Method and kit for detecting IgM (Immunoglobulin M) by light-activated chemiluminescence

Abstract

The invention provides a detection method and a kit for detecting IgM by using a light-activated chemiluminescence double-antigen sandwich method, the purpose of detecting an IgM antibody on a light-activated chemiluminescence platform by using the double-antigen sandwich method is achieved, the problem that the double-antigen sandwich method cannot distinguish IgG and IgM is solved, and the problems that the sensitivity is low and the detection sensitivity is low when the light-activated chemiluminescence indirect method is used for detecting the IgM antibody are solved. The method is easy to be interfered by irrelevant IgM or specific IgG in a sample, needs dilution and is long in time; meanwhile, the problem of interference of specific IgG is solved to a certain extent, and the detection sensitivity and the detection efficiency are improved.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly relates to a kit for detecting IgM by a light-activated chemiluminescence double-antigen sandwich method and a detection method thereof. Background Art

[0002] IgM is immunoglobulin M. Immunoglobulins are classified into five types, namely IgA, IgG, IgE, IgD, and IgM according to different structures; among them, IgM can form pentamers or hexamers, is the largest in molecular weight among Igs, and its molecular structure is circular. IgM mainly exists in the blood, accounting for 5% - 10% of the total serum immunoglobulin, and the serum concentration is about 1 mg / mL. IgM is the earliest synthesized antibody in individual development and is also the first antibody produced in the humoral immune response after antigen stimulation. Clinically, the serum IgM level is usually detected for the early diagnosis of infections.

[0003] Light-activated chemiluminescence immunoassay (amplified luminescent proximity homogeneous assay linked immunosorbent assay, AlphaLISA) is a new type of homogeneous immunoassay technology, which is an organic combination of the two disciplines of chemistry and biology. Chemically, this technology uses donor nanomaterials containing photosensitive materials (such as phthalocyanine substances) and acceptor nanomaterials containing luminescent substances (such as rubrene, europium chelates, etc.). Under the irradiation of an excitation light at 680 nm, the photosensitive material in the donor nanomaterial excites the surrounding oxygen molecules to be converted into high-energy 1 O2. 1 The half-life of O2 is about 4 μs, which determines that its diffusion diameter is about 200 nm. If it exceeds this range 1 O2 will fall back to the ground state. The generated 1 O2 diffuses outwards, and the acceptor nanomaterial in the immune sandwich complex can receive 1 O2, and reacts with the internal 1 O2-triggered luminescent reagent to generate energy. This energy is quickly transferred to the luminescent reagent in the acceptor nanomaterial. After receiving the energy, the luminescent reagent then generates emission light, and the number of photons is converted into a relative light signal through a single photon counter and mathematical fitting.

[0004] On the light-activated chemiluminescence platform, the indirect method for detecting IgM antibodies has low sensitivity, is easily interfered by irrelevant IgM or specific IgG in the sample, and requires dilution and takes a long time. The double-antigen sandwich method for detecting antibodies is simple to operate, has high sensitivity, does not require dilution, is not easily interfered, but cannot distinguish whether it is IgM or IgG that is sandwiched.

[0005] Therefore, there is an urgent need for a detection method that is highly sensitive, easy to operate, does not require dilution, is not easily interfered with, and can specifically detect IgM. Summary of the Invention

[0006] To solve at least one of the above problems, the present disclosure provides a method and a kit for detecting IgM antibodies in a sample to be tested.

[0007] According to a first aspect of the present disclosure, there is provided a method for detecting IgM in a sample to be tested, the method comprising the following steps:

[0008] 1) Add an equal amount of the sample to be tested to the detection well and the control well, and respectively add an equal amount of a treatment reagent and a control reagent;

[0009] 2) Add a detection reagent to the detection well and the control well, and detect the signal values of the detection well and the control well;

[0010] 3) Confirm whether the sample to be tested contains IgM antibodies according to the ratio of the signal values;

[0011] The treatment reagent contains a reducing agent, and the control reagent does not contain a reducing agent.

[0012] In some embodiments, the step 3) of confirming whether the sample to be tested contains IgM antibodies according to the ratio of the signal values comprises the following steps: the signal value of the detection well is the first signal value, the signal value of the control well is the second signal value, when the first signal value ≥ threshold M and the ratio of the first signal value / the second signal value < threshold N, it is determined that the IgM antibody in the sample to be tested is positive.

[0013] In some embodiments, if the first signal value < threshold M, it is determined that the IgM antibody in the sample to be tested is negative. If the first signal value ≥ threshold M, then calculate the ratio of the first signal value / the second signal value for a secondary determination.

[0014] In some embodiments, if the first signal value ≥ threshold M and the ratio of the first signal value / the second signal value ≥ threshold N, it is determined that the IgM antibody in the sample to be tested is negative.

[0015] In some embodiments, the threshold M and / or threshold N is determined by detecting known IgM-positive and IgM-negative samples, according to the detection results, through ROC curve analysis, and according to the Youden index.

[0016] In some embodiments, the Youden index of the threshold M and / or threshold N in the sample is the largest.

[0017] In some embodiments, the reducing agent is selected from one or more of dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol, 2-mercaptoethylamine, 2-dimethylaminoethyl mercaptan, 3-mercaptopropanol, cysteine, N-acetylcysteine, reduced glutathione, benzenethiol, ascorbic acid, sodium sulfite, sodium metabisulfite, hydroxylamine hydrochloride, sodium thiosulfate, carbohydrazide, hydrazine sulfate, sodium nitrite, tris(3-hydroxypropyl)phosphine (THPP), tributylphosphine (TBP), and tris(2-carboxyethyl)phosphine (TCEP).

[0018] In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine or dithiothreitol.

[0019] In some embodiments, the detection reagent includes:

[0020] A first reagent, which contains a first antigen coated on luminescent / sensitive microparticles, and the first antigen can specifically bind to IgM antibodies;

[0021] A second reagent, which contains a second antigen modified with a ligand, and the second antigen can specifically bind to IgM antibodies; and

[0022] A third reagent, containing receptor-modified luminescent / sensitive microparticles;

[0023] The ligand and the receptor specifically bind to each other.

[0024] In some embodiments, the concentration range of the luminescent / sensitive microparticles bound with the first antigen in the first reagent is 10-50 mg / L.

