Study On The Anti-Aflatoxin M₁ Monoclonal Antibody Preparation By HTS-ELISA Method

Aflatoxin M₁ is the hydroxylated metabolites of Aflatoxin B₁, because animals are fed with contaminated feeds by aflatoxin B₁. Aflatoxin M₁ is known to be cancerigenic, teratogenic and mutagenic and may be found in the milk with trace. Aflatoxin M₁ is difficult to remove due to its standing high temperature at the processing of milk. Therefore, the high sensitive detection method is used to monitor the polluted condition of aflatoxin M₁ by most countries around the world. It's a pity that there are not any advanced products of detecting aflatoxin M₁ instead of abroad detection kits in our countury. The technology of producting anti-AFM₁ monoclonal antibody has some problems to slove, which is the major reason,Three six-week Balb/C mice were immunized by aflatoxin M₁-bovine serum albumin from sigma. The titer of the mouse was beyond thirty-two thousand after the third immunity. The mouse of the number three which has the highest titer was fused and screened. The single clonal cell was segregated by the technology of sing-cell micromanipulation. Finally, twenty-one hybridoma cell lines which secrete anti-aflatoxin M₁ monoclonal antibody were selected. The heavy chain subtype of monoclonal antibody with the highest affinity which called 33-B-04 is IgG2b and the light chain isλ. The monoclonal antibody was produced ascites and cultivation without blood serum. The monoclonal antibody against aflatoxin M₁ was purified by ceramic bead of protein G. When the concentration of the protein is 5.14 milligram per milliliter, the titer of the putified monoclonal antibody against aflatoxin M₁ was 5000×29 and the affinity was 5.5×10¹⁰ per mole. After fifty generations, the titer of the antibody was still stabilization. The anti-aflatoxin M₁ monoclonal antibody with high affinity can be obtained by high throughout screening technology and it provides a good antibody material to establish the immune detection system of aflatoxin M₁。Using this antibody with high affinity, an indirect competitive ELISA method for detecting aflatoxin M₁ was developed. The best reaction condition lists as follows. 0.02 microgram per microliter aflatoxin M₁-bovine serum albumin coats twelve hours at four centidegree and five precent milk blocks two hour at thirty-seven centidegree. IgG-HRP which is diluted sixty thousand incubats one hour at thirty-seven centidegree and then it colored fifteen minutes by TMB at thirty-seven centidegree. Inter-plate competitive mode is used. The detection limit of indirect competitive ELISA was ten nanograms per litre. The linear range was from one tenth nanogram per millilitre to ten nanograms per millilitre. The accurate was from one hundred precent to a hundred and eighteen precent. The cross-reactivity of the monoclonal antibody was 21.5, 16.6 and less than 5 precent against aflatoxin B₁, aflatoxin G1 and others, respectively. The optimization indirect competitive ELISA developed was uniform, precise and stabilized, showing high practical application value.