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 mycoplasma pneumoniae antibody detection. The mycoplasma pneumoniae antibody detection kit comprises mixed mycoplasma pneumoniae antigens, magnetic microparticles and tracer-labeled antibodies. The kit is combined with three kinds of recombinant mycoplasma pneumoniae antigens as antigens connected with the magnetic microparticles, including a first recombinant mycoplasma pneumoniae antigen, a second recombinant mycoplasma pneumoniae antigen and a third recombinant mycoplasma pneumoniae antigen, and the working quality ratios of the three kinds of antigens to the magnetic microparticles are 2 μg / mg, 0.4 μg / mg and 0.4 μg / mg respectively. The mycoplasma pneumoniae antibody detection kit has the advantages of high sensitivity and strong specificity.

Description

TECHNICAL FIELD

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

[0002] Mycoplasma pneumoniae (MP) is the pathogen of human primary atypical pneumonia, which causes other respiratory tract infectious diseases and even base body multiple organ complications. The diagnosis of MP infection is of great significance for the correct treatment of the disease. Antibody detection is currently widely used for the diagnosis of MP infection. At present, whole bacterial antigens are more commonly used, but the glycolipid antigens on the cell membrane of MP have non-specific reactions with other microorganisms and collective tissues, and the sensitivity and specificity are not high enough. Therefore, improving the sensitivity and specificity of mycoplasma pneumoniae antibody detection is a problem to be solved at present.

[0003] Therefore, the present application is proposed. SUMMARY

[0004] The first object of the present application is to provide a mycoplasma pneumoniae antibody detection kit, which alleviates the technical problem of low sensitivity and specificity of mycoplasma pneumoniae antibody detection.

[0005] The second object of the present application is to provide an application of the above-mentioned mycoplasma pneumoniae antibody detection kit in detecting mycoplasma pneumoniae antibodies for non-diagnostic and therapeutic purposes.

[0006] To solve the above technical problems, the present application adopts the following technical solutions:

[0007] According to one aspect of the present application, the present application provides a mycoplasma pneumoniae antibody detection kit, comprising a mixed mycoplasma pneumoniae antigen, magnetic microparticles and a tracer-labeled antibody.

[0008] The mixed mycoplasma pneumoniae antigen comprises a first recombinant mycoplasma pneumoniae antigen, a second recombinant mycoplasma pneumoniae antigen and a third recombinant mycoplasma pneumoniae antigen.

[0009] The first recombinant mycoplasma pneumoniae antigen is a protein with an amino acid sequence as shown in SEQ No. 1, or a protein with an amino acid sequence as shown in SEQ No. 1, which is substituted, deleted or added with one or more amino acids and retains the activity and function of the first recombinant mycoplasma pneumoniae antigen.

[0010] The amino acid sequence of the second recombinant mycoplasma pneumoniae antigen is as shown in SEQ No. 2, or a protein with an amino acid sequence as shown in SEQ No. 2, which is substituted, deleted or added with one or more amino acids and retains the activity and function of the second recombinant mycoplasma pneumoniae antigen.

[0011] The third recombinant Mycoplasma pneumoniae antigen is a protein with an amino acid sequence as shown in SEQ No. 3, or a protein with an amino acid sequence as shown in SEQ No. 3 with one or more amino acids being substituted, deleted or added and retaining the activity and function of the third recombinant Mycoplasma pneumoniae antigen;

[0012] The working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 2 μg / mg;

[0013] The working mass ratio of the second recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg;

[0014] The working mass ratio of the third recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg.

[0015] Preferably, the Mycoplasma pneumoniae antibody detection kit further comprises an antigen buffer and / or an antigen working solution; the antigen working solution is an antigen buffer premixed with the mixed Mycoplasma pneumoniae antigens;

[0016] Preferably, the antigen buffer is a phosphate buffer with a pH of 6.0-8.0 and a concentration of 0.01 mol / L-0.2 mol / L, and contains the following components: 0.001-0.5 mg / ml of bovine serum albumin, 0.001-0.5 mg / ml of sodium chloride, 0.1-0.5 mg / ml of sucrose, and 0.005-0.05 ml / ml of proclin 300.

[0017] Preferably, in the antigen working solution, the concentration of the first recombinant Mycoplasma pneumoniae antigen is 1 μg / ml;

[0018] The concentration of the second recombinant Mycoplasma pneumoniae antigen is 0.2 μg / ml;

[0019] The concentration of the third recombinant Mycoplasma pneumoniae antigen is 0.2 μg / ml.

[0020] Preferably, the Mycoplasma pneumoniae antibody detection kit further comprises a magnetic microparticle buffer and / or a magnetic microparticle working solution; the magnetic microparticle working solution is a magnetic microparticle buffer premixed with magnetic microparticles;

[0021] Preferably, the magnetic microparticle buffer is a PBS buffer with a pH of 6.0-8.0 and a concentration of 0.01 mol / L-0.2 mol / L, and contains the following components: 0.05 mg / ml of bovine serum albumin, 0.005 mg / ml of Triton X-100, and 0.005 mg / ml of proclin 300.

[0022] Preferably, in the magnetic microparticle working solution, the concentration of the magnetic microparticles is 0.5 mg / mL.

[0023] Preferably, the tracer-labeled antibody is a tracer-labeled anti-human IgM antibody.

[0024] Preferably, the tracer-labeled antibody is an alkaline phosphatase-labeled IgG antibody.

[0025] Preferably, the mycoplasma pneumoniae antibody detection kit further comprises an antibody buffer and / or an antibody working solution; the antibody working solution is an antibody buffer premixed with anti-human antibodies.

[0026] Preferably, the antibody buffer is a phosphate buffer having a pH of 6.0-8.0 and a concentration of 0.01-0.2 mol / L, and contains the following components: 0.001-0.5 mg / ml of bovine serum albumin, 0.001-0.5 mg / ml of sodium chloride, 0.1-0.5 mg / ml of sucrose, and 0.005-0.05 ml / ml of proclin 300.

[0027] Preferably, the concentration of anti-human antibodies in the antibody working solution is 5-5000 ng / ml.

[0028] Preferably, the magnetic microparticles are connected with a first label, and the first, second, and third recombinant mycoplasma pneumoniae antigens are independently connected with a second label, and the second label specifically binds to the first label.

[0029] Preferably, the first label is streptavidin, and the second label is biotin.

