At present, in China's dairy farming industry, β-lactam antibiotics such as penicillin and cephalosporin play an important role in the control of diseases such as cow mastitis. Because some dairy farming merchants abuse antibiotics and do not strictly adhere to the drug withdrawal period, the antibiotic residues in milk exceed the standard, and the residual antibiotics in the milk seriously endanger food safety and human health. The Chinese government implements standard controls on raw milk that exceeds a certain limit of antibiotics. Most domestic dairy companies have adopted price cuts or no acquisitions for milk with excessive antibiotic residues, while some illegal milk stations use some biological agents to degrade residual antibiotics in milk to produce artificial "no anti-milk". After investigation, it is preliminarily judged that the main component of the commercially available biological preparation is β-lactamase, which is a family of enzymes composed of various enzymes produced and secreted by Gram-positive bacteria, and the relative molecular mass is between 28 and 32 ku. Selective decomposition of residual β-lactam antibiotics in milk. The β-lactamase is capable of destroying the penicillin lactam structure and rendering it inactive. The use of this enzyme masks the antibiotics actually contained in the milk. In 2007, Cui Shenghui and others collected 38 different types of milk samples produced by 5 manufacturers in Beijing retail supermarkets, of which 63.2% detected β-lactamase. Β-lactamase is one of the banned food additives in China, which can lead to increased resistance of antibiotics such as penicillin and cephalosporin, thereby greatly reducing people's ability to resist infectious diseases and harming consumers' physical and mental health. . In the list of illegally added non-edible substances in food and feed announced in China in 2009 and 2011, β-lactamase (Jinyulan enzyme preparation) was listed twice, and the detection method was determined by liquid chromatography. The detection methods of β-lactamase in raw milk include microbial cup and saucer method, rapid kit method, liquid chromatography and double flow enzyme-linked immunosorbent assay. The microbial cup and saucer method is inexpensive. The kit method and the enzyme-linked immunosorbent assay are simple in operation, fast in speed and specific in accordance with the basic characteristics of the antigen-antibody specific reaction. Liquid chromatography has the characteristics of short operating time, high sensitivity and accuracy, but the required experimental instruments are required. The polyclonal antibody and monoclonal antibody of β-lactamase were prepared in this study, and it is proposed to lay the foundation for the subsequent development of colloidal gold immunochromatographic test strip by double-anti-sandwich method. 1 Materials and methods 1.1 Materials and reagents Experimental animals: Japanese big-eared rabbits (female, two months old), Beijing Jinmuyang Experimental Animal Breeding Co., Ltd.; Balb/C mice (female, SPF, six weeks old), Kunming mice (18-22 g), purchased From the Experimental Animal Center of the Academy of Military Medical Sciences of the People's Liberation Army. Experimental reagents: β-lactamase, Sigma; penicillinase, China National Institute for the Control of Pharmaceutical and Biological Products; bovine serum albumin (BSA), skim milk powder, Beijing Jingri Jindian Technology Co., Ltd.; one-component TMB coloring solution, Beijing Suo Laibao Technology Co., Ltd.; horseradish peroxidase-labeled goat anti-rabbit IgG, horseradish peroxidase-labeled goat anti-mouse IgG, Beijing Youboao Biotechnology Co., Ltd.; HAT, HT, PEG2000, Freund's complete Adjuvant, Freund's incomplete adjuvant, Sigma; mouse SP2/0 cells, preserved in the laboratory; DMEM low sugar medium, GIBCO company; mouse monoclonal antibody subtype kit, Luoyang Saierwei experimental equipment limited the company. Instruments and equipment: 96-well polystyrene plate, cell culture plate, JETBIOFIL; enzyme-linked immunosorbent assay, gel imager, BIO-RAD; UV spectrophotometer, UNICO. 1.2 Selection of immune antigens The molecular mass and purity of both β-lactamase and penicillinase proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separation gel is 10%, the laminating glue is 4%, and the running buffer is glycine. 