Mouse anti-PBP2a hybridoma cell line, monoclonal antibody and application

Abstract

The invention relates to a mouse anti-PBP2a hybridoma cell line, a monoclonal antibody and an application thereof. The PBP2a protein of Staphylococcus aureus is extracted as an antigen. After obtaining a high-purity antigen, a good immune response is evoked in mice. Then, hybridoma technology is used to screen two cell lines with high affinity and specificity and prepare corresponding antibodies. The two antibodies are used as a gold-labeled antibody and a coating antibody in a colloidal gold immunoassay kit. The kit can be used to detect the PBP2a protein in methicillin-resistant Staphylococcus aureus and can be used for the development of related in vitro diagnostic reagents.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a mouse anti-PBP2a hybridoma cell line, a monoclonal antibody and applications. Background Art

[0002] In 1961, a strain of Staphylococcus aureus first showed resistance to methicillin. In the mid-1960s, methicillin-resistant Staphylococcus aureus (MRSA) gradually spread to many European countries and Canada. Since the 1980s, MRSA has increased dramatically worldwide and has become one of the most common nosocomial pathogens in the world. Now, methicillin-resistant Staphylococcus aureus (MRSA) is one of the most prevalent antibiotic-resistant pathogens causing hospital and community infections. The inherent resistance of MRSA is due to the acquisition of a new mobile genetic element called Staphylococcal chromosomalcassette mec (SCCmec) by Staphylococcus aureus through horizontal gene transfer. SCCmec carries the mecA gene, which encodes the expression of the membrane protein penicillin-binding protein (PBP) 2a. PBP2a is a penicillin-binding protein with low affinity for β-lactam antibiotics including methicillin. Its PBP2a has a very low affinity for β-lactam antibiotics, so it can avoid being destroyed by β-lactam antibiotics and show drug resistance. Immunodiagnostic reagents are the fastest-growing sub-area of ​​in vitro diagnostic reagents, which use the specific binding between antigens and antibodies for qualitative or quantitative detection. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides mouse anti-PBP2a hybridoma cell lines, monoclonal antibodies and applications.

[0004] The technical solution adopted by the present invention is: a mouse anti-PBP2a hybridoma cell line, named 2LE9, with a preservation number of CGMCCNo.46008, classified as a hybridoma cell, and preserved in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation time is June 27, 2024, and the detection is alive; or, named 2FE11, with a preservation number of CGMCCNo.46007, classified as a hybridoma cell, and preserved in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation time is June 27, 2024, and the detection is alive.

[0005] Mouse anti-PBP2a monoclonal antibody, antibody 2LE9 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises CDRL1 as shown in SEQ ID NO: 1, CDRL2 as shown in SEQ ID NO: 2, and CDRL3 as shown in SEQ ID NO: 3; the heavy chain variable region comprises CDRH1 as shown in SEQ ID NO: 4, CDRH2 as shown in SEQ ID NO: 5, and CDRH3 as shown in SEQ ID NO: 6;

[0006] or,

[0007] Antibody 2FE11 includes a light chain variable region and a heavy chain variable region, the light chain variable region includes CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12, and CDRL3 as shown in SEQ ID NO:13; the heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15, and CDRH3 as shown in SEQ ID NO:16.

[0008] Preferably, the amino acid sequence of the light chain variable region of antibody 2LE9 is SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9;

[0009] Alternatively, the amino acid sequence of the light chain variable region of antibody 2FE11 is SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:19.

[0010] Preferably, the antibody 2LE9 is produced by the mouse anti-PBP2a hybridoma cell line with a deposit number of CGMCC No.46008; or, the antibody 2FE11 is produced by the mouse anti-PBP2a hybridoma cell line with a deposit number of CGMCC No.46007.

[0011] A nucleic acid molecule comprising a polynucleotide encoding a mouse anti-PBP2a monoclonal antibody.

