title: 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.
abstract: Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the 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 sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.
authors.affiliation: Department of Porcine and Exotic Viral Diseases, Institute for Animal Science and Health ID-DLO, Lelystad, The Netherlands. TG.Kimman@RIVM.NL
authors.forename: T G
authors.initials: TG
authors.lastname: Kimman
authors.forename: O
authors.initials: O
authors.lastname: de Leeuw
authors.forename: G
authors.initials: G
authors.lastname: Kochan
authors.forename: B
authors.initials: B
authors.lastname: Szewczyk
authors.forename: E
authors.initials: E
authors.lastname: van Rooij
authors.forename: L
authors.initials: L
authors.lastname: Jacobs
authors.forename: J A
authors.initials: JA
authors.lastname: Kramps
authors.forename: B
authors.initials: B
authors.lastname: Peeters
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000737:chemistry; D000914:Antibodies, Viral / Q000032:analysis*; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics* / Q000276:immunology*; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005822:Genetic Vectors / Q000276:immunology*; D011558:Herpesvirus 1, Suid / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D019138:Antibodies, Blocking; D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C089580:glycoprotein E, Suid herpesvirus 1