Light chain and heavy chain variable regions of anti-mycoplasma pneumoniae monoclonal antibody and application of light chain and heavy chain variable regions

Abstract

The invention belongs to the technical field of biomedical engineering, and discloses light chain and heavy chain variable regions of an anti-mycoplasma pneumoniae monoclonal antibody and application of the light chain and heavy chain variable regions, and the light chain and heavy chain variable regions comprise light chain and heavy chain variable regions of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19. The mycoplasma pneumoniae antigen colloidal gold detection kit can be applied to detection method application scenes such as immunochromatography (latex particles, immunofluorescence and colloidal gold), immunoturbidimetry, (electrochemiluminescence) and enzyme-linked immunosorbent assay; the kit adopts a double-antibody sandwich method to detect mycoplasma pneumonia, has the advantages of high sensitivity, strong specificity, quick detection, simplicity and convenience in operation, macroscopic result judgment, convenience in storage and transportation and the like, and is suitable for on-site quick detection of early mycoplasma pneumonia infection of clinical respiratory tract patients in pediatrics, respiratory medicine and the like. And pathogenesis screening of screening, prognosis and the like of a mycoplasma pneumoniae carrier or an incubation period patient by a basic unit and clinical treatment guidance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to a light chain and a heavy chain variable region of an anti-Mycoplasma pneumoniae monoclonal antibody and an application thereof. Background Art

[0002] Mycoplasma pneumonia (MP) is a tiny pathogen without a cell wall, which is between bacteria and viruses. Clinically, it often presents as pharyngitis, bronchitis, and pneumonia, which can recur. Clinical and imaging examinations cannot distinguish infections caused by MP from those caused by other pathogens. Early laboratory diagnosis becomes the key to the diagnosis and treatment of MP infection.

[0003] Clinical detection methods for Mycoplasma pneumoniae are mainly divided into two categories: serological testing and etiological testing. Serological tests mainly detect antibodies that appear after infection with MP, such as IgA, IgG, IgM or total antibodies, and are indirect detection methods. Etiological testing is mainly divided into nucleic acid testing and antigen testing. Nucleic acid testing is relatively complex, time-consuming, labor-intensive, and expensive, so it is generally suitable for professional laboratories with experimental conditions. Antigen testing does not require sample processing, is simple to operate, and produces results in about 20 minutes. It is of great significance for the early and rapid diagnosis of Mycoplasma pneumoniae infection. The most important raw material for antigen testing is Mycoplasma pneumoniae antibody.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows: serological testing is prone to false negatives and requires clinical symptoms to be diagnosed; nucleic acid testing requires special equipment and technology, the operation is relatively complicated, and the cost is expensive, and it is generally suitable for professional laboratories with experimental conditions. Antigen testing with Mycoplasma pneumoniae monoclonal antibodies as the main raw material can better solve these shortcomings and has a very significant advantage in the rapid diagnosis of MP infection. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides light chain and heavy chain variable regions of an anti-Mycoplasma pneumoniae monoclonal antibody.

[0006] The present invention is achieved by providing a light chain and heavy chain variable region of an anti-Mycoplasma pneumoniae monoclonal antibody, wherein the light chain and heavy chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody include: the light chain and heavy chain variable region of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19.

[0007] Furthermore, the sequences of the three complementary determining regions (CDRs) of the light chain variable region of the monoclonal antibody SPPHMP-No.5 are:

[0008] CDR1: Lys-Ser-Val-Ser-Thr-Ser-Gly-Tyr-Ser-Tyr;

[0009] CDR2: Leu-Val-Ser;

[0010] CDR3: Gln-His-Ile-Arg-Glu-Leu-Thr-Arg.

[0011] Furthermore, the three complementary determining region (CDR) sequences of the heavy chain variable region of the monoclonal antibody SPPHMP-No.5 are:

[0012] CDR1: Gly-Tyr-Thr-Phe-Thr-Ser-Phe-Asp;

[0013] CDR2: Ile-Tyr-Pro-Gly-Asp-Gly-Ser-Thr;

[0014] CDR3: Thr-Arg-Lys-Gly-Gly-Trp-Gly-Ala-Gly-His-Ser-Ala-Met-Asp-Tyr.

[0015] Furthermore, the three complementary determining region (CDR) sequences of the light chain variable region of the monoclonal antibody SPPHMP-No.19 are:

[0016] CDR1: Glu-Asn-Val-Gly-Ser-Tyr;

[0017] CDR2: Gly-Ala-Ser;

[0018] CDR3: Gly-Gln-Thr-Tyr-Asn-Tyr-Pro-Tyr-Thr.

[0019] Furthermore, the three complementary determining region (CDR) sequences of the heavy chain variable region of the monoclonal antibody SPPHMP-No.19 are:

[0020] CDR1:Gly-Tyr-Thr-Phe-Thr-Thr-Tyr-Asn;

[0021] CDR2:Val-His-Pro-Gly-Tyr-Gly-Ile-Thr;

[0022] CDR3: Thr-Arg-Ser-Tyr-His-Gly-Tyr-Ser-Tyr-Trp-Phe-Phe-Asp-Val.

