Improved group determination of tetracycline antibiotics in competitive enzyme-linked immunosorbent assay

Seven tetracycline (TC) antibiotics were used as haptens for synthesis of conjugated antigens and as standards in indirect competitive enzyme-linked immunosorbent assay (ELISA). Seven conjugates were prepared by formaldehyde condensation (f) and another seven antigens were synthesised by linking TCs with periodate-oxidised transferrin (TF(pi)). Two gelatine-lymecycline (LC) conjugates were the products of glutaraldehyde and activated esters reactions. To estimate the influence of solid-phase antigen on assay specificity, all the conjugates were absorbed on polystyrene plates and using anti-bovine serum albumin-TC(f) sera, the ELISAs were developed and compared. The variant with immobilised TF-chlortetracycline (CTC(pi)) showed the best group specificity: recognition of seven TCs differed only in 5.3 times. This assay displayed sensitivity 0.1 ng/ml and cross-reactivity indexes: TC (100%), CTC (105%), oxytetracycline (19.8%), methacycline (23.6%), minocycline (70%), doxycycline (25%) and LC (50%). The dependence of ELISA specificity on immobilised antigen spatial structure was demonstrated. Solid-phase conjugate selection is a useful tool for modifying assay specificity.