Mouse anti-gram-positive bacterial peptidoglycan hybridoma cell line, monoclonal antibody and application

Abstract

The present invention provides a mouse anti-gram-positive bacterial peptidoglycan hybridoma cell line, a monoclonal antibody and an application thereof. A hybridoma cell line that stably secretes a mouse anti-gram-positive bacterial peptidoglycan monoclonal antibody is obtained by screening mouse hybridoma monoclonal antibodies and cloning Ig variable region genes by RT-PCR, and its variable region sequence is obtained by analysis, and the antibody binding specificity is identified by ELISA, proving that the obtained antibody has not only a high antibody titer, but also a strong specificity; the hybridoma cell line can be used to prepare a kit for detecting gram-positive bacterial peptidoglycan, and has good clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of antibody detection, and in particular relates to a mouse anti-Gram-positive bacteria peptidoglycan hybridoma cell line, a monoclonal antibody and an application thereof. Background Art

[0002] With the widespread use of broad-spectrum antimicrobial drugs and the increase in hospital infections, drug-resistant strains continue to emerge, increasing the cost of hospitalization and social burden on patients. Among them, drug-resistant Staphylococcus aureus and enterococcal infections are showing a gradual increase. Since the characteristics of bacterial resistance vary from region to region, they are even different in different periods in the same region. With the advancement of medical standards and the widespread use of invasive technologies such as deep venous catheterization and mechanical ventilation, patients are rescued while also accompanied by a high incidence of bloodstream bacterial infections. Bloodstream bacterial infections not only cause a large increase in medical expenses, but also seriously threaten the life and health of patients. Early diagnosis of bacterial infections is particularly important for timely control of infections and improving patient survival rates. At present, the diagnosis of bacterial infections can be divided into two categories according to the detection object: laboratory diagnosis for pathogens and laboratory diagnosis for infected hosts. Blood culture detection is the traditional "gold standard" for diagnosing bloodstream bacterial infections. It is a method of directly culturing and amplifying pathogens in the blood. Positive culture can be used as direct evidence of bloodstream bacterial infection. Peptidoglycan is the main component of bacterial cell walls, of which Gram-positive bacteria contain a relatively large proportion of peptidoglycan, while Gram-negative bacteria contain a relatively small proportion of peptidoglycan. Fungi, viruses, human cells and other pathogens do not contain this component. After bacteria invade the human body, they are phagocytosed and digested by phagocytes, and peptidoglycan can be released from the cell wall, increasing the content in the blood or other body fluids (urine, cerebrospinal fluid, ascites, pleural effusion, etc.). The peptidoglycan content in serum is detected to assess the degree of Gram-positive bacterial infection. Laboratory detection of Gram-positive bacteria is mainly through blood culture. However, blood culture technology has high operating requirements and a long cycle, which can easily lead to misdiagnosis, missed diagnosis and slow results. Summary of the invention

[0003] In order to solve the above technical problems, the present invention provides a mouse anti-Gram-positive bacteria peptidoglycan hybridoma cell line, monoclonal antibody and application.

[0004] The technical solution adopted by the present invention is: a mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line, one strain is named EB8, the preservation number is CGMCC No.46010, it is classified as a hybridoma cell, the preservation place is the General Microbiology Center of China Microorganism Culture Collection Administration, the address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation time is June 27, 2024, and the detection is alive; the other strain is named CG2, the preservation number is CGMCC No.46009, it is classified as a hybridoma cell, the preservation place is the General Microbiology Center of China Microorganism Culture Collection Administration, the address is 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-Gram-positive bacterial peptidoglycan monoclonal antibody, antibody EB8 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] SEQ ID NO:1 KSSQSLLNSGNQKNYLT (CDRL1)

[0007] SEQ ID NO:2 WASTRES (CDRL2)

[0008] SEQ ID NO:3 QNDYSFPLT (CDRL3)

[0009] SEQ ID NO:4 GYTFTNYGMN (CDRH1)

[0010] SEQ ID NO:5 WINTYTGEPKYVDDFKG (CDRH2)

