Development Of Hybridoma Cell Lines Excreting Monoclonal Antibodyies Against Fumonisin B₁ And Study Of ELISA-kit

ObjectiveFumonisins ( FB) are a group of structurally related mycotoxins produced by a number of Fusarium species. The most prevalent of these mycotoxin in contaminated corn is FB1, which is believed to be the most toxic. FB, has been detected in maize and maize-based products all over the world. FB1 is known to be the cause of equine leukoencephalomalacia and porcine pulmonary edema syndrome. Available epidemiological evidence has suggested a link between dietary fumoni-sin exposure and human oesophageal cancer in some locations with high disease rates.Several analytical methods have been used for the analysis of FB in food are sensitive and specific,but need too much time and money. Enzyme-linked im-munosorbant assay for detection of FB which is based on monclonal antibodies is simple, rapid, sensitive and can meet the demand of practical work. There are ELISA kits for FB, abroad which are expensive. To develop hybridoma cell lines excreting monoclonal antibodyies against FBt and develop rapid detection kit that possessing patent of our country are important.Methods1 Preparation monoclonal antibodyies against fumonisin B1FB1-KLH using as immunogen and FB1-BSA using as solid-phase antigenwere prepared with one-step glutaraldehyde method. BALB/c mice were immunized with FB1-KLH. Slpeen cells from the immunized mice were fused with SP2/0 cells. The hybridoma cells were screened and cloned and monoclonal antibodies were prepared. The subtypes of antibodies were identificated by subtype kit. The molecular weight was determined by SDS-PAGE. The titer and affinity constant were determined by indirect incompetitive enzyme-linked immunosor-bant assay. Sensitivity and specificity of the antibodies were determined by indirect competitive inhibition enzyme-linked immunosorbant assay.2 Development of the indirect competitive inhibition enzyme-linked immunosorbant assay (ELISA) for FB,Method of extraction and indirect competitive inhibition enzyme-linked immunosorbant assay (ELISA) for detection FB, in cereals were developed. The formula of calculating was also established.3 Study of parameters of the kitSensitivity, specificity, stability, recovery rate, within-laboratory repeatability and between-laboratory reproducibility were studied by indirect competitive inhibition enzyme-linked immunosorbant assay.4 Testifying ELISA kit with HPLCThe maize samples spiked with FB, were determined by the ELISA kit and HPLC respectively.Results1 Preparation of monoclonal antibodiesHybridoma cell lines excreting monoclonal antibodies were developed. Monoclonal antibodies produced by the hybridoma cells were tested for subtypes and designated as IgG1, the molecular weight was 150kD,the titer was 1: 2. 0 10~8 ,the affinity constant was 6.72 × 10~9 L/mol.2 Study of parameters of the kitThe limited concentration of detection of the ELISA-kit was 5ng/ml, which in sample was 250ng/g. There was no cross reaction with DON,ZEN,T-2 toxin. The kit can be stored at normal temperature over 225d. The recovery rate of...