Development and evaluation of an Epstein-Barr virus (EBV) immunoglobulin M enzyme-linked immunosorbent assay based on the 18-kilodalton matrix protein for diagnosis of primary EBV infection

Abstract

A new immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) based on the recombinant Epstein-Barr virus (EBV) matrix protein was developed. Compared to indirect immunofluorescence for the detection of IgM antibody to the EBV capsid antigen on clinical specimens, the sensitivity and specificity of the new IgM ELISA were 96 and 96%, respectively.

FIG. 1

SDS-PAGE analysis of affinity-purified GST-VCA fusion protein. The induced bacterial cells containing fusion protein were lysed by 0.1% Triton X-100 detergent in Tris-HCl buffer with mild sonication, clarified by centrifugation at 10,000 × g for 15 min, and purified by GST-glutathione affinity chromatography with glutathione-Sepharose 4B. The bound GST fusion proteins were eluted with glutathione elution buffer. The samples were loaded onto an SDS–10% polyacrylamide gel and stained with 0.5% Coomassie blue to visualize the fusion protein and control parental GST protein (made in control cells carrying the parental pGEX vector only). Masses are shown on the left, in kilodaltons.

FIG. 2

Characterization of VCA fusion protein by immunoblotting. Strips were stained with anti-GST antibody, antibody (Mo Ab) to VCA p-18 (rat antibody EBV.OT-15E, kindly supplied by J. M. Middeldorp), a serum pool from NPC patients, and EBV-negative serum.

FIG. 3

Reaction profiles of 36 sera, giving results suggestive of recent primary EBV infection by one or more tests (VCA IgM IF, VCA IgM ELISA, and VCA IgG-EBNA profiling).