Development of Enzyme-Linked Immunosorbent Assay with Tiramine Amplification for the Detection of Potato Virus X

Resumo

A method is proposed for reducing the detection limit of enzyme-linked immunosorbent assay (ELISA) of potato virus X, based on the multiple introduction of tyramine into immune complexes and the subsequent detection of an enzyme label. During ELISA, a step by step formation of sandwich complex containing immobilized antibodies – potato virus X – horseradish peroxidase conjugate with antibodies to the virus was carried out. Peroxidase catalyzed the multiple insertion of a tyramine-biotin label into protein molecules, providing signal amplification upon the addition of the streptavidin-polyperoxidase conjugate. The conditions of the assay that ensure a high degree of amplification and a minimum background signal were established. The use of tyramine amplification made it possible to lower the detection limit by more than 30 times (from 100 to 3 ng/ml) when assayed in the buffer and extracts of potato leaves, slightly increasing its duration. Tyramine amplification is based on the use of universal reagents and can be used to reduce the detection limit of ELISA for other antigens.