Carrier-Adjuvant로써 Cholera Toxin을 사용한 항 Fumonisin 항체생산과 이를 이용한 ELISA 개발

Production of Polyclonal Antibody to Fumonisin Using Cholera Toxin as a Carrier-Adjuvant and Development of an ELISA to Detect Fumonisin in Feedstuffs

Murine polyclonal antibody reactive with fumonisin B₁(FB₁) was produced by immunization with FB₁-cholera toxin(CT) conjugate as a carrier-adjuvant and an emzyme-linked immunosorbent assay (ELISA) was developed to detect fumonisin in feedstuffs. The immunogen, FB₁-CT, and coating antigen, FB₁-ovalbumin(OA), were prepared by coupling FB₁ to CT or OA with 2% glutaraldehyde. Mice were immunized by injecting FB₁-CT conjugate intraperitonially 3 times on 14 day intervals and a large volume of ascites containing antibody to fumonisins was produced by injecting pristane and myelorna cells into peritonium. Antibody titers were measured by indirect ELISA after each immunization and antibody in ascites was partially purified by ammonium sulfate precipitation and gel filtration. A standard curve of FB₁(n=8) was aquired by competitive indirect ELISA using the partially purified ascitic antibody and FB₁-OA as a coating antigen, and the standard curve showed that the lowest detection limit of FB₁ is 100ng/mL level(p<0.01). The ascitic antibody cross-reacted with FB₂(97%), but no cross-reactivities were observed with tricarballylic acid, aflatoxins(B₁, B₂, G₁, and G₂), zearalenone, T-2 toxin, and ochratoxin A Competitive ELISA curves in the presence of organic solvents used to extract FB₁ from feedstuffs showed that the concentrations of organic solvents up to 18.5% methanol and 12.5% acetonitrile had no prominent effects on the ELISA system developed, suggesting that the diluted organic solvent extract of the sample may be used for the ELISA without solvent evaporation. When FB₁ from spiked corns was extracted with 50% acetontrile and subjected to the ELISA after the concentration of acetonitrile was reduced to 12.5% by dilution with PBS, the recovery rates of FB₁ in corns containing 1, 2.5 and 5 μg/g of FB₁ were 194%, 107% and 82.1%, respectively. Therefore, the ELISA system developed in this study using the polyclonal antibody to FB1 could be applied to determine fumonisins in feedstuffs.