[0025] In some embodiments, the concentration of the luminescent / sensitive microparticles bound with the first antigen in the first reagent is 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L, 31 mg / L, 32 mg / L, 33 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 37 mg / L, 38 mg / L, 39 mg / L, 40 mg / L, 41 mg / L, 42 mg / L, 43 mg / L, 44 mg / L, 45 mg / L, 46 mg / L, 47 mg / L, 48 mg / L, 49 mg / L, or 50 mg / L.

[0026] In some embodiments, the concentration of the luminescent / sensitive microparticles bound with the first antigen in the first reagent is 25 mg / L.

[0027] In some embodiments, the first reagent further comprises HEPES buffer, BSA, dextran, TritronX-405, sodium chloride, EDTA and Proclin300.

[0028] In some embodiments, the first reagent further comprises 0.05M HEPES buffer with a pH of 7.0, containing 1.6% BSA, 0.1% dextran, 0.5% TritronX-405, 0.3M sodium chloride, 0.025M EDTA and 0.05% Proclin300.

[0029] In some embodiments, the concentration range of the ligand-modified second antigen in the second reagent is 1-10 mg / L.

[0030] In some embodiments, the concentration of the second antigen coated on the photosensitive / light-emitting particles in the second reagent is 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, 5 mg / L, 5.5 mg / L, 6 mg / L, 6.5 mg / L, 7 mg / L, 7.5 mg / L, 8 mg / L, 8.5 mg / L, 9 mg / L, 9.5 mg / L or 10 mg / L.

[0031] In some embodiments, the concentration of the ligand-modified second antigen in the second reagent is 2.5 mg / L.

[0032] In some embodiments, the second reagent further comprises HEPES buffer, BSA, dextran, TritronX-405, sodium chloride, EDTA and Proclin300.

[0033] In some embodiments, the second reagent further comprises 0.1M Tris-HCl buffer with a pH of 8.0, containing 0.1% BSA, 0.2% bovine gamma globulin, 0.1% dextran, 0.1% TritronX-405, 0.3M sodium chloride, 0.03M EDTA and 0.05% Proclin300.

[0034] In some embodiments, the concentration range of the receptor-modified photosensitive / light-emitting particles in the third reagent is 5-50 mg / L.

[0035] In some embodiments, the concentration of the receptor-modified photosensitive / photoluminescent particles in the third reagent is 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, 31 mmol / L, 32 mmol / L, 33 mmol / L, 34 mmol / L, 35 mmol / L, 36 mmol / L, 37 mmol / L, 38 mmol / L, 39 mmol / L, 40 mmol / L, 41 mmol / L, 42 mmol / L, 43 mmol / L, 44 mmol / L, 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L or 50 mmol / L.

[0036] In some specific embodiments, the concentration of the receptor-modified photosensitive / photoluminescent particles in the third reagent is 20 mg / L.

[0037] In some embodiments, the third reagent further comprises a PBS buffer.

[0038] In some embodiments, the third reagent further comprises a 0.02 M PBS buffer.

[0039] In some embodiments, the ligand and the receptor combination is selected from at least one of biotin / biotin analog - avidin / streptavidin, nanogold / iodacetyl - thiol, amino - aldehyde / carboxyl / isothiocyanato, and silyl - acrylamide.

[0040] In some embodiments, the first antigen or the second antigen is selected from at least one of a natural antigen, a recombinant antigen, or a specific polypeptide.

[0041] In some embodiments, the first antigen or the second antigen is selected from at least one of a Toxoplasma antigen, a rubella antigen, a cytomegalovirus antigen, a herpes simplex virus - 1 antigen or a herpes simplex virus - 2 antigen, parvovirus B19, Coxsackievirus, Treponema pallidum, hepatitis B virus, and Chlamydia.

[0042] In some embodiments, the first antigen and the second antigen are the same.

[0043] In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine or dithiothreitol.

[0044] In some embodiments, the reducing agent in the treatment reagent is dissolved in a solvent.

[0045] In some embodiments, the solvent is selected from one or more of water, physiological saline, or neutral buffer solution.

[0046] In some embodiments, the solvent is selected from at least one of phosphate buffer solution, physiological saline, carbonate buffer solution, or Tris / HCl buffer solution with a pH of about 6-8.

[0047] In some embodiments, the solvent is selected as physiological saline.

[0048] In some embodiments, the control reagent is selected from one or more of water, physiological saline, or neutral buffer solution.

[0049] In some embodiments, the water is selected from one or more of purified water, ultrapure water, deionized water, or distilled water.

[0050] In some embodiments, the control reagent is selected from at least one of phosphate buffer solution, physiological saline, carbonate buffer solution, or Tris / HCl buffer solution with a pH of about 6-8.

[0051] In some embodiments, the control reagent is selected from physiological saline.

[0052] In some embodiments, the control reagent is the same as the solvent in the treatment reagent.

[0053] In some embodiments, the sample to be tested includes at least one of blood, plasma, serum, urine, semen, saliva, cell culture, or tissue extract.

[0054] In some embodiments, the incubation temperature in step 1) and / or step 2) is selected from 20-40°C.

[0055] In some embodiments, the incubation temperature in step 1) and / or step 2) is selected from 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0056] In some embodiments, the incubation temperature in step 1) and / or step 2) is 37°C.

[0057] In some embodiments, the incubation time in step 1) and / or step 2) is selected from 5 to 20 minutes.

[0058] In some embodiments, the incubation time in step 1) and / or step 2) is selected from 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes or 20 minutes.

[0059] In some embodiments, the incubation temperature in step 1) and / or step 2) is selected from 20 to 40 °C, and the incubation time is 5 to 20 minutes.

[0060] According to a second aspect of the present invention, there is provided a kit for detecting IgM antibodies, comprising the following reagents:

[0061] A treatment reagent, comprising a reducing agent;

[0062] A control reagent, not containing a reducing agent; and,

[0063] A detection reagent.

[0064] In some embodiments, the detection reagent comprises:

[0065] A first reagent, which comprises a first antigen coated on luminescent / photosensitive particles, and the first antigen can specifically bind to IgM antibodies;

[0066] A second reagent, which comprises a second antigen modified with a ligand, and the second antigen can specifically bind to IgM antibodies; and

[0067] A third reagent, comprising receptor-modified photosensitive / luminescent particles;

[0068] The ligand and the receptor specifically bind to each other.

[0069] In some embodiments, the concentration range of the luminescent / photosensitive particles bound to the first antigen in the first reagent is 10 to 50 mg / L.

