Mycoplasma pneumoniae antibody detection kit and application thereof

Abstract

The application provides a mycoplasma pneumoniae antibody detection kit and application thereof, and relates to the technical field of biology.The kit comprises a mycoplasma pneumoniae fusion antigen working solution containing mycoplasma pneumoniae fusion antigen and a diluent; a tracer-labeled antibody working solution containing antibody IgG, IgM or IgA and a diluent; wherein the mycoplasma pneumoniae fusion antigen has an amino acid sequence as shown in SEQ ID No.3; and the diluent is a phosphate buffer containing 0.02-1 wt% fatty alcohol polyoxyethylene ether sulfate salt.The kit uses mycoplasma pneumoniae fusion antigen, has stronger specificity, higher sensitivity and better thermal stability; in addition, the application adds the sulfate salt type anionic surfactant fatty alcohol polyoxyethylene ether sulfate salt in the diluent, which provides a stable and good microenvironment for the fusion antigen, and further improves the thermal stability of the antigen of the kit.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Mycoplasma pneumoniae antibody detection kit and application thereof. BACKGROUND

[0002] Mycoplasma pneumoniae (MP) is a prokaryotic cell without cell wall, which can bind to the host respiratory mucosa epithelial cell mannitol pyruvic acid receptor and produce toxin, resulting in pneumonia. Serum epidemiological studies show that 20.7% to 38.9% of community-acquired pneumonia patients in China are caused by MP, ranking first among adult community-acquired pneumonia pathogens. In addition to respiratory symptoms, 20% to 25% of cases can have extrapulmonary manifestations such as meningitis, myocarditis, hemolytic anemia, and thrombocytopenic purpura. Timely and effective diagnosis can effectively control the aggravation of MP infection and reduce mortality. Therefore, accurate diagnosis of MP has important clinical significance.

[0003] At present, the main methods for detecting MP are isolation and culture, PCR detection, and serum specific antibody detection, each with advantages and disadvantages. Pathogen isolation and culture is considered the gold standard for disease diagnosis, but the culture requires high conditions and takes a long time (about 6 weeks), so it is not practical for the treatment of clinical patients, so few laboratories diagnose whether patients are infected with MP by culture. The PCR method detects P1 protein gene, 16S rRNA and other protein genes of MP, but the PCR method is complex to operate, and the experimenters must be specially trained, so it is difficult to promote in some primary units. Serological detection is the most widely used. Among them, ELISA and CLIA are simple to operate and can quantitatively detect MP-IgM, but they take a long time, and the entire reaction time requires more than one hour, and ELISA cannot meet the needs of on-site testing. Fluorescent immunochromatography and colloidal gold immunochromatography methods have the advantages of simplicity and rapidness, but they are mainly qualitative and semi-quantitative and have poor sensitivity. Fully automatic chemiluminescence immunoassay is based on enzyme immunoassay combined with high-sensitivity chemiluminescence detection technology and magnetic micro-particle separation technology, which greatly improves the detection sensitivity; due to the use of fully automatic instruments and matching reagents, human factors are minimized, improving the stability of the method and the repeatability of the results.

[0004] Stability as an important indicator of in vitro diagnostic reagent to maintain product safety and effectiveness, has important guiding significance to the production, transportation, storage and use of the product. In vitro diagnostic reagents are biological reagents, and some active groups have different chemical instability trends, which are prone to hydrolysis, enzymolysis and oxidation, etc. reactions, thereby affecting the quality and stability of the reagent. At present, the stability of mycoplasma pneumoniae kit mainly reflects on MP antigen, however, the existing kit has poor thermal stability of antigen, so it is difficult to store, and needs to be stored at low temperature to ensure that the active ingredients in it do not lose effectiveness.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a mycoplasma pneumoniae antibody detection kit and its application, to solve the problem of poor thermal stability of mycoplasma pneumoniae in vitro detection reagent and difficult storage in the prior art. The kit of the present application uses mycoplasma pneumoniae fusion antigen, which further improves the heat resistance of the antigen without affecting the high specificity and high sensitivity of the fusion antigen.

[0007] In order to achieve the above purpose, the present application adopts the following technical scheme:

[0008] In one aspect, the present application provides a mycoplasma pneumoniae antibody detection kit, comprising:

[0009] Mycoplasma pneumoniae fusion antigen working solution containing mycoplasma pneumoniae fusion antigen and diluent;

[0010] Tracer labeled antibody working solution containing antibody IgG, IgM or IgA and diluent;

[0011] Among them, the mycoplasma pneumoniae fusion antigen has an amino acid sequence as shown in SEQ ID No. 3; the diluent is phosphate buffer containing 0.02-1wt% fatty alcohol polyoxyethylene ether sulfate (AES).

[0012] Different from the commonly used P1M, P1C and MP whole bacteria antigens, the kit of the present application uses a fusion antigen (P1M / P30A antigen) with high thermal stability, which comprises a fusion protein (also referred to as P1M / P30A antigen) of a P1M antigen fragment (the amino acid sequence of which is shown as SEQ ID No. 1) and a P30A antigen fragment (the amino acid sequence of which is shown as SEQ ID No. 2) (the amino acid sequence of which is shown as SEQ ID No. 3). The nucleic acid molecule encoding the Mycoplasma pneumoniae fusion antigen has a DNA sequence shown as SEQ ID No. 6. Compared with the existing Mycoplasma pneumoniae whole bacteria antigen, the kit has higher specificity, higher sensitivity and better thermal stability, as verified by immunological serological detection technology.

[0013] Meanwhile, the kit of the present application further improves the thermal stability of the antigen after adding a sulfate salt type anionic surfactant (preferably a fatty alcohol polyoxyethylene ether sulfate salt (AES)) in the diluent, without affecting the sensitivity and specificity of the kit.

[0014] In the kit of the present application, the antibody in the tracer-labeled antibody working solution is a secondary antibody, and the type of the secondary antibody can be adjusted according to the type of the antibody to be detected. For example, anti-human IgM antibody, anti-human IgG antibody and anti-human IgA antibody.

[0015] In particular, when the AES concentration in the working solution is within a certain range, the effect of maintaining the thermal stability of the fusion antigen is optimal, and even after 7 days of acceleration at 37°C, the signal value deviation of the kit is controlled within 5%. The optimal concentration range of AES is 0.02-1 wt%, including but not limited to 0.03, 0.05, 0.07, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 wt%, and the optimal concentration is 0.5 wt%.