[0011] SEQ ID NO:6 LAFTTVVPTNAMDY(CDRH3)

[0012] or,

[0013] Antibody CG2 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;

[0014] SEQ ID NO:11 KSSQSLLNSGNQKNFLT (CDRL1)

[0015] SEQ ID NO:12 WASSRES(CDRL2)

[0016] SEQ ID NO:13 QNDYRFPLT(CDRL3)

[0017] SEQ ID NO:14 GYTFTNYGMN (CDRH1)

[0018] SEQ ID NO:15 WINTYTGEPKYVDDFKG (CDRH2)

[0019] SEQ ID NO:16 LAFITELPTNAMDY(CDRH3)

[0020] Preferably, the amino acid sequence of the light chain variable region of antibody EB8 is SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 9;

[0021] SEQ ID NO:7

[0022] DIQMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISGVQAEDLAVYYCQNDYSFPLTFGAGTKLELK

[0023] SEQ ID NO:9

[0024] GPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPKYVDDFKGRFAFSLETSANTAYLQINNLKNEDMATYFCARLAFTTVVPTNAMDYWGQGTSVTVS

[0025] Alternatively, the amino acid sequence of the light chain variable region of antibody CG2 is SEQ ID NO: 17, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 19;

[0026] SEQ ID NO:17

[0027] DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNFLTWYQQRPGQPPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISGVQAEDLSVYYCQNDYRFPLTFGAGTKLELK

[0028] SEQ ID NO:19

[0029] GPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPKYVDDFKGRFAFSLETSASTAYLQINNLKDEDMATYFCARLAFITELPTNAMDYWGQGTSVTVS

[0030] Preferably, the antibody EB8 is produced by the mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line with a deposit number of CGMCC No.46010;

[0031] The antibody CG2 is produced by a mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line with a deposit number of CGMCC No.46009.

[0032] A nucleic acid molecule comprising a polynucleotide encoding a mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibody.

[0033] Preferably, the nucleotide sequence encoding the variable region of the light chain of antibody EB8 is as shown in SEQ ID NO:8, and the nucleotide sequence encoding the variable region of the heavy chain of antibody EB8 is as shown in SEQ ID NO:10;

[0034] SEQ ID NO:8

[0035] GACATTCAGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAGACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGAATGATTATAGTTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0036] SEQ ID NO:10

[0037] GGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAAAATATGTTGATGACTTCA AGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAACACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGACTCGCCTTTTACTACGGTAGTACCTACGAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC

[0038] The nucleotide sequence encoding the variable region of the light chain of antibody CG2 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the variable region of the heavy chain of antibody CG2 is shown in SEQ ID NO: 20;

[0039] SEQ ID NO:18

[0040] GACATTTGTCATGACCCAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTTCTTGACCTGGTACCAGCAGAGACCAGGGCAGCCTCCTAAATTGTTGATTTATTGGG CATCCAGTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGTCCGTTTTACTGTCAGAATGATTATAGATTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0041] SEQ ID NO:20

[0042] GGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAAAATATGTTGATGACTTCA AGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAGATGAGGACATGGCTACATATTTCTGTGCAAGACTCGCCTTTTATTACGGAACTACCTACGAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC

[0043] A kit for determining peptidoglycan of gram-positive bacteria comprises mouse anti-gram-positive bacteria peptidoglycan monoclonal antibody.

[0044] 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.

[0045] Preferably, antibody EB8 is used in the magnetic bead coating, and antibody CG2 is used in the enzyme-labeled conjugate;

[0046] Alternatively, antibody CG2 can be used in magnetic bead coatings and antibody EB8 can be used in enzyme-labeled conjugates.

[0047] Application of Gram-positive bacterial peptidoglycan assay kit in detecting Gram-positive bacterial infection for non-disease diagnosis and treatment purposes.

