Application of Denatured, Electrophoretically Separated, and Immobilized Lysates of Herpes Simplex Virus-Infected Cells for Detection of Monoclonal Antibodies and for Studies of the Properties of Viral Proteins

Abstract

Herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICP) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose were used to detect monoclonal antibodies to viral polypeptides and to study the properties of the proteins with the monoclonal antibodies. The results were as follows. When the antigens were electrophoretically separated in denaturing cells and then immobilized on nitrocellullose strips, a greater diversity of monoclonal antibodies to viral proteins was detected than when the technique of immune precipitation of soluble, nondenatured viral antigens was used. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not avialable for reaction in preparatios cleared by high-speed centrifugation before immune reaction. Studies of the viral polypeptides reactive with 3 monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monovclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 .times. HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of 4 antigens, 39,000-46,500 in apparent MW, reactive with each of several monoclonal antibodies. These studies showed that 2 polypeptides partition in the cytoplasm and 2 in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the 4 polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.