[0016] In one embodiment, the phosphate buffer further contains bovine serum albumin, sodium chloride, sucrose and proclin 300; the concentration of the bovine serum albumin is 0.01-5 wt%, the concentration of the sodium chloride is 0.5-10 wt%, the concentration of the sucrose is 1-5 wt% and the concentration of the proclin 300 is 0.05-0.5 wt%.

[0017] In one embodiment, in the phosphate buffer, the concentration of the fatty alcohol polyoxyethylene ether sulfate salt (AES) is 0.5 wt%, the concentration of the bovine serum albumin is 1 wt%, the concentration of the sodium chloride is 8 wt%, the concentration of the sucrose is 1 wt% and the concentration of the proclin 300 is 0.05 wt%.

[0018] The present application found in the research that adding fatty alcohol polyoxyethylene ether sulfate (AES) as a surfactant in the working solution of the fusion antigen can improve the thermal stability of the kit, indicating that AES can protect the active groups of P1M / P30A fusion antigen, so that it is not easy to occur hydrolysis, enzymolysis and oxidation and other reactions in high heat environment.

[0019] In one embodiment, the phosphate buffer (PBS buffer) has a pH of 6.0-8.0 (including but not limited to 6.5, 7, 7.5, 8) and a concentration of 0.01-0.2 mol / L, including but not limited to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15 and 0.2 mol / L.

[0020] Preferably, the phosphate buffer has a pH of 7 and a concentration of 0.05 mol / L.

[0021] In a preferred embodiment, the phosphate buffer contains 1 wt% bovine serum albumin, 8 wt% sodium chloride, 1 wt% sucrose, 0.5 wt% fatty alcohol polyoxyethylene ether sulfate (AES), and 0.05 wt% proclin 300.

[0022] In one embodiment, the concentration of the mycoplasma pneumoniae fusion antigen in the mycoplasma pneumoniae fusion antigen working solution is 0.2-1 mg / L, and the concentration of the antibody in the tracer-labeled antibody working solution is 0.2-1 mg / L.

[0023] In a preferred embodiment, the concentration of the mycoplasma pneumoniae fusion antigen in the mycoplasma pneumoniae fusion antigen working solution is 0.5 mg / L, and the concentration of the antibody in the tracer-labeled antibody working solution is 0.5 mg / L.

[0024] The mycoplasma pneumoniae fusion antigen working solution and the tracer-labeled antibody working solution are prepared by diluting the fusion antigen and the tracer-labeled antibody to a certain concentration with a phosphate buffer (PBS buffer).

[0025] In one embodiment, the tracer is a luminescent label or a chemiluminescent catalyst;

[0026] Preferably, the luminescent label is selected from any one of adamantane, luminol and its derivatives, isoluminol and its derivatives, acrid ester and its derivatives, or trispyridine ruthenium;

[0027] Preferably, the chemiluminescent catalyst is selected from any one of alkaline phosphatase and horseradish peroxidase.

[0028] In one embodiment, a chemiluminescent substrate solution is further included; preferably, the chemiluminescent substrate solution comprises NaOH and H2O2.

[0029] When the tracer is a chemiluminescent catalyst, at least one of adamantane, luminol and its derivatives, isoluminol or its derivatives is further included in the chemiluminescent substrate solution.

[0030] In one embodiment, in the chemiluminescent substrate solution, the NaOH and H2O2 luminescent system, AMPPD is a luminescent substrate.

[0031] In one embodiment, the Mycoplasma pneumoniae antibody kit comprises: a Mycoplasma pneumoniae fusion antigen (P1M / P30A antigen) working solution and a tracer-labeled IgG, IgM or IgA antibody working solution. In a preferred embodiment, the Mycoplasma pneumoniae antibody kit comprises: a Mycoplasma pneumoniae fusion antigen working solution, a tracer-labeled antibody working solution, and a magnetic / magnetic bead microparticle / magnetic bead working solution.

[0032] In one embodiment, in the Mycoplasma pneumoniae antibody kit, the Mycoplasma pneumoniae fusion antigen working solution and the tracer-labeled antibody working solution are prepared from a phosphate buffer containing fatty alcohol polyoxyethylene ether sulfate (AES); and the magnetic / magnetic bead microparticle working solution is prepared from a phosphate buffer.

[0033] In one embodiment, the Mycoplasma pneumoniae fusion antigen is directly or indirectly coated on a solid support; more preferably, the solid support comprises magnetic microspheres.

[0034] Further, the magnetic microspheres or microparticles include carboxyl magnetic beads, amino magnetic beads, hydroxyl magnetic beads or epoxy magnetic beads, tosyl magnetic beads or epoxy magnetic beads, etc.

[0035] In one embodiment, the Mycoplasma pneumoniae fusion antigen is a biotin-labeled Mycoplasma pneumoniae fusion antigen; and the kit further comprises a streptavidin-coupled magnetic bead working solution.

[0036] Preferably, the streptavidin-coupled magnetic bead working solution is prepared from a phosphate buffer with a pH of 6.0-8.0 and a concentration of 0.01-0.2 mol / L;

[0037] The concentration of the streptavidin magnetic beads is 0.1-2 mg / mL.

[0038] In a preferred embodiment, the streptavidin-coupled magnetic bead working solution is prepared from a phosphate buffer with a pH of 7.4 and a concentration of 0.05 mol / L; and the concentration of the streptavidin-coupled magnetic beads is 0.5 mg / mL.

[0039] In one embodiment, the kit comprises a biotin-labeled P1M / P30A antigen working solution, a tracer-labeled IgG, IgM or IgA antibody working solution and a streptavidin-coupled magnetic bead working solution. In the detection, the sample to be detected, the biotin-labeled antigen and the streptavidin-coupled magnetic bead are mixed, incubated and washed, and then the tracer-labeled antibody is added to form a magnetic bead-streptavidin-biotin-antigen-antibody-secondary antibody complex, and then the detection of the sample to be detected is realized through the luminescence intensity.

[0040] In one embodiment, the present application detects Mycoplasma pneumoniae antibodies by using a full-automatic chemiluminescence immunoassay combined with enzyme immunoassay and chemiluminescence assay technology and magnetic micro-particle separation technology.

