A simplified multiwell plate assay for the measurement of hepatitis A virus infectivity

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.