[0030] According to another aspect of the present application, the present application also provides use of the mycoplasma pneumoniae antibody detection kit for detecting mycoplasma pneumoniae antibodies for non-diagnostic and therapeutic purposes.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The mycoplasma pneumoniae antibody detection kit provided by the present application uses the following three recombinant mycoplasma pneumoniae antigens as antigens connected with magnetic microparticles: P1M-linker-P30A, P1-LQ-P30B1, and P116A-(EAAAK)2-P1A-(EAAAK)2-P30B1. Due to containing multiple antigenic determinants, the three antigens complement each other in the detection of antibodies in the human population, and can cover as many specific and effective antigen epitopes as possible.

[0033] The mixing ratio of the three antigens when used simultaneously is particularly important. Only when the detection results of the mixed antigens are adjusted to the signal value level close to each other while maintaining the same specificity, the positive judgment values of the three antigens are close, and the sensitivity is the highest. The Mycoplasma pneumoniae antibody detection kit provided by the application preferably uses the working quality ratio of the magnetic microparticles and the coupling reaction of each antigen, so that the sensitivity is improved to 96.67%, the specificity can still be maintained at more than 91%, and the false negative is as low as 3.33%, and the specificity and sensitivity are good.

[0034] The Mycoplasma pneumoniae antibody detection kit has the advantages of high sensitivity and strong specificity when used for detecting Mycoplasma pneumoniae antibodies for non-diagnostic and therapeutic purposes. DETAILED DESCRIPTION

[0035] The technical solutions of the application will be described below in combination with the embodiments. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0036] According to one aspect of the application, the application provides a Mycoplasma pneumoniae antibody detection kit, which comprises mixed Mycoplasma pneumoniae antigens, magnetic microparticles and tracer-labeled antibodies.

[0037] The mixed Mycoplasma pneumoniae antigens comprise a first recombinant Mycoplasma pneumoniae antigen, a second recombinant Mycoplasma pneumoniae antigen and a third recombinant Mycoplasma pneumoniae antigen. The first recombinant Mycoplasma pneumoniae antigen is P116A-(EAAAK)2-P1A-(EAAAK)2-P30B1; the second recombinant Mycoplasma pneumoniae antigen is P1M-linker-P30A; and the third recombinant Mycoplasma pneumoniae antigen is P1-LQ-P30B1. P1A and P1M are different segments of the sequence fragment of P1, and the sequences of P1A and P1M are different.

[0038] The first recombinant Mycoplasma pneumoniae antigen is a protein with the amino acid sequence shown in SEQ No. 1, or a protein with the amino acid sequence shown in SEQ No. 1 with one or more amino acids substituted, deleted or added and retaining the activity and function of the first recombinant Mycoplasma pneumoniae antigen. The sequence of SEQ No. 1 is as follows:

[0039] VGTTAVVVPTTITLVNKTHQVEHESEQSDFQDIRFGLNSVKLPKAQPAAATRITVENGTDKLVNYKSSPQQLFLAKNALKDKLQGEFDKFLSDAKAFPALTADLQEWVDQQLFNPNQSFFDLSAPRSNFTLSSDKKASLDFIFRFTN FTESVQLLKLPEGVSVVVDSKQSFDYYVNASAQKLLVLPLSLPDEAAAKEAAAKSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPEAAAKEAAAKPIVKRKEKRLLEEKERQEQLAEQLQRISAQQEEQQALEQQAAAEAHAEAEVEPAPQPVPVPPQPQVQINFGPRTGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR

[0040] The second recombinant M. pneumoniae antigen is a protein having an amino acid sequence as set forth in SEQ No. 2, or a protein in which one or several amino acids in the amino acid sequence set forth in SEQ No. 2 are substituted, deleted, or added, and which retains the activity and function of the second recombinant M. pneumoniae antigen. The sequence of SEQ No. 2 is as follows:

[0041] WLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLQPIVKRKEKRLLEEKERQEQLAEQLQRISAQQEEQQALEQQAAAEAHAEAEVEPAPQPVPVPPQPQVQINFGPRTGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR

[0042] The third recombinant Mycoplasma pneumoniae antigen is a protein having an amino acid sequence as set forth in SEQ No. 3, or a protein in which one or more amino acids in the amino acid sequence as set forth in SEQ No. 3 are substituted, deleted, or added, and which retains the activity and function of the third recombinant Mycoplasma pneumoniae antigen. The sequence of SEQ No. 3 is as follows:

[0043] WLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLQRTGFPPQPGM

[0044] Specific examples of the above protein in which one or more amino acids are substituted, deleted, or added, and which retains the activity and function of the third recombinant Mycoplasma pneumoniae antigen can be, but are not limited to, substitution, deletion, or addition of one or more amino acids resulting from replacement of Linker connecting different fragments in the recombinant Mycoplasma pneumoniae antigen, while the structure of the protein after connection is substantially maintained; or substitution or deletion in which the ability of the antigen to bind to an antibody is substantially maintained, and the like.

[0045] The natural Mycoplasma pneumoniae is a multivalent antigen, after invading the body, the body produces a mixture of antibodies (polyclonal antibodies) to different antigen epitopes. The types of antibodies produced by different individuals and the proportion of antibodies are different. The artificially synthesized antigen / gene engineered expressed antigen only contains part of the antigenic determinant, and the use of a single antigen for immunological examination is extremely easy to cause missed detection, resulting in low sensitivity. The application uses multiple antigens in combination, as much as possible to cover more specific and effective antigen epitopes.

[0046] The three antigens selected by the application all contain multiple antigenic determinants, and the three antigens have complementary relationship in the detection of antibodies in the population. The mixing ratio of the three antigens used at the same time is particularly important. Only when the specificity is the same, the detection results of the mixed antigens are adjusted to the signal value level close to each other, the positive judgment value of the three antigens is close, and the sensitivity is the highest. The Mycoplasma pneumoniae antibody detection kit provided by the application is prepared according to the following working quality ratio, which can achieve the highest working sensitivity and maintain the specificity above 90%:

[0047] The working quality ratio of the first recombinant Mycoplasma pneumoniae antigen to magnetic microparticles is 2 μg / mg;

[0048] The working quality ratio of the second recombinant Mycoplasma pneumoniae antigen to magnetic microparticles is 0.4 μg / mg;

[0049] The working quality ratio of the third recombinant Mycoplasma pneumoniae antigen to magnetic microparticles is 0.4 μg / mg.

[0050] The working quality ratio is the mass ratio of each recombinant antigen in the mixed Mycoplasma pneumoniae antigen actually participating in the reaction to the magnetic microparticles in the coupling reaction. Because there is a dissociation equilibrium between the coupling reaction of the antigen and the magnetic microparticles, and because the magnetic microparticles and the antigen cannot always maintain sufficient contact, the coupling of the magnetic microparticles on the antigen is uneven and the number difference is large. Therefore, only the amount of amino acids existing in the buffer solution during coupling can be calculated after dissociation, so as to calculate the amount of each antigen coupled on the magnetic microparticles. In the case of neglecting a small number of magnetic microparticles with less coupled antigen, the calculated average ratio of antigen and magnetic microparticles is the only standard for representing the amount of antigen coupled on the magnetic microparticles.