[0048] The advantages and positive effects of the present invention are: providing a mouse anti-gram-positive bacterial peptidoglycan monoclonal antibody, the antibody titer for gram-positive bacterial peptidoglycan reaches 1:1280000; having no cross reaction with other sugars and having strong specificity; after being prepared into a detection reagent strip, a chemiluminescent immunoassay can be used to qualitatively detect the gram-positive bacterial peptidoglycan in a serum sample, thereby solving the problem of misdiagnosis, missed diagnosis and slow result generation due to high operation requirements and long cycle in blood culture to a certain extent;

[0049] It is of great help in the rapid identification of Gram-positive bacterial infections and greatly shortens the detection time; the test kit developed using this antibody as raw material has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Protein electrophoresis after purification of antibody CG2 and antibody EB8;

[0051] Figure 2 Ascites titer assay results of antibody CG2;

[0052] Figure 3 Ascites titer determination results of antibody EB8;

[0053] Figure 4 Results of cross-reactivity assay of antibody CG2;

[0054] Figure 5 Results of cross-reactivity assay of antibody EB8;

[0055] Biological materials: EB8, classified as hybridoma cells, 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: CGMCC No. 46010;

[0056] Biological material: CG2, 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. 46009. DETAILED DESCRIPTION

[0057] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0058] The present invention relates to a mouse anti-gram-positive bacterial peptidoglycan hybridoma cell line, a monoclonal antibody and an application. Two mouse anti-gram-positive bacterial peptidoglycan hybridoma cell lines were prepared and screened, one of which was named EB8, with a deposit number of CGMCC No. 46010, classified as a hybridoma cell, and deposited at the General Microbiology Center of China National Committee for the Preservation of Microorganisms and Cultures, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit time of June 27, 2024, and was detected as alive; the other was named CG2, with a deposit number of CGMCC No. 46009, classified as a hybridoma cell, and deposited at the General Microbiology Center of China National Committee for the Preservation of Microorganisms and Cultures, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit time of June 27, 2024, and was detected as alive. The present invention obtains hybridoma cell lines that stably secrete mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibodies through mouse hybridoma monoclonal antibody screening and RT-PCR cloning of Ig variable region genes, analyzes the variable region sequences, and identifies the antibody binding specificity using ELISA.

[0059] The antibody EB8 produced by the mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line EB8 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.

[0060] SEQ ID NO:1 KSSQSLLNSGNQKNYLT (CDRL1)

[0061] SEQ ID NO:2 WASTRES (CDRL2)

[0062] SEQ ID NO:3 QNDYSFPLT (CDRL3)

[0063] SEQ ID NO:4 GYTFTNYGMN (CDRH1)

[0064] SEQ ID NO:5 WINTYTGEPKYVDDFKG (CDRH2)

[0065] SEQ ID NO:6 LAFTTVVPTNAMDY(CDRH3)

[0066] The amino acid sequence of the light chain variable region of antibody EB8 is SEQ ID NO:7, and the nucleotide sequence encoding the light chain variable region of antibody EB8 is shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region 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;

[0067] SEQ ID NO:7

[0068] DIQMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQRPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISGVQAEDLAVYYCQNDYSFPLTFGAGTKLELK

[0069] SEQ ID NO:8

[0070] GACATTCAGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAGACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGG CATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGAATGATTATAGTTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0071] SEQ ID NO:9

[0072] GPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPKYVDDFKGRFAFSLETSANTAYLQINNLKNEDMATYFCARLAFTTVVPTNAMDYWGQGTSVTVS

[0073] SEQ ID NO:10

[0074] GGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAAAATATGTTGATGACTTCA AGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAACACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGACTCGCCTTTTACTACGGTAGTACCTACGAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC

[0075] The antibody CG2 produced by the mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line CG2 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;

[0076] SEQ ID NO:11 KSSQSLLNSGNQKNFLT (CDRL1)

[0077] SEQ ID NO:12 WASSRES(CDRL2)

[0078] SEQ ID NO:13 QNDYRFPLT(CDRL3)

[0079] SEQ ID NO:14 GYTFTNYGMN (CDRH1)

[0080] SEQ ID NO:15 WINTYTGEPKYVDDFKG (CDRH2)

[0081] SEQ ID NO:16 LAFITELPTNAMDY(CDRH3)

