Milton R. Tam

The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses

Abstract Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of C57BL/6 mice that were immunized with the AKR leukemia K36. Approximately 10% of the hybrid cells produced immunoglobulins that reacted in antibody binding assays with AKR MuLV. By the combined use of low-density passage and cloning, seven independent cell lines were isolated. These cells produced antiviral antibodies at a level of 3–15 μg/ml of culture fluid. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of tumors (hybridomas) that secreted extremely high levels of monoclonal antibodies (5–15 mg/ml) into the serum or ascites fluid. Five of the hybrid cell lines produced immunoglobulins of the IgM subclass, one produced IgG2a, and one produced IgG2b. In high-resolution two-dimensional polyacrylamide gels, these immunoglobulins showed the limited heterogeneity in heavy and light chains that would be expected for monoclonal products. Radioimmune precipitation assays demonstrated that the monoclonal antiviral antibodies reacted with the p15(E) protein of ecotropic MuLV; these antibodies did not react with the p15(E) protein of xenotropic MuLV. In contrast, rabbit antiserum prepared against purified p15(E) reacted equally well with ecotropic and xenotropic MuLV. The sera or ascites fluids from hybridoma-bearing mice had antibody titers 75- to 100-fold higher than the sera from conventionally immunized mice or rabbits. Serological analysis demonstrated that the monoclonal antibodies reacted with the cell surface of virus-producing leukemia cells, but not with normal thymocytes. Furthermore, monoclonal anti-pl5(E) antibodies of the IgG2a subclass mediated lysis of the virion of ecotropic MuLV in the presence of complement.

Monoclonal antibodies against murine leukemia viruses: Identification of six antigenic determinants on the p15(E) and gp70 envelope proteins

Abstract Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.

Monoclonal Antibody Therapy of Mouse Leukemia

Antibody treatment of neoplastic disease has long been of interest. Under certain limited conditions, significant inhibition of tumor growth has been achieved (reviewed in Wright and Bernstein, 1980). The therapeutic effects obtained with antisera have suggested a potential role for antibody therapy, but the success of this approach has so far not been impressive. Limitations in the effectiveness of serum treatment may have resulted from insufficient quantities of high-titered antibody of appropriate class, avidity, and specificity.

APPLICATION OF A THERMALLY-REVERSIBLE POLYMER-ANTIBODY CONJUGATE IN A NOVEL MEMBRANE-BASED IMMUNOASSAY

We have developed a novel method to immobilize antibodies onto a cellulose acetate membrane using a conjugate of an N-isopropylacrylamide polymer covalently bound to the antibody. When compared with the unconjugated antibody, over 30-fold increase in retention of the antibody on the membrane was observed when it was conjugated to poly (N-isopropylacrylamide). Studies of the polymer-membrane interaction suggest a combination of hydrophobic and ionic forces, especially the former, is responsible for the high retention. We applied this novel immobilization technology in the development of a membrane-based immunoassay.

Cytologic Manifestations of Cervical and Vaginal Infections: II. Confirmation of Chlamydia trachomatis Infection by Direct Immunofluorescence Using Monoclonal Antibodies

We found inflammatory patterns of transparent lymphocytes on increased numbers of histiocytes suggestive of chlamydial infection in 68 (56%) of 121 cervical cytologic smears. Chlamydia trachomatis was isolated from 36 (53%) of those with and only two (4%) of those without such inflammatory patterns. Direct stain with fluorescein-conjugated monoclonal antibodies demonstrated elementary bodies of C trachomatis in 30 (79%) of the 38 culture-positive patients, including 29 of the culture-positive patients who had an inflammatory cytologic pattern suggestive of C trachomatis infection. Thus, Papanicolaou smears can be screened for inflammatory pattern, and separate endocervical smears from patients with a pattern suggestive of chlamydial infection can then be stained by immunofluorescence to confirm the presence of C trachomatis infection. This two-step approach detected 29 of 38 infections confirmed by culture in the present study, giving a sensitivity of 76%, a specificity of 100%, and a positive predictive value of 100% in a population having a 31% prevalence of C trachomatis infection.

Monoclonal mouse antibodies as probes for antigenic polymorphism in murine leukemia viruses

Abstract Hybrid cell lines that produce monoclonal antibodies against the proteins of murine leukemia virus (MuLV) have been prepared by the fusion of mouse myeloma cells with the lymphocytes of mice that were immunized with MuLV-producing leukemia cells. Preliminary screening of culture fluids from these hybrid cells for reactivity on a panel of viruses led to the selection of cell lines that produced antibodies against highly polymorphic antigenic sites of the viral proteins. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of ascites fluids that contained high titers of monoclonal antibodies. These ascites fluids demonstrated 100,000-fold variations in reactivity with the proteins of different isolates of MuLV. Monoclonal antibodies of this type now provide new tools for the precise serological classification and detection of murine leukemia viruses.

Mapping of Viral Proteins with Monoclonal Antibodies

The mouse leukemia viruses (MuLV) are a highly polymorphic group of agents that occur as endogenous infections in inbred and feral mice. All mice contain some genetic equivalents of MuLV, although considerable variation occurs in the particular viruses that are inherited by one mouse strain or another. On the basis of host range characteristics the endogenous MuLV have been classified into three major groups: (1) ecotropic MuLV efficiently infect murine cells, but not cells of other species (Hartley et al., 1970), (2) xenotropic MuLV efficiently infect the cells of other species, but not murine cells (Levy, 1973), and (3) amphotropic MuLV efficiently infect cells of both murine and heterologous origin (Rasheed et al., 1976; Hartley and Rowe, 1976). Viruses of each of these groups can be further subclassified by more defined biological characteristics [e.g., N-ecotropism vs. B-ecotropism (Hartley et al., 1970) and inducible vs. noninducible xenotropic properties (Barbacid et al., 1978)] and recombinant viruses with the prop-erties of two different parental viruses have also been found to occur at high frequency in vivo (Hartley et al., 1977; Elder et al., 1977).