[0051] For example, the working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 2 μg / mg, i.e. in the reaction system, the real reaction ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 2 μg of the first recombinant Mycoplasma pneumoniae antigen per mg of the magnetic microparticles during the coupling reaction of the first recombinant Mycoplasma pneumoniae antigen and the magnetic microparticles. After mixing the mixed Mycoplasma pneumoniae antigens and the magnetic microparticles, the amount of the magnetic microparticles, the amount of the buffer and the amount of the first recombinant Mycoplasma pneumoniae antigen are adjusted according to the ratio so that the working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles in the reaction system is still 2 μg / mg after mixing the mixed Mycoplasma pneumoniae antigens and the magnetic microparticles.

[0052] In some alternative embodiments, the magnetic microparticles are connected with a first label, the first, second and third recombinant Mycoplasma pneumoniae antigens are independently connected with a second label, and the second label specifically binds to the first label. The purpose of connecting the labels is to connect the mixed Mycoplasma pneumoniae antigens with the magnetic microparticles, and thus any pair of molecules that can specifically bind in the art can be used as the first and second labels, respectively. In some alternative embodiments, the magnetic microparticles are labeled with streptavidin and the mixed Mycoplasma pneumoniae antigens are connected with biotin, so as to connect the mixed Mycoplasma pneumoniae antigens with the magnetic microparticles.

[0053] The role of the tracer-labeled antibody in the Mycoplasma pneumoniae antibody detection kit is to bind to the human Mycoplasma pneumoniae antibody in the sample to be detected after the antigen coated on the magnetic microparticles captures the human Mycoplasma pneumoniae antibody in the sample to be detected, and then the human Mycoplasma pneumoniae antibody in the sample to be detected is quantitatively and / or qualitatively evaluated by detecting the tracer on the antibody. The present application does not limit the type of tracer label, and any general tracer label acceptable in the art can be used as the tracer label of the anti-human antibody. In some preferred embodiments, alkaline phosphatase is used as the tracer label.

[0054] The source species of the antibody in the tracer-labeled antibody include, but are not limited to, mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey or human, and the antibody species can be, but are not limited to, IgM, IgG, IgE, IgA or IgD.

[0055] In an alternative embodiment, the Mycoplasma pneumoniae antibody detection kit detects human Mycoplasma pneumoniae IgM antibody, and the tracer-labeled antibody is alkaline phosphatase-labeled IgG antibody.

[0056] The Mycoplasma pneumoniae antibody detection kit can further comprise a buffer.

[0057] Optionally, the magnetic particle buffer is included, and the composition of the magnetic particle buffer is preferably as follows: the pH is 6.0-8.0, for example, but not limited to, 6.0, 6.5, 7, 7.5, or 8.0; the concentration is 0.01 mol / L-0.2 mol / L, for example, but not limited to, 0.01, 0.05, 0.1, 0.15, or 0.2 mol / L of PBS buffer, and contains the following components: 0.05 mg / ml of bovine serum albumin, 0.005 mg / ml of Triton X-100, and 0.005 mg / ml of proclin 300.

[0058] Optionally, the Mycoplasma pneumoniae antibody detection kit includes a magnetic particle working solution, and the magnetic particle working solution is a magnetic particle buffer premixed with magnetic particles, wherein the concentration of the magnetic particles in the magnetic particle working solution is preferably 0.5 mg / mL.

[0059] Optionally, the antigen buffer is included, and the composition of the antigen buffer is preferably as follows: the pH is 6.0-8.0, for example, but not limited to, 6.0, 6.5, 7, 7.5, or 8.0; the concentration is 0.01 mol / L-0.2 mol / L, for example, but not limited to, 0.01, 0.05, 0.1, 0.15, or 0.2 mol / L of phosphate buffer, and contains the following components: 0.001-0.5 mg / ml of bovine serum albumin, for example, but not limited to, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 mg / ml; 0.001-0.5 mg / ml of sodium chloride, for example, but not limited to, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 mg / ml; 0.1-0.5 mg / ml of sucrose, for example, but not limited to, 0.1, 0.2, 0.3, 0.4, or 0.5 mg / ml; and 0.005-0.05 ml / ml of proclin 300, for example, but not limited to, 0.005, 0.01, 0.02, 0.03, 0.04, or 0.05 ml / ml.

[0060] Optionally, the Mycoplasma pneumoniae antibody detection kit includes an antigen working solution, and the antigen working solution is an antigen buffer premixed with the mixed Mycoplasma pneumoniae antigens, wherein the concentration of the first recombinant Mycoplasma pneumoniae antigen in the antigen working solution is preferably 1 μg / ml, the concentration of the second recombinant Mycoplasma pneumoniae antigen is preferably 0.2 μg / ml, and the concentration of the third recombinant Mycoplasma pneumoniae antigen is preferably 0.2 μg / ml.

[0061] Optionally, the antibody buffer solution is included, and the composition of the antibody buffer solution is preferably as follows: the pH is 6.0-8.0, for example, but not limited to, 6.0, 6.5, 7, 7.5 or 8.0; the concentration is 0.01 mol / L-0.2 mol / L, for example, but not limited to, 0.01, 0.05, 0.1, 0.15 or 0.2 mol / L of phosphate buffer solution, and contains the following components: 0.001-0.5 mg / ml of bovine serum albumin, for example, but not limited to, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5 mg / ml; 0.001-0.5 mg / ml of sodium chloride, for example, but not limited to, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4 or 0.5 mg / ml; 0.1-0.5 mg / ml of sucrose, for example, but not limited to, 0.1, 0.2, 0.3, 0.4 or 0.5 mg / ml; and 0.005-0.05 ml / ml of proclin 300, for example, but not limited to, 0.005, 0.01, 0.02, 0.03, 0.04 or 0.05 ml / ml.

[0062] Optionally, the mycoplasma pneumoniae antibody detection kit includes an antibody working solution, which is an antibody buffer solution premixed with anti-human antibodies. The concentration of anti-human antibodies in the antibody working solution is preferably 5-5000 ng / ml, for example, but not limited to, 5, 10, 25, 50, 100, 200, 500, 1000, 2000, 3000, 4000 and 5000 ng / ml.

[0063] Examples of specific compositions of the mycoplasma pneumoniae antibody detection kit include, but are not limited to:

[0064] The kit includes separately packaged magnetic microparticles, a first recombinant mycoplasma pneumoniae antigen, a second recombinant mycoplasma pneumoniae antigen, a third recombinant mycoplasma pneumoniae antigen and a tracer-labeled antibody, and magnetic microparticle buffer solution, antigen buffer solution and antibody buffer solution, which are mixed to the target amount in the corresponding buffer solution to obtain a working solution when used;

[0065] Alternatively, the kit includes separately packaged magnetic microparticles, a premixed mixture of three recombinant antigens of mycoplasma pneumoniae and a tracer-labeled antibody, and magnetic microparticle buffer solution, antigen buffer solution and antibody buffer solution, which are mixed to the target amount in the corresponding buffer solution to obtain a working solution when used;

[0066] Alternatively, the kit includes separately packaged magnetic microparticle working solution, antigen working solution and antibody working solution.

[0067] The Mycoplasma pneumoniae antibody detection kit can also include conventional reagents and consumables for detection, such as, but not limited to, negative controls, positive controls, blank controls, or reagents for dissolving or pretreating samples.

[0068] According to another aspect of the present application, the present application also provides use of the above-mentioned Mycoplasma pneumoniae antibody detection kit for detecting Mycoplasma pneumoniae antibodies for non-diagnostic and therapeutic purposes. The use of the above-mentioned Mycoplasma pneumoniae antibody detection kit for detecting Mycoplasma pneumoniae antibodies has the advantages of high sensitivity and strong specificity.

[0069] The technical solutions and beneficial effects of the present application are further described below in combination with preferred embodiments.

[0070] The enzyme-labeled IgG antibody and the streptavidin magnetic bead microspheres used in the following examples and comparative examples are commercially available raw materials. The biotin-labeled Mycoplasma pneumoniae antigens (whole bacterial antigen, first recombinant Mycoplasma pneumoniae antigen, second recombinant Mycoplasma pneumoniae antigen, and third recombinant Mycoplasma pneumoniae antigen) are purchased from Zhuhai Lihuo Medical Diagnostics Products Co., Ltd.

[0071] Example 1

[0072] The present embodiment provides a Mycoplasma pneumoniae antibody detection kit, which comprises the following reagents:

[0073] (1) Streptavidin-coupled magnetic microparticle working solution: The streptavidin-coupled magnetic microparticle suspension is magnetically separated to remove the supernatant, resuspended to a concentration of 0.5 mg / mL with PBS buffer having a pH of 7.0 and a concentration of 0.1 mol / L, and the PBS buffer further contains 0.05 mg / ml of bovine serum albumin, 0.005 mg / ml of Triton X-100, and 0.005 mg / ml of proclin 300.

[0074] (2) Antigen working solution: The antigen working solution is a mixed antigen working solution containing biotin-labeled first recombinant Mycoplasma pneumoniae antigen, biotin-labeled second recombinant Mycoplasma pneumoniae antigen, and biotin-labeled third recombinant Mycoplasma pneumoniae antigen:

[0075] A phosphate buffer having a pH of 6.5 and a concentration of 0.1 mol / L is prepared, and the phosphate buffer further contains 0.2 mg / ml of bovine serum albumin, 0.1 mg / ml of sodium chloride, 0.1 mg / ml of sucrose, and 0.005 mg / ml of proclin 300. Then, the first recombinant Mycoplasma pneumoniae antigen is diluted to 1 μg / ml, the second recombinant Mycoplasma pneumoniae antigen is diluted to 0.2 μg / ml, and the third recombinant Mycoplasma pneumoniae antigen is diluted to 0.2 μg / ml with the buffer to obtain the mixed antigen working solution.

[0076] (3) Alkaline phosphatase labeled anti-human IgM antibody working solution: a phosphate buffer solution with pH 6.5 and a concentration of 0.1 mol / L, containing 0.2 mg / ml of bovine serum albumin, 0.1 mg / ml of sodium chloride, 0.1 mg / ml of sucrose and 0.005 mg / ml of proclin 300. Then the alkaline phosphatase labeled anti-human IgM antibody was diluted to 0.008 μg / ml with the buffer solution.

[0077] (4) 0-value control working solution / sample diluent: a phosphate buffer solution with pH 7.0 and a concentration of 0.01 mol / L, containing 0.05 mg / ml of bovine serum albumin, 0.05 mg / ml of sodium chloride and 0.01 mg / ml of Tween 20.

[0078] Kit assembly: 5.0 mL / bottle of streptavidin-conjugated magnetic microparticle working solution, 10 mL / bottle of biotin-labeled mixed Mycoplasma pneumoniae antigen working solution, 10 mL / bottle of alkaline phosphatase labeled anti-human IgM antibody working solution, and 1.0 mL / bottle of 0-value control working solution, assembled together and stored at 2-8 °C.

[0079] The working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to magnetic microparticles is 2 μg / mg; the working mass ratio of the second recombinant Mycoplasma pneumoniae antigen to magnetic microparticles is 0.4 μg / mg; and the working mass ratio of the third recombinant Mycoplasma pneumoniae antigen to magnetic microparticles is 0.4 μg / mg.

[0080] Comparative Examples 1-7

[0081] Comparative Examples 1-7 and Example 1 differ in that the antigen working solution contains only the first recombinant Mycoplasma pneumoniae antigen, with concentrations of 10 μg / ml, 5 μg / ml, 1 μg / ml, 0.2 μg / ml, 0.04 μg / ml, 0.008 μg / ml and 0.002 μg / ml, respectively. The working mass ratios of the first recombinant Mycoplasma pneumoniae antigen to magnetic microparticles in Comparative Examples 1-7 are 20 μg / mg, 10 μg / mg, 2 μg / mg, 0.4 μg / mg, 0.08 μg / mg, 0.016 μg / mg and 0.004 μg / mg, respectively.

[0082] Comparative Examples 8-14

[0083] Comparative Examples 8 to 14 and Example 1 differ in that the antigen working solution contains only the third recombinant M. pneumoniae antigen at concentrations of 10 μg / ml, 5 μg / ml, 1 μg / ml, 0.2 μg / ml, 0.04 μg / ml, 0.008 μg / ml and 0.002 μg / ml, respectively. The working mass ratio of the third recombinant M. pneumoniae antigen to the magnetic microparticles in Comparative Examples 8 to 14 is 20 μg / mg, 10 μg / mg, 2 μg / mg, 0.4 μg / mg, 0.08 μg / mg, 0.016 μg / mg and 0.004 μg / mg, respectively.

[0084] Comparative Example 15 to Comparative Example 21

[0085] Comparative Examples 15 to 21 and Example 1 differ in that the antigen working solution contains only the second recombinant M. pneumoniae antigen at concentrations of 10 μg / ml, 5 μg / ml, 1 μg / ml, 0.2 μg / ml, 0.04 μg / ml, 0.008 μg / ml and 0.002 μg / ml, respectively. The working mass ratio of the second recombinant M. pneumoniae antigen to the magnetic microparticles in Comparative Examples 15 to 21 is 20 μg / mg, 10 μg / mg, 2 μg / mg, 0.4 μg / mg, 0.08 μg / mg, 0.016 μg / mg and 0.004 μg / mg, respectively.

[0086] Comparative Example 22 to Comparative Example 28

[0087] Comparative Examples 22 to 28 and Example 1 differ in that the antigen working solution contains only the M. pneumoniae whole cell antigen at concentrations of 10 μg / ml, 5 μg / ml, 1 μg / ml, 0.2 μg / ml, 0.04 μg / ml, 0.008 μg / ml and 0.002 μg / ml, respectively. The working mass ratio of the M. pneumoniae whole cell antigen to the magnetic microparticles in Comparative Examples 22 to 28 is 20 μg / mg, 10 μg / mg, 2 μg / mg, 0.4 μg / mg, 0.08 μg / mg, 0.016 μg / mg and 0.004 μg / mg, respectively.

[0088] The M. pneumoniae whole cell antigen working solution was prepared as follows: a phosphate buffer having a pH of 6.5 and a concentration of 0.1 mol / L was prepared, and the phosphate buffer further contained 0.2 mg / ml of bovine serum albumin, 0.1 mg / ml of sodium chloride, 0.1 mg / ml of sucrose and 0.005 mg / ml of proclin 300. Then, each of the biotin-labeled M. pneumoniae whole cell antigens was diluted to 10 μg / ml, 5 μg / ml, 1 μg / ml, 0.2 μg / ml, 0.04 μg / ml, 0.008 μg / ml and 0.002 μg / ml using the buffer.

[0089] Comparative Example 29

[0090] The difference between Comparative Example 29 and Example 1 is that only the first recombinant Mycoplasma pneumoniae antigen and the second recombinant Mycoplasma pneumoniae antigen are contained, and the antigen working solution is a mixed antigen working solution with the first recombinant Mycoplasma pneumoniae antigen working concentration of 1 μg / ml and the second recombinant Mycoplasma pneumoniae antigen working concentration of 0.2 μg / ml. The working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles in the kit is 2 μg / mg; and the working mass ratio of the second recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg.

[0091] Comparative Example 30

[0092] The difference between Comparative Example 30 and Example 1 is that only the first recombinant Mycoplasma pneumoniae antigen and the third recombinant Mycoplasma pneumoniae antigen are contained, and the antigen working solution is a mixed antigen working solution with the first recombinant Mycoplasma pneumoniae antigen working concentration of 1 μg / ml and the third recombinant Mycoplasma pneumoniae antigen working concentration of 0.2 μg / ml. The working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles in the kit is 2 μg / mg; and the working mass ratio of the third recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg.

[0093] Comparative Example 31

[0094] The difference between Comparative Example 31 and Example 1 is that only the second recombinant Mycoplasma pneumoniae antigen and the third recombinant Mycoplasma pneumoniae antigen are contained, and the antigen working solution is a mixed antigen working solution with the second recombinant Mycoplasma pneumoniae antigen working concentration of 0.2 μg / ml and the third recombinant Mycoplasma pneumoniae antigen working concentration of 0.2 μg / ml. The working mass ratio of the second recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg; and the working mass ratio of the third recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg.

[0095] Comparative Examples 32-40

[0096] The difference between Comparative Examples 32-40 and Example 1 is that the concentrations of the antigens in the antigen working solution are different, and the working mass ratios of the antigens to the magnetic microparticles are different. The concentrations of the antigens in the antigen working solution of the kits of Comparative Examples 32-40 are shown in Table 1-1; and the working mass ratios of the antigens to the magnetic microparticles in the kits of Comparative Examples 32-40 are shown in Table 1-2.

[0097] Table 1-1 Concentrations of antigens in antigen working solution of Comparative Examples 32-40

[0098]

[0099] Table 1-2 Working mass ratios of antigens to magnetic microparticles in Comparative Examples 32-40

[0100]

[0101] Example 1

[0102] The kits in Comparative Examples 1-28 were used to detect 0-value samples, positive samples, and negative samples. According to the signal-to-noise ratio, the degree of distinguishing between positive and negative samples, and the cost of reagents, the optimal working mass ratio of the antigen to the magnetic microparticles was selected. The test results are shown in Tables 2-5.

[0103] The full-automatic chemiluminescence immunoassay analyzer SMART 6500H produced by Chongqing Cosmex Scientific Co., Ltd. was used in the experiment.

[0104] Step 1: After the instrument absorbs 10 μl of the sample, it is automatically diluted by 15 times.

[0105] Step 2: 30 μl of the diluted sample is taken, 50 μl of streptavidin-coupled magnetic microsphere working solution is added, 50 μl of biotin-labeled antigen working solution is added, and incubation is performed at 37°C for 10 min. The working mass ratio of each antigen to the magnetic microparticles is adjusted to meet the working mass ratio of the corresponding kit by adjusting the amount of antigen and buffer. After incubation, the supernatant is removed by magnetic separation and washing is performed three times.

[0106] Step 3: 100 μl of alkaline phosphatase-labeled anti-human IgM antibody working solution is added, and incubation is performed at 37°C for 10 min. After incubation, the supernatant is removed by magnetic separation and washing is performed three times.

[0107] Step 4: The substrate solution is added, and the signal value is read.

[0108] Table 2

[0109]

[0110] According to the signal-to-noise ratio, the higher the value of the positive sample and the lower the value of the negative sample, the better the effect. As shown in Table 2, the optimal working mass ratio of the whole bacterial antigen to the magnetic microparticles is 2 μg / mg, the upper limit of the working mass ratio is 10 μg / mg, and the lower limit is 0.4 μg / mg.

[0111] Table 3

[0112]

[0113]

[0114] According to the signal-to-noise ratio, the higher the value of the positive sample and the lower the value of the negative sample, the better the effect. As shown in Table 3, the optimal working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 2 μg / mg, the upper limit of the working mass ratio is 10 μg / mg, and the lower limit is 0.4 μg / mg.

