Immunofluorescent detection and quantitation of hepatitis A virus in sewage treatment effluent and on agri-food surfaces using scanning confocal microscopy

Abstract An immunofluorescent (IF) assay was developed for specific detection and quantitation of hepatitis A virus (HAV) in sewage treatment effluent or attached to stainless steel, copper, high density polyethylene and polyvinyl chloride surfaces. Polyclonal antibodies were produced in rabbits and characterized for specificity to HAV. Purified anti-HAV antibodies were used in combination with ALEXA-anti-rabbit conjugate and confocal microscopy for detection and quantitation of HAV in effluent samples. Using this immunological approach, as little as 2×10 5 PFU/ml were detected and the signal generated was proportional to the concentration of HAV up to 2×10 8 PFU/ml. Counts obtained by IF assay were highly correlated with those of the SYBR Green II nucleic acid labelling method ( r 2 =0.995) and conventional plaque assay ( r 2 =0.988). The IF assay described is rapid (3 h), sensitive, specific to HAV and suitable for qualitative as well as quantitative studies.