[0041] In one embodiment, the sample to be detected by the kit of the present application includes whole blood, serum or plasma.

[0042] In another aspect, the present application provides the use of the kit in the in vitro detection of Mycoplasma pneumoniae antibodies for non-disease diagnosis purposes.

[0043] Compared with the prior art, the present application has the following advantages:

[0044] The Mycoplasma pneumoniae antibody detection kit provided in the present application uses a Mycoplasma pneumoniae fusion antigen (P1M / P30A antigen), which has higher detection specificity and sensitivity and higher thermal stability than other antigen types (such as MP whole bacteria antigen, P1M antigen or P1C antigen). The present application further improves the thermal stability of the kit by adding a fatty alcohol polyoxyethylene ether sulfate salt (AES) as a surfactant in the working solution of the fusion antigen and the antibody (secondary antibody). The present application greatly improves the stability of the kit and the reliability of the detection results, and can realize the quantitative detection of Mycoplasma pneumoniae antibodies.

[0045] The present application uses tracer-labeled antibodies to form an antibody-antigen-secondary antibody complex with the fusion antigen and the antibodies in the test sample, and initiates the luminescence performance of the substrate through the tracer, and then the luminescence value is detected by the instrument, and the concentration of the antibodies in the test sample is calculated. Since the chemiluminescence immunoassay method is used, the cost is saved, the detection time is short, and the method has the advantages of simple operation, high precision, etc.

[0046] The kit of the present application is widely applicable, especially when it is used in a full-automatic chemiluminescence system. The steps of sample addition, incubation, washing and detection can be automated, which avoids the result deviation caused by human operation, improves the work efficiency, and through the built-in calibration curve in the test software, the Mycoplasma pneumoniae antibodies in the sample can be quantitatively detected only by testing the sample, which makes the detection faster, more reliable and more stable. DETAILED DESCRIPTION

[0047] The technical solutions of the present application will be described clearly and completely in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0048] 1. Raw material: fusion antigen

[0049] The Mycoplasma pneumoniae antigen used in the present application is Mycoplasma pneumoniae fusion antigen (P1M / P30A antigen).

[0050] The antigen epitope P1M (residues 1341-1518) (SEQ ID No. 1) and the antigen epitope P30A (residues 170-182) (SEQ ID No. 2) of P30 antigen are used, and a short peptide is used to connect to form P1M / P30A antigen fusion antigen (SEQ ID No. 3). The specific preparation method has been reported in the published patent literature.

[0051] WLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQT (SEQ ID No. 1).

[0052] RTGFPPQPGMAPR (SEQ ID No. 2).

[0053] WLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLQRTGFPPQPGMAPR (SEQ ID No. 3).

[0054] The present application adopts E. coli preference codon to reverse translate the antigen amino acid sequence of the present application, to obtain a recombinant antigen gene nucleic acid molecule composed of E. coli preference codon. The nucleic acid molecule can express the Mycoplasma pneumoniae fusion antigen of the present application completely and accurately.

[0055] The nucleic acid molecule is preferably a DNA sequence shown in SEQ ID No. 6:

[0056] TGGCTGGTTGGCCAGCTGCCGAGCACCAGCGATGGTAACACCAGCAGCACCAACAACCTGGCGCCGAACACCAACACCGGCAACGACGTGGTTGGTGTGGGCCGTCTGAGCGAAAGCAACGCGGCGAAAATGAACGATGACGTGGACGGTATCGTTCGTACCCCGCTGGCGGAGCTGCTGGATGGCGAGGGTCAGACCGCGGACACCGGTCCGCAGAGCGTGAAGTTTAAAAGCCCGGATCAAATCGACTTCAACCGTCTGTTTACCCACCCGGTTACCGACCTGTTCGACCCGGTGACCATGCTGGTTTACGATCAGTATATTCCGCTGTTTATCGACATTCCGGCGAGCGTTAACCCGAAGATGGTGCGTCTGAAAGTTCTGAGCTTCGATACCAACGAGCAAAGCCTGGGTCTGCGTCTGGAGTTCTTCAAACCGGATCAAGACACCCAGCCGAACAACAACGTGCAGGTTAACCCGAACAACGGTGACTTTCTGCCGCTGCTGACCGCGAGCAGCCAAGGTCCGCAGACCCTGCAGCGTACCGGTTTTCCGCCGCAGCCGGGTATGGCGCCGCGTTAA(SEQ ID No. 6).

[0057] In the DNA sequence shown in SEQ ID No. 6, the DNA sequence encoding the P1M antigen fragment is:

[0058] TGGCTGGTTGGCCAGCTGCCGAGCACCAGCGATGGTAACACCAGCAGCACCAACAACCTGGCGCCGAACACCAACACCGGCAACGACGTGGTTGGTGTGGGCCGTCTGAGCGAAAGCAACGCGGCGAAAATGAACGATGACGTGGACGGTATCGTTCGTACCCCGCTGGCGGAGCTGCTGGATGGCGAGGGTCAGACCGCGGACACCGGTCCGCAGAGCGTGAAGTTTAAAAGCCCGGATCAAATCGACTTCAACCGTCTGTTTACCCACCCGGTTACCGACCTGTTCGACCCGGTGACCATGCTGGTTTACGATCAGTATATTCCGCTGTTTATCGACATTCCGGCGAGCGTTAACCCGAAGATGGTGCGTCTGAAAGTTCTGAGCTTCGATACCAACGAGCAAAGCCTGGGTCTGCGTCTGGAGTTCTTCAAACCGGATCAAGACACCCAGCCGAACAACAACGTGCAGGTTAACCCGAACAACGGTGACTTTCTGCCGCTGCTGACCGCGAGCAGCCAAGGTCCGCAGACC (SEQ ID No. 4).

[0059] The DNA sequence encoding the P30A antigen fragment in the DNA sequence shown in SEQ ID No. 6 is:

[0060] CGTACCGGTTTTCCGCCGCAGCCGGGTATGGCGCCGCGT (SEQ ID No. 5).

[0061] 2. Preparation of biotin-labeled P1M / P30A antigen:

[0062] (1) Preparation of 0.1 mol / L carbonate buffer (dialysis solution):

[0063] In a 5000 mL beaker, add Na2CO314.31 g and NaHCO326.46 g, and add water to a constant volume of 4500 mL to obtain 0.1 mol / L carbonate buffer (dialysis solution); the prepared dialysis solution is placed on a magnetic stirrer for standby.

