Preparation and uses of immunoabsorbent monolayers in the purification of virus proteins and separation of cells on the basis of their cell surface antigens.
A method is described for forming monolayers of Staphylococcus aureus Cowan strain A on plastic tissue culture plates (A plates). Bacteria remain bound to such plates even under strong protein denaturing conditions. The specific binding of antibody to A plates provides a rapid method for immune precipitation on a solid matrix. Antibody can be covalently crosslinked to staphylococcus monolayers by fixation with paraformaldehyde without significant loss of specific antigen binding capacity. Furthermore antigen-antibody complexes may be efficiently disrupted with 9 M urea and non-ionic detergent and both antibody and antigen renatured by removal of urea, antibody regaining the ability to bind fresh antigen. In the presence of 9 M urea and 1% Nonidet P-40 fixed antibody remains bound to plates while antigen is released into the urea solution, providing a method for the immunological purification of proteins. Plates with such fixed antibody may be used multiple times to bind antigen. The use of this method is illustrated by the purification of 2 adenovirus structural proteins and a herpes simplex virus glycoprotein, by means of specific monoclonal antibodies to these proteins crosslinked to A plates. A method is also described to enrich for functional antibody from immune precipitates by dissociating antibody-antigen complexes with 9 M urea and 1% Nonidet P-40 and isolating the antigen free antibody on Staphylococcus aureus Cowan strain A. A plates have also been used to separate suspension cells on the basis of their cell surface antigens (e.g., lymphocyte subpopulations or cells expressing virus antigens on their surface). Cells were either (1) reacted with specific antisera to cell surface determinants and panned over A plates, cells with antibody on their surface binding to the monolayer, or (2) panned over A plates to which specific antibody had been coupled. Separated cells may be probed directly for certain properties, such as their ability to support virus replication. The staphylococcus monolayer does not prevent the growth of tissue culture cells on the plastic surface and the selected cell population can be examined for cells giving rise to infectious centres by the subsequent addition of permissive cells.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis*; D001666:Binding Sites, Antibody; D002469:Cell Separation; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques; D008214:Lymphocytes / Q000145:classification / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D018139:Simplexvirus / Q000276:immunology; D013205:Staphylococcal Protein A / Q000378:metabolism; D013211:Staphylococcus aureus / Q000254:growth & development / Q000378:metabolism; D014764:Viral Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D013205:Staphylococcal Protein A; D014764:Viral Proteins