[0012] Preferably, the nucleotide sequence encoding the light chain variable region of antibody 2LE9 is as shown in SEQ ID NO:8, and the nucleotide sequence encoding the heavy chain variable region of antibody 2LE9 is as shown in SEQ ID NO:10;

[0013] Alternatively, the nucleotide sequence encoding the light chain variable region of antibody 2FE11 is shown as SEQ ID NO:18, and the nucleotide sequence encoding the heavy chain variable region of antibody 2FE11 is shown as SEQ ID NO:20.

[0014] A detection kit comprises mouse anti-PBP2a monoclonal antibody.

[0015] Preferably, it is made into a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit or a fluorescent immunoassay kit, or is made into a microfluidic chip.

[0016] Preferably, when a colloidal gold immunoassay kit is prepared, antibody 2LE9 is used as a coating antibody and antibody 2FE11 is used as a gold-labeled antibody.

[0017] Application of the detection kit in detecting methicillin-resistant Staphylococcus aureus infection for non-disease diagnosis and treatment purposes.

[0018] The advantages and positive effects of the present invention are: providing two mouse anti-PBP2a antibodies, which can specifically bind to Staphylococcus aureus PBP2a protein, and the antibody titer can reach above 1:1280000, and have better performance in sensitivity, specificity and stability; it can be made into a kit for detecting bacterial infection containing PBP2a protein; for example, for detecting methicillin-resistant Staphylococcus aureus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the electrophoresis diagram of PBP2a protein in Example 1;

[0020] Figure 2 is the protein electrophoresis diagram of antibody 2LE9 and antibody 2FE11 prepared in Example 2;

[0021] Figure 3 This is the test result of the PBP2a test card sample prepared in Example 4;

[0022] Biological materials: 2LE9, classified as hybridoma cells, deposited on June 27, 2024, deposited at China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit number is CGMCC No. 46008;

[0023] Biological material: 2FE11, classified as hybridoma cell, deposited on June 27, 2024, deposited at China General Microbiology Center (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit number is CGMCC No. 46007. DETAILED DESCRIPTION

[0024] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0025] The present invention relates to mouse anti-PBP2a hybridoma cell lines, monoclonal antibodies and applications. After extracting Staphylococcus aureus PBP2a protein as an antigen and obtaining a high-purity antigen, a good immune response is evoked in mice, and then hybridoma technology is used to screen high-affinity and specific cell lines and prepare corresponding antibodies, and a colloidal gold immunochromatography-based methicillin-resistant Staphylococcus aureus PBP2a detection kit is developed, which can be used for the development of related in vitro diagnostic reagents.

[0026] After screening, two mouse anti-PBP2a hybridoma cell lines were obtained, one of which was named 2LE9, with a deposit number of CGMCC No. 46008, and a deposit place of the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit time of June 27, 2024, and the test was alive; the other was named 2FE11, with a deposit number of CGMCC No. 46007, and a deposit place of the General Microbiology Center of the China Microorganism Culture Collection Administration Committee, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit time of June 27, 2024, and the test was alive. The present invention obtains a hybridoma cell line that stably secretes mouse anti-PBP2a monoclonal antibodies by screening mouse hybridoma monoclonal antibodies and cloning Ig variable region genes by RT-PCR, and analyzes its variable region sequence, and identifies the antibody binding specificity by ELISA.

[0027] The antibody 2LE9 produced by the mouse anti-PBP2a hybridoma cell line 2LE9 includes a light chain variable region and a heavy chain variable region. The light chain variable region includes CDRL1 as shown in SEQ ID NO:1, CDRL2 as shown in SEQ ID NO:2, and CDRL3 as shown in SEQ ID NO:3; the heavy chain variable region includes CDRH1 as shown in SEQ ID NO:4, CDRH2 as shown in SEQ ID NO:5, and CDRH3 as shown in SEQ ID NO:6.

