Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli.
Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.
Pai
Wan Chain
WC
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 403, Taiwan.
Shien
Jui Huang
JH
Liu
Hung Jen
HJ
Lee
Long Huw
LH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics; D001667:Binding, Competitive; D002642:Chick Embryo; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007132:Immunoglobulin Isotypes; D051379:Mice; D008807:Mice, Inbred BALB C; D030101:Orthoreovirus, Avian / Q000254:growth & development / Q000276:immunology*; D011201:Poultry Diseases / Q000175:diagnosis / Q000821:virology*; D011233:Precipitin Tests / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012088:Reoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D013047:Specific Pathogen-Free Organisms; D013710:Tendons / Q000821:virology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral; D007132:Immunoglobulin Isotypes; D011994:Recombinant Proteins