TCID50 determination by an immuno dot blot assay as exemplified in a study of storage conditions of infectious pancreatic necrosis virus

• Published in: Journal of virological methods Vol. 80; no. 1; pp. 17 - 24
• Main Authors: SVENSSON, L, HJALMARSSON, A, EVERITT, E
• Format: Journal Article
• Language: English
• Published: London Elsevier 01.06.1999; Amsterdam; New York, NY
• Subjects: Animals; Animals Antibodies; Antibodies, Viral - immunology; Antigens, Viral - immunology; Biologi; Biological and medical sciences; Biological Sciences; Cell Division; Cell Line; Fundamental and applied biological sciences. Psychology; Immunoblotting - methods; Infectious pancreatic necrosis virus - immunology; Infectious pancreatic necrosis virus - pathogenicity; Microbiology; Natural Sciences; Naturvetenskap; Oncorhynchus mykiss; Rabbits; Reproducibility of Results; Techniques used in virology; Viral/immunology Antigens; Viral/immunology Cell Division Cell Line Immunoblotting/methods Infectious pancreatic necrosis virus/immunology/pathogenicity Oncorhynchus mykiss Rabbits Reproducibility of Results; Virology; Birnaviridae; Aquabirnavirus; In vitro; Virus; Dot blotting; Vertebrata; Cell line; Gonad; Pisces; Infectivity; Pisciculture; Infected cell; Infectious pancreatic necrosis virus; Quantitative analysis
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/s0166-0934(99)00018-x

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.