1.3 Preparation of polyclonal antibodies Three Japanese big ears rabbits were immunized with β-lactamase. For each rabbit, take 700 μg β-lactamase, dilute to 500 μL with 0.1 mol/LTris-HCL at pH 7, mix with an equal volume of Freund's complete adjuvant, fully emulsified and subcutaneously and bilaterally groin on the back of the rabbit. Point injection. Subsequent booster immunization was performed with the same dose of immunogen and an equal volume of Freund's incomplete adjuvant, and multiple injections were made subcutaneously at the back, with each immunization interval of two weeks. From the third immunization, blood was collected from the rabbit ear vein after 4 days of immunization, and serum was separated to detect the immune effect. Blood was collected from the heart 3 days after the fifth release, and serum was separated. 1.4 Establishment of ELISA screening method 1.4.1 Determination of the original concentration of the coating using the square matrix method, the 96-well microtiter plate was laterally coated with a series of diluted penicillinase, and the primary antibody diluted by the ratio was added longitudinally, and the best antigen coating was determined by indirect ELISA. concentration. 1.4.2 Determination of coating conditions Select 4°Covernight, 37°C1h, 37°C2h3 methods to determine the optimal coating conditions. 1.4.3 Selection of blocking solution 0.2% gelatin, 5% skim milk powder, 3% goat serum, 5% calf serum, 0.75% milk powder + 2% BSA + 5% sucrose, 3% BSA + 0.05% Tween + 3 The 7% blocking solution of % sucrose, 5% milk powder + 0.05% Tween + 3% sucrose was closed to determine the best blocking solution. 1.4.4 Optimization of the blocking time The blocking time was chosen to be 0.5, 1, 2 h, and the optimal blocking time was determined by indirect ELISA. 1.5 Preparation of monoclonal antibodies 1.5.1 Animal Immunization 9 female Balb/C mice were divided into 3 groups, and the high, medium and low dose groups were each immunized at 200, 100 and 50 μg/head each. The first dose of β-lactamase was firstly immunized, dissolved in 50 μl of 0.1 mol/L Tris-HCL (pH 7), and fully emulsified in an equal volume of Freund's complete adjuvant. Two weeks later, the same dose of immunogen was emulsified with Freund's incomplete adjuvant for the second immunization, and subcutaneously injected into the abdominal cavity. First After 3 immunizations, the titer of mouse antiserum was measured by ELISA. The mice with the highest titer were selected for fusion in the three groups, and the same dose of antigen solution was intraperitoneally injected for the first 3 days of fusion for booster immunization. 1.5.2 Culture of SP2/0 myeloma cells Three generations of SP2/0 myeloma cells cultured in vitro were treated with 8-azaguanine (20 μg/mL), and two of the back of Balb/C mice were injected subcutaneously in logarithmic growth phase. On the side, each mouse was injected with 5 x 105 to 1 x 106 cells. When the diameter of the solid tumor is as long as 2 to 3 cm, it is aseptically anatomically extracted, filtered through a 200-mesh sieve, and re-cultured until the cells grow stably. 1.5.3 Cell fusion The cultured feeder cells were cultured in Kunming rats 1 day before fusion. SP2/0 myeloma cells in logarithmic growth phase and spleen cells of immunized mice were fused with 50% PEG2000 according to a conventional method, and the culture supernatant was detected by indirect ELISA using established penicillinase. Strong positive wells were selected and serially subcloned 4 to 5 times by limiting dilution until the cloned wells secreted 100% of the anti-beta-lactamase antibody. The established hybridoma cell strain stably and efficiently secreted with anti-β-lactamase monoclonal antibody was expanded and cultured, and the hybridoma cell strain which stably secreted the antibody was still transmitted after 3 generations of cryopreservation and resuscitation, and then placed in liquid nitrogen. save. 1.5.4 Preparation of ascites antibodies Ascites was prepared by in vivo induction. Balb/C mice were intraperitoneally injected with 0.5 mL of sterile paraffin oil, and two weeks later, 5×105/β of hybridoma cells resistant to β-lactamase were injected. After 1 week, the peritoneal fluid of the mice was collected. 1.6 Characterization of antibodies The titer of polyclonal antibody and polyclonal antibody in ascites was determined by indirect ELISA. The polyclonal antibody was negatively controlled with blank serum, and the monoclonal antibody was negatively treated with SP2/0 myeloma cell culture supernatant. The affinity of the monoclonal antibody was determined by an enzyme immunoassay; the hybridoma cell line was subtyped using a subtype identification kit. Hybridoma cell lines were subjected to chromosome analysis using colchicine lysis method. The molecular mass and specificity of the monoclonal antibodies were determined using Western blotting. 