[0070] In some embodiments, the concentration of the luminescent / photosensitive particles that bind to the first antigen in the first reagent is 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L, 31 mg / L, 32 mg / L, 33 mg / L, 34 mg / L, 35 mg / L, 36 mg / L, 37 mg / L, 38 mg / L, 39 mg / L, 40 mg / L, 41 mg / L, 42 mg / L, 43 mg / L, 44 mg / L, 45 mg / L, 46 mg / L, 47 mg / L, 48 mg / L, 49 mg / L or 50 mg / L.

[0071] In some embodiments, the concentration of the luminescent / photosensitive particles that bind to the first antigen in the first reagent is 25 mg / L.

[0072] In some embodiments, the first reagent further includes HEPES buffer, BSA, dextran, TritronX-405, sodium chloride, EDTA and Proclin300.

[0073] In some embodiments, the first reagent further includes 0.05 M HEPES buffer with a pH of 7.0, containing 1.6% BSA, 0.1% dextran, 0.5% TritronX-405, 0.3 M sodium chloride, 0.025 M EDTA and 0.05% Proclin300.

[0074] In some embodiments, the concentration range of the second antigen modified with the ligand in the second reagent is 1-10 mg / L.

[0075] In some embodiments, the concentration of the second antigen modified with the ligand in the second reagent is 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, 5 mg / L, 5.5 mg / L, 6 mg / L, 6.5 mg / L, 7 mg / L, 7.5 mg / L, 8 mg / L, 8.5 mg / L, 9 mg / L, 9.5 mg / L or 10 mg / L.

[0076] In some embodiments, the concentration of the second antigen modified with the ligand in the second reagent is 2.5 mg / L.

[0077] In some embodiments, the second reagent further comprises HEPES buffer, BSA, dextran, Tritron X-405, sodium chloride, EDTA and Proclin 300.

[0078] In some embodiments, the second reagent further comprises 0.1 M Tris-HCl buffer with a pH of 8.0, containing 0.1% BSA, 0.2% bovine gamma globulin, 0.1% dextran, 0.1% Tritron X-405, 0.3 M sodium chloride, 0.03 M EDTA and 0.05% Proclin 300.

[0079] In some embodiments, the concentration range of the receptor-modified photosensitive / photoluminescent particles in the third reagent is 5-50 mg / L.

[0080] In some embodiments, the concentration of the receptor-modified photosensitive / photoluminescent particles in the third reagent is 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, 31 mmol / L, 32 mmol / L, 33 mmol / L, 34 mmol / L, 35 mmol / L, 36 mmol / L, 37 mmol / L, 38 mmol / L, 39 mmol / L, 40 mmol / L, 41 mmol / L, 42 mmol / L, 43 mmol / L, 44 mmol / L, 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L or 50 mmol / L.

[0081] In some embodiments, the ligand and the receptor combination is selected from at least one of biotin / biotin analog - avidin / streptavidin, nanogold / iodacetyl - thiol, amino - aldehyde / carboxyl / isothiocyanato, and silyl - acrylamide.

[0082] In some embodiments, the first antigen or the second antigen is selected from at least one of natural antigens, recombinant antigens or specific polypeptides.

[0083] In some embodiments, the first antigen or the second antigen is selected from at least one of Toxoplasma antigen, rubella antigen, cytomegalovirus antigen, herpes simplex virus-1 antigen or herpes simplex virus-2 antigen, parvovirus B19, Coxsackie virus, Treponema pallidum, hepatitis B virus, and Chlamydia.

[0084] In some embodiments, the first antigen and the second antigen are the same.

[0085] In some embodiments, the luminescent particles comprise a reagent capable of being triggered to luminesce by reactive oxygen species and a first carrier.

[0086] In some embodiments, the reagent capable of being triggered to luminesce by reactive oxygen species comprises a lanthanide metal and an olefin compound.

[0087] In some embodiments, the lanthanide metal comprises at least one of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0088] In some more specific embodiments, the lanthanide metal is europium.

[0089] In some embodiments, the olefin compound includes dimethylthiophene, diketone compounds, dioxacyclohexene, enol ethers, enamines, 9-alkylidenearane, 9-alkylidene-N-9,10-dihydroacridine, aryl ether olefins, aryl imidazoles, and lucigenin, and their derivatives.

[0090] In some more specific embodiments, the olefin compound is selected from dimethylthiophene and its derivatives.

[0091] In some embodiments, the lanthanide metal is loaded on the first carrier.

[0092] In some embodiments, the first carrier coating contains an olefin compound.

[0093] In some embodiments, the surface of the luminescent particles is further connected with chemical groups, and the chemical groups include at least one of epoxy group, chloromethyl group, mercapto group, amino group, hydroxyl group, maleimide group, sulfonic acid group, carboxyl group, and aldehyde group.

[0094] In some embodiments, the luminescent particles and the antigen are combined with each other through chemical groups.

[0095] The photosensitive particles in the third reagent comprise a reagent capable of activating oxygen molecules into reactive oxygen species and a second carrier.

[0096] In some embodiments, the reagent capable of activating oxygen molecules into reactive oxygen species includes phthalocyanine.

[0097] In some embodiments, the reagent capable of promoting the activation of oxygen molecules into reactive oxygen species is loaded on the second carrier.

[0098] In some embodiments, a chemical group is further connected to the surface of the photosensitive particles, and the chemical group includes at least one of epoxy group, chloromethyl group, mercapto group, amino group, hydroxyl group, maleimide group, sulfonic acid group, carboxyl group and aldehyde group.

[0099] In some embodiments, the loading method includes at least one of binding, embedding or semi-embedding.

[0100] In some embodiments, the first carrier and the second carrier are of the same type of carrier, and the carrier is magnetic or non-magnetic.

[0101] In some embodiments, the carrier is non-magnetic.

[0102] In some embodiments, the shape of the carrier is selected from at least one of strip, sheet, rod, tube, pore, microtiter plate, bead, particle and microsphere.

[0103] In some embodiments, the shape of the carrier is a microsphere.

[0104] In some embodiments, the material of the carrier is selected from natural, synthetic or modified natural polymers.

[0105] In some embodiments, the material of the carrier is a synthetic polymer.

[0106] In some embodiments, the material of the carrier is selected from agarose, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, poly(4-methylbutene), polyacrylamide, polymethacrylate, polyethylene terephthalate, nylon, polyvinyl butyrate or polyacrylate.

[0107] In some embodiments, the material of the carrier is polystyrene microspheres.

[0108] In some embodiments, the average particle size of the carrier is 5 nm to 1000 nm.

[0109] In some embodiments, the photosensitive / luminescent particles and the first antigen / second antigen are bound to each other through chemical groups.

