Monoclonal antibody-defined epitope map of expressed rubella virus protein domains.
An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.
Wolinsky
J S
JS
Department of Neurology, University of Texas Health Science Center, Houston 77225.
McCarthy
M
M
Allen-Cannady
O
O
Moore
W T
WT
Jin
R
R
Cao
S N
SN
Lovett
A
A
Simmons
D
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000032:analysis*; D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010957:Plasmids; D011233:Precipitin Tests; D012412:Rubella virus / Q000235:genetics / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D014764:Viral Proteins