0143] 2. Experimental Results Table 10

[0144] 3. Experimental Data Analysis As the concentration of pepsin increases, its effect on antigen-antibody reactions and its ability to destroy interfering proteins also increase. When the concentration of pepsin is 0.1 mg / mL, the signal values ​​of positive and negative samples are most differentiated, and at this point, the effect of pepsin in destroying interfering proteins is most obvious.

[0145] Therefore, 0.1 mg / mL pepsin can significantly improve the accuracy of detection.

[0146] Example 11: Effect of pH of hydrolysis reagent on homogeneous detection of Rubella IgM 1. Experimental Procedure Based on Example 10, the pepsin concentration in reagent R3 was prepared to be 0.1 mg / mL, and the hydrochloric acid concentration was adjusted to 0, 10, 50, and 100 mM, with corresponding pH values ​​of 5, 2, 1.3, and 1. Samples were tested, and the effect of pH changes in reagent R3 on the IgM detection signal value was analyzed. The detection results are shown in the table below.

[0147] 2. Experimental Results Table 11

[0148] 3. Experimental Data Analysis Hydrochloric acid is an activator of pepsin, and the optimal pH for pepsin is 1-4. When the pH exceeds 4, pepsin activity is significantly inhibited, and the anti-interference effect is significantly reduced. When the pH is between 1 and 2, pepsin can significantly eliminate interfering proteins, and the signal values ​​of positive and negative samples are basically consistent. As the concentration of hydrochloric acid increases, the signal value also decreases overall, indicating that high concentrations of hydrochloric acid also have an inhibitory effect on antigen-antibody reactions.

[0149] Therefore, under suitable pH conditions, adding pepsin can improve the accuracy of detection; by adjusting the hydrochloric acid concentration, the overall signal value trend can be controlled.

[0150] Based on the above experimental results, adding a hydrolysis reagent containing proteolytic enzymes when detecting samples in the photo-induced chemiluminescence platform can remove most of the interfering proteins that cause false negatives or false positives, thereby further improving the accuracy of homogeneous photo-induced chemiluminescence detection of IgM.

[0151] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting IgM antibodies, characterized in that, Includes the following steps: S1. Mix the hydrolysis reagent containing proteolytic enzymes and the immunoassay reagent that can specifically bind to the IgM antibody in the sample to be tested with the sample to be tested to obtain the test reaction solution. S2, the signal value of the detection reaction solution; The proteolytic enzyme can destroy the structure of interfering proteins in the test sample.

2. The detection method according to claim 1, characterized in that, The immunoassay reagent includes a luminescent reagent and a labeling reagent; the steps specifically include: S1-1. Mix the sample to be tested with the luminescent reagent, labeling reagent and hydrolysis reagent, and incubate for the first time to obtain the first reaction solution; S1-2. Add the donor reagent to the first reaction solution, and after a second incubation, obtain the second reaction solution; S2. The second reaction liquid is irradiated with excitation light, and the light signal is detected. The luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles can react with reactive oxygen species to generate a detectable light signal; the labeling reagent includes a second biomolecule labeled with a tag; the donor reagent can generate reactive oxygen species under laser irradiation of a certain wavelength; both the first biomolecule and the second biomolecule can specifically bind to the IgM antibody to be tested in the sample.

3. The detection method according to claim 1 or 2, characterized in that, Step S1 specifically includes: first adding hydrolysis reagent to the sample to be tested, and then adding immunoassay reagent.

4. The detection method according to claim 2, characterized in that, The incubation temperature is 25~45℃; And / or, the first incubation time is 5-25 min; preferably 15-25 min; And / or, the second incubation time is 5 to 20 minutes; preferably 5 to 15 minutes.

5. The method according to claim 1, characterized in that, The proteolytic enzyme includes at least one of papain, pepsin, trypsin, bromelain, and figase; preferably papain and pepsin.

6. The method according to claim 5, characterized in that, The hydrolysis reagent also includes a diluent and / or an enzymatic reaction promoter corresponding to the proteolytic enzyme; Preferably, the enzyme reaction promoter includes at least one of an acid, a base, a metal salt, and an oligopeptide; Preferably, the diluent includes at least one of physiological saline and buffer solution; more preferably, the buffer solution is selected from at least one of phosphate, citrate, Tris-HCl, and HEPES.

7. The method according to claim 5, characterized in that, The hydrolysis reagent includes papain and glutathione; preferably, the papain content is 0.05~5 mg / mL and the reduced glutathione content is 1~50 mM; more preferably, the papain content is 0.1~2 mg / mL and the reduced glutathione content is 5~20 mM; even more preferably, the pH of the hydrolysis reagent is 5~7.

8. The method according to claim 5, characterized in that, The hydrolysis reagent includes pepsin and hydrochloric acid; preferably, the pepsin content is 0.01~1 mg / mL and the hydrochloric acid content is 0.1~100 mM; more preferably, the pepsin content is 0.05~0.5 mg / mL and the hydrochloric acid content is 10~50 mM; even more preferably, the pH of the hydrolysis reagent is 1~4.

9. A detection kit for IgM antibodies, characterized in that, Includes hydrolysis reagents containing proteolytic enzymes as described in any one of claims 1 to 8, and immunoassay reagents for detecting IgM antibodies to be tested.

10. The detection method according to any one of claims 1 to 8, or the kit according to claim 9, for the homogeneous detection of the presence and / or content of specific IgM antibodies in blood samples.

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