[0028] SEQ ID NO:1 RTSENIYRNLA (CDRL1)

[0029] SEQ ID NO:2 TATNLAD(CDRL2)

[0030] SEQ ID NO:3 QHFWGTPFT (CDRL3)

[0031] SEQ ID NO:4 GFTVSTYAMS(CDRH1)

[0032] SEQ ID NO:5 TISSGGGYTYYTDSVKG (CDRH2)

[0033] SEQ ID NO:6 QYYGSESYFDY(CDRH3)

[0034] The amino acid sequence of the light chain variable region of antibody 2LE9 is SEQ ID NO:7, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region of antibody 2LE9 is SEQ ID NO:9, and the nucleotide sequence encoding the heavy chain variable region of antibody EB8 is shown in SEQ ID NO:10;

[0035] Light chain variable region amino acid sequence, SEQ ID NO: 7

[0036] DIVMTQTPASLSVSVGETVTITCRTSENIYRNLAWYQQKQGKSPQLLLYTATNLADGVPSRFSGSGSGTHFSLKINSLQSEDFGSYYCQHFWGTPFTFGGGTKLEIK

[0037] Light chain variable region base sequence, SEQ ID NO: 8

[0038] GACATTTGTCATGACCCAGACTCCAGCCTCCCTATCTGTTTCTGTGGGAGAAACTGTCACCATCACATGTCGAACAAGTGAGAATATTTATAGAAATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAACTCCTGCTCTATACTGCAACAAACT TAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACATTTCTCCCTCAAGATCAACAGCCTGCAGTCTGAAGATTTTGGGAGTTATTACTGTCAACATTTTTGGGGAACTCCGTTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0039] Heavy chain variable region amino acid sequence, SEQ ID NO: 9

[0040] GGGLVKPGGSLRLSCAATGFTVSTYAMSWLRQTPEKTLEWVATISSGGGYTYYTDSVKGRFTISKDNAKNSLYLQMSSLRSEDTAMYYCTRQYYGSESYFDYWGQGATLTVSSAKT

[0041] Heavy chain variable region base sequence, SEQ ID NO: 10

[0042] GGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCACTGGATTCACTGTCAGCACTTATGCCATGTCTTGGCTTCGACAGACTCCGGAGAAGACGCTGGAGTGGGTCGCAACCATAAGTAGTGGTGGTGGTTACACATACTACACAGACAGTGTGAAG GGGCGATTCACCATCTCCAAAGACAATGCCAAGAACTCCCTGTACCTGCAAATGAGTAGTCTGAGGTCTGAGGACACGGCCATGTATTGTACAAGACAATACTACGGTAGTGAGTCATACTTTGACTACTGGGGCCAAGGCGCCACTCTCACAGTCTCCTCAGCCAAAACG

[0043] The antibody 2FE11 produced by the mouse anti-PBP2a hybridoma cell line 2FE11 includes a light chain variable region and a heavy chain variable region, the light chain variable region includes CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12, and CDRL3 as shown in SEQ ID NO:13; the heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15, and CDRH3 as shown in SEQ ID NO:16;

[0044] SEQ ID NO:11 KSSQSLLDTDGKTYLN (CDRL1)

[0045] SEQ ID NO:12 VVSKKDS(CDRL2)

[0046] SEQ ID NO:13 WQGTHFPLT (CDRL3)

[0047] SEQ ID NO:14 GFNIKDIYIN(CDRH1)

[0048] SEQ ID NO:15 RIDPANGNTIYDPKFLG (CDRH2)

[0049] SEQ ID NO:16 SSRY (CDRH3)

[0050] The amino acid sequence of the light chain variable region of antibody 2FE11 is SEQ ID NO: 17, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO: 18; the amino acid sequence of the heavy chain variable region of antibody 2FE11 is SEQ ID NO: 19, and the nucleotide sequence encoding the heavy chain variable region of antibody CG2 is shown in SEQ ID NO: 20;

[0051] Light chain variable region amino acid sequence, SEQ ID NO: 17

[0052] DIVMTQSPLTLSVTIGQPASISCKSSQSLLDTDGKTYLNWLLQRPGQSPKRLIYVVSKKDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPLTFGAGTKLELK

[0053] Light chain variable region base sequence, SEQ ID NO: 18

[0054] GACATTTGTGATGACCCAGTCTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATACTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATTTATGTGGTG TCTAAAAAGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAACTGAAA

