An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies

An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, pH 7.2, The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization of virus read microscopically correlated with ELISA absorbance of <0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations. (C) 1996 The International Association of Biological Standardization