[0110] In some embodiments, the reducing agent is selected from one or more of dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol, 2-mercaptoethylamine, 2-dimethylaminoethanethiol, 3-mercaptopropanol, cysteine, N-acetylcysteine, reduced glutathione, benzenethiol, ascorbic acid, sodium sulfite, sodium metabisulfite, hydroxylamine hydrochloride, sodium thiosulfate, carbohydrazide, hydrazine sulfate, sodium nitrite, tris(3-hydroxypropyl)phosphine (THPP), tributylphosphine (TBP), and tris(2-carboxyethyl)phosphine (TCEP).

[0111] In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol.

[0112] In some embodiments, the concentration range of tris(2-carboxyethyl)phosphine in the treatment reagent is 5 to 50 mmol / L.

[0113] In some embodiments, the concentration of tris(2-carboxyethyl)phosphine in the treatment reagent is 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, 23 mmol / L, 24 mmol / L, 25 mmol / L, 26 mmol / L, 27 mmol / L, 28 mmol / L, 29 mmol / L, 30 mmol / L, 31 mmol / L, 32 mmol / L, 33 mmol / L, 34 mmol / L, 35 mmol / L, 36 mmol / L, 37 mmol / L, 38 mmol / L, 39 mmol / L, 40 mmol / L, 41 mmol / L, 42 mmol / L, 43 mmol / L, 44 mmol / L, 45 mmol / L, 46 mmol / L, 47 mmol / L, 48 mmol / L, 49 mmol / L, or 50 mmol / L.

[0114] In some specific embodiments, the concentration of tris(2-carboxyethyl)phosphine in the treatment reagent is 15 to 30 mmol / L.

[0115] In some embodiments, the concentration range of dithiothreitol in the treatment reagent is 2 to 20 mmol / L.

[0116] In some embodiments, the concentration of dithiothreitol in the treatment reagent is 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L or 20 mmol / L.

[0117] In some specific embodiments, the concentration of tris(2-carboxyethyl)phosphine in the treatment reagent is 4 - 10 mmol / L.

[0118] In some embodiments, the reducing agent in the treatment reagent is dissolved in a solvent.

[0119] In some embodiments, the solvent is selected from purified water, physiological saline, or neutral buffer solution.

[0120] In some embodiments, the solvent is selected from at least one of phosphate buffer solution, physiological saline, carbonate buffer solution or Tris / HCl buffer solution with a pH of about 6 - 8.

[0121] In some embodiments, the solvent is selected as physiological saline.

[0122] In some embodiments, the kit further includes a control reagent.

[0123] In some embodiments, the control reagent is selected from purified water, physiological saline, or neutral buffer solution.

[0124] In some embodiments, the water is selected from one or more of purified water, ultrapure water, deionized water, and distilled water.

[0125] In some embodiments, the control reagent contains at least one of phosphate buffer solution, physiological saline, carbonate buffer solution or Tris / HCl buffer solution with a pH of about 6 - 8.

[0126] In some specific embodiments, the control reagent is physiological saline.

[0127] In some embodiments, the control reagent is the same as the solvent in the treatment reagent.

[0128] In some embodiments, the kit further includes a reference product.

[0129] In some embodiments, the reference product includes a positive reference product and a negative reference product.

[0130] In some embodiments, the positive reference contains IgM antibodies, and the negative reference does not contain IgM antibodies.

[0131] In some embodiments, the positive reference and the negative reference also contain Proclin.

[0132] According to a third aspect of the present disclosure, there is provided an application of the method described in the first aspect or the kit described in the second aspect in detecting IgM antibodies in a sample to be tested.

[0133] According to a fourth aspect of the present disclosure, there is provided an analyzer for detecting IgM antibodies in a sample to be tested by using the method described in the first aspect and the kit described in the second aspect. The chemiluminescence analyzer at least includes the following parts:

[0134] An incubation module for providing a suitable temperature environment for the chemical reaction between the sample to be tested and the microsphere composition;

[0135] A detection module for detecting the chemiluminescence signal generated by the reaction of the luminescent particles with reactive oxygen species;

[0136] A processor for judging whether there are IgM antibodies in the sample to be tested according to the chemiluminescence signal detected by the detection module.

[0137] The present disclosure realizes the detection of IgM antibodies by the double-antigen sandwich method on the photoactivated chemiluminescence platform, solves the pain point that the double-antigen sandwich method cannot distinguish between IgG and IgM, and avoids the problems of low sensitivity, susceptibility to interference by irrelevant IgM or specific IgG in the sample, and the need for dilution and long time in the indirect photoactivated chemiluminescence method for detecting IgM antibodies; at the same time, to a certain extent, it solves the problem of interference by specific IgG, and improves the detection sensitivity and detection efficiency. Description of the Drawings

[0138] Figure 1 Exemplarily shows the IgM structure. Detailed Embodiments

[0139] To make the objectives, technical solutions and advantages of the present invention clearer, the following further describes the present invention in detail with reference to embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0140] Definition

[0141] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will be applied, and where appropriate, terms used in the singular form will also include the plural form, and vice versa.

[0142] Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural referents.

[0143] The expression "about" as used herein is as understood by those of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If those of ordinary skill in the art do not understand the use of this term according to the context in which it is used, "about" will mean a specific value plus or minus 10%.

[0144] The term "IgM antibody" as described in this disclosure has a pentameric structure, and its pentamer is composed of 5 radially arranged subunits connected by inter-subunit disulfide bonds. The two heavy chains and light chains in each subunit are also connected by inter-chain disulfide bonds (such as Figure 1 ). When a certain concentration of reducing agent is used to act on the disulfide bonds, the disulfide bonds within the subunits can resist this cleavage, but the inter-subunit disulfide bonds are easily broken to form IgM monomers.

[0145] The term "pathogen antigen" as described in this disclosure refers to a substance 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.

[0146] The term "reducing agent" as described in this disclosure refers to any compound or substance that keeps the sulfhydryl group in a reduced state and reduces the intermolecular disulfide bonds. In some embodiments, the "reducing agent" can reduce the disulfide bonds of each monomer in IgM, but will not reduce the disulfide bonds between the subunits within each monomer of IgM or between the subunits in IgG.

[0147] As an example, reducing agents suitable for the methods of this invention include thiol reducing agents or phosphine reducing agents. Examples of thiol reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), β-mercaptoethanol, or reduced L-glutathione. Examples of phosphine reducing agents include tris(3-hydroxypropyl)phosphine (THPP), tributylphosphine (TBP), and tris(2-carboxyethyl)phosphine (TCEP).

[0148] The terms "ligand" or "receptor" as used in the present disclosure are used interchangeably herein and refer to two different molecules, where one molecule has a specific spatial or polar configuration on the surface or in the cavity of the molecule that specifically attracts or binds to another molecule, resulting in a region where the two molecules have an affinity for each other. The components of a specific binding pair can be referred to as a ligand and a receptor (receptor and ligand). The terms ligand and receptor are intended to cover the entire ligand or receptor or a portion thereof sufficient to effect binding between the ligand and the receptor. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor receptor, and interferon and interferon receptor; streptavidin and biotin; enzymes and substrates; drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; a nucleic acid strand and a complementary oligonucleotide; interleukin and interleukin receptor; stimulatory factors and their receptors, nanogold / iodacetyl-thiol, amino-aldehyde / carboxyl / isothiocyanato or silyl-acrylamide, etc.

[0149] The term "binding" as used in the present disclosure refers to the direct association between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding interactions, including but not limited to interactions such as salt bridges and water bridges.

[0150] The term "specific binding" as used in the present disclosure refers to the mutual discrimination and selective binding reaction between two substances, which in terms of three-dimensional structure is the conformational correspondence between the corresponding reactants. Under the technical concept disclosed in the present invention, the detection methods for specific binding reactions include, but are not limited to: sandwich ELISA, competitive method, neutralization competition method, indirect method, or capture method.

[0151] The term "reactive oxygen species" as used in the present disclosure refers to the general term for substances composed of oxygen, containing oxygen and having active properties in the body or in the natural environment, mainly an excited state of oxygen molecules, including the one-electron reduction product superoxide anion (O2·□), two-electron reduction product hydrogen peroxide (H2O2), three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen ( 1 O2), etc.

[0152] The reactive oxygen species can be provided by "photosensitive particles", which are nanoparticles capable of generating reactive oxygen species in an excited state. Preferably, the photosensitive particles can be polymer particles coated with functional groups on a matrix and filled with a photosensitive compound, which can generate singlet oxygen under light excitation. At this time, the photosensitive particles can also be referred to as oxygen-supplying particles or donor particles.

[0153] In the present invention, the term "luminescent particle" refers to a nanoparticle that can react with reactive oxygen species to produce a detectable chemiluminescent signal, and it can also be referred to as an oxygen acceptor particle or a receptor particle. Preferably, the luminescent particle can be a polymer particle filled with a luminescent composition formed by filling a matrix with functional groups, and the luminescent composition contains a chemiluminescent compound that can react with reactive oxygen species. In some specific embodiments of the present invention, the chemiluminescent compound undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, and the metastable intermediate can decompose, emitting light simultaneously or subsequently.

[0154] The term "sample to be tested" in the present invention refers to a mixture that is to be tested and contains or is suspected of containing a target molecule to be tested. Samples to be tested that can be used in the present disclosure include body fluids such as blood (which can be anticoagulated blood as commonly seen in collected blood samples), plasma, serum, urine, semen, saliva, cell cultures, tissue extracts, etc. The sample to be tested can be diluted with a diluent as needed before use. For example, in order to avoid the HOOK effect, the sample to be tested can be diluted with a diluent before being detected on a detection instrument and then detected on the detection instrument.

[0155] The term "Youden index" in the present disclosure is a method for evaluating the authenticity of a screening test, indicating the total ability of the screening method to detect true patients and non-patients. The Youden index is the sum of sensitivity and specificity minus 1. The cut-off point corresponding to the maximum Youden index is used as the threshold to ensure the optimal accuracy of the kit.

[0156] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention and do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0157] Examples

[0158] Example 1 Detection of Cytomegalovirus IgM Antibody by Photoactivated Chemiluminescence Sandwich Immunoassay

[0159] 1. Main Experimental Raw Materials and Equipment

[0160] CMV IgM negative serum without interference, CMV IgM negative serum with interference, CMV IgM positive serum, ProClin TM Preservative, cytomegalovirus pp150 recombinant antigen (Nanjing Jingda Biotechnology Co., Ltd.), aldehyde group sphere luminescent microspheres (Shanghai Suoxin Biotechnology Co., Ltd.), aldehyde group photosensitive microspheres (Shanghai Suoxin Biotechnology Co., Ltd.), dithiothreitol (Shanghai Reagent Co., Ltd. of Sinopharm). Among them, the interference is rheumatoid factor.

[0161] Other consumables: reaction cups, disposable pipette tips

[0162] Test equipment: fully automatic photochemiluminescence detector 500

[0163] 2. Experimental procedures (1) Preparation of the photochemiluminescence double antigen sandwich assay kit for detecting IgM antibodies

[0164] 1) Preparation of Reagent 1

[0165] Aldehyde group luminescent microspheres and cytomegalovirus pp150 recombinant antigen are mixed at a mass ratio of 10:0.5 in an environment of 0.05M carbonate buffer solution (CB solution), reacted at 37°C for 4h, then sodium borohydride is added for reduction, reacted at 2 - 8°C for 1h, washed twice with Reagent 1 diluent (0.05M HEPES buffer solution with pH 7.0, containing 1.6% BSA, 0.1% dextran, 0.5% TritronX - 405, 0.3M sodium chloride, 0.025M EDTA and 0.05% Proclin300), and made up to a microsphere concentration of 10mg / mL. The luminescent particles coated with antigen are diluted to 25mg / L with Reagent 1 diluent to prepare Reagent 1.

[0166] 2) Preparation of Reagent 2

[0167] The recombinant antigen PP150 is diluted to 1mg / mL and dialyzed in 0.1M NaHCO3 solution at room temperature for 2 - 4h, with the solution changed twice or more. The dialyzed biotin is transferred to a centrifuge tube, and biotin dissolved in DMSO is added at a molar ratio of 1:10, reacted at 2 - 8°C for 2h, then dialyzed in 0.02M PBS buffer solution at 2 - 8°C for at least 6h, with the solution changed 4 times or more. The biotinylated recombinant antigen is diluted to 2.5mg / L with Reagent 2 diluent (0.1M Tris - HCl buffer solution with pH 8.0, containing 0.1% BSA, 0.2% bovine gamma globulin, 0.1% dextran, 0.1% TritronX - 405, 0.3M sodium chloride, 0.03M EDTA and 0.05% Proclin300) to prepare Reagent 2.

