Application of oriented and random antibody immobilization methods in immunosensor design

An immunosensor for the determination of bovine leukemia virus (BLV) antigen (gp51) using surface plasmon resonance (SPR) equipment and SPR-chip modified by oriented and randomly immobilized antibodies (anti-gp51) was developed. Different conditions of anti-gp51 reduction by 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) solutions were compared and explored. The best gp51 detection sensitivity was monitored using SPR-chip modified with oriented antibody fragments (frag-anti-gp51) obtained after reduction with DTT at 50 mM concentration. The SPR sensor could detect gp51 antigen in a range from 0.01 to 0.5 mg/mL. The developed SPR sensor offered the limit of detection (LOD) as low as 0.0028 mg/mL, while the limit of quantification was as low as 0.0092 mg/mL with very good repeatability during the three detection-regeneration cycles (1–4%). Oriented immobilization of frag-anti-gp51 was found to be more suitable for the design of SPR-immunosensor for the detection of gp51 if compared with random immobilization of anti-gp51. This method was successfully applied for the detection of gp51 in blood serum samples in a rapid, reliable and selective manner.