[0023] Furthermore, the gene sequence and amino acid sequence of the light chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.5 are:

[0024] Nucleotide sequence:

[0025] GACATTTGTGATGTCACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGT ATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATGAAA

[0026] Amino Acid Sequence:

[0027] DIVMSQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK

[0028] Furthermore, the gene sequence and amino acid sequence of the heavy chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.5 are:

[0029] Nucleotide sequence:

[0030] AGCTTGAGGAGTCTGGGGCTTTAGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAGCTTCGATATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATACATTTATCCTGGAGATGGTAGTACTAAATACAATGAGAAATTTAAGGG CAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGTTCAGCAGCCTGACTTCTGAGAACTCTGCAGTCTATTTCTGCACAAGAAAGGGAGGTTGGGGGGCCGGACACTCTGCTATGGATTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0031] Amino Acid Sequence:

[0032] LEESGALVKISCKASGYTFTSFDINWVKQRPGQGLEWIGYIYPGDGSTKYNEKFKGKATLTADKSSSTAYMQFSSLTSENSAVYFCTRKGGWGAGHSAMDYWGQGTTVTVSS

[0033] Furthermore, the gene sequence and amino acid sequence of the light chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.19 are:

[0034] Nucleotide sequence:

[0035] ATTGTGCTCACCCAGTCTCCCAAATCCGTGTCCATGTCAGTAGGAGAGGGTCACCTTGAACTGCAAGGCCAGTGAGAATGTGGGTTCTTATGTGGCCTGGTATCAACAGAAACCAGACCGGTCTCCTAAACTTCTGATACACGGCGCATCCAAC CGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTACAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGACTTACAACTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA

[0036] Amino Acid Sequence:

[0037] IVLTQSPKSVSMSVGERVTLNCKASENVGSYVAWYQQKPDRSPKLLIHGASNRYTGVPDRFTGSGSTTDFTLTISSVQAEDLADYHCGQTYNYPYTFGGGTKLE

[0038] Furthermore, the gene sequence and amino acid sequence of the heavy chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.19 are:

[0039] Nucleotide sequence:

[0040] AGCTGGTGGAGTCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCACTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGATATGTTCATCCTGGATATGGTATTACTTACTACAGTCAGAAATTTAAG GGCAAGGCCACATTGACTGCAGACACATCCTCCAGCACAGCCTACATGCTGATCAGCAGCCTGACATCTGAAGACTCAGCGGTCTATTTCTGTACAAGATCTTACCACGGCTACTCGTACTGGTTCTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0041] Amino Acid Sequence:

[0042] LVESGASVKMSCKASGYTFTTYNMHWVKQTPGQGLEWIGYVHPGYGITYYSQKFKGKATLTADTSSSTAYMLISSLTSEDSAVYFCTRSYHGYSYWFFDVWGQGTTVTVSS

[0043] An application of the light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody in the preparation of Mycoplasma pneumoniae paired antibody diagnostic reagents targeting different antigenic epitopes.

[0044] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0045] First, the anti-MP monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 provided by the present invention have high affinity to MP and can specifically recognize MP and conduct immune response.

[0046] The light chain and heavy chain variable region genes and amino acid sequences of the anti-MP monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 of the present invention are confirmed by sequence analysis to be unique in their antibody sequences.

[0047] The present invention analyzes and obtains the CDR regions of the light chain and heavy chain variable regions of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19, and on this basis provides support for constructing high-affinity and high-specificity anti-MP paired antibody detection reagents targeting different antigen epitopes.

[0048] Second, the Mycoplasma pneumoniae colloidal gold antigen detection kit of the present invention is used. It uses a double antibody sandwich method to detect Mycoplasma pneumoniae and has the advantages of high sensitivity, strong specificity, rapid detection, simple operation, visible results, convenient storage and transportation, etc. It is suitable for on-site rapid detection of early Mycoplasma pneumoniae infection in clinical respiratory patients such as pediatrics and respiratory medicine, as well as for screening of Mycoplasma pneumoniae carriers or latent patients by grassroots units, prognosis, etc., and for etiology screening and guidance of clinical treatment.

[0049] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0050] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0051] Mycoplasma pneumoniae is one of the important pathogens of respiratory tract infection, which can cause a variety of clinical manifestations, including nasopharyngitis, tonsillitis, trachea, bronchitis and pneumonia. Clinically, the prevention and treatment of Mycoplasma pneumoniae infection is strengthened. Mycoplasma pneumoniae detection in patients with acute respiratory tract infection is extremely helpful for clinical treatment and is an important indicator for diagnosing Mycoplasma pneumoniae infection. Roughly estimated, the domestic market demand for Mycoplasma pneumoniae detection can reach more than 2 billion yuan per year. After the technical solution of the present invention is converted, it is conservatively estimated that the market share is only 1%, and its expected income can also reach about 20 million yuan, which has considerable social and economic benefits.

[0052] (2) The technical solution of the present invention breaks the monopoly of large foreign multinational companies on the Mycoplasma pneumoniae detection market in the field of immunodiagnosis. The transformed detection products can fully replace foreign products and actively promote the healthy development of related industries in the domestic biomedicine field. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0054] Figure 1 This is a graph of antibody electrophoresis results provided by an embodiment of the present invention;

[0055] Figure 2 is a graph showing the results of purified antibody titer detection provided by an embodiment of the present invention;

[0056] Figure 3 This is a schematic diagram of a Mycoplasma pneumoniae monoclonal antibody double antibody sandwich experiment provided in an embodiment of the present invention;

[0057] Figure 4 It is a schematic diagram of the PCR products of the light and heavy chain variable region genes of the monoclonal antibody SPPHMP-No.5 provided in the embodiment of the present invention;

[0058] Figure 5 It is a schematic diagram of the PCR products of the light and heavy chain variable region genes of the monoclonal antibody SPPHMP-No.19 provided in the embodiment of the present invention;

[0059] Figure 6 The embodiment of the present invention provides a comparison result of the nucleotide sequence of the light chain variable region of monoclonal antibody SPPHMP-No.5;

[0060] Figure 7 The embodiment of the present invention provides a result diagram of the amino acid sequence alignment of the light chain variable region of monoclonal antibody SPPHMP-No.5;

[0061] Figure 8 The embodiment of the present invention provides a comparison result of the nucleotide sequence of the heavy chain variable region of monoclonal antibody SPPHMP-No.5;

[0062] Fig. 9 The embodiment of the present invention provides a comparison result of amino acid sequences of heavy chain variable regions of monoclonal antibody SPPHMP-No.5;

[0063] Fig.10 The embodiment of the present invention provides a comparison result of the nucleotide sequence of the light chain variable region of monoclonal antibody SPPHMP-No.19;

[0064] Fig.11 The embodiment of the present invention provides a result diagram of the amino acid sequence alignment of the light chain variable region of monoclonal antibody SPPHMP-No.19;

[0065] Fig.12The embodiment of the present invention provides a comparison result of the nucleotide sequence of the heavy chain variable region of monoclonal antibody SPPHMP-No.19;

[0066] Fig.13 The embodiment of the present invention provides a comparison result of amino acid sequences of heavy chain variable regions of monoclonal antibody SPPHMP-No.19;

[0067] Fig.14 It is a schematic diagram of assembling the colloidal gold chromatography test strip provided by an embodiment of the present invention;

[0068] Fig.15 It is a schematic diagram of determining the detection results of the colloidal gold test kit provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0069] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0070] In view of the problems existing in the prior art, the present invention provides a light chain and heavy chain variable region of an anti-Mycoplasma pneumoniae monoclonal antibody. The present invention is described in detail below in conjunction with the accompanying drawings.

[0071] The present invention uses natural mycoplasma pneumonia (MP) antigen to immunize Balb / c mice, prepares a group of mouse anti-monoclonal antibodies (mAb), screens out two monoclonal antibody hybridoma cell lines, SPPHMP-No.5 and SPPHMP-No.19, which can stably secrete high-affinity anti-MP, and prepares ascites to obtain high-affinity mAbs SPPHMP-No.5 and SPPHMP-No.19; and confirms the uniqueness of its gene sequence and corresponding protein sequence and its complementarity determining region (CDR) sequence; and provides support for the preparation of highly specific mycoplasma pneumonia antigen detection reagents. The present invention is described in detail below in conjunction with the preparation method of specific monoclonal antibodies, the identification of antibody activity and specificity, and the detection of sequences and the determination of uniqueness, which is an explanation of the present invention rather than a limitation. The present invention is specifically implemented according to the following steps:

[0072] 1. Preparation of high affinity anti-MP monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19

[0073] 1.1 Preparation and purification of monoclonal antibodies

[0074] According to the monoclonal antibody preparation method (refer to the first edition of experimental techniques in cellular and molecular immunology, P9-P17), natural MP was used as an immunogen to immunize female Balb / c mice (purchased from the Experimental Animal Center of Xi'an Jiaotong University). The immunization dose was 50 μg / mouse / each immunization. Freund's complete adjuvant was used as the first immunization emulsifier, and Freund's incomplete adjuvant was used as the second immunization emulsifier. The immunization interval was 4 weeks, and the immunization site was multiple subcutaneous injections on the back; 3 weeks later, 30 μg / mouse was directly immunized into the abdominal cavity of mice; 10 days later, the tail vein blood of mice was drawn to determine the titer and detect the immune effect.

[0075] Before fusion, the immunized mice were injected with antigens intraperitoneally to enhance immune shock. The animals were killed 3 days later, and the spleens of the immunized mice were aseptically removed, ground and sieved, washed three times with serum-free 1640 medium, centrifuged, and the cells were counted. The number of spleen cells reached 10 8 1640 medium without serum. Take the mouse myeloma cells Sp2 / 0 in the logarithmic growth phase and count them to prepare immune spleen cell suspension. Mix the myeloma cells with the spleen cell suspension for cell fusion. After fusion, add the cell suspension to a 96-well culture plate containing feeder cells (normal Balb / c mouse peritoneal macrophages), about 100 μL / well, and place at 37°C and 5% CO. 2 Incubate the cells. After 4 days, half of the cells were replaced with the medium. After 8 days, the spleen cells and myeloma cells were observed under the microscope to be dead, and the clone piles appeared. The cells were round, with good light transmittance and clear outlines. The medium was replaced half again. After 10 days, the positive cells that secreted antibodies were screened by indirect enzyme-linked immunosorbent assay. The cells containing the positive clone wells were cloned by limiting dilution method until the positive hybridoma cell lines that could stably secrete antibodies were obtained, which were numbered as SPPHMP-No.5 and SPPHMP-No.19.

