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Preparation of Monoclonal Antibodies Anainst Chloramphenicol and One-Step Chemiluminescence Enzyme Immunoassay for Chloramphenicol

Author: ZhangQiMo
Tutor: WuJianMin
School: Guangxi University
Course: Preventive Veterinary Medicine
Keywords: chloramphenicol residue detection monoclonal antibodies chemiluminescence enzyme immunoassay
CLC: S852.43
Type: Master's thesis
Year: 2011
Downloads: 47
Quote: 1
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Abstract


As an effective broad-spectrum antibiotic, Chloramphenicol(CAP) was once widely used in animal husbandry. Yet, because of its toxic and side effects, it has been baned in the use of food-producing animals by many countries. In order to monitor and reduce the potential incidence of CAP residues in the food chain, a effective, sensitive and specific method to detect CAP is of primary importance. Chemiluminescence immunoassay, containing high sensitivity of the chemiluminescence measuring technique and high specificity in immune repsonse, applys to all the clinical examination items for enzyme immunity analysis and radioimmunoassay, and has become a promising improved subsittute for these two techniques.BALB/c mice were immunized with CAP-HS-BSA. After the third immunization, the serum titer of the mice were detected by indirect ELISA, getting a result of 1:1.28×105, indicating good immunity in mice. Serum with the highest titre were used to detect the sensitivity which can be reflected by 50% inhibiting concentration, and the result is between 100μg/L and 1000μg/L. The mice’s immune spleen cells with the highest titre were fused with Sp2/0 cells by cytomixis technique. And the fusion rate was 22.9%. A hybridona cell line named 5D7 secreting monoclonal antibody against chloramphenicol(CAP) was established after screening the positive supernatant of cells using indirect ELISA. After 3 times-subclones, the hybridoma was stable.The supernatant of hybridoma cell line was used to detect the titres, in which the supernatant titre was 1:512 and the subtype was an IgG1(κ). Ascitic fluids in mice was produced to characterize the immunological traits of the McAb such as titer, affinity and specificity. The results showd that the ELISA titers of ascites were 1:1.2×105 and the affinity constant(Ka) was 3.6×108 L/mol. Besides, the McAb has no cross-reactivity with other antibiotics.The McAb of 5D7 was applied to establish the one-step chemiluminescence immunoassay(CLIA) for detecting chloramphenicol, and experiment parameters such as the antigen coating conditions, working concentration of the antibody and the competitive reaction time were optimized, from which the equation of calibration curve derived was y=-12.188x+62.988 with R2=0.9909 and IC50=1.066μg/L. The assay showed good linearity between 0.001 to 10μg/L.The lowest detection limit was 11.63μg/L, and the average recovery was 95.61～108.46%. Compared to the regular two-step approach, this method shortened the reaction time by two hours, better meeting the demands of rapid detection.In this study, we successfully established one-step indirect competitive CLEIA for the detection of chloramphenicol on the base of preparation of monoclonal antibody with high affinity, sensitivity and specificity. And this method can be applied to the detection of chloramphenicol in fishery products in our country. It will be used for the development of CLEIA kits for chloramphenicol.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Immunology > Antigen and antibody
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