Abstract
A genus-specific antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection orthopox viruses (OPV) was established, testing various combinations of monoclonal antibodies (MAbs) and rabbit hyper-immune sera. The most sensitive assay was based on capturing antigen with a non-neutralizing MAb against a highly conserved antigenic site of the OPV fusion protein. Bound antigen was detected by a mixture of the two broadly-reactive rabbit anti-monkey-pox- and rabbit anti-vaccinia virus hyper-immune sera. The reaction was visualized by the corresponding anti-species-horseradish-peroxidase- (HRP) conjugate and 3,3',5,5'-tetramethylbenzidine (TMB) as substrate. This system was able to identify all OPV-species and isolates which were investigated. The ELISA demonstrated complete agreement with conventional procedures undertaken for qualitative and quantitative OPV-detection. In parallel, 16 defined OPV-strains were analysed by quantitative electron microscopy (calculation of the number of physical particles; PN) and infectivity titration (TCID50/ml) on tissue cultures. When ELISA data were compared with infectivity titers, the detection limits in the ELISA varied in a wide range of 10(2)-10(4) TCID50/ml., depending on different replicative potencies of the individual virus strains. However, when PN was the basis for calculation, a minimum of 10(5) physical virus particles/ml could be detected. Gradient-purified virus was detectable up to a protein content of 15 ng/ml. Maximum optical densities in the sample (OD450nm) reached 2.0, while mock infected tissue cultures of negative samples offered corresponding values of < 0.01.
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