[0055] Heavy chain variable region amino acid sequence, SEQ ID NO: 19

[0056] GAELVKPGASVKLSCIASGFNIKDIYINWVKQRPEQGLEWIGRIDPANGNTIYDPKFLGKATITADTFSNTAYLHLSSLTSEDTAVYYCARSSRYWGQGTTLTVSSAKT

[0057] Heavy chain variable region base sequence, SEQ ID NO: 20

[0058] GGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCATAGCTTCTGGCTTCAACATTAAAGACATCTATAAACTGGGTGAAACAGAGGCCTGAGCAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTATTTATGACC CGAAGTTCCTGGGCAAGGCCACTATAACAGCAGACACATTTTCCAACACAGCCTACCTGCACCTCAGCAGCCTGACTTCTGAGGACACTGCCGTCTATTACTGTGCTAGATCCTCTCGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACG

[0059] The two mouse anti-PBP2a monoclonal antibodies have passed a systematic evaluation, including the evaluation of antibody subtypes and titers, kit sensitivity, specificity and stability. The mouse anti-PBP2a monoclonal antibodies have performed well in all aspects. The titers of both antibodies have reached more than 1:1280000, making them suitable as immunodiagnostic reagents for the preparation of in vitro diagnostic kits. They can be made into colloidal gold immunochromatography kits, chemiluminescence kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits or fluorescent immunoassay kits, or made into microfluidic chips; the prepared kits can detect Staphylococcus aureus PBP2a protein. For example, antibody 2LE9 and antibody 2FE11 can be used as gold-labeled antibodies and coating antibodies in colloidal gold immunochromatography test strips, respectively.

[0060] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0061] Example 1: Preparation of mouse anti-PBP2a monoclonal antibody

[0062] PBP2a protein preparation

[0063] The amino acid fragment of the mecA transpeptidase region was synthesized by full gene, and the recombinant plasmid was transformed into Escherichia coli for induced expression. The purification method was nickel column affinity chromatography. The wet weight of the bacteria was about 2g and resuspended in equilibrium buffer (pH 7.4). Ultrasonication was performed in an ice water bath until the bacterial solution was clear. The supernatant was passed through a membrane and then a nickel column to elute the target protein. The molecular weight was consistent with the expected value. SDS-PAGE was as follows Figure 1 shown.

[0064] 1.2 Immunization of mice

[0065] Female Balb / c mice about 6 weeks old were immunized with purified PBP2a protein for antibody preparation. They were divided into 2 groups according to the immunization dose, with 5 mice in each group. According to the antigen content, the immunization dose of the first group was 25ug / mouse, and the immunization dose of the second group was 50ug / mouse. For the first immunization, an appropriate amount of PBP2a protein was diluted to 500ul with physiological saline, and an equal amount of Freund's complete adjuvant 500ul was added to emulsify evenly, and the mice were immunized subcutaneously at multiple points. Two weeks later, the same dose was taken for the second immunization, and the adjuvant was replaced with Freund's incomplete adjuvant for the second immunization, and the mice were immunized intraperitoneally. Two weeks later, the mice were immunized again, and the mice were immunized intraperitoneally. After 7 days, the tail blood was collected, and the mouse serum titer was determined by ELISA. The specific steps are: PBP2a protein 0.2ug / ml, 100ul / well, ELISA plate was coated overnight at 4℃, dried, and washed 3 times with PBST. 5% skim milk powder, 200ul / well, blocked at 37℃ for 2h. Blood was collected from the tail of mice at 3000 rpm, and serum was collected after centrifugation. Serum was diluted from 1:1000 to 1:512000 with PBS for later use. Drying, washing 3 times with PBST, adding primary antibody diluted with PBS at 1:1000, 100ul / well, 37℃, 1h. Drying, washing 3 times with PBST, adding goat anti-mouse secondary antibody diluted with PBS at 1:10000, 100ul / well, 37℃, 45min. Drying, washing 5 times with PBST, adding 100ul TMB / well, 37℃, color development for 10min, stopping, and reading.

