Serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scFvs in inhibition ELISAs.
Two bluetongue virus (BTV) serotype 10-specific single-chain Fv chicken antibody fragments (scFvs) were evaluated in a competitive ELISA. The binding of one (F3) to purified BTV was only inhibited by antibodies against the homologous serotype. The binding of the other (F10) was blocked by antisera to each of the 24 BTV serotypes. F10 recognised VP7, a major structural protein of the BTV core, but not if the protein was directly adsorbed to a plastic surface. It did, however, bind to recombinant VP7 that had been captured from suspension by rabbit IgG. This made it possible to develop an scFv based inhibition ELISA for BTV antibodies using recombinant VP7 without prior purification. The resulting immunoassay detected antibodies to 24 BTV serotypes, but not those directed against three serotypes of the related epizootic haemorrhagic disease virus. A phage library displaying fusion peptides expressed by fragments of the BTV genome segment 7 cDNA was constructed and screened using F10. Comparing selected peptides with the amino acid sequence of VP7 showed that recognition by the scFv required at least 131 residues representing the protein's upper domain. By providing well-characterised immunological reagents, recombinant antibody technology can contribute to the development of improved immunoassays for BTV diagnosis.
Fehrsen
J
J
Immunology Division, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa.
van Wyngaardt
W
W
Mashau
C
C
Potgieter
A C
AC
Chaudhary
V K
VK
Gupta
A
A
Jordaan
F A
FA
du Plessis
D H
DH
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis*; D000956:Antigens, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001820:Bluetongue virus / Q000145:classification* / Q000235:genetics; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D012703:Serotyping; D014758:Viral Core Proteins / Q000276:immunology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus