ELISA of streptomycin in buffer and milk: Effect of reagents' structure and analysis format on assay performance

Abstract

The influence of immunoreagents' structure on assay performance was investigated and a range of ELISAs for streptomycin in direct and indirect format was developed. Streptomycin was conjugated with proteins (bovine serum albumin (BSA) for immunization and ovalbumin (OVA) for immobilization on a plate) by two different methods. Streptomycin was derivatized with carboxymethoxylamine (CMO) and then coupled to a protein or the protein was activated with adipic acid dihydrazide (ADH) and then coupled with streptomycin. A conjugate with horse-radish peroxidase was synthesized using streptomycin-ADH derivative. With the indirect ELISA the most sensitive assay for polyclonal antisera against streptomycin-oxime-BSA in combination with homologous and heterologous conjugates (limit of detection 2.5 ng ml-1) was developed, whereas for a combination 'antisera against streptomycin-ADH-BSA/heterologous conjugate' higher background level of a calibration curve was observed. Besides the level was very high (about 60%) for homologous conjugate. In a direct ELISA similar sensitivity was achieved only for antisera against streptomycin-oxime-BSA (limit of detection 3.0 ng ml-1). Chemiluminescent detection allowed to increase the assay sensitivity by several times (limit of detection 0.5 ng ml-1) but led to the worse reproducibility (CV 16%). A sensitive and simple direct ELISA for analysis of streptomycin in milk products without preliminary sample preparation was developed (limit of detection 3.2 ng ml-1). In the indirect ELISA an influence of fat content of a milk product on assay performance was observed.