[0066] 1.3 Cell fusion

[0067] Three days before fusion, mice were boosted with the same inoculation volume as the previous immunization, without adjuvant, and injected intraperitoneally. One day before fusion, feeder cells were prepared. One 6-8 week old Balb / c mouse was taken, and the eyeball was bled and then killed by cervical dislocation. The mouse was placed in 75% alcohol for disinfection for 5 minutes, fixed on a plate, and the abdominal skin was aseptically cut in the clean bench. 10 ml of HAT selection culture medium was drawn with a sterile syringe and injected into the mouse peritoneal cavity. The abdomen was gently rubbed with an alcohol cotton ball and the culture medium was withdrawn. Add 40 ml of HAT culture medium and spread into 4 96-well cell culture plates, 100 μL / well, and cultured in a cell culture incubator at 37°C and 5% CO2. One week before fusion, myeloma cells (Sp2 / 0 cells) were resuscitated and cultured in PRMI-1640 culture medium containing 10% fetal bovine serum, and subcultured in a 37°C and 5% CO2 incubator. Cells in the logarithmic growth phase were collected into a centrifuge tube, the cells were counted, and the cells were diluted to 10 7 / ml for later use. Take Balb / c mice that have been boosted for 3 days, remove the eyeballs and bleed to prepare positive serum, then kill them by dislocating the cervical vertebrae, disinfect with 75% alcohol for 5 minutes, remove the spleen aseptically on a clean bench, wash it several times in a sterile dish, and peel off the connective tissue. Place the spleen on a microporous copper mesh, add fresh RPMI-1640 culture medium, first use a syringe to draw the culture medium and inject it from a section of the spleen, blow off the spleen cells, repeat several times, and gently grind the remaining spleen with the inner plug of the syringe until there are no obvious red tissue blocks. Gently blow the spleen cell suspension in the dish and transfer it to a 50ml centrifuge tube, centrifuge it at 1000r / min for 5 minutes, collect the spleen cells, count them and set aside. Mix the immune mouse spleen cells and Sp2 / 0 cells at a cell number of 10:1, add them to a 50ml centrifuge tube, centrifuge at 1000r / min for 5min, discard the supernatant, gently rub the two cells in the palm of your hand to fully mix, put the centrifuge tube into a 100mL blue-capped bottle filled with 37℃ hot water, add the preheated 1ml DMSO / PEG dropwise into the fusion tube within 1min, first slowly and then quickly, and gently rotate the centrifuge tube while adding. Then immediately add the anti-blood-free RPMI-1640 culture medium to terminate the reaction, add 1ml in the first minute, add 2ml in the second minute, add 3ml in the third minute, and add 4ml in the fourth minute. Place in a 37℃ water bath for 5min, then centrifuge at 800r / min for 5min, discard the supernatant, suspend the precipitate with HAT, mix it in 40ml HAT selection medium containing 20% ​​calf serum preheated at 37℃, and plate it in a 96-well cell plate with feeder cells, 100μL / well, and place the culture plate in a 37℃, 5% CO2 incubator for culture. After 7d, half of the cell plate will be replaced with fresh HAT medium, and after 10 days, the medium will be replaced with HT medium. The cells tested positive in the 96-well plate are subcloned by limiting dilution method: first, prepare the feeder layer cells according to the above method, take the hybridoma cells to be cloned for cell counting, dilute the cells to 5-8 cells / ml with HT medium, add them to the 96-well cell plate with feeder cells, 100μL / well, and clone one 96-well cell plate for each hybridoma cell, and culture them in a 37℃, 5% CO2 cell incubator. After about 5 days, count the number of clones in the cell wells and mark them. After 7 days, replace the culture medium with new one. When the cells cover 1 / 3 to 1 / 2 of the entire bottom of the well, test. After 2-3 cloning, when all the cell wells of the 96-well plate are positive, they can be expanded, fixed, and frozen. The hybridoma cells that are positive and fixed are expanded and frozen. The specific process is as follows: The hybridoma cells that are growing vigorously and in good condition are gently blown off the cell bottle with antibody-free and blood-free DMEM, centrifuged at 1000 r / min for 5 minutes, and the supernatant is discarded. Add freezing solution (containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, and 10% DMSO), blow the cells apart, and then divide them into cell cryopreservation tubes.Place the cryovials in a freezing box in a -70°C refrigerator. One day later, transfer the cryovials into liquid nitrogen and keep records.