[0076] After obtaining a hybridoma cell line that can stably secrete antibodies, healthy female BALB / c mice were selected and the monoclonal antibody cell line numbered SPPHMP-No.5 was cultured at a cell density of 10 8 When the cells were resuspended in 1 mL of normal saline, they were injected into the peritoneal cavity of mice. After about 10 days, when the abdominal cavity of the mice was obviously bulging and the movements were slow, aseptically collected the ascites under the clean bench, collected the supernatant by centrifugation, and packaged it in aliquots and stored in a refrigerator at -20°C.

[0077] In order to obtain high-titer and high-specificity monoclonal antibodies, the ascites was precipitated with saturated ammonium sulfate, treated with different concentrations of saturated ammonium sulfate and dialyzed. To ensure the effectiveness of the antibody, it needs to be collected and freeze-dried for storage, and the protein content is determined by ultraviolet spectrophotometer, and the purity is detected by electrophoresis. After 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis purity detection, the purity of the purified mAb SPPHMP-No.5 and SPPHMP-No.19 reached more than 95%. The relative molecular mass of the SPPHMP-No.5 light chain is about 22kD, and the relative molecular mass of the heavy chain is about 50kD; the relative molecular mass of the SPPHMP-No.19 light chain is about 25kD, and the relative molecular mass of the heavy chain is about 50kD. Figure 1 shown.

[0078] 1.2 Determination of the potency of anti-MP monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19

[0079] The relative affinity of mAb purified from ascites was determined by indirect ELISA. The immune antigen Mycoplasma pneumoniae was coated with coating solution at a concentration of 2 μg / mL and kept in a refrigerator at 4°C overnight; 5% bovine serum albumin was blocked at 37°C for 1 hour; the plate was washed three times, and serially diluted monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 were added, with dilutions ranging from 10 to 10 7 The plate was diluted 7 times in total and incubated at 37°C for 1 h. The plate was washed 3 times, and a 2500-fold diluted goat anti-mouse-horseradish peroxidase (HRP) enzyme-labeled secondary antibody was added and the plate was incubated at 37°C for 1 h. The plate was washed 5 times and color developing solutions A and B were added. After color development for 5 min, 2 mol / L sulfuric acid was used to terminate the color development and the OD value was read at 450 nm using a microplate reader.

[0080] The ascites titer of the high-affinity SPPHMP-No.5 mAb screened was 1×10 -7 After purification, the potency reaches 10 6 , the ascites titer of SPPHMP-No.19 mAb was 1×10 -6 After purification, the potency reaches 10 6 The indirect ELISA method is generally used to detect ascites titer of 1×10 -5 The above antibodies can be used, such as Figure 2 shown.

[0081] 1.3 Identification of monoclonal antibody subtypes

[0082] The double antibody sandwich method in the enzyme-linked immunosorbent assay was used to identify the subtype of the monoclonal antibody. The specific method was to coat the standard antiserum of the sheep anti-mouse Ig type provided in the kit with 2 μg / mL coating solution and keep it in a refrigerator at 4°C overnight; wash the plate with 0.1 mol / L pH7.4 carbonate buffer containing 0.05% Tween-20, add the SPPHMP-No.5 and SPPHMP-No.19 cell culture supernatant stock solution, and incubate at 37°C for 1 hour; wash the plate, add diluted HRP-labeled anti-mouse Ig types IgG1, IgM, IgG3, IgG2b, IgG2a, IgA, κ chain and λ chain respectively, and incubate at 37°C for 1 hour; wash the plate, develop color with TMB substrate, and stop the color development with 2 mol / L sulfuric acid, and finally determine the subclass and subtype of the monoclonal antibody.

[0083] After identification, the Ig subclass determination results of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 were both the most stable IgG1 subclass, κ-type light chain, as shown in Table 1.

[0084] Table 1 Immunoglobulin subclasses and light chain subtypes of antibodies

[0085]

[0086] 1.4 Antibody specificity and cross-reaction with other related microorganisms

[0087] The indirect ELISA method was used to coat the immune antigens Mycoplasma pneumoniae, echovirus, enterovirus EV71, syncytial virus, adenovirus, H1N1 influenza virus, parainfluenza virus, Hantavirus, Chlamydia, hemoglobin, Vero cells and MDCK cells at a concentration of 2 μg / mL with coating solution, and placed in a 4°C refrigerator overnight; the plate was washed 3 times with PBST, 100 μL of blocking solution was added for blocking, and the plate was incubated in a 37°C water bath for 1 hour; the plate was washed 3 times with PBST, and monoclonal antibody S was added respectively. PPHMP-No.5 and SPPHMP-No.19 supernatant stock solution, and Sp2 / 0 culture supernatant as negative control, were added to the corresponding wells with 100 μL respectively, and incubated at 37℃ for 1h; the plate was washed 3 times with PBST, and 100 μL of 2500-fold diluted goat anti-mouse enzyme-labeled secondary antibody was taken, and incubated at 37℃ for 1h; the plate was washed 5 times with PBST, and color development solution A and solution B were added; 2 mol / L sulfuric acid was used to terminate the color development after 5 minutes of color development, and the OD value was read at 450nm with a microplate reader. The results showed that monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 only reacted specifically with Mycoplasma pneumoniae, and had no cross-reaction with other microorganisms such as influenza virus and syncytial virus, as shown in Table 2.