[0064] (2) Choose the cut-off volume of 14000 dialysis bag, measuring the appropriate size, take 1 ~ 2 mg P1M / P30A antigen with dialysate adjusted to 1 ~ 2 mL, put into dialysate, room temperature stirring dialysis 2 ~ 3 hours;

[0065] The activated biotin is dissolved in dimethylformamide (DMF), and the biotin and P1M / P30A antigen are mixed according to a molar ratio of 5:1 ~ 20:1, and reacted at 37°C for 2 hours; then the reacted liquid is dialyzed with 0.1 mol / L PBS at 4°C for 24 hours, to obtain a biotin-labeled P1M / P30A antigen solution.

[0066] (3) The biotin-labeled P1M / P30A antigen obtained in the above reaction is purified by G-25 gel column.

[0067] 3. Preparation of streptavidin-coated magnetic microspheres:

[0068] (1) Preparation of acetic acid buffer with pH 3.6: 2.55 g of sodium acetate trihydrate is dissolved in 4500 mL of purified water, and then 14 mL of acetic acid is added and mixed, and then diluted to 5000 mL to obtain acetic acid buffer with pH 3.6.

[0069] (2) Magnetic microsphere connection (magnetic microsphere connection CMC method):

[0070] The magnetic microspheres are suspended in an equal volume of the above-mentioned pH 3.6 acetic acid buffer, and the concentration of the magnetic microspheres is 20 mg / mL, and then 1-cyclohexyl-2-morpholinoethyl carbodiimide p-toluenesulfonate (CMC) with a concentration of 10 mg / mL is added, and 1 mg of magnetic microspheres is added with 12 μg of streptavidin (SA) to form a reaction system. The above reaction system is placed in a constant temperature shaking water bath at 37°C for 24 hours.

[0071] (3) Washing of magnetic microspheres:

[0072] Preparation of magnetic bead washing solution: BSA is dissolved in 0.05M PBS with pH 7.4 to make the concentration of BSA 0.5 g / mL, which is the magnetic bead washing solution.

[0073] Washing: the reacted reaction system is poured into a beaker, and then placed on a magnet to precipitate, and then the supernatant is poured out, and 5 times the volume of magnetic bead washing solution is added and stirred to wash, and then placed on a magnet, and then the supernatant is poured out when the supernatant is clear.

[0074] (4) Suspension of magnetic microspheres:

[0075] Preparation of magnetic bead suspension: Dissolve BSA and methyl cellulose (MC) in 0.05M PBS with pH 7.4, so that the concentration of BSA is 0.5g / mL and the concentration of MC is 0.4g / mL, which is the magnetic bead suspension. After washing, add the coated volume of magnetic bead suspension, and the suspension concentration is 20mg / mL, so that the magnetic microsphere solution coated with streptavidin is obtained.

[0076] 4. A method for preparing enzyme-labeled IgG, IgM or IgA antibody, comprising the following steps:

[0077] A: Mix 1mL of 10-15mg / mL NaIO4 aqueous solution with 1mL of 5mg / mL ALP (alkaline phosphatase) or HRP (horseradish peroxidase) aqueous solution at 2-8℃ for 30-60min to prepare an activated ALP or HRP solution;

[0078] Then, add 0.5mL of 90%(V / V) ethylene glycol aqueous solution to the activated ALP or HRP solution, and react at room temperature for 30-60min in the dark to obtain an ALP or HRP mixed solution;

[0079] B: Mix IgG, IgM or IgA antibody dialyzed with 0.05-0.1moL / L sodium carbonate-sodium bicarbonate buffer with pH 8.5-9.5 with the ALP or HRP mixed solution obtained in step A at a mass ratio of 1:1-1:2, and dialyze overnight at 2-8℃ to obtain dialyzed IgG, IgM or IgA antibody;

[0080] C: Add 80-120μL of 2-8mg / mL NaBH4 solution to the dialyzed IgG, IgM or IgA antibody obtained in step B, and react at 2-8℃ for 2-4h, then load into a dialysis bag, and dialyze overnight in 10-50mM PBS solution with pH 7.4 at 2-8℃;

[0081] D: Under stirring, add an equal volume of saturated ammonium sulfate dropwise to the antibody solution obtained in step C, and react at 2-8℃ for 1-2h; centrifuge at 3000-5000rpm for 30-40min to discard the supernatant, and wash the precipitate with half-saturated ammonium sulfate twice; then dissolve the precipitate in 1mL of 10-50mM PBS solution; after dialysis in 10-50mM PBS solution, centrifuge at 10000-12000rpm to remove the precipitate, and the supernatant is the enzyme-labeled IgG, IgM or IgA antibody.

[0082] 5. Preparation of reagent kit

[0083] In a preferred scheme of the present application, the mycoplasma pneumoniae antibody kit comprises: biotin-labeled P1M / P30A antigen working solution, streptavidin-coupled magnetic microsphere working solution, tracer-labeled IgG, IgM or IgA antibody working solution. Preferably, the tracer-labeled IgG, IgM or IgA antibody working solution is alkaline phosphatase-labeled IgM antibody.

[0084] The biotin-labeled P1M / P30A antigen working solution and the tracer-labeled IgG, IgM or IgA antibody working solution are respectively prepared by using phosphate buffer solution with pH of 6.0-8.0 and concentration of 0.01-0.2 mol / L. The phosphate buffer solution contains 0.01-5 wt% of bovine serum albumin, 0.5-10 wt% of sodium chloride, 1-5 wt% of sucrose, 0.05-0.5 wt% of proclin 300 and 0.02-1 wt% of fatty alcohol polyoxyethylene ether sulfate (AES). The working concentration of P1M / P30A antigen is 25-5000 ng / mL, and the working concentration of IgG, IgM or IgA antibody is 50-5000 ng / mL.

[0085] In the marker system, the working concentration of the labeling tracer is preferably 5-500 ng / mL, and the working concentration of IgG, IgM or IgA antibody is preferably 50-5000 ng / mL. The working concentration of the magnetic microsphere in the magnetic microsphere system is preferably 0.1-2 mg / mL. By setting the concentration of each reagent component in the above range, the sensitivity of the reagent detection can be improved without causing cost waste due to excessively high concentration.

