Development Of Immunoaffinity Clumn Of Anti-Aflatoxin M₁ Monoclonal Antibody

Aflatoxin (Aflatoxin, AFT) is a class of strong carcinogens, produced by A.falvus and A.parasiticus during their growthing and breeding, discovered in the suburbs of London in 1960 first. It destroys human and animal liver tissue, severe cases can cause liver cancer and even death, in 1993, classified by WHO-International Agency for Research on Cancer (IARC) as a Group1 human carcinogen(IARC,1993). Aflatoxin M₁ (AFM₁) is the hydroxylated metabolites of AFB₁ (aflatoxin B₁) in vivo and may be detected in the milk of mammals that are fed AFB₁ contaminated feeds and food, such as cattle, sheep and other mammals .Although the toxicity of AFM₁ is an order of magnitude lower than AFB₁, still more highly toxic than Potassium Cyanide and arsenic, for the strong carcinogenic agent, serious threat to human health and safety. In 2002, classified by WHO-International Agency for Research on Cancer (IARC) as a Group 1 human carcinogen (IARC,2002).At present, the detection methods of commonly used are tedious,time consuming and other shortcomings during the pretreatment. Therefore, it is important to develop efficient separation, accurate and quantitative, sensitive and reliable immune affinity column for protecting human and animal food safety.This main results of research are as follows :1. The hybridoma of excreting monoclonal antibodies against AFM₁ was cultivated by Serum-free culture medium, when 96% of the cell was dead, the culture medium were purified,by using protein G affinity chromatography, the antibody was detected at 260nm and 280nm by UV spectrophotometer. protein concentration was 3.533mg/mL by calculating; the titer wasmeasure by indirect ELISA was 3.2×10⁶, and the cross-reactivity of the monoclonal antibody clone was 100%, 15.9%, 6.8% and 0% against AFM₁, AFB₁, AFG₁and DON,The affinity is 6.0×10⁸ L/mol; which met the requirements of developing a immunoaffinity column;Through SDS-PAGE analysis, AFM₁ monoclonal antibody has a good purity.2. High-purity anti-aflatoxin M₁ monoclonal antibody with serum-free medium coupled with CNBrActivated Sepharose 4B, blocked and packed, which produced a coupling rate of 94%; An indirect competitive ELISA and HPLC method for the detection of AFM₁ was developed, the corresponding standard curve was established, the equation of linear regression is y = -9.0249x+96.272(coefficient correlation is R² = 0.977), y = 326.08x-122.17(coefficient correlation is R² =0.9995), the column has been established the optimum reaction conditions through IC-ELISA and evaluated. The sample conditions: pH7.2, flow rate: 3mL/min, elution conditions: 90% methanol-water solution, flow rate: 3mL/min,. In optimum conditions, the column capacityof the immune affinity column was 256ng, can be used 3 times, if the immunoaffinity column not used in one day, it should be stored at 4℃.The recoveries were determinated by recovery experiment for 92.5%～95.6%,standard was added to blanksamples from 0.5～200ng/mL, the recovery rate between 90.7%⁹3.5%,coefficient of variation was less than 8%.3. The 30 milk samples from the Daqing City in Heilongjiang supermarket were randomly selected were tested by indirect competitive ELISA and IAC-HPLC and all samples were notnegative and have lower level than 0.5ng/mL for AFM₁.