[0088] Table 2 Cross-reaction of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 with other related microorganisms detected by indirect ELISA

[0089]

[0090] Note: "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0091] 1.5 Mycoplasma pneumoniae monoclonal antibody double antibody sandwich experiment

[0092] The double antibody sandwich method was used to coat the antibody SPPHMP-No.5 (1:1000) at a concentration of 2 μg / mL with the coating solution as the primary antibody, and the plate was kept in a refrigerator at 4°C overnight; the plate was washed with 0.1 mol / L pH7.4 carbonate buffer containing 0.05% Tween-20, and Mycoplasma pneumoniae, irrelevant antigen Coxsackievirus A group 16 (CA16) and phosphate buffer (PBS) were added at 1:100 and 1:1000 dilutions, respectively, and incubated at 37°C for 1 hour; the plate was washed, and diluted HRP-labeled SPPHMP-No.19 was added as the enzyme-labeled secondary antibody, and the incubation continued at 37°C for 1 hour; the plate was washed 5 times with PBST, and solution A and solution B were added for color development for 5 minutes, and 2 mol / L sulfuric acid was used to stop the color development, and the OD value was read at 450nm with an enzyme reader. The test results are as follows: Figure 3 shown.

[0093] 2. Cloning of antibody light chain and heavy chain variable region genes

[0094] 2.1 Cultivation of SPPHMP-No.5 and SPPHMP-No.19 mAb hybridoma cells

[0095] Hybridoma cells secreting SPPHMP-No.5 and SPPHMP-No.19 mAbs were revived according to conventional methods and cultured in RPMI 1640 medium containing 10% calf serum at 37°C and 5% CO. 2 Culture in an incubator until the logarithmic growth phase.

[0096] Total RNA extraction and preparation and cDNA synthesis:

[0097] Collect hybridoma cells with good growth status, the number of cells is about 1×10 6 Total RNA was extracted and cDNA was synthesized using the cDNA first-strand kit from Tiangen Company using the extracted RNA as a template.

[0098] Cloning of antibody light chain and heavy chain variable region genes:

[0099] The extracted cDNA was used as a template and the designed degenerate primers were used to amplify the light chain and heavy chain variable region genes of the SPPHMP-No.5 and SPPHMP-No.19 antibodies, respectively. The PCR reaction system and amplification conditions of the light chain and heavy chain variable region genes were consistent.

[0100] The PCR system included 2.5 μL of 10×Ex buffer (added with Mg2+), 2 μL of dNTPs (2.5 μmol / L each), 1 μL of upstream and downstream primers (20 μmol / L), 0.3 μL of ExTaq polymerase, 1 μL of cDNA, 17.2 μL of distilled water, and a total volume of 25 μL.

[0101] PCR reaction conditions: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, for a total of 30 cycles; 72°C for 10 min.

[0102] The nested PCR method was used to identify the light chain variable region gene amplified by PCR, and then the light chain variable region gene and heavy chain variable region gene fragments obtained by PCR were recovered by gel and connected to the pMD19-T vector. The ligation product was transformed into Escherichia coli DH5α competent cells, and 6 to 8 clones were picked for bacterial liquid PCR and plasmid enzyme digestion identification.

[0103] The upstream and downstream primer sequences of SPPHMP-No.5 light and heavy chain variable regions are:

[0104]

[0105] (IUB standard merged base code: w: a / t; b: g / t, c; k: g / t; y: c / t; s: c / g; m: a / c; r: a / g)

[0106] The upstream and downstream primer sequences of SPPHMP-No.19 light and heavy chain variable regions are:

[0107]

[0108] (IUB standard merged base code: s: c / g; w: a / t; b: g / t,c; k: g / t; y: c / t; m: a / c; r: a / g)

[0109] Colony PCR identification of target gene fragments:

[0110] PCR amplified the target band (94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles, and finally 72°C extension for 10 min), took 10 μL of the reaction solution, and identified the PCR product by 1.5 g / L agarose gel electrophoresis.

[0111] The amplified products were identified by 1.5% agarose gel electrophoresis. The VL gene of SPPHMP-No.5 and SPPHMP-No.19 mAbs was about 320 bp, and the VH gene was about 350 bp. Figure 4 and 5 shown.

[0112] DNA sequencing and analysis:

[0113] The obtained recombinant positive E. coli clones were shaken and the bacterial suspension was sent to Beijing Liuhe BGI Genomics Co., Ltd. to complete gene sequence determination. The sequencing results were compared and analyzed using DNAMAN and BLAST software.

[0114] 4 to 6 positive clones were selected for each of the amplified antibody light chain genes and 3 to 4 positive clones were selected for each of the heavy chain genes for sequencing. The sequencing results were compared with the IgBLAST database and the results showed that the light chain and heavy chain variable region genes of the two antibody strains were consistent with the characteristics of the mouse immunoglobulin variable region genes (such as Figure 6 , 8 , 10 and 12).

