Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18
More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of...
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| description | More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18. |
| doi_str_mv | 10.1371/journal.pone.0229672 |
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Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0229672</identifier><identifier>PMID: 32214362</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Antibodies ; Antigen-antibody reactions ; Antigens ; Biology and life sciences ; Cancer ; Carcinogenesis ; Carcinoma ; Cervical cancer ; Characterization ; Clinical trials ; Compensation ; Cross-reactivity ; Dilution ; Diseases ; Dynamic range ; Employees ; Enzyme-linked immunosorbent assay ; Head & neck cancer ; Head and neck cancer ; Human papillomavirus ; Immunoassay ; Immunoglobulin G ; Immunoglobulins ; Malignancy ; Medicine and Health Sciences ; Methods ; Motor vehicle drivers ; Multiplexing ; Oncoproteins ; Papilloma ; Papillomavirus infections ; Pharmaceutical industry ; Protein purification ; Proteins ; Rankings ; Research and Analysis Methods ; Respiratory system agents ; Serology ; Squamous cell carcinoma ; Technology ; Tumorigenesis ; Viruses ; Wages & salaries</subject><ispartof>PloS one, 2020-03, Vol.15 (3), p.e0229672-e0229672</ispartof><rights>COPYRIGHT 2020 Public Library of Science</rights><rights>2020 Layman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2020 Layman et al 2020 Layman et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-43f507c1c8984bf62a64b7ee97b51465a6082fe79f51fb81a18b14647851db013</citedby><cites>FETCH-LOGICAL-c692t-43f507c1c8984bf62a64b7ee97b51465a6082fe79f51fb81a18b14647851db013</cites><orcidid>0000-0003-4480-5153</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098588/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098588/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2095,2914,23846,27903,27904,53769,53771,79346,79347</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/32214362$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Langevin, Scott M.</contributor><creatorcontrib>Layman, Hans</creatorcontrib><creatorcontrib>Rickert, Keith W</creatorcontrib><creatorcontrib>Wilson, Susan</creatorcontrib><creatorcontrib>Aksyuk, Anastasia A</creatorcontrib><creatorcontrib>Dunty, Jill M</creatorcontrib><creatorcontrib>Natrakul, Dusit</creatorcontrib><creatorcontrib>Swaminathan, Nithya</creatorcontrib><creatorcontrib>DelNagro, Christopher J</creatorcontrib><title>Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>More than 170 types of human papilloma viruses (HPV) exist with many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. 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many causing proliferative diseases linked to malignancy in indications such as cervical cancer and head and neck squamous cell carcinoma. Characterization of antibody levels toward HPV serology is challenging due to complex biology of oncoproteins, pre-existing titers to multiple HPV types, cross-reactivity, and low affinity, polyclonal responses. Using multiplex technology from MSD, we have developed an assay that simultaneously characterizes antibodies against E6 and E7 oncoproteins of HPV16 and 18, the primary drivers of HPV-associated oncogenesis. We fusion tagged our E6 and E7 proteins with MBP via two-step purification, spot-printed an optimized concentration of protein into wells of MSD 96-well plates, and assayed various cynomolgus monkey, human and HPV+ cervical cancer patient serum to validate the assay. The dynamic range of the assay covered 4-orders of magnitude and antibodies were detected in serum at a dilution up to 100,000-fold. The assay was very precise (n = 5 assay runs) with median CV of human serum samples ~ 5.3% and inter-run variability of 11.4%. The multiplex serology method has strong cross-reactivity between E6 oncoproteins from human serum samples as HPV18 E6 antigens neutralized 5 of 6 serum samples as strongly as HPV16 E6. Moderate concordance (Spearman's Rank = 0.775) was found between antibody responses against HPV16 E7 in the multiplex assay compared to standard ELISA serology methods. These results demonstrate the development of a high-throughput, multi-plex assay that requires lower sample quantity input with greater dynamic range to detect type-specific anti-HPV concentrations to E6 and E7 oncoproteins of HPV16 and 18.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>32214362</pmid><doi>10.1371/journal.pone.0229672</doi><tpages>e0229672</tpages><orcidid>https://orcid.org/0000-0003-4480-5153</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Antibodies Antigen-antibody reactions Antigens Biology and life sciences Cancer Carcinogenesis Carcinoma Cervical cancer Characterization Clinical trials Compensation Cross-reactivity Dilution Diseases Dynamic range Employees Enzyme-linked immunosorbent assay Head & neck cancer Head and neck cancer Human papillomavirus Immunoassay Immunoglobulin G Immunoglobulins Malignancy Medicine and Health Sciences Methods Motor vehicle drivers Multiplexing Oncoproteins Papilloma Papillomavirus infections Pharmaceutical industry Protein purification Proteins Rankings Research and Analysis Methods Respiratory system agents Serology Squamous cell carcinoma Technology Tumorigenesis Viruses Wages & salaries |
| title | Development and validation of a multiplex immunoassay for the simultaneous quantification of type-specific IgG antibodies to E6/E7 oncoproteins of HPV16 and HPV18 |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-23T16%3A19%3A51IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Development%20and%20validation%20of%20a%20multiplex%20immunoassay%20for%20the%20simultaneous%20quantification%20of%20type-specific%20IgG%20antibodies%20to%20E6/E7%20oncoproteins%20of%20HPV16%20and%20HPV18&rft.jtitle=PloS%20one&rft.au=Layman,%20Hans&rft.date=2020-03-26&rft.volume=15&rft.issue=3&rft.spage=e0229672&rft.epage=e0229672&rft.pages=e0229672-e0229672&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0229672&rft_dat=%3Cgale_plos_%3EA618633934%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2383488996&rft_id=info:pmid/32214362&rft_galeid=A618633934&rft_doaj_id=oai_doaj_org_article_1cc40719378244fdb98fe4a48e91e7fc&rfr_iscdi=true |