Biopanning Of Citrinin Mimotope By Phage Displayed Peptide Library

Citrinin (CIT) is a toxic metabolite produced by several filamentous fungi of thegenera Penicillium Aspergillus and Monascus, which has been encountered as anatural contaminant in grains, foods and feedstuffs. This mycotoxin ishepato-nephrotoxic and implicated in disease outbreaks in animals and humans. Thecurrently used methods to detect CIT are high-performance liquid chromatography,thin layer chromatography, enzyme-linked immunosorbent assay(ELISA), and theELISA is widely used. But ELISA needs to use CIT standard which is expensive andharmful to the personnel. Phage display libraries allow s.electing peptide moleculescapable of mimicking the structural and functional features of native proteins or lowmolecule weight chemicals, which can serve as surrogates of original objects. So thistechnique can be applied to overcoming such problems. We used McAb as target tobiopanning CIT mimotope. And CIT mimotope was used as substitute of CITstandard in ELISA. The monoclonal-antibodies against CIT was used as target forbiopanning from a phage random 7 peptide library and a phage random 12 peptidelibrary, which were displayed as a fusion protein with the coat proteinⅢoffilamentous phage M13. We reduced the concentration of antibodies stepwise, anddifferent blocking buffer was used between every round. After 3 rounds ofbiopanning, 30 clones of the phage random 7 peptide library and 38 clones ofthe phage random 12 peptide library were detected by ELISA and. CITcompetition experiments to indentify the postive clones. 20 positive clonescould bind to the antibody, and the binding could be blocked by freecitrinin in the phage random7 peptide library, 33 positive clones couldbind to the antibody, and the binding could be blocked by free citrinin in the phage random 12 peptide library. Then the inserted amino sequencesof the positive clones were deduced by DNA sequencing. The common aminosequence of the mimicking epitope was XHKXXXX. A competitive ELISAimmunoassay was established with clone P10 of the phage random 7 peptidelibrary, the linear range of the inhibition curves was between 15 ng/mLand 325 ng/mL; the detecting limitation was 10 ng/mL. In detection of thefeeds, corn and rice samples mixed with CIT(50—500 ng/g)by competitiveindirect ELISA, the recovery rate of CIT in feeds, corn and rice sampleswere 80.6-102.1％,86.4-93.4％and 84.8-94.8％respectively, the coefficients ofvariation were 1.0-7.4％,1.5-7.8％and 2.2-6.7％respectively. A competitiveELISA immunoassay was established with clone P1 of the phage random 12peptide library, the linear range of the inhibition curves was between15 ng/mL and 439 ng/mL; the detecting limitation was 10 ng/mL. In detectionof the feeds,corn and rice samples mixed with CIT(50—500 ng/g)by competitiveindirect ELISA,the recovery rate of CIT in feeds, corn and rice samples were64.2-108.4％,81.4-100.04％and 84.1-94.4％respectively,the coefficients of variationwere 1.1-5.6％, 1.1-4.0％and 2.4-5.8％respectively. The phage ELISA was comparedwith conventional ELISA, the data. suggested the new method is applicable. Theresults indicated that the acquired mimotope peptides could be used as the surrogateof CIT. to establish ELISA for detecting citrinin without the mycotoxin, themechanism of the interaction between mimotope peptide and antibody might bemimicking hydrophobic interaction of original CIT and antibody.