TCID 50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus

Abstract

Some cell lines are difficult to grow in confluent monolayers and, therefore, the plaque assay cannot be applied since plaques may be hard to distinguish from other blank areas of the cell monolayers. To avoid this problem a rapid and sensitive immuno dot blot TCID 50 method was developed using antibodies against virus antigens to detect infection and virus production. An alternative statistical method was developed to treat the scoring data and thereby obtained a coefficient of variation of 10%. To speed up the total procedure and to increase the proliferation rate of cells grown in 96-well cell culture plates, the plates were pretreated for 4 h at 4°C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID 50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at −70°C with a cryoprotective agent (10% glycerol) preserved the TCID 50 level even after as many as ten cycles of freezings and thawings, whereas the infectivity decreased by four orders of magnitude after storage at 4–8°C for 2 months in the salt buffer used commonly.