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Epstein-Barr (EBV) infection and presence of a nasopharyngeal cancer (NPC) case in the family increases the risk of developing NPC. Aberrant anti-EBV immunoglobulin A (IgA) antibodies (EBV-IgA) may be present in the sera of non-cancer individuals and predict NPC. Limited studies report the presence of EBV-IgA antibodies within non-cancer individuals in Indonesia where the disease is prevalent. This study aimed at exploring whether EBV-IgA was found more frequently among first degree relatives of NPC patients and individuals presenting with chronic symptoms in the head and neck area compared to healthy controls. A total of 967 non-cancer subjects were recruited, including 509 family members of NPC cases, 196 individuals having chronic complaints in the head and neck region, and 262 healthy donors of the local blood bank. Sera were analyzed using a standardized peptide-based EBV-IgA ELISA. Overall, 61.6% of all individuals had anti-EBV IgA reactivity equal to or below cut off value (CoV). Seroreactivity above CoV was significantly higher in females (38.7%) compared to males (28.7%) (p = 0.001). Older individuals had more seroreactivity above CoV (42.5%) than the younger ones (26.4%) (p< 0.001). Seroprevalence was significantly higher in family members of NPC patients (41.7%), compared to 32.7% of individuals with chronic head and neck problems (p = 0.028) and 16.4% healthy blood donors (p< 0.001). As conclusion, this study showed a significant higher seroprevalence in healthy family members of NPC cases and subjects presenting with chronic symptoms in the head and neck area compared to healthy individuals from the general community. This finding indicates that both groups have elevated risk of developing NPC and may serve as targets for a regional NPC screening program.

Epstein-Barr virus (EBV) infection and family history are significant risk factors associated with undifferentiated nasopharyngeal carcinoma. The presence of aberrant immunoglobulin A (IgA) antibodies against specific EBV antigens in healthy individuals can be predictive of the disease. Very limited reports explored the EBV IgA antibody presence within families of sporadic cases of nasopharyngeal carcinoma. This study aimed to determine whether EBV IgA was observed more frequently among family members of sporadic cases of nasopharyngeal carcinoma compared to community controls and evaluated the non-viral factors as determinants of antibody level. First-degree relatives of nasopharyngeal carcinoma patients (n = 520) and case-matched community controls (n = 86) were recruited. Sera from all individuals were tested in standardized peptide-based EBV IgA ELISA. Data on demographic variables and other exogenous factors were collected using a questionnaire through face-to-face interviews. A similar frequency of EBV IgA (cut-off value/CoV 0.354) was observed in the first-degree relatives of cases and in community controls (41.2% vs. 39.5%, P = 0.770). However, with a higher antibody level (OD(450)  = 1.000; about three times standard CoV), the relatives showed significantly higher frequency (36.9% vs. 14.7%, P = 0.011). When adjusted for all exogenous factors, the strongest factors associated with seropositivity are being a father (odds ratio/OR = 4.36; 95% confidence interval/CI = 1.56-12.21) or a sibling (OR = 1.89; 95% CI = 1.06-3.38) of a case of nasopharyngeal carcinoma. The higher level of EBV IgA seroreactivity in first-degree relatives of sporadic cases of nasopharyngeal carcinoma compared to the general population supports the use of EBV IgA ELISA for screening among family members., (Copyright © 2012 Wiley Periodicals, Inc.)

Undifferentiated nasopharyngeal carcinoma (NPC; WHO type III) is 100% associated with Epstein-Barr virus (EBV) infection and the fourth most prevalent cancer in Indonesian males. Therapy failure is high, since most patients come to the hospital at an advanced stage of disease. Screening for early-stage NPC is needed. Here, a simple and economical two-step enzyme-linked immunosorbent assay (ELISA) system is proposed for diagnosing NPC in high-risk populations, employing the peptide-based immunoglobulin A (IgA) EBNA1 plus viral capsid antigen p18 ELISA as an initial screening test and the IgA early antigen (EA) ELISA using a different set of EBV antigens as a confirmation test. A total of 151 NPC patients and 199 regional healthy EBV carriers were used to evaluate the two-step ELISA approach. Routinely, EBV IgG immunoblotting is used as a standard confirmation test. The sensitivity and specificity for diagnosing NPC by the two-step ELISA approach increased from 85.4% to 96.7% and 90.1% to 98%, respectively, with positive predictive values and negative predictive values increasing from 78.7 and 93.9% to 97.3 and 97.5%, respectively, relative to the immunoblotting confirmation system. On discrepant samples, additional testing was done by EBV DNA load quantification in blood. Results showed that 5/11 discrepant NPC samples with an elevated IgA EA ELISA also had elevated an EBV DNA load in the circulation (range, 3,200 to 25,820 copies/ml). Therefore, the IgA EA ELISA is proposed as a confirmation test in first-line NPC serological screening studies. This two-step EBV ELISA system provides a standardized approach for NPC screening and may be used in combination with dried blood sampling in future field studies for identification of early-stage NPC in high-risk regions.

Dried-blood (DB) samples on filter paper are considered clinical specimens for diagnostic use because of the ease of collection, storage, and transport. We recently developed a synthetic-peptide-based immunoglobulin A (IgA) (EBNA1 plus viral capsid antigen [VCA]-p18) enzyme-linked immunosorbent assay (ELISA) for nasopharyngeal carcinoma (NPC) screening. Here, we evaluate the use of two filter papers for DB sampling, i.e., Schleicher & Schuell (S&S) no. 903 and Whatman no. 3; the DB samples were either taken directly from a finger prick or spotted from a Vacutainer blood collector. The elution of DB samples on filter paper was optimized and tested for IgG and IgA reactivity by ELISA (EBNA1 plus VCA-p18) and compared to simultaneously collected plasma samples. The results showed that both types of filter paper can be used for sample collection in NPC diagnosis by using either finger prick or blood spot sampling. Both DB sampling methods produced comparable ELISA (EBNA1 plus VCA-p18) results for IgG and IgA reactivity in 1:100-diluted plasma samples. DB samples of whole blood or finger prick blood show correlation coefficients (r(2)) of 0.825 to 0.954 for IgA on S&S no. 903 filter paper, 0.9133 to 0.946 for IgA on Whatman no. 3 filter paper, 0.807 to 0.886 for IgG on S&S no. 903 filter paper, and 0.819 to 0.934 for IgG on Whatman no. 3 filter paper. Using plasma IgA as a reference, DB sampling showed sensitivities and specificities of 75.0 to 96.0% and 93.5 to 100%, respectively. DB samples could be stored at 37 degrees C for 1 to 4 weeks on S&S no. 903 filter paper and 1 to 6 weeks on Whatman no. 3 filter paper without a significant loss of reactivity, with provision of transport options for tropical conditions. IgA proved to be more stable than IgG. Whatman no. 3 filter paper is a more economical yet diagnostically comparable alternative to S&S no. 903 filter paper. Finger prick DB sampling is proposed for NPC diagnosis, particularly for remote hospitals and field screening studies.

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.

Epstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and viral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (n=150) showed IgG responses to few EBV proteins--VCA-p18, VCA-p40, EBNA1, and Zebra--and sporadically weak IgA reactivity to EBNA1 and VCA-p18. Patients with NPC stage 1 (n=6) had similar response patterns. Patients with NPC stage 2-4 (n=132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins--VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, -thymidine kinase, and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.