[0068] 1.4 Ascites preparation

[0069] Female Balb / c mice aged 8-10 weeks were injected intraperitoneally with sterile liquid paraffin (0.5 mL / mouse). After 7 days, hybridoma cells cultured to the logarithmic phase were injected intraperitoneally with 5×10 6 Cells / mouse. Observe every day. After about 7-10 days, when the abdomen of the mouse is obviously bulging, disinfect the lower abdominal skin with 75% alcohol cotton balls, pierce the abdominal cavity with a No. 16 needle, and collect ascites. After the ascites regenerates and accumulates, collect it again. Centrifuge the collected ascites at 3000 r / min for 10 minutes, take the middle clear part, and store it at -70℃.

[0070] 1.5 Antibody Purification

[0071] Use Protein-G column to purify ascites, the steps are as follows: take 2ml of ascites, centrifuge at 10000g, take the clarified part, add 2ml of washing buffer, mix well, drain the column with 20% ethanol and balance with 8ml of washing solution, pass the sample through the column, repeat the loading 3 times, then wash with 15ml of washing buffer, elute with 10ml of elution buffer, adjust the pH to 7.4 with 1M Tris PH=9 after elution, concentrate and dialyze overnight in 50kd dialysis bag, PBS, 4℃.

[0072] Example 2: Identification of mouse anti-PBP2a monoclonal antibodies

[0073] 2.1 Antibody subclass identification

[0074] According to the instructions of the SIGMA kit, the subclass identification of monoclonal antibodies was performed by the capture ELISA method, as follows: dilute the monoclonal antibody subclass identification reagent 1:1000, add it to the enzyme-labeled well, 100 μL / well, incubate at 37°C for 1 hour; wash three times with PBST and pat dry; dilute the antibody 1:1000 and add it to the sample, 100 μL / well, incubate at 37°C for 1 hour; wash three times with PBST and pat dry; dilute the HRP enzyme-labeled goat anti-mouse IgG secondary antibody 1:10000 and add it to the sample, 100 μL / well, incubate at room temperature for 30 minutes; color development for 10 to 20 minutes. The subclass type of the monoclonal antibody is the one with a significantly higher OD450 reading than the subclass reagent added to other wells. The antibody subclass of antibodies 2FE11 and 2LE9 is IgG1.

[0075] 2.2 Antibody titer determination

[0076] The titer of purified antibodies was determined by indirect ELISA, and the steps were as follows: PBP2a protein was diluted to 0.2ug / mL, 100ul / well, and a non-coated control was set up at the same time. Coating was carried out overnight at 4℃, and PBST was washed 3 times; 5% skim milk powder, 200ul / well, blocked at 37℃ for 2h; PBST was washed 3 times, and antibodies (concentration of 1mg / ml) were added in a series of dilutions starting from 1:1000, with a total of 12 gradients, and a non-coated control 100ul / well was set up at 37℃ for 1h. PBST was washed 3 times, and PBS was added with a 1:10000 dilution of goat anti-mouse secondary antibody, 100ul / well, 37℃, 45min. PBST was washed 5 times, and 100ul TMB / well was added, 37℃, color development for 10min, stop, and read the value. The purified antibody was diluted to 1mg / ml, and the titer reached more than 1:1280000.

