| Penicillins, one important type of Beta-lactam antibiotics, are used commonly in the veterinary clinic because of their high efficiency and low toxicity. They are used so extensively that the penicillins residues in animal food become much more serious. Experimental studies and anecdotal evidence have shown that penicillins don't possess serious toxicity to organisms, but they can cause the allergic reactions easily, with the strongest ability of causing the allergic reactions in all of the allergic medicines. If these penicillins are taken in for a long time in low dosage, they will produce the properties including development of bacterial resistance, maladjustment of flora, low immunity of organism and substantial financial losses in the industry of animal food. Therefore, establishing the rapid detection system of penicillins residues and developing the kit with reasonable price will play a significant role in the controlling of the sanitation and quality of animal food and ensuring the consumers' healthy.In this study, the hapten ampicillin and penicillin-G were linked to the carrier protein Albumin Bovine Serum (BSA) by physiological binding and glutaraldehyde binding methods, and three immunogens were obtained. The successful coupling of the haptens and the BSA were proved by ultraviolet scan, infrared spectrogram scan and SDS-PAGE electrophoresis. Then three envelope antigens were synthesized with carrier protein Albumin Chicken egg (OVA) likewise. The three immunogens were used in order to immunize nine New Zealand Rabbits. After six times of booster injection, eight rabbits produced high titre polyclonal antibodies serum. When the three envelope antigens were used to detect the polyclonal antibodies serum, the results showed the immunized rabbits with immunogens by two synthesis methods both could produce specific antibodies against the penicillin group but the effect of the immunogens synthesized by the physiological binding was better than that synthesized by the glutaraldehyde binding comparatively, and immunogen synthesized by the physiological binding with hapten ampicillin was better than that with penicillin-G. Immune rabbits' spleens were chosen which had polyclonal antibodies with high titre and good affinity. Then they were maintained carefully in liquid nitrogen and would be used for the preparation of monoclonal antibodies.Due to the lack of time, mouse monoclonal antibodies accordant with the experiment were obtained from overseas co-operation corporation and they were prepared using the physiological binding. Relevant envelope antigens through the improved sodium periodate reacted with horseradish peroxidase prior to purification by ammonium sulfate sedimentation and then enzyme-labelled antigens were obtained. Mouse monoclonal antibodies, envelope antigens, enzyme-labelled secondary antibodies were used in order to establish the indirect competitive ELISA detection, and mouse monoclonal antibodies, enzyme-labelled antigens were used to establish the direct competitive ELISA detection. Competitive haptens were opened loops selectively in order to enhance the affinity of the anti-monoclonal antibodies. Considering the factors including sensitivity, linear range, detection process and detection time, direct competitive ELISA which was reasonable sensitivity and convenience was decided as the detection method for the kits products.After the analysis on mono-factor remarkably and optimization through orthogonal tests about certain ELISA detection method the condition of zymolyte were decided as followings: lOmL Buffer, 450 uL TMB, 2 uL H2O2 and 5% glycin was used as confining liquid;the dilution ratio of mouse monoclonal antibodies and enzyme-labelled antigens were 1: 800 and 1: 80 respectively with phosphatic buffer (PBS, 0.01M pH7.4);the antigens were placed in the refrigerator at 4°C over night to be enveloped;the mixture was incubated at 37°C for 2.5 h to be confined;the reaction condition was that the mixture was incubated at 37°C for 1.0 h;the mixture reacted at 37°C for 10 min to color develop and it could achieve the best coloration effect. For the lowest detection limits of penicillins detected by the optimized direct competitive ELISA were at 1.58ng/mL (ampicillin), 0.38ng/mL (penicillin G), 1.90ng/mL (amoxicillin), 0.13ng/mL (cloxacillin) and 0.44ng/mL (oxacillin). All these results achieved the detection standards of penicillins residues in food.It would cost less than 1.5 h to detect the samples with this kit. Under multiple repeated tests and averaging, a calibration curve with equation: Y=0.327X+0.2419 {X=lg (the concentration of penicillins (ng/mL)),Y was the inhibition rate} was constructed using buffer solutions with the lowest detection limit at 0.368 ng/mL and within the detection range of 0.5-100 ng/mL. The detection of urine specimen and milk were diluted for 10 multiples and refered to this equation. The detected samples recovery rate ranged from 70% to 120%. In consideration of the serious penicillins-residues in milk, a calibration curve with equation: Y=0.3288X+0.1666 {X=lg (the concentration of penicillins (ng/mL) ),Y was theinhibition rate} was constructed using 10% skimmed miJk powder solutions with the lowest detection limit at 0.627 ng/mL and within the detection range of 1.0~100 ng/mL. The milk at original concentration could refer to this equation and the detected milk samples recovery rate range from 70% to 120%. |
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