[0086] The streptavidin-coupled magnetic microsphere working solution is prepared by using phosphate buffer solution with pH of 6.0-8.0 and concentration of 0.01-0.2 mol / L. Preferably, the phosphate buffer solution with pH of 7.4 and concentration of 0.05 mol / L is used to prepare 0.5 mg / mL.

[0087] The magnetic microsphere of the present application is also called magnetic bead or magnetic ball, which can be the commonly used magnetic microsphere in the art. For example, nanoscale Fe2O3 or Fe3O4 magnetic particles and organic polymer materials are compounded to form micron-sized solid-phase microspheres with superparamagnetism and extremely large protein adsorption capacity, which can be rapidly magnetized under the action of an external magnetic field and have zero residual magnetism after the magnetic field is removed. The type of the organic polymer material is not particularly limited and can be selected as needed. The magnetic microsphere used should satisfy the diameter of 0.1-5 μm. The magnetic microsphere can also be modified on the surface to have various active functional groups, including but not limited to -OH, -COOH, -NH2, modified streptavidin, etc. The labeling tracer used in the present application includes the following:

[0088] 1. Directly luminescent markers used in chemiluminescent immunoassay, such as luminol and its derivatives, isoluminol or its derivatives, acridinium ester and its derivatives, and trispyridine ruthenium, etc.

[0089] 2. Markers used in chemiluminescent enzyme immunoassay, which can emit light in cooperation with corresponding substrates, such as alkaline phosphatase (ALP) or horseradish peroxidase (HRP), etc.

[0090] The oxidation system cooperating with the above luminescent markers includes at least one of H2O2-microperoxidase, H2O2-catalase, H2O2-lactoperoxidase, H2O2-hemin, H2O2-chloride hemin, hypochlorite-CoCl2, persulfate, potassium peroxide, sodium periodate, H2O2-K3Fe(CN)6, xanthine-oxidase, potassium tert-butoxide, and tripropylamine.

[0091] Chemiluminescent catalysts are selected from alkaline phosphatase and peroxidase. When used, they can emit light in cooperation with corresponding chemiluminescent substrates, which include NaOH and H2O2, and at least one of adamantane, luminol and its derivatives, isoluminol or its derivatives.

[0092] Preferably, the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution are prepared using a phosphate buffer with a pH of 7.4 and a concentration of 0.05 mol / L, wherein the phosphate buffer contains 1 wt% bovine serum albumin, 8 wt% sodium chloride, 1 wt% sucrose, 0.05 wt% proclin 300, and 0.5 wt% fatty alcohol polyoxyethylene ether sulfate (AES). The biotin-labeled P1M / P30A antigen and the alkaline phosphatase-labeled IgM antibody are diluted to 0.5 mg / L respectively using the phosphate buffer.

[0093] Detection principle:

[0094] In one embodiment, the sample to be tested, the biotin-labeled fusion antigen working solution, and the streptavidin-coupled magnetic bead working solution can be mixed and incubated, and then the tracer-labeled antibody working solution can be added and incubated. Alternatively, the streptavidin-coupled magnetic bead working solution can be mixed with the biotin-labeled fusion antigen working solution, incubated, and then the sample to be tested can be added and incubated, and then the tracer-labeled antibody working solution can be added and incubated. When the sample contains antibodies against Mycoplasma pneumoniae, a magnetic bead-streptavidin-biotin-antigen-antibody-secondary antibody complex is formed, and the luminescence intensity value of the sample to be tested is read by the signal substance labeled with the tracer on the secondary antibody.

[0095] Example 1

[0096] The reaction system of streptavidin-coated magnetic microspheres and biotin-labeled P1M / P30A antigen was used in the embodiment.

[0097] The working concentration of biotin was 100 ng / mL, and the working concentration of P1M / P30A antigen was 500 ng / mL. During the labeling of the antigen, 1.5 mg of P1M / P30A antigen was adjusted to 1.5 mL with a dialysis solution, and stirred at room temperature for 3 hours. The molar ratio of biotin to P1M / P30A antigen was 10:1. The labeling was performed according to the preparation process of the biotin-labeled P1M / P30A antigen described above.

[0098] The streptavidin-coated magnetic microspheres had a diameter of 1 μm and a working concentration of 0.5 mg / mL. The streptavidin had a working concentration of 10 μg / mL. The coating was performed according to the preparation process of the streptavidin-coated magnetic microspheres described above.

[0099] In the embodiment, alkaline phosphatase (ALP)-labeled anti-human IgM antibody, NaOH and H2O2 luminescence system, and AMPPD luminescent substrate were used.

[0100] The working concentration of ALP was 100 ng / mL, and the working concentration of anti-human IgM antibody was 500 ng / mL.

[0101] The ALP-labeled IgM antibody process was as follows:

[0102] A: 1 mL of 10 mg / mL NaIO4 aqueous solution was mixed with 1 mL of 5 mg / mL ALP (alkaline phosphatase) aqueous solution at 2-8°C for 60 min to prepare an activated ALP solution. Then, 0.5 mL of 90% (V / V) ethylene glycol aqueous solution was added to the activated ALP solution, and the mixture was reacted at room temperature for 60 min in the dark to obtain an ALP mixed solution;

[0103] B: IgM antibody dialyzed with 0.1 moL / L sodium carbonate-sodium bicarbonate buffer solution at pH 9.0 was mixed with the ALP mixed solution obtained in step A at a mass ratio of 1:2, and dialyzed at 2-8°C overnight to obtain dialyzed antigen;

[0104] C: 100 μL of 5 mg / mL NaBH4 solution was added to the dialyzed antigen obtained in step B, and the mixture was reacted at 2-8°C for 4 h. Then, the mixture was loaded into a dialysis bag and dialyzed in 25 mM PBS solution at pH 7.4 at 2-8°C overnight;

[0105] D: Under stirring, add saturated ammonium sulfate into the antibody solution obtained in step C drop by drop, and react at 2-8°C for 2 hours; centrifuge at 3000 rpm for 30 minutes to discard the supernatant, and wash the precipitate with half-saturated ammonium sulfate twice; then dissolve the precipitate in 1 mL of 20 mM PBS solution; after dialysis in 10-50 mM PBS solution, centrifuge at 12000 rpm to remove the precipitate, and take the supernatant as the enzyme-labeled IgM antibody.