[0077] 2.3 Antibody purity and molecular weight identification

[0078] The molecular weight and purity of the antibody were identified by SDS-PAGE. The separation gel was 12% and the concentration gel was 5%. Sample preparation was 20ul sample + 20ul buffer, mixed and boiled for 3min. 20ul sample was loaded into each well, and a protein pre-stained marker control was set up. The electrophoresis was performed at 80 volts for 30min and 120 volts for 2h. After electrophoresis, the antibody was stained with Coomassie brilliant blue solution. The purified monoclonal antibody was identified by SDS-PAGE, and the bands were clear and without any other bands. Figure 2 As shown, there are clear bands at 50 KDa and 25 KDa.

[0079] Example 3: Gene verification of mouse anti-PBP2a monoclonal antibody

[0080] Total RNA extraction and single-stranded cDNA synthesis:

[0081] The total RNA of 2FE11 and 2LE9 hybridoma cell lines was extracted using the Trizol method (the kit was purchased from Invitrogen), and the total RNA was reversed into a cDNA library using M-MLV reverse transcriptase (purchased from Invitrogen).

[0082] Heavy chain backbone region upstream primer

[0083] P1:5'SAGGTGGMAGCTKCASSARTCWGG3' (SEQ ID NO:21)

[0084] Heavy chain variable region downstream primer

[0085] P2: 5'TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3' (SEQ ID NO: 22)

[0086] Light chain leader peptide upstream primer

[0087] P3: 5'ATGGATTTTCAAGTGCAGATTTTCAG3' (SEQ ID NO:23)

[0088] Light chain variable region downstream primer

[0089] P4: 5'GGATACAGTTGGTGCAGCATCAGCCCGTTT3' (SEQ ID NO:24)

[0090] Prepare PCR reaction system (50 μl) as follows:

[0091] cDNA: 2μl; upstream primer (10μM): 2μl; downstream primer (10μM): 2μl; dNTP mixture: 2μl; pfuDNA polymerase (5U / μl): 1μl; 10X pfu Buffer Ⅱ: 5μl; ddH2O: make up to 50μl.

[0092] Reaction conditions: pre-denaturation at 95°C for 5 min; repeat the following cycle 35 times: 95°C for 30 s, 58°C for 30 s, 72°C for 1 min; finally, extension at 72°C for 10 min.

[0093] Agarose gel electrophoresis was used to separate and recover the VL and VH fragments. The recovered VL and VH fragments were connected to the pMD19-T (sKPCle) vector (Takara Company) respectively. The connection system is as follows:

[0094] 70 ng of VL PCR product / VH PCR product, 1 μl of pMD19-T (sKPCle) vector, 5 μl of Solution I ligation reaction solution; add ddH2O to 10 μl, and ligate at 37°C for 30 min.

[0095] The ligation product was transformed into E. coli DH5α competent bacteria, cultured overnight at 37°C, and a single colony was picked. After shaking at 37°C for 2 hours, PCR identification of the bacterial solution was performed, and the cDNA of the corresponding antibody was used as a positive control. The reaction system (25 μl) was prepared as follows:

[0096] Bacterial solution: 1 μl, upstream primer (10 μM): 1 μl; downstream primer (10 μM): 1 μl; dNTP Mixture (2.5 μm each) 2 μl; Taq DNA polymerase (5 U / μl): 0.5 μl; 10× Taq Buffer (Mg 2+ plus): 2.5 μl; add water to 25 μl. The reaction conditions are the same as before.

[0097] The clones with positive PCR were selected for expansion culture, and the plasmids of positive clones were extracted with a plasmid extraction kit (Takara) and sent for sequencing. At least 5 clone samples of each chain of each antibody were sent for testing, and the sequencing results of at least three samples were the same. The heavy chain and light chain variable region sequences of antibodies 2FE11 and 2LE9 were successfully cloned and matched the characteristics of typical antibody variable region sequences.

