TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus.
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 TCID50 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 degrees C with growth medium obtained from cell culturing flasks containing confluent cell monolayers. This immuno dot blot TCID50 method was applied for a study of the infectivity maintenance upon storage of infectious pancreatic necrosis virus (IPNV). Storage of IPNV at -70 degrees C with a cryoprotective agent (10% glycerol) preserved the TCID50 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 degrees C for 2 months in the salt buffer used commonly.
Department of Microbiology, Lund University, Sweden.
L
L
Svensson
A
A
Hjalmarsson
E
E
Everitt
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D002455:Cell Division; D002460:Cell Line; D015151:Immunoblotting / Q000379:methods*; D018061:Infectious pancreatic necrosis virus / Q000276:immunology* / Q000472:pathogenicity; D017686:Oncorhynchus mykiss; D011817:Rabbits; D015203:Reproducibility of Results
D000914:Antibodies, Viral; D000956:Antigens, Viral