AN INDIRECT DOUBLE-ANTIBODY SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) USING BACULOVIRUS-EXPRESSED ANTIGEN FOR THE DETECTION OF ANTIBODIES TO GLYCOPROTEIN-E OF PSEUDORABIES VIRUS AND COMPARISON OF THE METHOD WITH BLOCKING ELISAS

Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorb ent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs ) directed against gE, Our aim was to develop a confirmation assay whi ch is based on another principle and which is able to detect antibodie s directed against most potential binding sites on gE with high specif icity, Therefore, we developed an indirect double-antibody sandwich as say (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +10 20 of gE, coding for the major antigenic sites of gE but not the trans membrane region, was cloned behind the signal sequence of PRV gG and t he p10 promoter in a baculovirus vector. Immunoblot analysis showed th at the expressed protein reacted with MAbs directed against five of th e six antigenic sites on gE. Although the conformation of some antigen ic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodi es in porcine sera in Western blots (immunoblots) and ELISAs, For the IDAS, a coating MAb directed against the nonimmunodominant antigenic s ite A on gE was chosen. A major obstacle in binding ELISAs, such as th e IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of ser a in blocking ELISAs, The sensitivity and specificity of the newly dev eloped BacgE960 IDAS were evaluated and compared with those of five co mmercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnost ic specificity and a moderate sensitivity, The five blocking ELISAs di ffered remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation, We conclude that the B acgE960 IDAS is a useful and specific additional (confirmatory) test f or the detection of antibodies to gE.