[0082] The amino acid sequence of the light chain variable region of antibody CG2 is SEQ ID NO: 17, and the nucleotide sequence encoding the light chain variable region of antibody CG2 is shown in SEQ ID NO: 18; the amino acid sequence of the heavy chain variable region 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;

[0083] SEQ ID NO:17

[0084] DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNFLTWYQQRPGQPPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISGVQAEDLSVYYCQNDYRFPLTFGAGTKLELK

[0085] SEQ ID NO:18

[0086] GACATTTGTCATGACCCAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTTCTTGACCTGGTACCAGCAGAGACCAGGGCAGCCTCCTAAATTGTTGATTTATTGGG CATCCAGTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCGGTGTGCAGGCTGAAGACCTGTCCGTTTTACTGTCAGAATGATTATAGATTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0087] SEQ ID NO:19

[0088] GPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPKYVDDFKGRFAFSLETSASTAYLQINNLKDEDMATYFCARLAFITELPTNAMDYWGQGTSVTVS

[0089] SEQ ID NO:20

[0090] GGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAAAATATGTTGATGACTTCA AGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAGATGAGGACATGGCTACATATTTCTGTGCAAGACTCGCCTTTTATTACGGAACTACCTACGAATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC

[0091] The obtained mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibodies were systematically evaluated and were respectively of mouse IgG1 subtype and mouse IgG1 subtype, and the light chain was a κ chain. The titer of both antibodies can reach 1:1280000, and both have better performance in sensitivity, specificity and stability, and are suitable as immunodiagnostic reagents for the preparation of in vitro diagnostic kits. In certain embodiments of the present invention, the two antibodies can be made into colloidal gold immunoassay kits, chemiluminescence kits, radioimmunoassay kits, enzyme-linked immunosorbent assay kits or fluorescent immunoassay kits, or made into microfluidic chips; the prepared kits can detect anti-Gram-positive bacterial peptidoglycan antigens.

[0092] When preparing a chemiluminescent immunoassay kit, antibody EB8 can be used in the magnetic bead coating and antibody CG2 can be used in the enzyme-labeled conjugate; or, antibody CG2 can be used in the magnetic bead coating and antibody EB8 can be used in the enzyme-labeled conjugate, both of which can achieve better detection results. The use of chemiluminescent immunoassay to qualitatively detect Gram-positive bacterial peptidoglycan in serum samples is of great help in the rapid identification of Gram-positive bacterial infections.

[0093] 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.

[0094] Example 1: Screening of mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell lines

[0095] 1.1 Antigen preparation

[0096] Select penicillin-sensitive Staphylococcus aureus, take a single colony of Staphylococcus aureus and inoculate it in broth culture medium, shake and culture at 37℃ until the logarithmic growth phase of bacteria, centrifuge, collect bacterial precipitate, and wash with PBS. Transfer the precipitate to basic culture medium, place it in a 37℃ constant temperature electric incubator, add penicillin solution, shake, sterilize at 95℃, centrifuge, collect supernatant; centrifuge the supernatant again, collect the supernatant, ultrafilter the supernatant: filter and concentrate on a rotary evaporator, and store in a refrigerator at 4℃ for later use;

[0097] Dextran G-100 gel column chromatography separation: take 30ml of the reserved concentrated sample, filter it with a 0.22μm filter membrane, take 20ml of the filtrate for sample loading, the flow rate is 3ml / min, the ultraviolet detection wavelength is 215nm, elute with triple-distilled water, and collect each peak separately; the first main peak obtained is rotary concentrated, freeze-dried and stored, which is the soluble peptidoglycan.

[0098] 1.2 Screening of mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell lines

[0099] Immunize female Balb / c mice about 6 weeks old with purified soluble peptidoglycan. The first immunization dose is 50ug / mouse, and the adjuvant is Freund's complete adjuvant, which is injected subcutaneously at multiple points. The interval between the second immunization is 2 weeks; the second immunization dose is 50ug / mouse, and the adjuvant is Freund's incomplete adjuvant, which is injected intraperitoneally. The interval between the third immunization is 2 weeks; the third and subsequent immunization doses, routes, and adjuvants are the same as the second immunization method, and the interval between the two immunizations is 2 weeks until the fourth immunization. Blood is collected from the tail vein of the mouse to determine the titer, and cell fusion is performed when the titer reaches 1:8000 or above.

[0100] Immunize 50ug sugar 3 days before fusion, no adjuvant is required. After blood is collected from the mouse eyeball, spleen cells of the successfully immunized mice are taken for cell fusion with myeloma SP2 / 0 cells (at a ratio of 10:1). During the fusion, 50% PEG is added to the spleen cells and myeloma cell clusters that are mixed and the supernatant is discarded in a 37°C water bath within 1 minute, shaken in a 37°C water bath for 1 minute, and then 10ml of serum-free 1640 medium is added within 2 minutes. Centrifuge at 800rpm for 6 minutes, discard the supernatant, resuspend the cells with 1640 medium containing HAT, and pipette into a 96-well plate (2.5×10 7 Cells were cultured at 37°C and 5% CO2. The medium was half-changed on the third day after fusion and fully changed on the seventh day.

[0101] When the clones in the fusion plate are large enough, take 100 μl of supernatant from each well for testing, the method is the same as the titer detection. The OD value of the negative well is twice as much as the positive well, and the next cloning culture is carried out. The hybridoma clones that are screened positive are expanded from the 96-well plate to the 24-well plate for 3-5 days, and the culture supernatant is screened again. The clones that are tested positive are then subcloned for the next step, and the remaining cells are frozen. The hybridoma cells in the 24-well plate were collected, and the cells were counted to adjust the cell density to 10 cells / mL. The cells were plated into 96-well plates, with 100 μl per well, and cultured in an incubator at 37°C and 5% CO2. After about 10 days of culture, clone formation was observed. The wells with only a single clone were selected, and the culture supernatant was aspirated. The detection method was the same as before, and the positive clones were selected and expanded to 24-well plates for culture. After the supernatant was tested again, the positive clones were selected for the second round of subclone culture. Generally, multiple rounds of subclone culture were performed until all the test wells were positive, and stable hybridoma cell lines were obtained. The hybridoma cell lines obtained by screening were named CG2 and EB8.

[0102] The positive hybridoma culture supernatant was selected and the antibody subtype detection kit was used to detect the antibody subtype. The monoclonal antibodies CG2 and EB8 produced by the hybridoma cell lines screened were respectively of mouse IgG1 subtype and mouse IgG1 subtype, and the light chain was κ chain.

[0103] Example 2: Preparation of mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibodies

[0104] 2.1 Ascites preparation and purification

[0105] Female Balb / c mice aged 10-12 weeks were intraperitoneally injected with sterile liquid paraffin (0.5 mL / mouse). After 7 days, hybridoma cells cultured to the logarithmic phase were intraperitoneally injected with 5×10 6 Cells / mouse. Observe every day. After about 7-10 days, when the mouse abdomen shows obvious bulge, 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 clear part in the middle, filter it with filter paper, package it, and store it at -70℃.

[0106] 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 at a flow rate of 8S / drop, repeat the loading 3 times, then wash with 15mL of washing buffer at a flow rate of 8S / drop, elute with 10mL of elution buffer, adjust the pH to 7.4 with 1M Tris PH=9, then concentrate with concentration syringe, dialyze overnight in 50kd dialysis bag, PBS, 4℃. 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. Electrophoresis was performed at 80 volts for 30min and 120 volts for 2h. After electrophoresis, the antibody was stained with Coomassie brilliant blue solution. Decolorization was performed by boiling in deionized water for 5min each time for a total of 3 times. The purified monoclonal antibody was identified by SDS-PAGE, and the bands were clear and free of mixed bands. Figure 1 As shown, monoclonal antibodies CG2 / EB8 both have clear bands at 50 KDa and 25 KDa.

