Monoclonal Antibodies to Mouse Mammary Tumor Virus: Antigenic Analysis of Envelope Glycoprotein gp52

Abstract

Two sets of hybrid cell lines producing monoclonal antibodies against two different strains of mouse mammary tumor virus (MMTV) were established. The first set (designated MMTV VI and VII) was prepared by the fusion of mouse myeloma cells (NS-1) with the lymphocytes of BALB/c mice which had previously been immunized with the C3H strain of MMTV. Approximately 10% of the hybrid cells from MMTV VI and VII initially plated after cell fusion produced immunoglobulins that reacted in antibody binding assays with C3H MMTV. Forty of these were cloned and six eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from sera and ascites of syngeneic mice inoculated with the hybrid cells. All the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the IgM subclass and three produced IgG₂a. Three serologically distinct specificities were observed when these ascitic fluids were tested against different strains of MMTV. The antigenic reactivities detected were: (1) a type-specific determinant unique to the C3H strain of MMTVs; (2) class-specific determinants shared between C3H and OR MMTV; and (3) a group-specific determinant found on C3H, OR, RIII, and the endogenous C3H (C3Hf). MMTVs. The second set of hybridoma cells (designated MMTV XVII and XXI) was prepared by the fusion of NP-3 mouse myeloma cells with the lymphocytes from the C3Hf mice for XVII and BALB/c mice for XXI. For these fusions approximately 10% of the hybrid cells initially plated showed positive antibody production in binding assays with C3Hf MMTV. Fifty-four of these were cloned and five yielded stable cell lines. Again all monoclones were directed against gp52. One of these cell lines produced IgM, two produced IgG₃, one produced IgG₂b, and one was a direct binder but did not react in immunodiffusion studies. Only two distinct serological specificities were seen with these clones: (1) a type-specific determinant unique to the C3Hf MMTV; and (2) a group-specific determinant shared by C3H, GR, Rill, and C3Hf MMTVs was seen again. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule, and also clearly show that one strain of MMTV can be distinguished from other strains.