Peptide mimotopes of phomopsins: Identification, characterization and application in an immunoassay

Peptide mimotopes of plant-associated toxins offer the potential for improving analytical and diagnostic methodologies as well as providing candidates for potential protective vaccines against plant poisoning diseases. Monoclonal antibody (mAb) C₃C₁₁, which recognizes the antimicrotubule phomopsin mycotoxins, was used to isolate peptide mimics of phomopsin A from a random 15-mer phage display peptide library. A total of 46 clones were isolated that showed specific reactivity with the mAb. Amino acid sequence analysis revealed four different types of mimotope sequences, all of which contained a common motif V-A-L/V-C. Of the 46 clones isolated, 44 contained the motif V-A-L-C while 2 contained the V-A-V-C motif. All four types of phage clones inhibited the reactivity of the mAb with phomopsin A in a competition ELISA. The clone with the mimotope sequence CT VALCNMYFGAKLD demonstrated the strongest binding. It was further shown that synthetic peptides containing these mimotope amino acid sequences were able to inhibit the mAb-phomopsin A interaction, indicating that the peptide mimotopes were responsible for the specific binding, independent of the phage framework. The results also suggest that the mimotope peptides bind to mAb C₃C₁₁ at the same site as phomopsin A. The application of recombinant phage particles carrying phomopsin mimotopes in immunoassay was evaluated and the results demonstrated approximately 100-fold increase in sensitivity in comparison with a conventional immunoassay using a chemically linked phomopsin-horseradish peroxidase conjugate.