[0107] 2.2 Monoclonal antibody titer detection

[0108] The antibody titer was determined by the indirect ELISA method. Gram-positive bacterial peptidoglycan was coated at 2ug / ml, 100ul / well. 1% BSA was used as the blocking solution. The dilution was performed from 1:1000, with a total of 12 dilution gradients. The Promega enzyme-labeled antibody was used as the secondary antibody after dilution of 1:20000. The OD was read at 450nm. At the same time, the uncoated group was set as a control. The OD value when the antibody was diluted 1028000 times was more than 2.1 times that of the control group, indicating that the antibody titer had reached 1:1280000. After purification, the antibody was diluted to 1mg / ml, and the titer reached more than 1:1280000. The titer determination results are as follows Figure 2 Figure 3 shown.

[0109] 2.3 Cross-reactivity with other polysaccharides

[0110] The cross-reactivity of monoclonal antibodies with other sugars was detected by indirect ELISA. Cryptococcal capsular polysaccharide, Candida mannan, fungal (1-3)-β-D glucan, lipopolysaccharide, and Aspergillus galactomannan were coated at a concentration of 2ug / ml. The purified antibody was used as the primary antibody, and the Promega anti-mouse enzyme-labeled antibody was used as the secondary antibody. The results are shown in Figure 4 Figure 5It can be seen that antibody CG2 and antibody EB8 have no cross-reaction with other sugars, indicating that the two antibodies have strong specificity.

[0111] Example 3: Genetic verification of mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibodies

[0112] The Ig variable region gene was cloned by RT-PCR.

[0113] 1) Total RNA extraction and single-stranded cDNA synthesis:

[0114] The total RNA of 22CG2 and 27EB8 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).

[0115] Heavy chain backbone region upstream primer

[0116] SEQ ID NO:21 P1:

[0117] 5'SAGGTGGMAGCTKCASSARTCWGG3' (SEQ ID NO:21)

[0118] Heavy chain variable region downstream primer

[0119] SEQ ID NO:22 P2:

[0120] 5'TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3' (SEQ ID NO:22)

[0121] Light chain leader peptide upstream primer

[0122] SEQ ID NO:23 P3:

[0123] 5'ATGGATTTTCAAGTGCAGATTTTCAG3' (SEQ ID NO:23)

[0124] Light chain variable region downstream primer

[0125] SEQ ID NO:24 P4:

[0126] 5'GGATACAGTTGGTGCAGCATCAGCCCGTTT3' (SEQ ID NO:24)

[0127] Prepare PCR reaction system (50 μl) as follows:

[0128] cDNA: 2 μl; upstream primer (10 μM): 2 μl; downstream primer (10 μM): 2 μl; dNTP mixture: 2 μl; pfu DNA polymerase (5 U / μl): 1 μl; 10 X pfu Buffer Ⅱ: 5 μl; ddH2O: make up to 50 μl.

[0129] 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.

[0130] 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:

[0131] 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 4°C overnight.

[0132] 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:

[0133] 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.

[0134] 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 CG2 and EB8 were successfully cloned and matched the characteristics of typical antibody variable region sequences.

[0135] Example 4: Preparation and application of a kit for determining the peptidoglycan of gram-positive bacteria (chemiluminescent immunoassay)

[0136] The kit for detecting Gram-positive bacterial peptidoglycan comprises the mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibodies CG2 and EB8 prepared in Example 3, and is mainly used to detect Gram-positive bacterial peptidoglycan in human serum, which is of great significance for the early rapid detection of Gram-positive bacterial infection. Based on the antibody, a Gram-positive bacterial peptidoglycan determination kit (chemiluminescent immunoassay) was developed through the chemiluminescent technology and immunoassay sandwich principle, and clinical sample testing was carried out.

[0137] The preparation process of the kit is as follows:

[0138] 1. Preparation of magnetic bead coating

[0139] Use 0.1mg / mL EDAC solution to activate carboxylated magnetic beads, add anti-gram-positive bacterial peptidoglycan monoclonal antibody BE8, the scale is 2mg / mL, label for 1h, block for 1h, wash 3 times, precipitate with coupling buffer to 4 times the volume of magnetic beads added. Use PBS to dilute the magnetic bead-coated antibody 10 times, then add Tris powder to make the final concentration 0.1M, mix, package, and freeze-dry.

