Detection of Epstein-Barr virus early antigen-D and its antibodies by passive hemagglutination

These data describe the use of a passive hemagglutination test (PHAT) for the detection of antibodies to the Epstein-Barr virus (EBV) associated antigen, early antigen-D (EA-D). EA-D was obtained from EBV-superinfected RPMI 64-10 cell preparations which showed by immunofluorescent staining (IF) that 25-35% of the cells contained EA-D. Solubilization of EA-D was achieved by sonic oscillation, treatment of the pellet-associated material with triton X 100, ammonium sulfate precipitation, and chromatography on diethylaminoethyl celluiose. Successful use of the PHAT for detection of antibodies to EA-D was dependent on the degree of purity of the partially purified antigen. Throughout these studies preparations from uninfected 64-10 cells were used as control reagents. More than 100 human antisera from patients with EBV-associated diseases, which had been previously tested by standard IF procedures were assayed by PHAT. A good correlation was found between IF and PHAT titers for EA-D and, in addition, the PHAT was 50-100 times more sensitive. Most human sera gave no agglutination of red cells coated with control preparations. An inhibition (blocking) of PHAT was developed which was useful in the purification and characterization of EA-D. These data indicate that the PHAT may provide a rapid, reliable, objective and sensitive method for studying EA-D and possibly other virus-associated antigens.