[0106] The working solution of streptavidin-coated magnetic beads is prepared by using PBS buffer solution with pH of 7.4 and 0.05 mol / L, and the concentration of the magnetic beads is 0.5 mg / mL. The working solutions of biotin-labeled P1M / P30A antigen and alkaline phosphatase-labeled IgM antibody are prepared by using phosphate buffer solution with pH of 7.4 and 0.05 mol / L, and the phosphate buffer solution contains 1 wt% of bovine serum albumin, 8 wt% of sodium chloride, 1 wt% of sucrose, 0.5 wt% of fatty alcohol polyoxyethylene ether sulfate (AES), and 0.05 wt% of proclin 300. Then, the biotin-labeled P1M / P30A antigen and the alkaline phosphatase-labeled IgM antibody are diluted to 0.5 mg / L respectively by using the phosphate buffer solution.

[0107] Example 2

[0108] The concentration of AES in the working solutions of the antigen and the antibody in Example 1 is changed to 0.02 wt%, and the other conditions are the same as those in Example 1.

[0109] Example 3

[0110] The concentration of AES in the working solutions of the antigen and the antibody in Example 1 is changed to 1 wt%, and the other conditions are the same as those in Example 1.

[0111] Comparative Example 1

[0112] The biotin-labeled P1M / P30A antigen in Example 1 is replaced by biotin-labeled MP whole bacterial antigen, the fatty alcohol polyoxyethylene ether sulfate (AES) in the working solutions of the antigen and the antibody is removed, and the other conditions are the same as those in Example 1.

[0113] Comparative Example 2

[0114] The fatty alcohol polyoxyethylene ether sulfate (AES) in the working solutions of the biotin-labeled P1M / P30A antigen and the alkaline phosphatase-labeled IgM antibody in Example 1 is removed, and the other conditions are the same as those in Example 1.

[0115] Comparative Example 3

[0116] The fatty alcohol polyoxyethylene ether sulfate salt (AES) in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution in Example 1 was replaced with the same concentration of octadecyltrimethylammonium chloride (cationic surfactant), and the other conditions were the same as in Example 1.

[0117] Comparative Example 4

[0118] The fatty alcohol polyoxyethylene ether sulfate salt (AES) in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution in Example 1 was replaced with the same concentration of alkyl phosphate monoester salt (phosphate ester salt anionic surfactant), and the other conditions were the same as in Example 1.

[0119] Comparative Example 5

[0120] The fatty alcohol polyoxyethylene ether sulfate salt (AES) in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution in Example 1 was replaced with the same concentration of Tween 20 (nonionic surfactant), and the other conditions were the same as in Example 1.

[0121] Comparative Example 6

[0122] The biotin-labeled P1M / P30A antigen in Example 1 was replaced with biotin-labeled P1M antigen, and the fatty alcohol polyoxyethylene ether sulfate salt (AES) in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution was removed, and the other conditions were the same as in Example 1.

[0123] Comparative Example 7

[0124] The biotin-labeled P1M / P30A antigen in Example 1 was replaced with biotin-labeled P1M antigen, and the other conditions were the same as in Example 1.

[0125] Comparative Example 8

[0126] The biotin-labeled P1M / P30A antigen in Example 1 was replaced with biotin-labeled P1C antigen, and the fatty alcohol polyoxyethylene ether sulfate salt (AES) in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution was removed, and the other conditions were the same as in Example 1.

[0127] Comparative Example 9

[0128] The biotin-labeled P1M / P30A antigen in Example 1 was replaced with biotin-labeled P1C antigen, and the other conditions were the same as in Example 1.

[0129] Comparative Example 10

[0130] The AES concentration in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution in Example 1 was changed to 0.015 wt%, and the rest of the conditions were the same as in Example 1.

[0131] Comparative Example 11

[0132] The AES concentration in the biotin-labeled P1M / P30A antigen working solution and the alkaline phosphatase-labeled IgM antibody working solution in Example 1 was changed to 3 wt%, and the rest of the conditions were the same as in Example 1.

[0133] The kits in Example 1 and Comparative Examples 1 and 2 were prepared, and 50 samples of Mycoplasma pneumoniae IgM positive samples (numbered P1-P50) and 50 samples of healthy human negative samples (numbered N1-N50) collected from a hospital in Guangdong were detected to compare the sensitivity and specificity of the three kits.

[0134] Table 1. Sensitivity and specificity analysis of the determination results of the kit in Example 1 and clinical samples

[0135]

[0136] Result analysis:

[0137] Sensitivity 46 / 50; 92.00%, confidence interval (80.77%-97.78%);

[0138] Specificity 49 / 50; 98.00%, confidence interval (89.35%-99.95%).

[0139] Table 2. Sensitivity and specificity analysis of the determination results of the kit in Comparative Example 1 and clinical samples

[0140]

[0141] Result analysis:

[0142] Sensitivity 40 / 50; 80.00%, confidence interval (66.28%-89.97%);

[0143] Specificity 46 / 50; 92.00%, confidence interval (80.77%-97.78%).

[0144] Table 3. Analysis of the determination results of the kit in Comparative Example 2 and the positive and negative coincidence rate of clinical samples

[0145]

[0146] Result analysis:

[0147] Sensitivity 46 / 50; 92.00%, confidence interval (80.77%-97.78%);

[0148] Specificity 49 / 50; 98.00%, confidence interval (89.35%-99.95%).

[0149] Through comparative analysis of Tables 1, 2 and 3, the results show that the sensitivity of MP whole bacterial antigen to positive and negative clinical samples is 80%, and the specificity is 92%; the sensitivity of P1C / P30A antigen is 92%, and the specificity is 98%, which indicates that the P1M / P30A fusion antigen provided in the application has higher specificity and sensitivity. Moreover, AES does not affect the specific binding of P1M / P30A fusion antigen and antibody and the detection results of the kit.

[0150] Two sets of kits in Examples 1-3 and Comparative Examples 1-11 were prepared, and were respectively placed in a 2-8℃ cold storage environment and a 37℃ high-temperature environment. After 7 days of storage, three samples L1, L2 and L3 with increasing MP purified antibody concentrations were tested, and the signal rise and fall degree was compared.

[0151] Table 4. Comparative analysis of 14 kinds of kits after 2-8℃ cold storage and 37℃ heat acceleration

[0152]

[0153]

[0154] The results show that, according to the signal value rise and fall deviation of Examples 1, Comparative Example 1 and Comparative Example 2, the thermal stability of P1M / P30A fusion antigen is better than that of MP whole bacterial antigen, and the average signal value deviation is increased from-33.81% to-18.21%, and the signal value deviation is reduced by 46.1%. The addition of AES in the working solution of P1M / P30A fusion antigen can further improve the thermal stability of the kit, and the average signal value deviation is increased from-18.21% to-1.33%, and the signal value deviation is reduced by 92.70%, which produces an unexpected technical effect, indicating that AES can protect the active groups of P1M / P30A fusion antigen, so that it is not easy to occur hydrolysis, enzymatic hydrolysis and oxidation reactions in a high-temperature environment.

[0155] From the signal value fluctuation deviation of Example 1, Comparative Example 3, Comparative Example 4 and Comparative Example 5, it can be seen that the octadecyl trimethyl ammonium chloride (cationic surfactant) used in Comparative Example 3 reduces the thermal stability of the kit, and the mean signal value deviation is reduced from -18.21% to -51.41%, and the signal value deviation is increased by 182.3%; the alkyl phosphate monoester salt (phosphate ester salt anionic surfactant) used in Comparative Example 4 improves the thermal stability of the kit, and the mean signal value deviation is increased from -18.21% to -15.14%, and the signal value deviation is reduced by 16.86%, which is not obvious; the Tween 20 (non-ionic surfactant) used in Comparative Example 5 improves the thermal stability of the kit, and the mean signal value deviation is increased from -18.21% to -17.13%, and the signal value deviation is reduced by 5.96%, which is not obvious; the fatty alcohol polyoxyethylene ether sulfate AES (sulfate ester salt anionic surfactant) used in Example 1 improves the thermal stability of the kit, and the mean signal value deviation is increased from -18.21% to -1.33%, and the signal value deviation is reduced by 92.70%, which produces an unexpected technical effect. From Comparative Examples 3-5 compared with Example 1, it can be inferred that only the sulfate ester salt anionic surfactant AES can significantly improve the thermal stability of the P1M / P30A fusion antigen, and other types of anionic surfactants (phosphate ester salts) and non-ionic surfactants do not have obvious improvement on the thermal stability of the P1M / P30A fusion antigen. The cationic surfactant destroys the active groups of the protein, resulting in poor thermal stability of the P1M / P30A fusion antigen.

[0156] From the signal value fluctuation deviation of Example 1, Comparative Example 2, Comparative Example 6, Comparative Example 7, Comparative Example 8 and Comparative Example 9, it can be seen that the P1M antigen after using AES reduces the thermal stability of the kit, and the mean signal value deviation is reduced from -32.31% to -34.27%, and the signal value deviation is increased by 6.07%, which is not obvious; the P1C antigen after using AES reduces the thermal stability of the kit, and the mean signal value deviation is reduced from -39.82% to -41.55%, and the signal value deviation is increased by 4.34%, which is not obvious; therefore, it is inferred that AES can only improve the thermal stability of the P1M / P30A fusion antigen, and has no obvious effect on the thermal stability of other MP antigens P1C and P1M. It is shown that AES can specifically improve the thermal stability of the P1M / P30A fusion antigen, and may not produce this effect on other Mycoplasma pneumoniae antigens.

[0157] From the signal value fluctuation of Example 1, Comparative Example 10, Comparative Example 11, Example 2, Example 3, it can be seen that the signal value fluctuation of the kits of Example 2 and Example 3 after 7 days of 37℃ acceleration is within 5% of the kit of Example 1. The signal value fluctuation of the kits of Comparative Example 10 and Comparative Example 11 exceeds 10%, indicating that the optimal concentration range of AES is 0.02-1wt%, and the optimal concentration is 0.5wt%.

[0158] The present application utilizes the signal amplification system of streptavidin-biotin, uses the high-thermal-stability P1M / P30A fusion antigen to replace the MP whole-bacterium antigen, and after adding the fatty alcohol polyoxyethylene ether sulfate (AES) surfactant, further improves the thermal stability of the P1M / P30A fusion antigen.

