Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA.
A fixed-cell ELISA was developed using swine testicle (ST) cells infected with the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and purified biotinylated monoclonal antibodies (b-MAbs). Five of the b-MAbs were specific for the peplomer (E2), five reacted to the nucleocapsid (N), and one reacted to the E 1 protein of the Miller strain of TGEV. Protein A-Sepharose purification of MAbs yielded protein concentrations ranging from 0.40 to 3 mg per ml of ascites. Separate pools of N-MAbs and E 2-MAbs, and the E 1-MAb were used to monitor synthesis of TGE viral antigen in ST cells from 0 to 16 h post-infection at various multiplicities of infection (MOI). Epitopes of N proteins appeared sooner and at a lower MOI than those for the E 1 and E 2 proteins. The fixed-cell ELISA was also used to examine relative binding affinities of TGEV MAbs. Concentrations of b-MAbs producing a half-maximal signal ranged from 0.11 to 3.8 microgram/ml for E 2-MAbs, from 0.05 to 0.82 microgram/ml for N-MAbs, and 6 micrograms/ml for the E 1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E 2-MAbs (0.1 microgram/ml to 6.9 micrograms/ml) and one E 1-MAb (1.2 micrograms/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E 2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV.
Simkins
R A
RA
Ohio Agricultural Research and Development Center, Ohio State University, Wooster.
Saif
L J
LJ
Weilnau
P A
PA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001710:Biotin; D002213:Capsid / Q000276:immunology; D002478:Cells, Cultured; D003332:Coronaviridae / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D009500:Neutralization Tests; D010449:Peptide Mapping; D013997:Time Factors; D005760:Transmissible gastroenteritis virus / Q000276:immunology*; D014758:Viral Core Proteins / Q000276:immunology; D014759:Viral Envelope Proteins / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D001710:Biotin