Nasopharyngeal Carcinoma Sera Research Articles

Screening for nasopharyngeal carcinoma with an ELISA using the Epstein-Barr virus nuclear antigen, EBNA 1: a complementary test to the IgA/VCA immunofluorescence assay

An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.

Epstein-Barr virus nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors

The entire amino acid sequence of the unique region of the EBNA 1 protein was synthesized as a set of 41 20-residue peptides with an overlap of 10 amino acids. The peptides were tested in the enzyme-linked immunosorbent assay for reactivity with immunoglobulin A (IgA) and IgG in sera from 50 patients with nasopharyngeal carcinoma (NPC) as compared with 36 serum samples from healthy Epstein-Barr virus (EBV)-seropositive donors and 5 serum samples from EBV-negative donors. The most immunoreactive peptide for both IgA and IgG binding was localized to the glycine-alanine repeat domain of the antigen. In the unique regions, 16 immunoreactive peptides were found. Of these, four were reactive with IgG but not IgA and three peptides were reactive with IgA but not IgG in NPC sera. In addition, several IgA and IgG epitopes on the carboxy-terminal region were specifically reactive with NPC sera, but unreactive with sera from healthy EBV-positive donors. The results suggest that EBV serology specific for individual epitopes may provide additional useful information not available by conventional serology with whole antigens or the EBNA complex.

Immunological characterization of the Epstein-Barr virus phosphoprotein PP58 and deoxyribonuclease expressed in the baculovirus expression system.

The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells. The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E. coli. DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities. Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58. The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E. coli. The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate. The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0). Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera. Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC. In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

Antibody responses against polypeptide components of Epstein‐Barr virus‐induced early diffuse antigen in patients with connective tissue diseases

The specific humoral response against polypeptide components of Epstein-Barr virus (EBV), the induced early diffuse antigen (EA-D), in patients with connective tissue diseases, including systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD), was investigated by using the immunoblotting technique. The EA(D)-positive sera from patients with infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), immunocompromised patients (renal transplant recipients and patients with AIDS) as well as the EA(D)-negative sera from patients with Burkitt's lymphoma and from clinically healthy subjects served as controls. Seven major antigenic polypeptides with molecular weights of 33 kDa, 35 kDa, 52 kDa, 54 kDa, 56 kDa, 58 kDa, and 134 kDa were detected reproducibly by the EA(D)-positive reference sera and, in particular, by each of the NPC sera tested. The EA(D)-positive sera from the other groups showed various combinations of detection patterns and few samples reacted with all the major EA(D) polypeptides. Seventy-three percent of sera from SLE and 47% of sera from MCTD were found to react with EA(D). Sixty-one percent of sera from SLE vs. 5% from MCTD detected all the EA(D) polypeptides. These results could either reflect perturbations of the immune response linked to the autoimmune disease or suggest a possible pathogenic role of EBV.

Antibodies against the large subunit of the EBV-encoded ribonucleotide reductase in patients with nasopharyngeal carcinoma

The open reading frame corresponding to BORF2 and encoding the large subunit of the Epstein-Barr virus (EBV) ribonucleotide reductase has been inserted into the prokaryotic expression vector pUC19. A 90-kDa protein was produced when the intact plasmid was used as a template for in vitro DNA-directed protein synthesis. Using templates generated by restriction digests within the BORF2 open reading frame, in the same system, truncated polypeptides resulted confirming the identity of the 90-kDa protein. The protein was then produced in a heterologous expression system and used in protein immunoblotting to screen for antibodies in sera from nasopharyngeal carcinoma (NPC), Burkitt's lymphoma (BL) or control subjects. Twenty out of 33 NPC sera were positive for antibodies against the large subunit, 13 of these were positive for both IgG and IgA, whilst 7 were positive for IgG only. Out of 15 BL sera and 10 control sera, none were positive. These results are similar to those observed for other EBV-encoded enzymes, including the DNase which had been used as an early marker for the development of NPC. The results presented here indicate that antibodies against the large subunit of ribonucleotide reductase could serve as an additional marker for NPC.

Improved Sensitivity of Detection by Avidin—Biotin Complex (ABC) Immunocytochemistry in Epstein‐Barr Virus Serology

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.

Relationship between nasopharyngeal carcinoma and high antibody titers to epstein‐barr virus‐specific thymidine kinase

Epstein-Barr virus (EBV) has long been implicated in nasopharyngeal carcinoma (NPC). Recent studies in our and other laboratories have shown a correlation between the disease and high antibody titers to EBV-specific DNase. These data led us to also examine serial sera from healthy adults and patients with infectious mononucleosis or NPC, for their capacity to neutralize the EBV-specific thymidine kinase (TK) activity from chemically induced EBV-carrying human lymphoblastoid cells. Our results were the following: (i) sera were found that efficiently blocked the EBV-specific TK activity of induced-Raji TK- cell extracts, but not the host-cell TK activity from EBV-negative BJAB cells; (ii) a relationship appeared between high levels of EBV-specific TK-neutralizing activity in sera and NPC pathology, even though in this preliminary study the degrees of EBV-induced TK-blocking activity detected in sera were not significantly correlated with EBV-specific antibody titers; (iii) the EBV-induced TK-neutralizing activity was found in the main IgG fraction derived from NPC sera. These data must be compared with other known antibody responses to EBV for their clinical interest in NPC control.