[0168] 3) Preparation of the universal solution

[0169] Aldehyde group photosensitive microspheres and streptavidin are mixed at a mass ratio of 10:0.5 in an environment of 0.05M CB solution, reacted at 37°C for 4h, then sodium borohydride is added for reduction, reacted at 2 - 8°C for 1h, washed twice with 0.02M PBS buffer solution, and made up to a microsphere concentration of 20mg / L.

[0170] 4) Preparation of the control solution

[0171] Prepare 0.9% NaCl as the control solution.

[0172] 5) Preparation of the treatment agent

[0173] Use 0.9% NaCl to make the volume of dithiothreitol up to 5 mmol / L to prepare the treatment agent.

[0174] 6) Preparation of the negative control

[0175] Add the interference-free CMV IgM negative serum to 0.05% Proclin to prepare the negative control 1; add the CMV IgM negative serum with interference to 0.05% Proclin to prepare the negative control 2.

[0176] 7) Preparation of the positive control

[0177] Dilute the CMV IgM positive serum by a certain multiple with the CMV IgM negative serum, and add 0.05% Proclin to prepare the positive control.

[0178] 8) Aliquot each component and assemble it into a finished kit.

[0179] (2) Detect CMV IgM antibody in the sample

[0180] 1) Prepare the samples: Samples 1 and 2 are 2 replicates of CMV IgM and IgG positive, samples 3 and 4 are 2 replicates of CMV IgM positive and IgG negative, samples 5 and 6 are 2 replicates of CMV IgM negative and IgG positive, and samples 7 and 8 are 2 replicates of CMV IgM and IgG negative. For each test sample, make two wells, mix 25 μL of the test sample with 25 μL of the treatment agent or an equal amount of the control solution respectively, and react fully at 37 °C for 15 minutes;

[0181] 2) Add 25 μL of reagent 1 and 25 μL of reagent 2 to each well, add 175 μL of the universal solution after reacting for 10 minutes, and react at 37 °C for 15 minutes.

[0182] 3) Data analysis and give the test results:

[0183] Use a large number of samples with third-party measured values and detect them with the above detection mode. According to the sample measured values, find the optimal threshold through ROC curve analysis. The threshold when the Youden index is the largest is the optimal threshold. Determine the cut-off value for distinguishing positive and negative according to the above standard, that is, when the treatment agent is dithiothreitol, the fixed value of the treatment agent is 0.5, and the threshold is 2000.

[0184] Read the signal values of two wells. If the signal value of the sample in the control well to be tested < 2000, it is directly determined as negative for CMV IgM; if the signal value of the control well of the sample to be tested ≥ 2000, then calculate the ratio of the signal value of the treatment agent well to the signal value of the control well for secondary determination to confirm whether CMV IgM is negative or positive: when the ratio ≥ 0.5, CMV IgM is negative; when the ratio < 0.5, CMV IgM is positive. The test results are shown in Table 1.

[0185] Table 1. Test data

[0186]

[0187] Table 1 (continued)

[0188]

[0189] The signal values of the control wells of Samples 7 and 8 < 2000, indicating that the content of IgM to be tested is not sufficient to be detected, and it is directly determined as negative for CMV IgM.

[0190] The signal values of the control wells of Samples 1 - 6 > 2000, then calculate the ratio of the signal value of the treatment agent well to the signal value of the control well. The ratios of Samples 1 - 4 < 0.5, and it is determined that CMV IgM is positive; the ratios of Samples 5 and 6 > 0.5, and it is determined that CMV IgM is negative.

[0191] 4. Experimental conclusion

[0192] This kit can detect CMV IgM antibody by the double antigen sandwich method.

[0193] Example 2 Exploration of the concentration of dithiothreitol as the treatment agent

[0194] (1) Prepare samples: Samples 1 and 2 are positive for CMV IgM and IgG, Samples 3 and 4 are positive for CMV IgM and negative for IgG, Samples 5 and 6 are negative for CMV IgM and positive for IgG, and Samples 7 and 8 are negative for CMV IgM and IgG.

[0195] (2) Use 0.9% NaCl to prepare dithiothreitol at different concentrations to prepare the treatment agent, and use it with the kit prepared in Example 1 to detect the samples. The detection steps are the same as those in Example 1.

[0196] (3) The test data are shown in Table 2, which shows the signal values and ratios of Samples 1 - 8 when the concentration of dithiothreitol is 0 mmol / L, 2 mmol / L, 4 mmol / L, 10 mmol / L, and 20 mmol / L.

[0197] The determination results of CMV IgM detection are shown in Table 3, which shows the determination results of samples 1 to 8 when the dithiothreitol concentrations are 0 mmol / L, 2 mmol / L, 4 mmol / L, 10 mmol / L, and 20 mmol / L.

[0198] Table 2. Test data of Example 2

[0199]

[0200]

[0201] Table 3. CMV IgM test results

[0202]

[0203] According to the analysis of the experimental results, when the concentration of dithiothreitol used was 2mmol / L, the test result of sample 4 was a false negative, when the concentration of dithiothreitol used was 20mmol / L, the test results of samples 5 and 6 were false positives, and when the concentration of dithiothreitol used was 4-10mmol / L, it could be detected normally.

[0204] Example 3 Detection of Toxoplasma gondii IgM Antibody by Photoluminescence Double Antibody Sandwich Method

[0205] 1. Main experimental materials and equipment

[0206] No interference with Toxoplasma IgM negative serum, interference with Toxoplasma IgM negative serum, containing Toxoplasma IgM positive serum, ProClin TM Preservatives, Toxoplasma gondii pp25+pp30+pp35 recombinant chimeric antigen (Phipeng Biotechnology), carboxyl luminescent microspheres (Shanghai Suoxin Biotechnology Co., Ltd.), aldehyde photosensitive microspheres (Shanghai Suoxin Biotechnology Co., Ltd.), tris(2-carboxyethyl)phosphine (TCEP, Sinopharm Wokai). Among them, the interference is rheumatoid factor.

[0207] Other consumables: reaction cup, disposable pipette tip

[0208] Test equipment: Fully automatic photoluminescence detector 500

[0209] 2. Experimental steps (1) Preparation of photochemiluminescence double antigen sandwich method for detecting IgM antibody

[0210] 1) Preparation of Reagent 1

[0211] The carboxyl luminescent microspheres and the recombinant chimeric antigen of Toxoplasma gondii pp25+pp30+pp35 were mixed at a mass ratio of 10:0.5 in a 0.02M PBS buffer solution with a pH of 7.2. After reacting at 37°C for 4h, 100mg / ml BSA was added for blocking. After reacting at 37°C for 1h, it was washed twice with reagent 1 diluent (0.02M PBS buffer solution with a pH of 7.2, containing 1.6% BSA, 0.1% dextran, 0.03M EDTA and 0.05% Proclin300), and made up to a microsphere concentration of 10mg / mL. The luminescent particles after antigen coating were diluted to 12.5mg / L with reagent 1 diluent to prepare reagent 1.

