Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows.

Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle.