Electrochemical Detection of African Swine Fever Virus in Pig Serum With a Competitive Separation Flow Injection Analysis-immunoassay

Abstract

A competitive separation FIA-immunoassay was established to detect African Swine Fever Virus (ASFV) in infected pigs. The detection was based on the competition between a peroxidase (horseradish peroxidase, HRP) labelled monoclonal antibody and the specific pig polyclonals for the biotinylated virus protein VP73. The affinity reaction was performed in solution followed by electrochemical determination of the HRP activity in the FIA system. The sensitivity of this competitive separation assay was similar to the conventionally used microtiterplate ELISA for ASFV detection. For the monoclonal antibody 18BG3, the detection range was 1–80 ng ml ^{- 1} with a concentration of 50% inhibition (C ₅₀ ) at 6 ng ml ^{- 1} . The immunological complex formed during this homogeneous incubation was trapped in a small column containing glass beads with streptavidin on their surface. The amount of captured HRP labelled antibodies was determined by the enzymatic turnover of hydroquinone and hydrogen peroxide to p-benzoquinone and H ₂ O. The enzymatic product was electrochemically reduced at -100 mV vs. Ag/AgCl at a glassy carbon electrode. After each measurement the reactor was regenerated by a removal of the streptavidin bridge including the captured immunological complex (IC). The biotin modified glass beads could be reactivated with a new coating of streptavidin for the next affinity reaction. The determination could be performed automatically in the FIA system within 15 min. With this procedure more than 150 tests were performed with the same reactor without a significant loss of sensitivity.