Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA.

Archives of Virology
R A SimkinsP A Weilnau

Abstract

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 a...Continue Reading

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Citations

Nov 1, 1993·Veterinary Microbiology·L J Saif
Aug 13, 2005·Progress in Nucleic Acid Research and Molecular Biology·S Anuradha, K Muniyappa
Apr 1, 1996·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·D I ShoupL J Saif
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