[0115] Table 4

[0116]

[0117] The higher the positive sample value and the lower the negative sample value, the better the effect according to the signal-to-noise ratio. According to the signal-to-noise ratio, it can be seen from Table 4 that the optimal working mass ratio of the third recombinant Mycoplasma pneumoniae antigen and magnetic particles is 0.4 μg / mg, the upper limit of the working mass ratio is 2 μg / mg, and the lower limit is 0.08 μg / mg.

[0118] Table 5

[0119]

[0120]

[0121] The higher the positive sample value and the lower the negative sample value, the better the effect according to the signal-to-noise ratio. It can be seen from Table 5 that the optimal working mass ratio of the second recombinant Mycoplasma pneumoniae antigen and magnetic particles is 0.4 μg / mg, the upper limit of the working mass ratio is 2 μg / mg, and the lower limit is 0.08 μg / mg.

[0122] Effect Example 2

[0123] Prepare 60 samples clinically diagnosed as positive and 61 samples clinically diagnosed as negative. Use the kits provided in Example 1, Comparative Example 24, Comparative Example 3, Comparative Example 10, Comparative Example 17, and Comparative Examples 29-31 to detect the clinical samples, and the detection process is the same as that in Effect Example 1. The detection results are shown in Table 6 and Table 7.

[0124] Table 6 Positive and negative test results of 8 antigen combinations

[0125]

[0126]

[0127] Table 7 Specificity test results of 8 antigen combinations

[0128]

[0129]

[0130]

[0131] Table 8 Sensitivity and specificity test of Comparative Example 24 kit

[0132]

[0133] Table 9 Sensitivity and specificity test of Comparative Example 1 kit

[0134]

[0135] Table 10 Sensitivity and specificity test of kit of comparative example 10

[0136]

[0137] Table 11 Sensitivity and specificity test of kit of comparative example 17

[0138]

[0139] Table 12 Sensitivity and specificity test of kit of comparative example 30

[0140]

[0141] Table 13 Sensitivity and specificity test of kit of comparative example 29

[0142]

[0143] Table 14 Sensitivity and specificity test of kit of comparative example 31

[0144]

[0145] Table 15 Sensitivity and specificity test of kit of example 1

[0146]

[0147] Table 16 Sensitivity and specificity test of each kit for mycoplasma pneumoniae antibody

[0148]

[0149] From the data in Table 16, it can be seen that the kit provided by example 1 using the first recombinant mycoplasma pneumoniae antigen, the second recombinant mycoplasma pneumoniae antigen and the third recombinant mycoplasma pneumoniae antigen has better sensitivity and specificity for mycoplasma pneumoniae antibody than the whole cell antigen of mycoplasma pneumoniae, and has a greater improvement than the whole cell antigen.

[0150] From the results in Tables 6 and 7, it can be seen that the combined use of the first recombinant mycoplasma pneumoniae antigen, the third recombinant mycoplasma pneumoniae antigen and the second recombinant mycoplasma pneumoniae antigen can supplement the missed detection results of the first recombinant mycoplasma pneumoniae antigen, the third recombinant mycoplasma pneumoniae antigen or the second recombinant mycoplasma pneumoniae antigen used alone. Although the specificity is lower than that of the third recombinant mycoplasma pneumoniae antigen or the second recombinant mycoplasma pneumoniae antigen used alone, the specificity can still be maintained above 91%, the sensitivity is improved to 96.67%, the false negative rate is reduced from 13.33% to 3.33%, and the reduction is 75.19%, which is a significant effect.

[0151] The original explanation: natural mycoplasma pneumoniae is a multivalent antigen, after invading the body, the body produces a mixture of antibodies (polyclonal antibodies) to different antigen epitopes. The types and proportions of antibodies produced by different individuals are different. The artificially synthesized antigen / gene engineered antigen only contains part of the antigenic determinant, and the use of a single antigen for immunological testing is extremely easy to cause missed detection, resulting in low sensitivity. In the present application, three antigens are used in combination to cover as many specific and effective antigen epitopes as possible.

[0152] The combination of the first recombinant mycoplasma pneumoniae antigen, the third recombinant mycoplasma pneumoniae antigen and the second recombinant mycoplasma pneumoniae antigen has the highest sensitivity, and maintains a specificity of more than 90%, at which the working quality ratio of the three antigens to the magnetic microparticles is: the first recombinant mycoplasma pneumoniae antigen 2 μg / mg, the second recombinant mycoplasma pneumoniae antigen 0.4 μg / mg and the third recombinant mycoplasma pneumoniae antigen 0.4 μg / mg.

[0153] Effect example 3

[0154] The kits of Examples 1 and Comparative Examples 32-40 were used to detect the clinical samples of Effect Example 2, and the test process was the same as that of Effect Example 1. The specificity was set as a fixed value of 91.8%; the sensitivity and specificity test results are as follows:

[0155] Table 17 Sensitivity and specificity test of Comparative Example 32

[0156]

[0157] Table 18 Sensitivity and specificity test of Comparative Example 33

[0158]

[0159] Table 19 Sensitivity and specificity test of Comparative Example 34

[0160]

[0161] Table 20 Sensitivity and specificity test of Comparative Example 35

[0162]

[0163] Table 21 Sensitivity and specificity test of Comparative Example 36

[0164]

[0165] Table 22 Sensitivity and specificity test of Comparative Example 37

[0166]

[0167] Table 23 Sensitivity and specificity test of Comparative Example 38

[0168]

[0169] Table 24 Sensitivity and specificity test of comparative example 39

[0170]

[0171] Table 25 Sensitivity and specificity test of comparative example 40

[0172]

[0173]

[0174] Table 26 Sensitivity and specificity test results of different working quality ratio combinations of first recombinant Mycoplasma pneumoniae antigen & third recombinant Mycoplasma pneumoniae antigen & second recombinant Mycoplasma pneumoniae antigen mixed antigen

[0175]

[0176] Result analysis:

[0177] The sensitivity and specificity of comparative examples 32, 35 and 40 in Table 26, in which the first recombinant Mycoplasma pneumoniae antigen, the third recombinant Mycoplasma pneumoniae antigen and the second recombinant Mycoplasma pneumoniae antigen are mixed in different working quality ratios, are all decreased compared with the sensitivity and specificity of the first recombinant Mycoplasma pneumoniae antigen, the third recombinant Mycoplasma pneumoniae antigen and the second recombinant Mycoplasma pneumoniae antigen used alone. The sensitivity of comparative examples 37 and 39 is not obviously improved, and the specificity is decreased. The sensitivity and specificity of comparative examples 33, 34, 38 and comparative example 31 (second recombinant Mycoplasma pneumoniae antigen & third recombinant Mycoplasma pneumoniae antigen mixed) and comparative example 29 (second recombinant Mycoplasma pneumoniae antigen & first recombinant Mycoplasma pneumoniae antigen mixed) are all decreased.