[0098] Example 4: Preparation of PBP2a Detection Card by Colloidal Gold Immunochromatography

[0099] The double antibody sandwich immunocolloidal gold test strip is prepared as follows;

[0100] 0.1M K2CO3 solution was added to the colloidal gold solution while stirring, mouse anti-PBP2a monoclonal antibody 2FE11 was added after adjusting the pH value, 10% bovine serum albumin solution and 2% PEG20000 were added after stirring, supernatant was taken after low-speed centrifugation after stirring, precipitation was taken after high-speed centrifugation, and the volume was fixed with colloidal gold resuspension to form a gold-labeled antibody; the gold-labeled antibody was sprayed on a glass cellulose membrane and dried to form a gold-labeled pad; 1% sodium thimerosal solution was added to mouse anti-PBP2a monoclonal antibody 2LE9, mixed to form a detection line coating solution, and PBS and 1% sodium thimerosal solution were added to goat anti-mouse IgG, mixed to form a quality control line coating solution, the quality control line coating solution and the detection line coating solution were drawn on a nitrocellulose membrane, and the coating film was obtained after drying; the coating film was attached to a bottom plate, the gold-labeled pad and absorbent paper were placed on the coating film, and the PBP2a detection card prepared by the colloidal gold immunochromatography method was obtained by cutting after lamination.

[0101] The PBP2a test card was prepared by colloidal gold immunochromatography to test blank control, PBP2a antigen, and MRSA samples. The test results were as follows: Figure 3 As shown, the PBP2a detection card can accurately detect the presence of PBP2a in each sample.

[0102] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A mouse anti-PBP2a hybridoma cell line, characterized in that: Named as 2LE9, with a deposit number of CGMCC No.46008; or, named as 2FE11, with a deposit number of CGMCC No.46007.

2. Mouse anti-PBP2a monoclonal antibody, characterized in that: Antibody 2LE9 includes a light chain variable region and a heavy chain variable region, the light chain variable region includes CDRL1 as shown in SEQ ID NO: 1, CDRL2 as shown in SEQ ID NO: 2, and CDRL3 as shown in SEQ ID NO: 3; the heavy chain variable region includes CDRH1 as shown in SEQ ID NO: 4, CDRH2 as shown in SEQ ID NO: 5, and CDRH3 as shown in SEQ ID NO: 6; or, Antibody 2FE11 includes a light chain variable region and a heavy chain variable region, the light chain variable region includes CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12, and CDRL3 as shown in SEQ ID NO:13; the heavy chain variable region includes CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15, and CDRH3 as shown in SEQ ID NO:

16.

3. The mouse anti-PBP2a monoclonal antibody according to claim 2, characterized in that: The amino acid sequence of the light chain variable region of antibody 2LE9 is SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:9; Alternatively, the amino acid sequence of the light chain variable region of antibody 2FE11 is SEQ ID NO:17, and the amino acid sequence of the heavy chain variable region is SEQ ID NO:

19.

4. The mouse anti-PBP2a monoclonal antibody according to claim 3, characterized in that: The antibody 2LE9 is produced by the mouse anti-PBP2a hybridoma cell line with a deposit number of CGMCC No.46008; or, the antibody 2FE11 is produced by the mouse anti-PBP2a hybridoma cell line with a deposit number of CGMCC No.46007.

5. A nucleic acid molecule, characterized in that: It is a polynucleotide encoding the mouse anti-PBP2a monoclonal antibody according to claim 2 or 3.

6. The nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence encoding the light chain variable region of antibody 2LE9 is shown in SEQ ID NO:8, and the nucleotide sequence encoding the heavy chain variable region of antibody 2LE9 is shown in SEQ ID NO:10; Alternatively, the nucleotide sequence encoding the light chain variable region of antibody 2FE11 is shown as SEQ ID NO:18, and the nucleotide sequence encoding the heavy chain variable region of antibody 2FE11 is shown as SEQ ID NO:

20.

7. A detection kit, characterized in that: The invention comprises the mouse anti-PBP2a monoclonal antibody as described in any one of claims 2 to 4.

8. The detection kit according to claim 7, characterized in that: It can be made into a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit or a fluorescent immunoassay kit, or a microfluidic chip.

9. The detection kit according to claim 8, characterized in that: When the colloidal gold immunoassay kit is prepared, antibody 2LE9 is used as the coating antibody and antibody 2FE11 is used as the gold-labeled antibody.

Images