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 21 with a concentration of 50% inhibition (C 50 ) at 6 ng ml 21 . 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 2100 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.
A novel totally screen-printed flow-through cell for immunoanalysis is presented. It contained screen-printed carbonaceous electrodes, which allowed the determination of peroxidase activity through the electrochemical reduction of p-benzoquinone. As different electrode materials differ strongly in their electrochemical properties, electrodes resulting from various screen-printable carbonaceous pastes were characterized using the hydroquinone/ p-benzoquinone redox couple. For most of the electrodes, cyclic voltammogram peak separations of between 550 and 670 mV were observed indicating only quasi-reversible electrochemical behavior. This was confirmed by variation of the peak separation with scan rate. Heterogeneous electron transfer rates of ca. 0.5 - 1 x 10(-3) cm s(-1) and electrochemical activation energies of ca. 20 kJ mol(-1) were found. These flow-through cells were not only applied to electrochemical peroxidase activity determinations but also, in combination with a separate detector, as affinity reactors. After biotinylation of screen-printed layers, streptavidin and then biotinylated peroxidase could be bound. However, as signals were only 10-20% of those obtained with a column filled with biotinylated glass beads, only the screen-printed electrochemical detector was applied to the detection of antibodies against the African Swine Fever Virus.
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