1.7 Antibody purification 1.7.1 Purification of polyclonal antibodies The rabbit serum of anti-β-lactamase was purified by caprylic acid-saturated ammonium sulfate method, and the final volume fraction of saturated ammonium sulfate was 50% and 40%, respectively. The precipitate was dissolved in a small amount of PBS, transferred to a dialysis bag, dialyzed against a PBS solution for 48 hours, and the dialysate was detected by ultraviolet analysis until the dialysis was completed. 1.7.2 Purification of monoclonal antibody The ascites of Balb/C mice was purified by PEG6000 precipitation method. The precipitate was dissolved in 50mmol/LTris-HCL solution (pH 8.0), and the protein was removed in ice bath to obtain crude monoclonal antibody. The purity of the antibody was identified by SDS-PAGE. 2 Results and analysis 2.1 Immunogen The molecular mass and purity of the two proteins are analyzed by SDS-PAGE analysis of β-lactamase and penicillinase. Both proteins showed significant protein expression at a molecular mass of 30 ku, and the molecular mass data were in line with expectations. The β-lactamase was analyzed by SDS-PAGE and the purity was 100%, which was consistent with the specification. Penicillinase was analyzed by Bandscan software with a purity of 61.4% and contained a large amount of heteroprotein. A β-lactamase of better purity is used as the immunogen. 2.2 rabbit anti-β-lactamase polyclonal antibody titer After 4 exemptions, the titers of 3 Japanese big-eared rabbits were 1×106, 2×106, and 1×106, respectively (see Figure 2). Among them, No. 2 big ear rabbit had the highest titer, and the serum of No. 2 big ear rabbit was selected for ELISA optimization experiment. 2.3 ELISA screening method 2.3.1 The original concentration of the coating was square matrix method, and the 96-well microtiter plate was laterally coated with a series of diluted penicillinase, and the primary antibody diluted by the dilution was added longitudinally (indicated by "+"), and each group was added with 50 μg/ The penicillinase in mL was tested for inhibition (indicated by "-") and the optimal antigen coating concentration was determined using an indirect ELISA. When the original mass concentration was 5 μg/mL and the rabbit serum concentration was 1:64000, the OD450 was close to 1, and the inhibitory effect was the best. 5 μg/mL penicillinase was used as the coating. 2.3.2 Coating conditions were selected by 4°Covernight, 37°C1h, 37°C 2h in three ways to determine the optimal coating conditions. Based on the ratio of P/N, it is determined that 4°Covernight is the optimum coating concentration. 2.3.3 Penicillinase has a complex structure and contains heteroproteins. Among the 7 kinds of blocking solutions, 3% BSA + 0.05% Tween + 3% sucrose was used for blocking, the specificity was the best, and the blocking effect was the best. 2.3.4 Blocking time The indirect ELISA was used to determine the optimal blocking time. When the sealing time is 2h, the negative value is lower, the P/N ratio is the largest, and the sealing effect is the best. 2.4 Preparation of monoclonal antibodies 2.4.1 Immunization effect of BALB/C mice From the results of immunization, the low titer group (50 μg/mouse) of Balb/C mice had the highest antibody titer of 1:128,000. At the same time, the mental state of Balb/C mice was compared. The Balb/C mice in the low-dose group were more active and brighter than the middle and high-dose groups. Fusion was performed using the low dose group of Balb/C mouse spleens. 2.4.2 After cell screening and subcloning cell fusion, the fusion rate was 80% and the positive rate was 20.5%. After 4 subclones, the positive rate was 100%. Three positive cell lines stably secreting anti-β-lactamase antibodies were screened and named as C1, D1 and F8. After continuous passage of F8 for 3 weeks and cryopreservation, the ELISA titer was tested and the results were basically unchanged, indicating that the obtained hybridoma cell strain was stable. 2.5 Antibody characterization 2.5.1 Determination of titer and affinity of monoclonal antibody in ascites of mice The supernatant titer (1:32) to (1:512) of serum-free medium of 3 hybridoma cells was detected by indirect ELISA. Three hybridoma cells were intraperitoneally injected to obtain ascites, and the titer of F8 ascites was the highest among the three hybridoma cell lines. F8 was selected for follow-up experiments. The assay was performed using a non-competitive enzyme immunoassay. Calculated by the affinity constant formula, the affinity of F8 is 6.4×107 L/mol. 2.5.2 Identification of three hybridoma cell subtypes Using the monoclonal antibody subtype identification kit, the selected three monoclonal antibodies C1, D1, and F8 were subtyped, and the three hybridoma cells belonged to IgM. Subtype. 