Immunoprecipitation with two-dimensional pools as a hybridoma screening technique: Production and characterization of monoclonal antibodies against adenovirus 2 proteins

• Published in: Virology (New York, N.Y.) Vol. 110; no. 2; pp. 385 - 401
• Main Authors: Cepko, Constance L., Changelian, Paul S., Sharp, Phillip A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30.04.1981
• Subjects: Adenoviruses, Human - immunology; Antibodies, Viral - isolation & purification; Antibody Specificity; Capsid - immunology; Capsid Proteins; Clone Cells - immunology; Hybrid Cells - immunology; Myeloma Proteins - immunology; Precipitin Tests; Protein Conformation; Radioimmunoassay; Viral Proteins - immunology
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/0042-6822(81)90069-6

A screening technique based on immunoprecipitation has been developed and used to select α-adenovirus monoclonal antibodies. This immunoprecipitation procedure can be used as a rapid primary screening method for virtually any antibody—antigen system. This method employs in vivo [ 35S]methionine-labeled proteins as antigens and two-dimensional pools of hybrid culture supernatants as antibody, thus allowing identification of the specificity of a given well in the first screen. The α-adenovirus hybrids that have been cloned to date to yield stable, ascites fluid-producing tumors include two α-hexon clones, three α-fiber clones, one α-protein IX clone, and one clone that precipitates hexon plus two proteins of molecular weights 100,000 and 95,000 daltons. The α-protein IX clone as well as an α-hexon clone and an α-penton base clone were chosen for characterization of their specificities by immunoprecipitation analysis. The hybrid cell line secreting antibody against hexon, the major virion capsid protein, was characterized as being reactive with hexon trimers, both free in solution and assembled in groups of nine (“ninemers”). The α-hexon monoclonal antibody did not precipitate the monomer form of hexon, prepared by in vitro translation, or intact mature virions. These characteristics, as well as the ability to precipitate hexons prepared from all adenovirus serotypes tested to date, are the same characteristics exhibited by group-specific hexon serum. The monoclonal antibody specific for penton base, the virion protein found at the vertices of the icosohedral capsid, immunoprecipitated the monomer form of penton base prepared by in vitro translation or denaturation of mature virions. It was not reactive with the 10 S complete penton form of penton base assembled with fiber, nor did it precipitate mature virions. The α-protein IX antibody was reactive with the monomer form of protein IX as well as denatured protein IX. All three of the antibodies were also tested for the ability to neutralize virus infectivity and were found to be negative in this assay. In addition, infected cells were stained by indirect immunofluorescence using these antibodies. The α-hexon and α-penton base antibodies gave the expected pattern of bright nuclear fluorescence, while the α-protein IX antibody gave rise to an unusual pattern of bright nuclear sparkles.