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Study on the Anti-Aflatoxin M1 Monoclonal Antibody Preparation by HTS-ELISA Method

Author: HeNa
Tutor: PeiShiChun
School: Heilongjiang Bayi Agricultural University
Course: Of Food Science
Keywords: High-throughput enzyme screening Aflatoxin M1 Monoclonal antibodies
CLC: R155.5
Type: Master's thesis
Year: 2011
Downloads: 39
Quote: 0
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Abstract


The the aflatoxin M 1 the aflatoxin B 1 animals oxidase by hydroxylation formed, the toxin has a strong carcinogenic, teratogenic well as mutagenicity, trace amounts of this toxin in dairy, due Aflatoxin M 1 temperature capability, so in the processing of dairy products can not be directly removed by a high temperature sterilization process, so most of the countries in the world using a high-sensitivity detection method to monitor milk and dairy products the aflatoxin M 1 pollution levels, but there has not yet found that can completely replace the foreign advanced rapid detection of aflatoxin M 1 related products, mainly due to unresolved domestic secreting anti the aflatoxin M 1 antibody screening of hybridoma cell lines and related technical field there are some bottlenecks . In this study the aflatoxin M of purchased from Sigma immune antigen 1 and bovine serum albumin conjugates were immunized three on six weeks 齢 Balb / C mice after the third immunization titer 1:32 000 or more, take the highest titer Three mice after fusion, high-throughput screening, using single-cell micromanipulation technique a single cell colony isolated, and ultimately get 21 hybridoma cell lines Department, whichever the strongest affinity 33-B-04 carried out the biological activity test, and the heavy chain of the antibody-secreting hybridoma derived Isoform kit identification was IgG2b type, the strains of hybridoma cells to secrete The antibody light chain λ chain, respectively, using the mouse ascites preparation method, and a large number of serum-free culture method is the production of antibodies, and then serum-free cell culture supernatant by protein G ceramic beads for antibody purification by polyacrylamide gel electrophoresis Identification of the target antibody purity above 95%, the concentration of the purified antibody protein 5.14mg/mL when the antibody titer of 5000 × 29, the obtained anti-aflatoxin M 1 mAb affinity for 5.5 × 10 1 (0) M -1 50 generations of continuous culture measured antibody titers remained stable. Using high-throughput screening method can be prepared with a high affinity anti-aflatoxin M 1 monoclonal antibody, available for the detection of aflatoxin M 1 of the immune system to establish good antibodies material. The subject prepared strongest affinity antibody indirect competitive enzyme-linked immunosorbent assay method to establish the optimal reaction conditions: 0.02μg/mL AFM 1 -BSA at 4 ℃ 12h; 5% nonfat dry milk closed at 37 ℃ for 2 hours; 1:60 000 dilution IgG-HRP incubated for 1h at 37 ℃; using TMB 15min at 37 ℃ in the dark color; competitive mode to take board competition. The minimum detection limit for the eventual establishment of indirect competition detection the aflatoxin M 1 method 0.01ng/mL, the best detection range of 0.1 1 0ng/mL, accuracy ranging from 100% to 118%, the law and AFM 1 the the a similar structure AFB 1 AFG1 cross-reaction rates were 21.5%, 16.6%, and other structural analogues The cross-reactivity of less than 5%. The uniformity of the the indirect competition detection method established in this study, precision and stability are good, and has great application prospects.

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CLC: > Medicine, health > Preventive Medicine,Health > Nutrition, hygiene,food hygiene > Food hygiene and food inspection > Food hygiene and inspection
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