A dried blood spot protocol for high-throughput semi-quantitative analysis of Epstein-Barr Virus VCA IgG and EBNA IgG...

Epstein-Barr virus (EBV) is highly prevalent worldwide and has been linked to different cancers and autoimmune disorders, including Multiple sclerosis (MS). Primary infection typically occurs during early childhood or adolescence and often goes undetected. Serological studies allow the detection of EBV-specific antibodies after infection, but venous blood collection is resource-intense and thus limits sample size. To overcome these challenges, we developed a high-throughput, semi-automated serology protocol targeting antibodies against EBV in capillary blood samples (Dried Blood Spots, DBS).

Paired serum and DBS samples were obtained from 417 participants aged 18-25 years. We used Roche Elecsys^® EBV nuclear antigen-1 EBNA IgG and Elecsys^® EBV viral capsid antigen VCA IgG assays, established for serum, to detect antibodies against EBV anti-EBNA‑1 IgG and anti-VCA IgG in DBS. Cut-off Indices (COI) for the DBS assays were determined using a classification tree.

Of the 416 valid paired serum samples, 78.6% (327/416) were anti-VCA-positive and 76.4% (318/416) anti-EBNA-positive. Discrepant results were observed in 3.6% (15/416) of participants. Based on combined assay results, 20.7% (86/416) of the samples were classified as EBV-negative. The serum COI values revealed that most of the negative cases clustered well below the cutoff, while positive cases spanned a broad range of higher values. Overall, 98.8% (326/330) of participants classified as positive based on serum testing (n = 330) were also classified as positive in DBS (n = 326), corresponding to the sensitivity of DBS relative to serum results. Likewise, 96.5% (83/86) of participants classified as serum-negative participants (n = 86) were also negative in DBS (n = 83), corresponding to the specificity of DBS relative to serum results. Among the 416 participants, only 4/330 (1.2%) were false negatives and 3/86 (3.5%) were false positives in DBS. The newly established assays were validated in a self-sampling cohort of 295 participants.

We established DBS-specific cutoff values to detect antibodies against EBV in DBS, achieving high sensitivity (98.8%) and specificity (96.5%) relative to the corresponding venous blood assay. Furthermore, we derived a correction formula to convert semi-quantitative DBS values to serum-equivalents, enabling comparison with other studies and standardized datasets. The DBS-based approach allows sero-status assessment in a large population and thus e.g. simplifies the identification of suitable participants for clinical trials.