Background: More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity–purified recombinant HPV proteins. Methods: Twenty-seven HPV proteins were expressed as glutathione S-transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Spectrally distinct bead sets, each carrying one particular antigen, were mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer. Results: Glutathione S-transferase fusion proteins bound with high affinity to glutathione-displaying beads (Kd = 6.9 × 10−9 mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1 000 000. Imprecision (median CV) was ≤5.4%, and assay reproducibility was high (R2 = 0.97). Results on clinical samples showed high concordance with ELISA (κ = 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer (P <0.001). Conclusion: Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.

Certain infectious agents are recognised causes of cancer and other chronic diseases. To understand the pathological mechanisms underlying such relationships, here we design a Multiplex Serology platform to measure quantitative antibody responses against 45 antigens from 20 infectious agents including human herpes, hepatitis, polyoma, papilloma, and retroviruses, as well as Chlamydia trachomatis , Helicobacter pylori and Toxoplasma gondii , then assayed a random subset of 9695 UK Biobank participants. We find seroprevalence estimates consistent with those expected from prior literature and confirm multiple associations of antibody responses with sociodemographic characteristics (e.g., lifetime sexual partners with C. trachomatis ), HLA genetic variants (rs6927022 with Epstein-Barr virus (EBV) EBNA1 antibodies) and disease outcomes (human papillomavirus-16 seropositivity with cervical intraepithelial neoplasia, and EBV responses with multiple sclerosis). Our accessible dataset is one of the largest incorporating diverse infectious agents in a prospective UK cohort offering opportunities to improve our understanding of host-pathogen-disease relationships with significant clinical and public health implications.

BACKGROUND: Human papillomavirus 16 (HPV16) E6 antibodies may be an early marker of the diagnosis and recurrence of human papillomavirus–driven oropharyngeal cancer (HPV‐OPC).METHODS: This study identified 161 incident oropharyngeal cancer (OPC) cases diagnosed at the University of Pittsburgh (2003‐2013) with pretreatment serum. One hundred twelve had preexisting clinical HPV testing with p16 immunohistochemistry and HPV in situ hybridization (87 were dual‐positive [HPV‐OPC], and 25 were dual‐negative [HPV‐negative]); 62 had at least 1 posttreatment serum sample. Eighty‐six of the 161 tumors were available for additional HPV16 DNA/RNA testing (45 were dual‐positive [HPV16–OPC], and 19 were dual‐negative [HPV16–negative). HPV16 E6 antibody testing was conducted with multiplex serology. The following were evaluated: 1) the sensitivity and specificity of HPV16 E6 serology for distinguishing HPV‐OPC and HPV16–OPC from HPV‐negative OPC, 2) HPV16 E6 antibody decay after treatment with linear models accommodating correlations in variance estimates, and 3) pre‐ and posttreatment HPV16 E6 levels and the risk of recurrence with Cox proportional hazards models.RESULTS: Seventy‐eight of 87 HPV‐OPCs were HPV16 E6–seropositive (sensitivity, 89.7%; 95% confidence interval [CI], 81.3%‐95.2%), and 24 of 25 HPV‐negative OPCs were HPV16 E6–seronegative (specificity, 96.0%; 95% CI, 79.6%‐99.9%). Forty‐two of 45 HPV16–OPCs were HPV16 E6–seropositive (sensitivity, 93.3%; 95% CI, 81.7%‐98.6%), and 18 of 19 HPV16–negative OPCs were HPV16 E6–seronegative (specificity, 94.7%; 95% CI, 74.0%‐99.9%). Posttreatment HPV16 E6 antibody levels did not decrease significantly from the baseline (P = .575; median follow‐up, 307 days) and were not associated with the risk of recurrence. However, pretreatment HPV16 E6 seropositivity was associated with an 86% reduced risk of local/regional recurrence (hazard ratio, 0.14; 95% CI, 0.03‐0.68; P = .015).CONCLUSIONS: HPV16 E6 antibodies may have potential clinical utility for the diagnosis and/or prognosis of HPV‐OPC. Cancer 2017;123:4382‐90. © 2017 American Cancer Society.