Detection of hantavirus infection in hemolyzed mouse blood using alkaline phosphatase conjugate

Conventional methods such as ELISA and culture require a long time for the determination of viral infections. Fast and reliable instrumentation suitable for operation under field conditions that allows rapid detection of hantavirus in rodent populations in the wild, would be a substantial improvement over these methods. In response to this challenge, a prototype amperometric immunosensor based on highly dispersed immunoelectrode for rapid, sensitive and quantitative assay of hantavirus in mice blood has been developed. A sandwich scheme of immunoassay is used and the naphthol that is formed as a result of enzymatic hydrolysis of alpha-naphthyl phosphate in the presence of alkaline phosphatase (AP) label is quantified amperometrically. The main complication faced when testing the amperometric immunosensor for application to mouse blood samples under field conditions is the removal of the interference caused by the electroactive components due to the hemolysis of the samples. We studied the possibility of using other enzyme markers such as AP to replace the horseradish peroxidase (HRP) used earlier in testing for antibodies against hantavirus in human blood plasma. Best results were obtained when the reference electrode is covered with a thin Nafion layer. Further improvement of assay performance was achieved by the modification of the sensing element. The amperometric immunoelectrode showed significantly higher sensitivity (more than 10-fold) than the standard spectrophotometric detection ELISA method. It can also be used for rapid analysis in conventional and field conditions in biological, physiological and analytical practices.