[0178] As can be seen from Table 16 and Table 26, the detection is performed by using the first recombinant Mycoplasma pneumoniae antigen, the third recombinant Mycoplasma pneumoniae antigen and the second recombinant Mycoplasma pneumoniae antigen mixed, and the optimal working quality ratio combination of the three mixed antigens and the magnetic microparticles is 2 μg / mg of the first recombinant Mycoplasma pneumoniae antigen, 0.4 μg / mg of the third recombinant Mycoplasma pneumoniae antigen and 0.4 μg / mg of the second recombinant Mycoplasma pneumoniae antigen. At this time, the sensitivity and specificity of the detection of the Mycoplasma pneumoniae antibody are the highest, the sensitivity is 96.67%, the specificity is 91.8%, and the false positive rate is 3.33%. Compared with the false positive rate of 13.3% of comparative example 38 (the working quality ratio of the first recombinant Mycoplasma pneumoniae antigen and the magnetic microparticles is 0.4 μg / mg, the working quality ratio of the second recombinant Mycoplasma pneumoniae antigen and the magnetic microparticles is 2 μg / ml, and the working quality ratio of the third recombinant Mycoplasma pneumoniae antigen and the magnetic microparticles is 0.08 μg / mg), the false positive rate is reduced by 75.19%.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; 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 replacements 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> Mycoplasma pneumoniae antibody detection kit and application thereof <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 520 <212> PRT <213> Artificial sequence <400> 1 Val Gly Thr Thr Ala Val Val Val Pro Thr Thr Ile Thr Leu Val Asn 1 5 10 15 Lys Thr His Gln Val Glu His Glu Ser Glu Gln Ser Asp Phe Gln Asp 20 25 30 Ile Arg Phe Gly Leu Asn Ser Val Lys Leu Pro Lys Ala Gln Pro Ala 35 40 45 Ala Ala Thr Arg Ile Thr Val Glu Asn Gly Thr Asp Lys Leu Val Asn 50 55 60 Tyr Lys Ser Ser Pro Gln Gln Leu Phe Leu Ala Lys Asn Ala Leu Lys 65 70 75 80 Asp Lys Leu Gin Gly Glu Phe Asp Lys Phe Leu Ser Asp Ala Lys Ala 85 90 95 Phe Pro Ala Leu Thr Ala Asp Leu Gin Glu Trp Val Asp Gin Gin Leu 100 105 110 Phe Asn Pro Asn Gin Ser Phe Phe Asp Leu Ser Ala Pro Arg Ser Asn 115 120 125 Phe Thr Leu Ser Ser Asp Lys Lys Ala Ser Leu Asp Phe He Phe Arg 130 135 140 Phe Thr Asn Phe Thr Glu Ser Val Gin Leu Leu Lys Leu Pro Glu Gly 145 150 155 160 Val Ser Val Val Val Asp Ser Lys Gin Ser Phe Asp Tyr Tyr Val Asn 165 170 175 Ala Ser Ala Gin Lys Leu Leu Val Leu Pro Leu Ser Leu Pro Asp Glu 180 185 190 Ala Ala Ala Lys Glu Ala Ala Ala Lys Ser Ser Thr Asn Asn Leu Ala 195 200 205 Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly Val Gly Arg Leu Ser 210 215 220 Glu Ser Asn Ala Ala Lys Met Asn Asp Asp Val Asp Gly He Val Arg 225 230 235 240 Thr Pro Leu Ala Glu Leu Leu Asp Gly Glu Gly Gin Thr Ala Asp Thr 245 250 255 Gly Pro Gin Ser Val Lys Phe Lys Ser Pro Asp Gin He Asp Phe Asn 260 265 270 Arg Leu Phe Thr His Pro Val Thr Asp Leu Phe Asp Pro Val Thr Met 275 280 285 Leu Val Tyr Asp Gin Tyr He Pro Leu Phe He Asp He Pro Ala Ser 290 295 300 Val Asn Pro Lys Met Val Arg Leu Lys Val Leu Ser Phe Asp Thr Asn 305 310 315 320 Glu Gin Ser Leu Gly Leu Arg Leu Glu Phe Phe Lys Pro Glu Ala Ala 325 330 335 Ala Lys Glu Ala Ala Ala Lys Pro He Val Lys Arg Lys Glu Lys Arg 340 345 350 Leu Leu Glu Glu Lys Glu Arg Gin Glu Gin Leu Ala Glu Gin Leu Gin 355 360 365 Arg He Ser Ala Gin Gin Glu Glu Gin Gin Ala Leu Glu Gin Gin Ala 370 375 380 Ala Ala Glu Ala His Ala Glu Ala Glu Val Glu Pro Ala Pro Gin Pro 385 390 395 400 Val Pro Val Pro Pro Gin Pro Gin Val Gin He Asn Phe Gly Pro Arg 405 410 415 Thr Gly Phe Pro Pro Gin Pro Gly Met Ala Pro Arg Pro Gly Met Pro 420 425 430 Pro His Pro Gly Met Ala Pro Arg Pro Gly Phe Pro Pro Gin Pro Gly 435 440 445 Met Ala Pro Arg Pro Gly Met Pro Pro His Pro Gly Met Ala Pro Arg 450 455 460 Pro Gly Phe Pro Pro Gin Pro Gly Met Ala Pro Arg Pro Gly Met Pro 465 470 475 480 Pro His Pro Gly Met Ala Pro Arg Pro Gly Phe Pro Pro Gin Pro Gly 485 490 495 Met Ala Pro Arg Pro Gly Met Gin Pro Pro Arg Pro Gly Met Pro Pro 500 505 510 Gln Pro Gly Phe Pro Pro Lys Arg 515 520 <210> 2 <211> 357 <212> PRT <213> Artificial Sequence <400> 2 Trp Leu Val Gly Gin 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 Gln Ser Leu Gly Leu Arg Leu Glu Phe Phe 130 135 140 Lys Pro Asp Gln Asp Thr Gln Pro Asn Asn Asn Val Gln Val Asn Pro 145 150 155 160 Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr Ala Ser Ser Gln Gly Pro 165 170 175 Gln Thr Leu Gln Pro lie Val Lys Arg Lys Glu Lys Arg Leu Leu Glu 180 185 190 Glu Lys Glu Arg Gin Glu Gin Leu Ala Glu Gin Leu Gin Arg lie Ser 195 200 205 Ala Gin Gin Glu Glu Gin Gin Ala Leu Glu Gin Gin Ala Ala Ala Glu 210 215 220 Ala His Ala Glu Ala Glu Val Glu Pro Ala Pro Gin Pro Val Pro Val 225 230 235 240 Pro Pro Gin Pro Gin Val Gin lie Asn Phe Gly Pro Arg Thr Gly Phe 245 250 255 Pro Pro Gin Pro Gly Met Ala Pro Arg Pro Gly Met Pro Pro His Pro 260 265 270 Gly Met Ala Pro Arg Pro Gly Phe Pro Pro Gin Pro Gly Met Ala Pro 275 280 285 Arg Pro Gly Met Pro Pro His Pro Gly Met Ala Pro Arg Pro Gly Phe 290 295 300 Pro Pro Gin Pro Gly Met Ala Pro Arg Pro Gly Met Pro Pro His Pro 305 310 315 320 Gly Met Ala Pro Arg Pro Gly Phe Pro Pro Gin Pro Gly Met Ala Pro 325 330 335 Arg Pro Gly Met Gin Pro Pro Arg Pro Gly Met Pro Pro Gin Pro Gly 340 345 350 Phe Pro Pro Lys Arg 355 <210> 3 <211> 190 <212> PRT <213> Artificial Sequence <400> 3 Trp Leu Val Gly Gin 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 He 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 He 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 He Pro Leu Phe He 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 180 185 190

Claims

1. A Mycoplasma pneumoniae antibody detection kit, characterized in that, This includes antibodies containing a mixture of Mycoplasma pneumoniae antigen, magnetic microparticles, and tracer-labeled antibodies; The mixed Mycoplasma pneumoniae antigen includes a first recombinant Mycoplasma pneumoniae antigen, a second recombinant Mycoplasma pneumoniae antigen, and a third recombinant Mycoplasma pneumoniae antigen; The first recombinant Mycoplasma pneumoniae antigen is a protein with an amino acid sequence as shown in SEQ No. 1, or a protein with one or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ No. 1 while retaining the activity and function of the first recombinant Mycoplasma pneumoniae antigen; The second recombinant Mycoplasma pneumoniae antigen has the amino acid sequence shown in SEQ No. 2, or the amino acid sequence shown in SEQ No. 2 is modified by substitution, deletion or addition of one or more amino acids and retains the activity and function of the second recombinant Mycoplasma pneumoniae antigen. The third recombinant Mycoplasma pneumoniae antigen is a protein with an amino acid sequence as shown in SEQ No. 3, or a protein with one or more amino acids substituted, deleted, or added to the amino acid sequence shown in SEQ No. 3 while retaining the activity and function of the third recombinant Mycoplasma pneumoniae antigen; The working mass ratio of the first recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles was 2 μg / mg; The working mass ratio of the second recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles was 0.4 μg / mg; The working mass ratio of the third recombinant Mycoplasma pneumoniae antigen to the magnetic microparticles is 0.4 μg / mg.

2. The Mycoplasma pneumoniae antibody detection kit according to claim 1, characterized in that, It also includes an antigen buffer solution and / or an antigen working solution; the antigen working solution is an antigen buffer solution premixed with the mixed Mycoplasma pneumoniae antigen.

3. The Mycoplasma pneumoniae antibody detection kit according to claim 2, characterized in that, The antigen buffer is a phosphate buffer with a pH of 6.0-8.0 and a concentration of 0.01 mol / L-0.2 mol / L, containing the following components: 0.001-0.5 mg / ml bovine serum albumin, 0.001-0.5 mg / ml sodium chloride, 0.1-0.5 mg / ml sucrose, and 0.005-0.05 ml / ml proclin 300.

4. The Mycoplasma pneumoniae antibody detection kit according to claim 2, characterized in that, In the antigen working solution The concentration of the first recombinant Mycoplasma pneumoniae antigen was 1 μg / ml; The concentration of the second recombinant Mycoplasma pneumoniae antigen was 0.2 μg / ml; The concentration of the third recombinant Mycoplasma pneumoniae antigen is 0.2 μg / ml.

5. The Mycoplasma pneumoniae antibody detection kit according to claim 1, characterized in that, It also includes magnetic microparticle buffer solution and / or magnetic microparticle working solution; the magnetic microparticle working solution is a magnetic microparticle buffer solution premixed with magnetic microparticles.

6. The Mycoplasma pneumoniae antibody detection kit according to claim 5, characterized in that, The magnetic microparticle buffer is a PBS buffer with a pH of 6.0-8.0 and a concentration of 0.01 mol / L-0.2 mol / L, containing the following components: 0.05 mg / ml bovine serum albumin, 0.005 mg / ml Triton X-100 and 0.005 mg / ml proclin 300.

7. The Mycoplasma pneumoniae antibody detection kit according to claim 5, characterized in that, The concentration of the magnetic particles in the working solution is 0.5 mg / mL.

8. The Mycoplasma pneumoniae antibody detection kit according to claim 1, characterized in that, The tracer-labeled antibody is a tracer-labeled anti-human IgM antibody.

9. The Mycoplasma pneumoniae antibody detection kit according to claim 1, characterized in that, The tracer-labeled antibody is an alkaline phosphatase-labeled IgG antibody.

10. The Mycoplasma pneumoniae antibody detection kit according to claim 1, characterized in that, It also includes antibody buffer and / or antibody working solution; the antibody working solution is an antibody buffer premixed with anti-human antibodies.

11. The Mycoplasma pneumoniae antibody detection kit according to claim 10, characterized in that, The antibody buffer is a phosphate buffer with a pH of 6.0-8.0 and a concentration of 0.01 mol / L-0.2 mol / L, containing the following components: 0.001-0.5 mg / ml bovine serum albumin, 0.001-0.5 mg / ml sodium chloride, 0.1-0.5 mg / ml sucrose, and 0.005-0.05 ml / ml proclin 300.

12. The Mycoplasma pneumoniae antibody detection kit according to claim 10, characterized in that, The concentration of anti-human antibody in the antibody working solution is 5~5000 ng / ml.

13. The Mycoplasma pneumoniae antibody detection kit according to any one of claims 1-12, characterized in that, The magnetic microparticles are connected to a first marker, and the first recombinant Mycoplasma pneumoniae antigen, the second recombinant Mycoplasma pneumoniae antigen, and the third recombinant Mycoplasma pneumoniae antigen are each independently connected to a second marker, and the second marker specifically binds to the first marker.

14. The Mycoplasma pneumoniae antibody detection kit according to claim 13, characterized in that, The first marker is streptavidin, and the second marker is biotin.

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

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