The Establishment Of Direct Competition ELISA And Non-toxic System Of AFM

Aflatoxins (AF) are toxic secondary metabolites mainly prouduced by the fungiAspergillus flavus, Aspergillus parasiticus and Aspergillus nomius on foods andfeedstuffs. AF has been evaluated by the International Agency for Ressarch on Cancerof WHO as a classâ… human carcinogen in 1993 because of their toxic andcarcinogenic potentials to humans and animals. Among 20 different types ofaflatoxins identified, the major ones are aflatoxin B₁ (AFB₁), AFB₂, AFG₁, AFG₂,AFM₁, AFM₂ and so on, and AFB₁ is the most potent hepatocarcinogen known inmammals. Although AFM₁ is not as toxic as AFB₁, but compared with Potassiumcyanide, it still has strong toxic power. Many countries have established regulatorylimits for AFM₁ in foods. In order to avoid foods and foodstuffs which are highcontaminated by AFM₁ especially dairy products enter human food chain. So it isvery important to develop convenient operation method for rapid detecting AFM₁.The main results are as follows:1. The establishments of AFM₁ direct competition ELISA(1) AFM₁ oxime has to be synthesized first, then conjugate AFM₁ with HRP.(2) The best working concentration of AFM₁-HRP and anti AFM₁ monoclonalantibody were optimized, and the direct competitive ELISA standard curve wasestablished basis on the above conditions, the linear range of the inhibition curve wasfrom 0.1 ng/ml to 4 ng/ml. The IC₅₀ was 2.2 ng/ml and the detecting limit was 0.1ng/ml.2. Panning mimotopes of aflatoxin M₁ by phage display peptide library(1) The monoclonal antibody against the aflatoxin M₁ was used as ligand to panthe binding peptide from the Ph.D.-7^TM phage display peptide library. After fourrounds of panning, 20 clones were picked out randomly from the fourth round. Thosephage plaques were amplified and purified, respectively. Four of clones wereidentified positive by indirect ELISA. They were screened by indirect competitiveELISA and named phageA1, A2, A3, A4.(2) DNA of four phage clones was extracted, sequence and then translated into the amino acid sequence according to the genetic code. The results showed that theyrepresent 2 different sequencing (A1 and A2, A3 and A4 are the same sequencing).The peptide sequences of 4 phage clones were analyzed by sequencing and displayed.The peptide sequence of A1 and A2 is LTSFPRH, the peptide sequence of A3 and A4is MAPSSWR(3) The indirect competitive ELISA curves were established and the linear rangeof the inhibition curves were between 0.1 ng/mL and 5 ng/mL, IC₅₀ was 3 ng/mL, thedetecting limit was 0.1 ng/mL.