[0115] 3. Gene sequence and homology analysis of light and heavy chain variable regions of mAb SPPHMP-No.5 and SPPHMP-No.19

[0116] 3.1 After confirming that the sequencing is correct, perform nucleotide sequence homology analysis (Blastn) in the GenBank+EMBL+DDBJ+PDB+RefSeq database.

[0117] The SPPHMP-No.5 mAb light chain variable region gene has the highest homology with the mouse Ig light chain variable region gene numbered: MGI:1330815, reaching 288 / 294 (98%). Figure 6 shown.

[0118] The SPPHMP-No.5 mAb heavy chain variable region gene has the highest homology with the mouse Ig heavy chain variable region gene numbered: GenBank: X02465.1, which is 239 / 249 (96.0%). Figure 8 shown.

[0119] The SPPHMP-No.19 mAb light chain variable region gene has the highest homology with the mouse Ig light chain variable region gene numbered: MGI:1330836, reaching 264 / 281 (94%). Fig.10 shown.

[0120] The SPPHMP-No.19 mAb heavy chain variable region gene has the highest homology with the mouse Ig heavy chain variable region gene numbered: MGI:3646284, which is 242 / 266 (91.0%). Fig.12 shown.

[0121] Homology analysis showed that the nucleotide sequences encoding the light and heavy chain variable regions of mAb SPPHMP-No.5 and SPPHMP-No.19, although having more than 90% homology with the mouse Ig light and heavy chain gene sequences, no gene sequence completely identical to that of the present invention was found, indicating that the present invention is unique in gene sequence.

[0122] 3.2 Translation of SPPHMP-No.5 and SPPHMP-No.19 variable region genes into amino acid sequences and analysis

[0123] Amino acid sequence homology analysis (Blastp) was performed in the non-redundant Genbank CDS translations+PDB+SwissProt+PIR+PRF protein databases.

[0124] The analysis results showed that the amino acid sequence of the light chain variable region of mAb SPPHMP-No.5 had the highest homology with the light chain variable region of mouse Ig numbered BAD91565.1, reaching 103 / 107 (96%). Figure 7 shown.

[0125] The amino acid sequence of the heavy chain variable region of SPPHMP-No.5 mAb has the highest homology with the mouse Ig heavy chain variable region protein numbered: AAO23202.1, which is 93 / 112 (83%). Fig. 9 shown.

[0126] The analysis results showed that the light chain amino acid sequence of SPPHMP-No.19 mAb had the highest homology with the mouse Ig light chain variable region protein numbered CAA76239.1, reaching 94 / 104 (90%). Fig.11 shown.

[0127] The amino acid sequence of the heavy chain of SPPHMP-No.19 mAb has the highest homology with the mouse Ig heavy chain variable region protein numbered BEA71428.1, which is 90 / 111 (81%). Fig.13 shown.

[0128] Homology analysis showed that the amino acid sequences of the light and heavy chain variable regions of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19, although having certain homology with the amino acid sequences of other proteins, no amino acid sequence completely identical to that of the present invention was found, indicating that the present invention is also unique in terms of amino acid sequence.

[0129] 3.3 Use IgBLAST tool to analyze the variable region structure and determine the CDR region sequence

[0130] The sequenced light chain and heavy chain variable region sequences of SPPHMP-No.5 and SPPHMP-No.19 mAbs were analyzed on the IgBlast website (https: / / www.ncbi.nlm.nih.gov / igblast / ) to obtain their CDR region sequences.

[0131] The three complementary determining regions (CDR) sequences of the light chain variable region of SPPHMP-No.5 mAb, such as Figure 6 As shown, specifically:

[0132] CDR1: Lys-Ser-Val-Ser-Thr-Ser-Gly-Tyr-Ser-Tyr;

[0133] CDR2: Leu-Val-Ser;

[0134] CDR3: Gln-His-Ile-Arg-Glu-Leu-Thr-Arg;

[0135] The three complementary determining regions (CDR) sequences of the SPPHMP-No.5 mAb heavy chain variable region, such as Figure 8 As shown, specifically:

[0136] CDR1: Gly-Tyr-Thr-Phe-Thr-Ser-Phe-Asp;

[0137] CDR2: Ile-Tyr-Pro-Gly-Asp-Gly-Ser-Thr;

[0138] CDR3: Thr-Arg-Lys-Gly-Gly-Trp-Gly-Ala-Gly-His-Ser-Ala-Met-Asp-Tyr;

[0139] The three complementary determining regions (CDR) sequences of the SPPHMP-No.19 light chain variable region are as follows: Fig.10 As shown, specifically:

[0140] CDR1:Glu-Asn-Val-Gly-Ser-Tyr;

[0141] CDR2: Gly-Ala-Ser;

[0142] CDR3:Gly-Gln-Thr-Tyr-Asn-Tyr-Pro-Tyr-Thr;

[0143] The three complementary determining regions (CDR) sequences of the SPPHMP-No.19 heavy chain variable region are as follows: Fig.12 As shown, specifically:

[0144] CDR1:Gly-Tyr-Thr-Phe-Thr-Thr-Tyr-Asn;

[0145] CDR2:Val-His-Pro-Gly-Tyr-Gly-Ile-Thr;

[0146] CDR3: Thr-Arg-Ser-Tyr-His-Gly-Tyr-Ser-Tyr-Trp-Phe-Phe-Asp-Val.

