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

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 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.

• Authors: R A Simkins, L J Saif, P A Weilnau
• Journal: Archives of virology (Arch Virol) Vol. 107 Issue 3-4 Pg. 179-90 ( 1989) ISSN: 0304-8608 [Print] Austria
• PMID: 2479362 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Antibodies, Monoclonal
• Epitopes
• Viral Core Proteins
• Viral Envelope Proteins
• Viral Proteins
• Biotin

Topics

• Animals
• Antibodies, Monoclonal (immunology)
• Biotin
• Capsid (immunology)
• Cells, Cultured
• Coronaviridae (immunology)
• Enzyme-Linked Immunosorbent Assay (methods)
• Epitopes (analysis, immunology)
• Neutralization Tests
• Peptide Mapping
• Time Factors
• Transmissible gastroenteritis virus (immunology)
• Viral Core Proteins (immunology)
• Viral Envelope Proteins (immunology)
• Viral Proteins (immunology)