Development Of Colloidal Gold Immunochromatography Test Strip For FB₁ And Its Application

Fumonisins are water-soluble metabolites mainly produced by Fusarium moniliforme.They are most frequently found in corn,wheat,rice,other grains and their products.FB₁ is the major fumonisin found in culture of Fusarium moniliforme,and main factor for the toxic effect of fumonisins.Colloidal gold immunochromatographic method is a lateral chromatography technology which is developed based on monoclonal antibody?McAb?and colloidal gold material.Currently,it has become a new research direction of the mycotoxin detection.Colloidal gold immunochromatographic method is easy to operate and adapted to fast detection of a large amount of samples.In this experiment,colloidal gold immunochromatography test strip for detecting FB₁ was developed by immunochromatographic method and used for FB₁ fast detection in corn.Fistly,the hybridoma was made intermediate culture and injected into mice.After the ascites was gathered and purified,the antibodies against FB₁ were gained.The antibody purity and titer were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?and Ci-ELISA,respectively.In the experiment,the glutaraldehyde was used to conjugate FB₁ to ovalbumin?OVA?for preparing the complete antigen?FB₁-OVA?.The conjugate of FB₁-OVA was analyzed by SDS-PAGE and mass spectrum?MS?.The colloidal gold solutions were prepared by sodium citrate.The McAb and FB₁-OVA were used to develop the colloidal gold immunochromatography test strip.The concentrations of FB₁ McAb labeled with colloidal gold,detecting antigen and goat anti-mouse IgG were optimized.The clinical application of colloidal gold immunochromatography test strip was estimated.The results were as follows:1.The concentration of the McAb was 1.3 mg·mL^-1 and the titer detected by indirect ELISA method were 1:1024.The results indicated that the coupling method was successful,and the coupling ratio of FB₁ and OVA was 6.2:1,the concentration of the conjugate was 0.8 mg·mL^-1.2.The colloidal gold solutions with particle size of 25 nm,20 nm and 15 nm were prepared by sodium citrate.The optimum pH of colloidal gold labeled antibody was 7.0,in which 1 mL colloidal gold solution was added to 15 ?L 0.2 mol·L^-1 K2CO3.The best quantity of McAb combined with colloidal gold was that 1 mL colloidal gold solution was added to 7 ?L antibody proteins.The optimal coating colloidal gold labeled antibodies were 0.4 ?g·cm^-1,the optimal coating detection antigen was 0.3 ?g·cm^-1,and the optimal coating control line was 0.10 mg·mL^-1.The visible detection sensitivity of the test strip was 2 ng·mL^-1 and optimal measurable concentration was 40 ng·mL^-1.3.The colloidal gold immunochromatography test strip had no specific binding with AFB₁,DON,ZEN and OTA.It had characteristic of good stability and sample recovery.The results of testing corn samples by the test strip showed excellent correlation and consistency with high performance liquid chromatography?HPLC?method.In conclusion,the colloidal gold immunochromatography test strip cauld accurately,fast detect FB₁ in corn.