title: A simplified multiwell plate assay for the measurement of hepatitis A virus infectivity.
abstract: A standardized multiwell plate assay (MWPA) was developed to provide a simple in situ measurement of hepatitis A virus (HAV) infectivity titers. Following attachment (4 h, 35 degrees C) of serial 10-fold dilutions of HAV strain CR326 F (variant F') to confluent MRC-5 monolayers in 24-well plates, cultures were overlaid with maintenance medium and incubated for 35 days at 35 degrees C with weekly medium replacement. Cells were fixed with 90% acetone and HAV antigen was quantitated by reaction with a radio-iodinated anti-HAV serum. Measurement of virus infectivity was based on the amounts of specifically bound and eluted radiolabelled antibody. Virus titers (50% tissue culture infectious doses/ml, TCID50/ml) were calculated using the method of Reed & Muench (Am J Hyg. 1938; 27: 493-497), with wells producing cpm values greater than 2.1-times that of uninoculated controls considered positive. The use of a virus standard provided an estimate of the test variability (+/- 5% in Log10 TCID50 units). The MWPA offers a significant savings in time and reagents, and is proposed as a standard method for the simple and reliable measurement of the potency of HAV vaccine preparations.
authors.affiliation: Department of Cellular and Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: D L
authors.initials: DL
authors.lastname: Krah
mesh_terms: D000956:Antigens, Viral; D002460:Cell Line; D005069:Evaluation Studies as Topic; D006508:Hepatitis Antibodies; D006507:Hepatovirus / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D014765:Viral Vaccines / Q000032:analysis / Q000592:standards; D014773:Virology / Q000379:methods*
chemical_list: D000956:Antigens, Viral; D006508:Hepatitis Antibodies; D014765:Viral Vaccines