Carl Feit

An enzyme-linked immunosorbert assay (ELISA) for the detection of monoclonal antibodies recognizing surface antigens expressed on viable cells

A solid-phase enzyme-linked immunosorbent assay (ELISA) has been developed for detecting monoclonal antibodies binding to surface antigens expressed on viable adherent cells of tumor cell lines. This assay utilizes a sheep anti-mouse IgG to which a beta-galactosidase is linked. It is highly sensitive and permits quantification of IgG monoclonal antibody levels. In studies of monoclonal antibodies prepared against human tumors, the ELISA assay revealed the presence of antigens which were not seen using acetone-fixed cell immunofluorescence methods. This assay is safe, rapid, low cost, and gives reproducible quantitative results. As such it should prove useful to laboratories engaged in the study of antigens expressed on cell membranes.

Detection of retroviral particles in hybridomas secreting monoclonal antibodies

Electronmicroscopy of hybridoma clones derived by fusing BALB/c mouse spleen cells with P3U1 mouse plasmacytoma cells to generate monoclonal antibodies against human sarcoma antigens, revealed the presence of large number of viral particles. These particles were also seen budding from the cell surfaces. The intracytoplasmic particles were intracisternal and resembled type-A oncornavirus, while the budding and extracellular forms, with a centrally located nucleoid, resembled mature type-C oncornaviruses. Cells of the parental P3U1 palsmacytoma cell line and of the NS-1 myeloma cell line contained morphologically identical viral structures. The scientific and medical communities engaged in hybridoma research should be alert to the possible presence of viruses in hybridomas and their products. The question is raised as to whether it is safe to use mouse monoclonal antibodies for clinical purposes, both diagnostic and therapeutic.

The interaction of Kaposi's sarcoma with monoclonal antibodies to human sarcoma and connective tissue differentiation antigens

Four monoclonal antibodies (McAbs) previously generated against human soft tissue sarcomas and reacting with connective tissue differentiation antigens were evaluated for their interaction with tissues obtained from patients with classic Kaposis sarcoma. Biopsy was performed on active neoplastic lesions from the skin of 26 patients, frozen sections were prepared, and the binding of the McAbs was tested using the indirect immunofluorescence assay. Clinically uninvolved skin from the same patients as well as skin and muscle from eight non‐cancer patients were treated similarly and served as controls. McAbs IXG11, 23H7, IIIE5, and 15G5 interacted strongly with the Kaposis sarcoma lesions and weakly with the uninvolved skin in 22 of 26 (84%), 23 of 26 (88%), 12 of 14 (85%), and 1 of 6 (16%) of the patients, respectively. IXG11, 23H7, and IIIE5 interacted weakly with the skin of seven of eight non‐cancer patients. McAb 15G5 was found to bind strongly to tumor lesions, to the respective uninvolved skin in four of five Kaposis sarcoma patients, and also to skin and connective tissues of muscle from non‐cancer patients. The mode of interaction was morphologically different for each McAb. It is suggested that McAbs IXG11, 23H7 and IIIE5 identify markers whose expression is markedly increased in Kaposis sarcoma lesions as compared with uninvolved skin of the same patients. These markers may serve as immunologic probes for the investigation of this neoplastic process.

Modification of HAT Medium and Hybridoma Formation

Among the major challenges in the field of hybridoma formation is the need to find ways of increasing the efficiency of myeloma-spleen cell hybridization. HAT medium originally developed by Littlefield in 1964 (1) has been one of the key factors that has made hybridoma generation practical. The value of this medium is generally considered from the viewpoint of its ability to inhibit unfused myeloma cell proliferation. We have recently explored modifications of this selective medium to determine whether an augmented medium designed to further support hybridoma growth would be capable of enhancing and stabilizing the newly formed hybrid clones at their earliest stage, closest to their formation. Our approach was based on the assumption that the first hours and days after cell fusion are the most critical for the outcome of a given fusion, and therefore enhancement efforts should be concentrated on these early phases (2). Our strategy consisted of adding known biologically active molecules to HAT medium immediately following the PEG-mediated cell fusion, anticipating that the successful agent would enhance hybridoma formation (3). It was further assumed that an effective agent would ultimately cause an increase in the number of the relevant hybridomas produced per fusion, increase the average size of the clones formed, and consequently be associated with higher production of monoclonal antibodies, as compared to yields of fusions using conventional HAT medium alone.

Dexamethasone Effects on Hybridoma Formation

To assess the effects of sterioids on the formation of hybridomas, sheep red blood cell (SRBC) immunized spleen cells from Balb/c mice were fused with P3U1 myeloma cells. After fusion, cells were grown for 1 week in HAT medium containing 10-3, 10-5, 10-7, 10-9 mM dexamethasone (DM) or HAT alone. Subsequently spent media was removed and refeeding with HAT was commenced. Parameters measured were: clone number, size, antibody production and DNA content. At concentrations of DM given above, the number of clones produced was 16, 39, 24, 29 respectively as compared to 40 clones in control wells. At 10-3 and 10-5 mM DM parental cells were markedly suppressed. All clones were < 1 mm in size at 10-3 mM, whereas at 10-5 mM and in controls, 43% and 46% of the clones respectively were 1–4 mm. By micro hemagglutination it was found that only 5% of the clones at 10-3 mM and 10% at 10-5 mM produced anti-SRBC antibody. In contrast, at lower doses and in control plates 99% of clones were antibody producers. Computerized DNA-RNA flow cytometry using acridine orange indicated that DNA loss was more pronounced in clones exposed to DM than controls. This method also indicated a lower number of parental cells in cultures grown in the presence of DM.

A phase I clinical study of a serological test for detection of leukemia-associated antigens (LAA)

Abstract A series of antisera to leukemia-associated antigens (LAA) were prepared by immunization of monkeys and rabbits with leukemia cells or leukemia cell extracts and absorption of the resultant antisera with red blood cells and leukocytes from healthy donors. These antisera were used with a complement-dependent microcytotoxicity test procedure at two leukemia treatment centers in a controlled Phase I clinical study designed to evaluate the specificity and sensitivity of the antisera for the detection of LAA and the suitability of the test procedure for routine clinical use. Tests of mononuclear cells prepared from coded blood samples of patients with different types of leukemia in various stages, patients with other malignancies and benign disorders unrelated to leukemia, and healthy controls indicated that several antisera had sufficient specificity and sensitivity for the detection of LAA to be of potential clinical value. These studies also suggested that such antisera might be useful in monitoring patients with leukemia and evaluating the quality of disease control, although the test procedure had some disadvantages for routine use in a general hematology laboratory.