Enzyme-linked immunosorbent assay for IgG antibodies to Epstein-Barr virus-associated early antigens and viral capsid antigen.

An enzyme-linked immunosorbent assay has been developed for the detection of IgG antibodies to Epstein-Barr virus-associated early antigens and late antigens including the viral capsid antigen. The antibody titers of human sera determined in this way correlate well with those by indirect immunofluorescence. ELISA was more sensitive than the IF method. The assays described may be used for rapid and sensitive diagnosis of EBV-related diseases. In addition, the ELISA will be useful for the determination of antibody titers to isolated EBV-associated antigens, e.g., purified components of the EA complex.

Detection of IgA antibodies to Epstein-Barr virus-associated antigens by ELISA.

The detection of IgA antibodies to the Epstein-Barr virus (EBV)-associated viral capsid antigen (VCA) and early antigens (EA) is of diagnostic and prognostic importance for patients with nasopharyngeal carcinoma (NPC). An ELISA for the determination of serum IgG antibodies to these antigens has been developed which uses the double antibody method. 136 sera obtained from healthy donors and patients with non-EBV related tumors and lymphomas were tested by ELISA; only 3 sera, from patients with chronic lymphatic leukemia, hairy cell leukemia and Burkitt-like lymphoma, contained antibodies of IgA class to VCA and EA. Ninety-five sera from patients suspected of having NPC were tested. IgA anti-VCA was found in 28 sera (29.5%), 12 of which also contained IgA anti-EA. The assays described are suitable for diagnosis and follow-up of patients with EBV-associated nasopharyngeal carcinoma. Furthermore, isolated EA components may be tested for their reactivity with IgA antibodies, as was shown for the 60 kDa polypeptide associated with the EA complex.

Purification of a polypeptide complex (p52) belonging to the D-subspecificites of Epstein-Barr virus-induced early antigens.

A two-dimensional immunoblot analysis of chemically induced EBV DNA carrying Burkitt's lymphoma cell lines shows besides a large number of minor components at least two major groups of polypeptides: the most prominent group of polypeptides is observed in the range of 48 to 58 kDa (pI 4.5 to 8.5) and another group at 38/36 kDa (pI 4.4). A polypeptide complex (p52) belonging to the Epstein-Barr virus (EBV)-induced early antigen complex (EA) has been isolated from IdU-induced Raji and B95-8 cells as well as from n-butyrate-induced P3HR-1 cells. The p52 polypeptides have been purified by chromatography on Blue-, DEAE-, CM-, and Phenyl-Sepharose. The purification of these components of the EA complex was monitored by ELISA and by two-dimensional immunoblots using a well-characterized high anti-EBV positive human serum. The isolated polypeptides have an apparent mol wt of about 52,000 Da as determined under nondenaturing conditions by gel filtration chromatography on Sephacryl S-300. One- and two-dimensional immunoblots show a major group of polypeptides of 52 kDa (pI 8.5 to 5.5) with EA activity and some minor components with smaller size up to 40 kDa. The latter seem to be generated by limited proteolysis of p52 polypeptides. The EA activity of the isolated polypeptides could be confirmed by their reaction with IgG anti-EA positive as well as IgA anti-EA positive sera by ELISA. The purified polypeptide complex did not react with anti-EA-D negative, anti-EA-R positive sera obtained from patients with African Burkitt's lymphoma, suggesting that these polypeptides belong to the EA-D complex. The monoclonal antibody R3 reacted with the isolated 52 kDa components of EA suggesting a common epitope present on these polypeptides, the same result was obtained with three rabbit sera produced against the isolated polypeptide complex.

Enzyme-linked immunosorbent assay for antibodies to Epstein-Barr virus-associated early antigens and the viral capsid antigen.

The determination of serum antibody titers to Epstein-Barr virus (EBV)-associated antigens is of diagnostic and prognostic importance in the case of patients with EBV-associated tumors, ie, Burkitt's lymphoma and nasopharyngeal carcinoma, and in infectious mononucleosis. Enzyme-linked immunosorbent assays (ELISA) were developed for the detection of antibodies to EBV-associated early antigens (EA) and the viral-capsid antigen. The antibody titers obtained by the ELISA method correlate well with the antibody titers determined by the standard indirect immunofluorescence technique. An ELISA developed for the quantitation of early antigens in cell extracts was used to purify a protein of the EA complex. Interestingly, some of the high anti-EA titered sera, as determined by immunofluorescence, did not contain significant amounts of antibodies to this component detectable by the ELISA with immobilized partially purified protein. Anti-EA negative sera did not react with the isolated EA component.

Purification of a protein (60K/58K) associated with the Epstein-Barr virus-induced early antigen complex in Raji cells.

A double antibody sandwich ELISA has been established for the detection and quantitation of EBV-associated early antigens (EA) in IUdR-induced Raji cells. The EA complex extracted from Raji cells could be separated by ion exchange chromatography and isoelectric focusing into several components. One EA-associated subspecificity has been purified by DEAE-, CM-, and Blue-Sepharose chromatography followed by isoelectric focusing. The isolated protein has an apparent molecular weight of 240,000 +/- 20,000 daltons under non-dissociating conditions on Sephacryl S-300, an isoelectric point of 4.5, and seems to be composed of two polypeptides of 60,000 and 58,000 daltons as shown by SDS-gel electrophoresis and two-dimensional gel electrophoresis. Preliminary data indicate that the 58,000 polypeptide is generated by limited proteolysis of the 60,000 polypeptide. The EA activity of the isolated protein has been confirmed by the double antibody sandwich ELISA and its reactivity with anti-EA-positive sera in an ELISA for the detection of anti-EA antibodies.

[Demonstration of IgG- and IgA-antibodies against Epstein-Barr virus-associated antigens with a microtiter enzyme immunoassay system. The determination of serum antibodies against the viral capsid antigen and the early antigen complex in the sera of tumor and infectious mononucleosis patients]. / Nachweis von IgG- und IgA-Antikörpern gegen Epstein-Barr-Virus-assoziierte Antigene mit einem Enzymimmunoassay im Mikrotitersystem. Bestimmung der Serumantikörper gegen das Viruskapsidantigen und den Frühantigenkomplex in Seren von Tumorpatienten und Patienten mit infektiöser Mononukleose.

An enzyme-linked immunosorbent assay (ELISA) using micro-titre plates has been established for determination of IgG and IgA antibodies to the Epstein-Barr virus (EBV)-associated early antigen complex (EA) and the viral capsid antigen (VCA). The results obtained correlated well with the antibody titre determinations by the standard immunofluorescence technique. The ELISA was found to be about 15 to 30 times more sensitive than the immunofluorescence assay. Because of its high sensitivity, specificity and rapid performance the enzyme-immunoassay is a useful tool for large scale epidemiological studies of EBV-associated diseases as well as for the early diagnosis and monitoring of patients with EBV-associated tumours: Burkitt's lymphoma and nasopharyngeal carcinoma. IgG and IgA antibody titres to EA and VCA were determined in sera obtained from healthy controls, patients with EBV-associated tumours, infectious mononucleosis and various diseases known to be associated with EBV-reactivation, i.e. lymphomas and leukaemias. Except in sera of patients with nasopharyngeal carcinoma, IgA antibodies to VCA and EA could only be detected at a rather high frequency (36% IgA anti-VCA+, 21% IgA anti-EA+) and in substantial titres in sera obtained from patients with chronic lymphatic leukaemia.