[0140] 2. Preparation of enzyme-labeled conjugate

[0141] Sodium periodate oxidation was used to couple horseradish peroxidase (HRP) with the anti-Gram-positive bacterial peptidoglycan monoclonal antibody CG2. First, 10 mg of HRP was weighed, dissolved in deionized water, and 10 mM sodium periodate solution was added. The reaction was carried out at 4 ° C in the dark for 30 minutes. After ethylene glycol was added to terminate, 10 mg of the antibody was added, and it was placed in a 50KD dialysis bag. It was reacted in a potassium carbonate solution with pH = 9.6 for 6 hours. After taking it out, sodium borohydride was added. After reacting for 2 hours, saturated ammonium sulfate was slowly added for precipitation. After the precipitation was allowed to stand for 1 hour, it was centrifuged at high speed, and the supernatant was discarded. The precipitate was resuspended with phosphate buffer, dialyzed with PBS overnight, and taken out the next day. After high-speed centrifugation, the supernatant was discarded, and an equal volume of glycerol was added and stored at -20 ° C. When used, the HRP-labeled anti-Gram-positive bacterial peptidoglycan monoclonal antibody was diluted 1000 times with HRP antibody diluent and packaged.

[0142] 3. Assemble the reagent strips

[0143] Assemble the components according to regulations to prepare a reagent strip.

[0144] 4. Clinical sample testing

[0145] The Gram-positive bacteria peptidoglycan assay kit (chemiluminescent immunoassay) was compared with clinical blood culture samples, 196 clinical samples were measured, and consistency analysis was performed. The results showed that the prepared reagent strip had a sensitivity of 90.16%, a specificity of 95.56%, an overall compliance rate of 93.88%, and a Kappa value of 0.86.

[0146] This method uses chemiluminescent immunoassay to qualitatively detect Gram-positive bacterial peptidoglycan in serum samples, which can to a certain extent solve the problems of misdiagnosis, missed diagnosis and slow results caused by blood culture due to high operating requirements and long cycle. It is of great help in the rapid identification of Gram-positive bacterial infection and greatly shortens the detection time. The kit developed with this antibody as raw material has good clinical application value.

[0147] 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-Gram-positive bacterial peptidoglycan hybridoma cell line, characterized in that: Named as EB8, with the deposit number CGMCC No.46010; or, named as CG2, with the deposit number CGMCC No.46009.

2. Mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibody, characterized in that: The antibody EB8 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; or, Antibody CG2 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-Gram-positive bacterial peptidoglycan monoclonal antibody according to claim 2, characterized in that: The amino acid sequence of the light chain variable region of antibody EB8 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 CG2 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-Gram-positive bacterial peptidoglycan monoclonal antibody according to claim 3, characterized in that: Antibody EB8 is produced by the mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line with the deposit number CGMCC No.46010; The antibody CG2 is produced by a mouse anti-Gram-positive bacterial peptidoglycan hybridoma cell line with a deposit number of CGMCC No.46009.

5. A nucleic acid molecule, characterized in that: The invention comprises a polynucleotide encoding the mouse anti-Gram-positive bacterial peptidoglycan 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 variable region of the light chain of antibody EB8 is shown in SEQ ID NO:8, and the nucleotide sequence encoding the variable region of the heavy chain of antibody EB8 is shown in SEQ ID NO:10; The nucleotide sequence encoding the light chain variable region of antibody CG2 is shown in SEQ ID NO:18, and the nucleotide sequence encoding the heavy chain variable region of antibody CG2 is shown in SEQ ID NO:

20.

7. A kit for determining peptidoglycan of gram-positive bacteria, characterized in that: The invention comprises the mouse anti-Gram-positive bacterial peptidoglycan monoclonal antibody as described in any one of claims 2 to 4.

8. The Gram-positive bacteria peptidoglycan assay 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 Gram-positive bacteria peptidoglycan assay kit according to claim 7, characterized in that: Antibody EB8 was used in magnetic bead coatings, and antibody CG2 was used in enzyme-labeled conjugates; Alternatively, antibody CG2 can be used in magnetic bead coatings and antibody EB8 can be used in enzyme-labeled conjugates.

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