[0212] 2) Preparation of reagent 2

[0213] The recombinant antigen of Toxoplasma gondii was diluted to 1mg / ml and dialyzed in 0.1m NaHCO3 solution at room temperature for 2 - 4h, with the solution changed 2 times or more. The dialyzed biotin was transferred to a centrifuge tube, and biotin dissolved in DMSO was added at a molar ratio of 1:10. After reacting at 2 - 8°C for 2h, it was dialyzed in 0.02M PBS buffer solution at 2 - 8°C for at least 6h, with the solution changed 4 times or more. The biotinylated recombinant antigen was diluted to 2.5mg / L with reagent 2 diluent (0.02M PBS buffer solution with a pH of 7.2, containing 1.6% BSA, 0.1% dextran, 0.03M EDTA and 0.05% Proclin300) to prepare reagent 2.

[0214] 3) Preparation of universal solution

[0215] The aldehyde - group photosensitive microspheres and avidin were mixed at a mass ratio of 10:0.5 in a 0.05M CB solution. After reacting at 37°C for 4h, sodium borohydride was added for reduction. After reacting at 2 - 8°C for 1h, it was washed twice with 0.02M PBS buffer solution and made up to a microsphere concentration of 20mg / L.

[0216] 4) Preparation of control solution

[0217] 0.9% NaCl was prepared as the control solution.

[0218] 5) Preparation of treatment agent

[0219] TCEP was diluted to 20mmol / L with 0.9% NaCl to prepare the treatment agent.

[0220] 6) Preparation of negative control

[0221] The Toxoplasma gondii IgM - negative serum without interference was added with 0.05% Proclin to prepare negative control 1, and the Toxoplasma gondii IgM - negative serum with interference was added with 0.05% Proclin to prepare negative control 2.

[0222] 7) Preparation of positive control

[0223] Dilute the positive serum of Toxoplasma IgM with negative serum of Toxoplasma IgM by a certain multiple, and add 0.05% Proclin to prepare the positive control.

[0224] 8) Dispense each component and assemble them into a finished kit.

[0225] (2) Detection of Toxoplasma IgM antibody in test samples

[0226] 1) Make two holes for the test sample. Mix 25 μL of the test sample, 25 μL of the treatment agent or an equal amount of the control solution, and react fully at 37°C for 15 minutes;

[0227] 2) Add 25 μL of Reagent 1 and 25 μL of Reagent 2, then react at 37°C for 10 minutes. After the reaction, add 175 μL of the universal solution and react at 37°C for 15 minutes;

[0228] 3) Analyze the data and give the test result

[0229] Collect 100 positive and 500 negative serum samples, and detect them using the above detection mode according to the third-party measured values (indirect method) of the samples. The test results are shown in Table 4. According to the sample measured values, find the optimal threshold through ROC curve analysis. The standard for the optimal threshold is the maximum Youden index. Determine the cut-off value for distinguishing positive and negative according to the above standard. That is, when the treatment agent is TCEP, the fixed value of the treatment agent is 0.6, and the threshold is 2571.

[0230] Read the signal values of the two holes. If the signal value of the control hole of the test sample < 2571, it is directly determined as negative for Toxoplasma IgM; if the signal value of the control hole of the test sample ≥ 2571, calculate the ratio of the signal value of the treatment agent hole to the signal value of the control hole for secondary determination to confirm whether Toxoplasma IgM is negative or positive: when the ratio ≥ 0.6, Toxoplasma IgM is negative; when the ratio < 0.6, Toxoplasma IgM is positive.

[0231] Table 4. Test data

[0232] TCEP (mmol / L) Indirect detection result Control well Treatment agent well Ratio Discrimination Sample 1 IgM+IgG+ 38362 2665 0.07 Positive Sample 2 IgM-IgG+ 9948 6097 0.61 Negative Sample 3 IgM-IgG+ 29752 24532 0.82 Negative Sample 4 IgM+IgG- 7832 4564 0.58 Positive Sample 5 IgM+IgG- 6720 1096 0.16 Positive Sample 6 IgM-IgG+ 13017 10897 0.84 Negative Sample 7 IgM-IgG+ 40351 38956 0.97 Negative Sample 8 IgM-IgG- 831 672 / Negative

[0233] The signal value of the control hole of sample 8 < 2571, and it is directly determined as negative for Toxoplasma IgM; the signal values of the control holes of other samples are all > 2571, then calculate the ratio of the signal value of the treatment agent hole to the signal value of the control hole. The ratios of samples 1, 4, and 5 < 0.6, and it is determined that Toxoplasma IgM is positive; the ratios of samples 2, 3, 6, and 7 > 0.6, and it is determined that CMV IgM is negative.

[0234] 4. Experimental conclusion

[0235] This kit can detect Toxoplasma gondii IgM antibody by the double-antigen sandwich method.

[0236] Example 4 Exploration of the Concentration of the Treatment Agent TCEP

[0237] (1) Prepare samples: Samples 1 and 2 are positive for Toxoplasma gondii IgM and IgG, samples 3 and 4 are positive for Toxoplasma gondii IgM and negative for IgG, samples 5 and 6 are negative for Toxoplasma gondii IgM and positive for IgG, and samples 7 and 8 are negative for Toxoplasma gondii IgM and IgG.

[0238] (2) Use 0.9% NaCl to prepare TCEP at different concentrations to form a treatment agent, and combine it with the kit prepared in Example 3 to detect the samples. The detection steps are the same as those in Example 3.

[0239] (3) The experimental data are shown in Table 5, which shows the signal values and ratios of samples 1-8 when the TCEP concentration is 0 mmol / L, 5 mmol / L, 15 mmol / L, 30 mmol / L, and 50 mmol / L.

[0240] The determination results of Toxoplasma gondii IgM detection are shown in Table 6, which shows the determination results of samples 1-8 when the TCEP concentration is 0 mmol / L, 5 mmol / L, 15 mmol / L, 30 mmol / L, and 50 mmol / L.