[0147] 4. Design and application of Mycoplasma pneumoniae antigen detection kit

[0148] Based on the expression, purification and sequence analysis of anti-Mycoplasma pneumoniae mAbs SPPHMP-No.5 and SPPHMP-No.19, the following immunodiagnostic products were designed and constructed. The products use immunolateral flow chromatography technology and a double antibody sandwich method to detect Mycoplasma pneumoniae antigens.

[0149] 4.1 Preparation of gold-labeled antibody solution

[0150] Gold nanoparticles were prepared by trisodium citrate reduction method, and the size and morphology of gold nanoparticles were observed and determined. The pH value of the colloidal gold solution was adjusted to be slightly alkaline to the isoelectric point of the protein to be labeled (MP antibody). The optimal amount of protein that can stabilize the colloidal gold was selected and then 20% was added as the optimal amount of labeled protein. A colloidal gold diluent was prepared (its composition is: 1% bovine serum albumin; 2.5% sucrose; 0.05% (v / v) Tween-20; 0.01 mol / L, pH 7.4 phosphate buffer; final concentration of 0.1% sodium azide) to dilute the antibody (SPPHMP-No.5 monoclonal antibody) to prepare a gold-labeled antibody solution (maximum absorption peak wavelength range, 532-534nm; OD greater than 0.4Abs).

[0151] 4.2 The assembly materials of colloidal gold chromatography test strips mainly include: sample pad (containing chemical reagents for processing sample components), gold pad (containing colloidal gold-labeled Mycoplasma pneumoniae monoclonal antibody (SPPHMP-No.5) and related chemical reagents, showing wine red), preparation of chromatography membrane containing two color bands (preparing detection antibody (SPPHMP-No.19) spotting solution and control antibody (sheep anti-mouse IgG) spotting solution, and then using a spotting machine to draw the detection antibody spotting solution and the control line antibody spotting solution to the corresponding positions on the chromatography membrane), absorption pad (absorb and store excess liquid in the reaction system after the reaction is complete). Assemble the sample pad, gold pad, chromatography membrane, absorption pad, backing and other parts, and finally obtain the MP colloidal gold chromatography test strip.

[0152] 4.3 Sandwich ELISA for detection of Mycoplasma pneumoniae

[0153] This method uses monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.19 to bind to different antigenic epitopes of Mycoplasma pneumoniae, and can effectively identify Mycoplasma pneumoniae antigens in samples. That is, one of the antibodies (SPPHMP-No.19) fixes the antigen on a solid phase carrier, i.e., the capture antibody, and the other antibody (SPPHMP-No.5) is labeled with colloidal gold and directly measures the amount of the antigen, i.e., the detection antibody. Its characteristics are non-competitive binding reactions, and its advantages are high sensitivity and high specificity, and the antigen does not need to be purified in advance.

[0154] During the test, take a nasopharyngeal swab specimen and pre-treat it in a collection tube containing a sample diluent. The treated extract is dripped into the sample well of the test strip. When the sample to be tested contains Mycoplasma pneumoniae antigen, the Mycoplasma pneumoniae antigen first forms a reaction complex with the colloidal gold labeled antibody (SPPHMP-No.5), and then under the action of chromatography, the reaction complex moves forward along the nitrocellulose membrane and is captured by the Mycoplasma pneumoniae monoclonal antibody (SPPHMP-No.19) pre-coated in the detection area (T) on the nitrocellulose membrane, and finally forms a red / pink reaction line in the detection area, and the result is positive at this time; on the contrary, when the sample does not contain Mycoplasma pneumoniae antigen or the antigen concentration is lower than the minimum detection limit, no red / pink reaction line appears in the detection area, and the result is negative at this time.

[0155] The sensitivity of the colloidal gold chromatography test strip can reach 8ng / mL, and there is no cross-reactivity with microorganisms such as influenza A, influenza B, parainfluenza, syncytial virus, adenovirus, and Chlamydia pneumoniae.

[0156] 4.4 Validation of Mycoplasma pneumoniae antigen detection kit

[0157] 205 clinical samples were tested using the colloidal gold detection method for Mycoplasma pneumoniae and the fluorescent quantitative PCR detection method for Mycoplasma pneumoniae nucleic acid, and the test results are shown in Table 3. Compared with the fluorescent quantitative PCR method, the sensitivity and specificity of the colloidal gold test strip were 83.5% and 96.0%, respectively, indicating that the developed colloidal gold test strip has a certain detection efficacy and can be used for initial screening and diagnosis of patients suspected of being infected with Mycoplasma pneumoniae.

[0158] Table 3 Comparison results of Mycoplasma pneumoniae antigen detection kit (colloidal gold method) and PCR method

[0159]

[0160] The Mycoplasma pneumoniae antigen colloidal gold detection kit of the present invention uses a double antibody sandwich method to detect Mycoplasma pneumoniae, and has the advantages of high sensitivity, strong specificity, rapid detection, simple operation, visible result judgment, convenient storage and transportation, etc., and is suitable for on-site rapid detection of early Mycoplasma pneumoniae infection in clinical respiratory patients in pediatrics, respiratory medicine, etc., as well as screening of Mycoplasma pneumoniae carriers or patients in the latent period, prognosis, etc., and guiding clinical treatment in grassroots units.

