Martin H. Moar

Autoradiographic Detection of Epstein-Barr Virus (EBV)-Associated Early Antigen in a Variety of EBV DNA-Containing Lymphoblastoid Cell Lines Previously Designated as Nonproducers

By using autoradiography combined with 125I-labeled IgG prepared from human sera containing high anti-EA (early antigen) antibody titers, EA has been detected in a low fraction of cells in a variety of EBV DNA-containing cell lines previously designated as negative for this antigen. These positive cell lines are highly inducible for EA following treatment with iododeoxyuridine (IUDR) and also contain multiple copies of the virus genome on average. The correlation between IUDR inducibility and spontaneous expression of EA, in particular, suggests that the ability to express EA in both situations is interrelated.

Demonstration of complement receptors on lymphoblastoid cells by radiolabeled antibodies and in situ autoradiography

Abstract Exposure of established human lymphoid lines to fresh human complement, followed by radiolabeled anti-C3 antibody and subsequent counting of radioactivity or autoradiography gave strong membrane-associated reactions with some lines but not with others. There was a good correspondence between complement membrane fluorescence (CMF) staining and the autoradiographic reaction. In all probability, these reactions detect a combination of alternate pathway activation and binding of the activated complement products to preformed complement receptors, but the autoradiographic reaction appears to be more sensitive. It also permits the localization and quantitation of the receptors on single cells. Raji cells were found to express the reactive receptor in 95% of the cells, with a high surface density. Daudi and Ramos contained 66 and 45% reactive cells, respectively. The receptor density was considerably lower than on Raji cells, in agreement with previous EAC-rosette tests. P3HR-1 cells that were negative for C3 receptors by both EAC rosetting and complement membrane fluorescence showed only trace reactivity in the autoradiographic test in a low percentage of the cells. Trypsin treatment, known to remove complement receptors without affecting alternate pathway activation, reduced or abolished the autoradiographic reaction.

Detection localization of Epstein-Barr virus-associated early antigens in single cells by autoradiography using 125I-labeled antibodies. (With 1 color plate).

A characteristic feature of virus-transformed cells is the expression of virus-specific antigens usually detectable by immunological or radioimmune procedures. We report here an autoradiographic method for the detection and localization of such antigens in individual cells which combines the cellular specificity and radiolabeling sensitivity of the above procedures. As a test system we have studied the reaction between virus antigen in some Epstein-Barr virus (EBV) DNA-containing cell lines and anti-virus antibody specificities in certain human sera. EBV DNA-containing cell lines express an EBV-determined nuclear antigen (EBNA) and some lines also express the EBV-associated antigens, early antigen (EA) and virus capsid antigen (VCA), in a minority of cells in the population. By employing appropriate 125I-IgG-labeled human sera, we clearly show that EA can be detected without difficulty in cell lines known to spontaneously express this antigen. Moreover, the specificity and sensitivity of the present method is such that low levels of EA cand be detected at cellular concentrations which remain undetectable by conventional immunofluorescence.

Detection of virus and cellular-determined antigens in sity using [125I]protein A and autoradiography

The present paper describes the use of [125I]Protein A, isolated from Staphylococcus aureus, in detecting antigen-antibody complexes by autoradiography on single cells. the method is relatively quick, reproducible, potentially more sensitive than immunofluorescence, and should be useful in combination with conventional radioimmuno-assays. We have used it to detect the cellular expression of IgM, kappa, lambda, and beta 2-microglobulin, as well as the expression of Epstein-Barr Virus (EBV)-associated antigens expressed in human lymphoblastoid cell lines.

Effect of Mitomycin C and Hydroxyurea on the Expression of the Epstein-Barr Virus Cycle Following P3HR-1 Superinfection

Epstein-Barr virus (EBV) DNA synthesis following EBV P3HR-1 superinfection of the Burkitt lymphoma cell line, Daudi, is refractory to mitomycin C and hydroxyurea at concentrations which inhibit cell DNA synthesis to greater than 98% of control values. Further, neither mitomycin C nor hydroxyurea inhibit virus antigen synthesis following P3HR-1 superinfection.

Autoradiographic Detection of the Epstein-Barr Virus (EBV)-Associated Early Antigen (EA) in Lymphoblastoid Cell Lines

Epstein-Barr Virus (EBV) is considered to be a strong candidate as the causative agent in certain human malignancies, particularly African Burkitt’s Lymphoma and Nasopharyngeal Carcinoma (5). Good circumstantial evidence supports this view: in patients suffering from these diseases there are anti-viral antibody titers which are much higher than those found in normal individuals (e.g. 3), and although the virus has never been detected in tumor biopsies directly the tumor cells consistently possess EBV-DNA and express a virus-determined nuclear antigen, EBNA, and in some cases, an EBV-associated membrane antigen as well (5). Moreover, the EBV-DNA in these tumor cells must posses a large coding potential since the synthesis of other EBV antigens can be induced in the cells by 1) explantation of biopsy material in culture, 2) by treatment of in vitro-established tumor cell lines under a variety of physiological conditions, 3) by somatic cell fusion and 4) by the use of DNA synthesis inhibitors (1, 5, 9). Further, in some such induction experiments frequently virus DNA synthesis and virus particles can be demonstrated indicating that the virus DNA coding potential in the original tumor cell is complete. More direct evidence for an oncogenic potential for the virus comes from observations such as the virus’s ability to cause tumors in heterologous hosts (as do some in vitro-established EBV-DNA containing cell lines) (7), and the ability of the virus to “immortalize” blood lymphocytes which have an otherwise limited lifespan in culture (6) and which, after EBV infection, express a variety of transformation characteristics in addition to “immortalization”.