[0159] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application. SEQUENCE LISTING <110> Zhuhai Lzbio-Reagent Co., Ltd. <120> A mycoplasma pneumoniae antibody detection kit and application thereof <130> PA22005097 <160> 6 <170> PatentIn version 3.3 <210> 1 <211> 178 <212> PRT <213> P1M <400> 1 Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser 1 5 10 15 Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly 20 25 30 Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp Asp Val 35 40 45 Asp Gly lie Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly Glu Gly 50 55 60 Gln Thr Ala Asp Thr Gly Pro Gin Ser Val Lys Phe Lys Ser Pro Asp 65 70 75 80 Gln lie Asp Phe Asn Arg Leu Phe Thr His Pro Val Thr Asp Leu Phe 85 90 95 Asp Pro Val Thr Met Leu Val Tyr Asp Gin Tyr lie Pro Leu Phe lie 100 105 110 Asp lie Pro Ala Ser Val Asn Pro Lys Met Val Arg Leu Lys Val Leu 115 120 125 Ser Phe Asp Thr Asn Glu Gin Ser Leu Gly Leu Arg Leu Glu Phe Phe 130 135 140 Lys Pro Asp Gin Asp Thr Gin Pro Asn Asn Asn Val Gin Val Asn Pro 145 150 155 160 Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr Ala Ser Ser Gin Gly Pro 165 170 175 Gln Thr <210> 2 <211> 13 <212> PRT <213> P30A <400> 2 Arg Thr Gly Phe Pro Pro Gln Pro Gly Met Ala Pro Arg 1 5 10 <210> 3 <211> 193 <212> PRT <213> P1M / P30A <400> 3 Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser 1 5 10 15 Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly 20 25 30 Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp Asp Val 35 40 45 Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly Glu Gly 50 55 60 Gln Thr Ala Asp Thr Gly Pro Gln Ser Val Lys Phe Lys Ser Pro Asp 65 70 75 80 Gln Ile Asp Phe Asn Arg Leu Phe Thr His Pro Val Thr Asp Leu Phe 85 90 95 Asp Pro Val Thr Met Leu Val Tyr Asp Gln Tyr Ile Pro Leu Phe Ile 100 105 110 Asp Ile Pro Ala Ser Val Asn Pro Lys Met Val Arg Leu Lys Val Leu 115 120 125 Ser Phe Asp Thr Asn Glu Gin Ser Leu Gly Leu Arg Leu Glu Phe Phe 130 135 140 Lys Pro Asp Gin Asp Thr Gin Pro Asn Asn Asn Val Gin Val Asn Pro 145 150 155 160 Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr Ala Ser Ser Gin Gly Pro 165 170 175 Gln Thr Leu Gin Arg Thr Gly Phe Pro Pro Gin Pro Gly Met Ala Pro 180 185 190 Arg <210> 4 <211> 534 <212> DNA <213> DNA sequence encoding the P1M antigen fragment <400> 4 tggctggttg gccagctgcc gagcaccagc gatggtaaca ccagcagcac caacaacctg 60 gcgccgaaca ccaacaccgg caacgacgtg gttggtgtgg gccgtctgag cgaaagcaac 120 gcggcgaaaa tgaacgatga cgtggacggt atcgttcgta ccccgctggc ggagctgctg 180 gatggcgagg gtcagaccgc ggacaccggt ccgcagagcg tgaagtttaa aagcccggat 240 caaatcgact tcaaccgtct gtttacccac ccggttaccg acctgttcga cccggtgacc 300 atgctggttt acgatcagta tattccgctg tttatcgaca ttccggcgag cgttaacccg 360 aagatggtgc gtctgaaagt tctgagcttc gataccaacg agcaaagcct gggtctgcgt 420 ctggagttct tcaaaccgga tcaagacacc cagccgaaca acaacgtgca ggttaacccg 480 aacaacggtg actttctgcc gctgctgacc gcgagcagcc aaggtccgca gacc 534 <210> 5 <211> 39 <212> DNA <213> DNA sequence encoding the P30A antigen fragment <400> 5 cgtaccggtt ttccgccgca gccgggtatg gcgccgcgt 39 <210> 6 <211> 582 <212> DNA <213> DNA sequence encoding the Mycoplasma pneumoniae fusion antigen <400> 6 tggctggttg gccagctgcc gagcaccagc gatggtaaca ccagcagcac caacaacctg 60 gcgccgaaca ccaacaccgg caacgacgtg gttggtgtgg gccgtctgag cgaaagcaac 120 gcggcgaaaa tgaacgatga cgtggacggt atcgttcgta ccccgctggc ggagctgctg 180 gatggcgagg gtcagaccgc ggacaccggt ccgcagagcg tgaagtttaa aagcccggat 240 caaatcgact tcaaccgtct gtttacccac ccggttaccg acctgttcga cccggtgacc 300 atgctggttt acgatcagta tattccgctg tttatcgaca ttccggcgag cgttaacccg 360 aagatggtgc gtctgaaagt tctgagcttc gataccaacg agcaaagcct gggtctgcgt 420 ctggagttct tcaaaccgga tcaagacacc cagccgaaca acaacgtgca ggttaacccg 480 aacaacggtg actttctgcc gctgctgacc gcgagcagcc aaggtccgca gaccctgcag 540 cgtaccggtt ttccgccgca gccgggtatg gcgccgcgtt aa 582

Claims

1. A Mycoplasma pneumoniae antibody detection kit, characterized in that, Include: Mycoplasma pneumoniae fusion antigen working solution contains Mycoplasma pneumoniae fusion antigen and diluent; A tracer-labeled antibody working solution containing antibody IgG, IgM or IgA and a diluent; The Mycoplasma pneumoniae fusion antigen has an amino acid sequence as shown in SEQ ID No. 3; the diluent is a phosphate buffer containing 0.02~1 wt% of fatty alcohol polyoxyethylene ether sulfate.

2. The reagent kit according to claim 1, characterized in that, The phosphate buffer also contains 0.01–5 wt% bovine serum albumin, 0.5–10 wt% sodium chloride, 1–5 wt% sucrose, and 0.05–0.5 wt% proclin 300.

3. The reagent kit according to claim 2, characterized in that, In the phosphate buffer, the concentration of fatty alcohol polyoxyethylene ether sulfate is 0.5 wt%; the concentration of bovine serum albumin is 1 wt%; the concentration of sodium chloride is 8 wt%; the concentration of sucrose is 1 wt%; and the concentration of proclin 300 is 0.05 wt%.

4. The reagent kit according to claim 1, characterized in that, In the working solution of Mycoplasma pneumoniae fusion antigen, the concentration of Mycoplasma pneumoniae fusion antigen is 0.2~1 mg / L; in the working solution of tracer-labeled antibody, the concentration of antibody is 0.2~1 mg / L.

5. The reagent kit according to claim 1, characterized in that, The tracer is a luminescent marker or a chemiluminescent catalyst.

6. The reagent kit according to claim 5, characterized in that, The luminescent marker is selected from any one of adamantane, luminol and its derivatives, isoluminol and its derivatives, acridine ester and its derivatives, or ruthenium terpyridine.

7. The reagent kit according to claim 5, characterized in that, The chemiluminescent catalyst is selected from either alkaline phosphatase or horseradish peroxidase.

8. The reagent kit according to any one of claims 1, 5-7, characterized in that, It also includes chemiluminescent substrate liquids.

9. The reagent kit according to claim 8, characterized in that, The chemiluminescent substrate solution includes NaOH and H2O2.

10. The kit according to claim 1, characterized in that, The Mycoplasma pneumoniae fusion antigen is directly or indirectly coated on a solid support.

11. The reagent kit according to claim 10, characterized in that, The solid support includes magnetic microspheres.

12. The kit according to claim 1, characterized in that, The Mycoplasma pneumoniae fusion antigen is a biotin-labeled Mycoplasma pneumoniae fusion antigen, and the kit also includes a streptavidin-conjugated magnetic bead working solution.

13. The reagent kit according to claim 12, characterized in that, The working solution of the streptavidin-conjugated magnetic beads was prepared from a 0.01-0.2 mol / L phosphate buffer solution at pH 6.0-8.

0.

14. The kit according to claim 13, characterized in that, The concentration of the streptavidin-conjugated magnetic beads is 0.1~2 mg / mL.

15. The use of the kit according to any one of claims 1-14 in the in vitro detection of Mycoplasma pneumoniae antibodies for non-disease diagnostic purposes.

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