[0161] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A light chain and heavy chain variable region of an anti-Mycoplasma pneumoniae monoclonal antibody, characterized in that: The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody include: the light chain and heavy chain variable regions of monoclonal antibodies SPPHMP-No.5 and SPPHMP-No.

19.

2. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The three complementary determining region (CDR) sequences of the light chain variable region of the monoclonal antibody SPPHMP-No.5 are: CDR1: Lys-Ser-Val-Ser-Thr-Ser-Gly-Tyr-Ser-Tyr; CDR2: Leu-Val-Ser; CDR3: Gln-His-Ile-Arg-Glu-Leu-Thr-Arg.

3. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The three complementary determining regions (CDR) sequences of the heavy chain variable region of the monoclonal antibody SPPHMP-No.5 are: CDR1: Gly-Tyr-Thr-Phe-Thr-Ser-Phe-Asp; CDR2: Ile-Tyr-Pro-Gly-Asp-Gly-Ser-Thr; CDR3: Thr-Arg-Lys-Gly-Gly-Trp-Gly-Ala-Gly-His-Ser-Ala-Met-Asp-Tyr.

4. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The three complementary determining region (CDR) sequences of the light chain variable region of the monoclonal antibody SPPHMP-No.19 are: CDR1: Glu-Asn-Val-Gly-Ser-Tyr; CDR2: Gly-Ala-Ser; CDR3: Gly-Gln-Thr-Tyr-Asn-Tyr-Pro-Tyr-Thr.

5. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The three complementary determining regions (CDR) sequences of the heavy chain variable region of the monoclonal antibody SPPHMP-No.19 are: CDR1:Gly-Tyr-Thr-Phe-Thr-Thr-Tyr-Asn; CDR2:Val-His-Pro-Gly-Tyr-Gly-Ile-Thr; CDR3: Thr-Arg-Ser-Tyr-His-Gly-Tyr-Ser-Tyr-Trp-Phe-Phe-Asp-Val.

6. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The gene sequence and amino acid sequence of the light chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.5 are: Nucleotide sequence: GACATTTGTGATGTCACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGT ATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATGAAA Amino Acid Sequence: DIVMSQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPR LLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEG GPSWK.

7. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The gene sequence and amino acid sequence of the heavy chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.5 are: Nucleotide sequence: AGCTTGAGGAGTCTGGGGCTTTAGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAGCTTCGATAAACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATACATTTATCCTGGAGATGGTAGTACTAAATACAATGAGAAATTTAAGGG CAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGTTCAGCAGCCTGACTTCTGAGAACTCTGCAGTCTATTTCTGCACAAGAAAGGGAGGTTGGGGGGCCGGACACTCTGCTATGGATTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Amino Acid Sequence: LEESGALVKISCKASGYTFTSFDINWVKQRPGQGLEWIGYIYPGDGSTKY NEKFKGKATLTADKSSSTAYMQFSSLTSENSAVYFCTRKGGWGAGHSAMD YWGQGTTVTVSS.

8. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The gene sequence and amino acid sequence of the light chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.19 are: Nucleotide sequence: ATTGTGCTCACCCAGTCTCCCAAATCCGTGTCCATGTCAGTAGGAGAGGGTCACCTTGAACTGCAAGGCCAGTGAGAATGTGGGTTCTTATGTGGCCTGGTATCAACAGAAACCAGACCGGTCTCCTAAACTTCTGATACACGGCGCATCCAAC CGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTACAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGACTTACAACTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA Amino Acid Sequence: IVLTQSPKSVSMSVGERVTLNCKASENVGSYVAWYQQKPDRSPKLLIHG ASNRYTGVPDRFTGSGSTTDFTLTISSVQAEDLADYHCGQTYNYPYTFGGGT KLE.

9. The light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claim 1, characterized in that: The gene sequence and amino acid sequence of the heavy chain variable region of the anti-Mycoplasma pneumoniae monoclonal antibody SPPHMP-No.19 are: Nucleotide sequence: AGCTGGTGGAGTCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCACTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGATATGTTCATCCTGGATATGGTATTACTTACTACAGTCAGAAATTTAAG GGCAAGGCCACATTGACTGCAGACACATCCTCCAGCACAGCCTACATGCTGATCAGCAGCCTGACATCTGAAGACTCAGCGGTCTATTTCTGTACAAGATCTTACCACGGCTACTCGTACTGGTTCTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA Amino Acid Sequence: LVESGASVKMSCKASGYTFTTYNMHWVKQTPGQGLEWIGYVHPGYGI TYYSQKFKGKATLTADTSSSTAYMLISSLTSEDSAVYFCTRSYHGYSYWFFD VWGQGTTVTVSS.

10. Use of the light chain and heavy chain variable regions of the anti-Mycoplasma pneumoniae monoclonal antibody according to claims 1 to 9 in the preparation of Mycoplasma pneumoniae paired antibody diagnostic reagents targeting different antigenic epitopes.

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