2.5.3 Analysis of the chromosomes of F8 hybridoma cell lines The total number of 100 bodies is basically the same (the number of chromosomes in SP2/0 cells is known to be in the middle of mitosis, and the number of chromosomes is 62 to 68, and the spleen cells of Balb/C mice. The number of chromosomes is 96-104 (Fig. 7), and 40 staining with SP2/0 cells and spleen cells). 2.5.4 Western-Blotting detection of F8 specific Western blotting identification showed that F8 ascites can specifically react with β-lactamase and penicillinase (Fig. 8), indicating that F8 is specific for β-lactamase and penicillinase. Sexual antibodies. Purification of 2.6 F8 hybridoma cell line monoclonal antibody Compared with the octanoic acid-cold alcohol method, the octanoic acid-saturated ammonium sulfate method and the PEG60003 method, the monoclonal antibody purified by the PEG6000 method is not high in purity, but the antibody titer is best preserved. 3 Discussion Metal β-lactamases are a class of β-lactamases that depend on divalent metal cations, which are widely hydrolyzed (including carbapenems, broad-spectrum cephalosporins, etc.) and various β-lactam antibiotics. It is not sensitive to commonly used β-lactamase inhibitors (such as clavulanic acid, sulbactam, etc.). According to molecular biology, Bush divided the 12 metalloenzymes with sequence data into three subgroups in 1997: B1, B2, B3. Most metalloenzymes belong to the B1 subgroup, and the molecular weight of the various enzymes of this subgroup is similar (about 28 ku), and the sequence identity is greater than 23%. The B2 subpopulation is similar in size to the B1 subpopulation but has lower homology. Only 9 amino acids in the B3 subgroup are significantly different from other metalloenzymes, and the molecular mass of the monomer is 31.7 ku. Although the amino acid sequence homology between the subgroups of metalloenzymes is not high, the steric structure of the enzyme protein formation is very similar, and the key residues in the metalloenzyme, especially the amino acid residues forming the metal bond, are highly conserved. In view of the structural characteristics of β-lactamase, a β-lactamase of better purity is selected as an immunogen. In order to allow the selected monoclonal antibody to better recognize the β-lactamase, the penicillinase of the laboratory was used as the coating. At present, most of the commercially available β-lactamase test colloidal gold test strips are indirect. The indirect method uses the principle of β-lactamase to digest penicillin, and mixes the milk sample with a trace amount of penicillin. If a certain concentration of β-lactamase is present in the milk, the concentration of penicillin is reduced after enzymatic hydrolysis by β-lactamase. The test strip is used to detect penicillin to determine whether β-lactamase is present in the milk, but the sample to be tested may be subjected to multiple centrifugation, incubation, etc., and then tested using a test strip. In this study, monoclonal antibodies and polyclonal antibodies against β-lactamase were developed, and the double-anti-sandwich method was used to directly detect the colloidal gold test strip. The advantage of this method is to reduce the reaction time and optimize the reaction steps. In addition, bacterial resistance can also produce endogenous β-lactams. There are two main sources of endogenous β-lactamase in milk: one is produced by the bovine body itself, and the other is that some resistant microorganisms infected by the bovine itself continuously secrete and express β-lactamase. There is no report on whether the β-lactamase contained in the product is artificially added or resistant to bacteria, which is a new problem in regulating β-lactamase. Only strengthening the management of the whole process of animal husbandry and dairy production and improving the safety responsibility of the farmers is the basis for eliminating antibiotic residues and ensuring the safety of milk and dairy products.
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Preparation of β-lactamase monoclonal antibody and polyclonal antibody
Preparation of β-lactamase monoclonal antibody and polyclonal antibody