Abstract: An immunosensor for the detection of the African Swine Fever (ASF) disease in infected pigs is presented. A sensitive, direct immunoassay for measurements in diluted pig sera was established using the virus protein 73 (VP73) as a highly specific receptor layer for a mass sensitive piezoelec. quartz crystal. The fundamental sensor effect of this transducer is based on the linear dependence of the resonance frequency of an oscillating quartz crystal upon the binding of mass on the coated surface during the measurement. A quartz crystal which is coated with VP73 is an highly specific sensor and allows to detect the ASF-antibodies in the sample through mass accumulation on the surface of the quartz crystal. An appropriate immobilization technique for VP73 was established and a suitable carrier buffer for the flow injection analysis system was developed during this study. Regeneration of the receptor layer is possible for about ten times. With this quartz crystal microbalance, results were available within a few minutes and with a selectivity comparable to a licensed microtiterplate ELISA. Measurements with real pig serum samples have proven the suitability of the quartz crystal biosensor for the classification ofpositive and negative pig serum samples.

Abstract: Pig serum was diluted 50-fold with PBS containing 0.05% Tween 20. A portion (1 ml) of the resulting solution was incubated with 50 ng biotinylated virus protein VP73 (preparation described) and 250 ng horse-radish peroxidase-labelled mAb 18BG3 (Ingenasa) for 25 min. A portion of the reaction mixture was injected into a carrier stream of 0.1 M phosphate buffer of pH 5.5 (buffer A) and passed through a column of biotinylated glass beads coated with streptavidin (preparation described) where the immunological complex formed during the incubation reaction was trapped. The peroxidase activity of the captured labelled antibodies was determined by passing a solution of H2O2 and hydroquinone (each 2 mM) in buffer A through the column and detecting the enzymatic product amperometrically at a vitreous C electrode at -100 mV vs. Ag/AgCl. The calibration graph was linear from 1-80 ng/ml mAb 18BG3 with a concentration of 50% inhibition at 6 ng/ml.

Abstract: A direct piezoelec. flow injection analysis immunoassay for the detection of African Swine Fever virus and antibodies is presented. The peptide-specific monoclonal antibody 18BG3 and the virus protein 73 were used for detection with a quartz crystal microbalance. Accumulation of the analyte on the surface of this mass-sensitive biosensor resulted in a shift of the resonant frequency. Highly selective receptor layers were applied on the sensing electrode of the quartz crystal for detection of the complementary analyte. Different immobilization methods proved to be appropriate for coating of the monoclonal antibody 18BG3. A quartz crystal covalently coated with the antibody 18BG3 detected virus protein VP73 samples more than 20 times and was stable for more than 30 days. The coating of virus protein was performed by physisorption. A sensor with a virus protein receptor layer detected antibody 18BG3 samples 10 times within one day. The sensor device was able to perform one measurement cycle including blocking and regeneration within 30 min. With the help of a suitable carrier liq., measurements with serum samples were performed. The calibration curves for measurements in buffer and in serum could be determined and the detection limits for virus protein detection were 0.31 and 1 µg/mL, and for antibody detection 0.1 and 0.2 µg/mL, respectively.