Nasopharyngeal Carcinoma in Japan —Clinical Evaluation of EBV Serology and EBV-Associated Nuclear Antigens Test—

This study was aimed at the possible clinical application of two Epstein-Barr virus (EBV) immunological tests to Japanese nasopharyngeal carcinoma (NPC). One of them was serological test to measure antibody titers to EBV-associated antigens (viral capsid antigens (VCA) and virus-induced early antigens (EA)) of 42 NPC sera. The other was for the detection of EBV-associated nuclear antigens (EBNA) in 22 tumour cells of NPC. The results showed that significant elevation of antibody titers to VCA and EA which resided particularly in IgA class as well as a high incidence in the presence of EBNA were found in undifferentiated NPC. Apart from its importance in etiology, EBV serology and the EBNA test may help clinicians to establish a diagnosis of undifferentiated NPC in Japan and there is little doubt that these tests should eventually become a routine biologic test in ENT practice.

A technique for the identification of glycoprotein antigens in immune complexes and its application to the detection of a common glycomprotein in sera of patients with burkitt's lymphoma and nasopharyngeal carcinoma

A new technique for the detection of glycoprotein antigens in immune complexes (IC) isolated from serum is described. The technique was developed with a model IC system consisting of ovalbumin (OVA)-rabbit anti-ovalbumin antibodies (aOVA), at 3 times antigen excess. OVA-aOVA IC added to normal human serum (NHS) were purified by absorption onto and elution from tubes coated with rheumatoid factor (RF) and were subjected to electrophoresis in polycrylamide gels. Concanavalin A (Con A) binding proteins were detected by treating the gels with radioiodinated Con A 125Con A), followed by autoradiography. IC isolated from sera of patients with Burkitt's lymphoma (BL) and Nasopharyngeal Carcinoma (NPC) were analyzed before and after reduction with dithioreitol. Two closely spaced proteins of about 40 kdalton were identified in the reduced samples in 26 of 30 BL sera (86%) and in 24 of 30 NPC sera (80%) but were not seen in 30 sera of African patients with a variety of unrelated tumors nor in 12 sera of European blood bank donors.

Frequency and levels of antibodies to Epstein-Barr virus-specific DNase are elevated in patients with nasopharyngeal carcinoma.

Sera from healthy individuals and patients with infectious mononucleosis, Burkitt lymphoma, nasopharyngeal carcinoma, or other malignancies were examined for their capacity to neutralize Epstein-Barr virus (EBV)-induced DNase activity. Sera were found that neutralized the EBV DNase but not herpes simplex virus type 1 or type 2 DNases, and vice versa. Sera from 46 of the 49 patients with nasopharyngeal carcinoma examined (94%) neutralized > 6 units of EBV DNase per ml of serum. In contrast, only 19% of 47 patients with Burkitt lymphoma, 12% of 183 patient with other malignancies, 4% of 58 patients with infectious mononucleosis, and none of 101 healthy individuals had such levels of neutralizing activity. The neutralizing factor was found in the IgG fraction derived from nasopharyngeal carcinoma sera. There was no correlation between the concentration of these antibodie and the titers of IgG ad IgA antibodies to the EBV capsid antigen, the early antigen complex, or the EBV-associated nuclear antigen.

Epstein-Barr-virus-specific IgA and IgG serum antibodies in nasopharyngeal carcinoma

The sera of 73 patients with nasopharyngeal carcinoma (NPC), 28 patients with other carcinomas (OC) and 89 healthy subjects (HS) were tested for IgG and IgA antibodies to Epstein-Barr virus (EBV) viral capsid antigen (VCA). The majority of the NPC sera had IgG titres of 160 or above, whereas the majority of the other sera had titres below 160. For IgA reactivity to EBV-VCA, 68 of 73 (93-2%) NPC sera had titres of greater than or equal to 10. In contrast, only 6 of 28 (21-4%) OC sera and none of the HS sera had such titres. The mean serum concentrations of IgG, IgA, IgM and C3' were also determined in 55 NPC and 20 OC patients and 18 HS. They were all significantly higher in the NPC sera than in the HS. Although the concentrations of IgG and C3' were not significantly different in the two carcinoma groups, the concentrations of IgA and IgM were significantly higher in the NPC group than in OC. These findings appear to reflect the intensity of EBV-specific antigenic stimulation in NPC, and the EBV-specific serum IgA reactivity may be a sueful aid to the diagnosis of NPC, especially in cases with an occult primary tumour. It may be also of value as a screening test in people at high risk.