PRODUCTION AND APPLICATION OF MONOCLONAL-ANTIBODIES SPECIFIC TO PYRROLOQUINOLINE QUINONE

We produced five monoclonal antibodies (mAbs 1, 2, 6, 7, and 9) that a re specific to pyrroloquinoline quinone (PQQ) PQQ-conjugated hemocyani n was used for the immunization of mice and the hybridomas were select ed using PQQ-conjugated BSA in an enzyme-linked immunosorbent assay. M Abs 2 and 9 were of the IgG1 isotype. Both could recognize free PQQ, t he former probably at the o-quinone and the latter at the opposite sid e of the molecule. They did not bind with trihydroxyphenylalanine, dih ydroxyphenylalanine, 1,2,4-trihydroxybenzene, ascorbic acid, riboflavi n, or menadione. In contrast to the IgGs, mAbs 1, 6, and 7 (IgMs) did not bind with free PQQ. Using mAb 2, a competitive enzyme-linked immun osorbent assay was developed, which enabled us to determine 50 nM-1 mu M free PQQ. Furthermore, we analyzed the covalently bound prosthetic groups of two quinoproteins (amine oxidase from Aspergillus niger and amine dehydrogenase from Pseudomonas putida) by Western analysis using these mAbs. However, the result was negative, indicating that the pro sthetic groups are not PQQ.