AN IMMUNOASSAY FOR DETERMINING AFLATOXIN B1 USING A RECOMBINANT PHAGE AS A NONTOXIC COATING CONJUGATE

Immunoassay for determining aflatoxin B1 (AFB1) involves using the toxic mycotoxin in a conjugated form. To replace the toxic conjugate in an immunoassay, four mimotope peptides of AFB1 were acquired by a panning-elution selection from a phage library. Because the phage library was displayed on the N-terminus of the minor coat protein gIIIp of the filamentous phage M13, the copy number of the expressed mimotope was too low to being used in an immunoassay. To solve such problem, one mimotope peptide (HPSDPRH) was fused with the major coat protein gVIIIp by the pC89 phagemid display system, which led to a high copy number expression in a recombinant phage. The recombinant phage bearing the AFB1 mimotope peptide was identified by an anti-AFB1 monoclonal antibody, and confirmed by DNA sequencing. An immunoassay was set up with the recombinant phage. The new method was compared with a conventional immunoassay. The calibration curves and the results of accuracy and precision were almost identical in both methods, which demonstrated the possibility of using the recombinant phage replacing the AFB1-protein conjugate to set up immunoassay. PRACTICAL APPLICATIONS The recombinant phages with mimotope peptide-fused gVIIIp can be used in hazards detection concerning food safety in immunoassays, when haptens are unstable, difficult to conjugate, valuable, dangerous or toxic in practice.