[0241] Table 5. Experimental Data

[0242]

[0243] Table 6. Determination Results of Toxoplasma gondii IgM Detection

[0244]

[0245] According to the analysis of the experimental results this time, when the usage concentration of TCEP is 5 mmol / L, the detection results of samples 1, 2, and 4 are false negatives; when the usage concentration of TCEP is 50 mmol / L, the detection results of samples 5 and 6 are false positives; when the usage concentration of TCEP is 15-30 mmol / L, normal detection can be achieved.

[0246] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A method for detecting IgM antibodies in a sample to be tested, characterized in that, The method includes the following steps: 1) Add an equal amount of the sample to be tested into the detection well and the control well, and respectively add an equal amount of the treatment reagent and the control reagent; 2) Add a detection reagent to the detection well and the control well, and detect the signal values of the detection well and the control well; 3) Confirm whether the sample to be tested contains IgM antibody according to the ratio of the signal values; The treatment reagent contains a reducing agent, and the control reagent does not contain a reducing agent.

2. The method according to claim 1, wherein In step 3), the confirmation of whether the sample to be tested contains IgM antibody according to the ratio of the signal values includes the following steps: the signal value of the detection well is the first signal value, and the signal value of the control well is the second signal value. When the first signal value ≥ threshold M and the ratio of the first signal value / the second signal value < threshold N, it is determined that the IgM antibody in the sample to be tested is positive; Preferably, if the first signal value < threshold M, it is determined that the IgM antibody in the sample to be tested is negative; If the first signal value ≥ threshold M, then calculate the ratio of the first signal value / the second signal value for secondary determination; Preferably, if the first signal value ≥ threshold M and the ratio of the first signal value / the second signal value ≥ threshold N, it is determined that the IgM antibody in the sample to be tested is negative; Preferably, the threshold M and / or threshold N are determined according to the detection of known IgM-positive and IgM-negative samples, and according to the detection results, through ROC curve analysis, and according to the Youden index to determine the threshold M and / or threshold N.

3. The method according to claim 1, wherein The reducing agent is selected from one or more of dithiothreitol, dithioerythritol, β-mercaptoethanol, 2-mercaptoethylamine, 2-dimethylaminoethyl mercaptan, 3-mercaptopropanol, cysteine, N-acetylcysteine, reduced glutathione, benzenethiol, ascorbic acid, sodium sulfite, sodium metabisulfite, hydroxylamine hydrochloride, sodium thiosulfate, carbohydrazide, hydrazine sulfate, sodium nitrite, tris(3-hydroxypropyl)phosphine, tributylphosphine, and tris(2-carboxyethyl)phosphine; Preferably, the reducing agent is tris(2-carboxyethyl)phosphine or dithiothreitol; Preferably, the reducing agent in the treatment reagent is dissolved in a solvent, and the solvent is selected from water, physiological saline or a neutral buffer solution, more preferably at least one of a phosphate buffer solution, physiological saline, carbonate buffer solution or Tris / HCl buffer solution with a pH of about 6-8, and further preferably physiological saline.

4. The method according to claim 1, characterized in that The control reagent is selected from purified water, deionized water, physiological saline or a neutral buffer solution, more preferably at least one of a phosphate buffer solution, carbonate buffer solution or Tris / HCl buffer solution with a pH of about 6-8, and further preferably physiological saline; Preferably, the solvent in the control reagent is the same as the solvent in the treatment reagent.

5. A kit for detecting IgM antibodies, characterized in that, The kit is used to implement the method according to any one of claims 1 to 4, and it includes the following reagents: A treatment reagent containing a reducing agent; A control reagent not containing a reducing agent; and, A detection reagent.

6. The kit according to claim 5, characterized in that, The detection reagent includes: A first reagent, which contains a first antigen coated on a luminescent / photosensitive particle, and the first antigen can specifically bind to IgM antibody; A second reagent, which contains a second antigen modified with a ligand, and the second antigen can specifically bind to IgM antibody; and A third reagent, comprising receptor-modified photosensitive / photoluminescent microparticles; The ligand and the receptor specifically bind to each other.

7. The kit according to claim 6, wherein The concentration range of the luminescent / photosensitive microparticles bound to the first antigen in the first reagent is 10-50 mg / L, more preferably 25 mg / L; Preferably, the concentration range of the ligand-modified second antigen in the second reagent is 1-10 mg / L, more preferably 2.5 mg / L; Preferably, the concentration range of the receptor-modified photosensitive / photoluminescent microparticles in the third reagent is 5-50 mg / L, more preferably 20 mg / L; Preferably, the ligand and receptor combination is selected from at least one of biotin / biotin analog - avidin / streptavidin, nano gold / iodoacetyl-thiol, amino-aldehyde / carboxyl / isothiocyanato, and silyl-acrylamide.

8. The kit according to claim 5, wherein The reducing agent is tris(2-carboxyethyl)phosphine or dithiothreitol, Preferably, the concentration range of tris(2-carboxyethyl)phosphine in the treatment reagent is 5-50 mmol / L, preferably 15-30 mmol / L; Preferably, the concentration range of dithiothreitol in the treatment reagent is 2-20 mmol / L, preferably 4-10 mmol / L; Preferably, the reducing agent in the treatment reagent is dissolved in a solvent; Preferably, the solvent is selected from purified water, physiological saline, or neutral buffer solution, more preferably at least one of phosphate buffer solution, physiological saline, carbonate buffer solution, or Tris / HCl buffer solution with a pH of about 6-8, and further preferably physiological saline; 9. The kit according to claim 5, wherein The control reagent is selected from water, physiological saline, or neutral buffer solution, more preferably at least one of phosphate buffer solution, carbonate buffer solution, or Tris / HCl buffer solution with a pH of about 6-8, and further preferably physiological saline; Preferably, the control reagent is the same as the solvent in the treatment reagent; Preferably, the kit further includes reference samples. Preferably, the reference samples include a positive reference sample and a negative reference sample. The positive reference sample contains IgM antibodies, and the negative reference sample does not contain IgM antibodies.

10. An analyzer, characterized in that, Using the method according to any one of claims 1-4 or the kit according to any one of claims 5-9 to detect IgM antibodies in a test sample, the chemiluminescence analyzer at least includes the following parts: An incubation module, which is used to provide a suitable temperature environment for the chemical reaction between the test sample and the microsphere composition; A detection module, which is used to detect the chemiluminescence signal generated by the reaction of the luminescent microparticles with reactive oxygen species; A processor, which determines whether the IgM antibodies in the test sample are positive or negative according to the chemiluminescence signal detected by the detection module.

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