{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:24:16Z","timestamp":1792805056279,"version":"4.5.0"},"reference-count":22,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2021,3,6]],"date-time":"2021-03-06T00:00:00Z","timestamp":1614988800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Innovative Medical Research\u201c of the University of Muenster Medical School","award":["LE221801"],"award-info":[{"award-number":["LE221801"]}],"id":[{"id":"https:\/\/ror.org\/00pd74e08","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Male patients with Fabry disease (FD) are at high risk for the formation of antibodies to recombinant \u03b1-galactosidase A (AGAL), used for enzyme replacement therapy. Due to the rapid disease progression, the identification of patients at risk is highly warranted. However, currently suitable references and standardized protocols for anti-drug antibodies (ADA) determination do not exist. Here we generate a comprehensive patient-derived antibody mixture as a reference, allowing ELISA-based quantification of antibody titers from individual blood samples. Serum samples of 22 male patients with FD and ADAs against AGAL were pooled and purified by immune adsorption. ADA-affinities against agalsidase-\u03b1, agalsidase-\u03b2 and Moss-AGAL were measured by quartz crystal microbalance with dissipation monitoring (QCM-D). AGAL-specific immune adsorption generated a polyclonal ADA mixture showing a concentration-dependent binding and inhibition of AGAL. Titers in raw sera and from purified total IgGs (r2 = 0.9063 and r2 = 0.8952, both p &lt; 0.0001) correlated with the individual inhibitory capacities of ADAs. QCM-D measurements demonstrated comparable affinities of the reference antibody for agalsidase-\u03b1, agalsidase-\u03b2 and Moss-AGAL (KD: 1.94 \u00b1 0.11 \u00b5M, 2.46 \u00b1 0.21 \u00b5M, and 1.33 \u00b1 0.09 \u00b5M, respectively). The reference antibody allows the ELISA-based ADA titer determination and quantification of absolute concentrations. Furthermore, ADAs from patients with FD have comparable affinities to agalsidase-\u03b1, agalsidase-\u03b2 and Moss-AGAL.<\/jats:p>","DOI":"10.3390\/ijms22052680","type":"journal-article","created":{"date-parts":[[2021,3,7]],"date-time":"2021-03-07T20:20:43Z","timestamp":1615148443000},"page":"2680","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Generation and Characterization of a Polyclonal Human Reference Antibody to Measure Anti-Drug Antibody Titers in Patients with Fabry Disease"],"prefix":"10.3390","volume":"22","author":[{"given":"Malte","family":"Lenders","sequence":"first","affiliation":[{"name":"Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, Interdisciplinary Fabry Center Muenster (IFAZ), University Hospital Muenster, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Scharnetzki","sequence":"additional","affiliation":[{"name":"Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, Interdisciplinary Fabry Center Muenster (IFAZ), University Hospital Muenster, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1010-8894","authenticated-orcid":false,"given":"Ali","family":"Heidari","sequence":"additional","affiliation":[{"name":"Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9672-9773","authenticated-orcid":false,"given":"Daniele","family":"Di Iorio","sequence":"additional","affiliation":[{"name":"Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9072-0858","authenticated-orcid":false,"given":"Seraphine Valeska","family":"Wegner","sequence":"additional","affiliation":[{"name":"Institute of Physiological Chemistry and Pathobiochemistry, University of Muenster, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva","family":"Brand","sequence":"additional","affiliation":[{"name":"Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology, Interdisciplinary Fabry Center Muenster (IFAZ), University Hospital Muenster, 48149 Muenster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1016\/S0140-6736(08)61589-5","article-title":"Fabry\u2019s disease","volume":"372","author":"Zarate","year":"2008","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1056\/NEJM200107053450102","article-title":"Safety and efficacy of recombinant human a-galactosidase A replacement therapy in Fabry\u2019s disease","volume":"345","author":"Eng","year":"2001","journal-title":"N. Engl. J. Med."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2743","DOI":"10.1001\/jama.285.21.2743","article-title":"Enzyme replacement therapy in Fabry disease: A randomized controlled trial","volume":"285","author":"Schiffmann","year":"2001","journal-title":"JAMA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2265","DOI":"10.1681\/ASN.2018030329","article-title":"Effects of enzyme replacement therapy and antidrug antibodies in patients with Fabry disease","volume":"29","author":"Lenders","year":"2018","journal-title":"J. Am. Soc. Nephrol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1589","DOI":"10.1111\/j.1523-1755.2004.00924.x","article-title":"Enzyme therapy for Fabry disease: Neutralizing antibodies toward agalsidase alpha and beta","volume":"66","author":"Linthorst","year":"2004","journal-title":"Kidney Int."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Rombach, S.M., Aerts, J.M., Poorthuis, B.J., Groener, J.E., Donker-Koopman, W., Hendriks, E., Mirzaian, M., Kuiper, S., Wijburgm, F.A., and Hollak, C.E. (2012). Long-term effect of antibodies against infused alpha-galactosidase A in Fabry disease on plasma and urinary (lyso)Gb3 reduction and treatment outcome. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0047805"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1681\/ASN.2014121226","article-title":"Serum- mediated inhibition of enzyme replacement therapy in Fabry disease","volume":"7","author":"Lenders","year":"2016","journal-title":"J. Am. Soc. Nephrol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2879","DOI":"10.1681\/ASN.2018070740","article-title":"Dose-Dependent Effect of Enzyme Replacement Therapy on Neutralizing Antidrug Antibody Titers and Clinical Outcome in Patients with Fabry Disease","volume":"29","author":"Lenders","year":"2018","journal-title":"J. Am. Soc. Nephrol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.ymgme.2018.11.008","article-title":"Antibodies against recombinant alpha-galactosidase A in Fabry disease: Subclass analysis and impact on response to treatment","volume":"126","author":"Hollak","year":"2019","journal-title":"Mol. Genet. Metab."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1016\/j.ymgme.2008.03.003","article-title":"Treatment of Fabry disease with different dosing regimens of agalsidase: Effects on antibody formation and GL-3","volume":"94","author":"Vedder","year":"2008","journal-title":"Mol. Genet. Metab."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2289","DOI":"10.1016\/j.jaci.2017.12.1001","article-title":"Characterization of drug-neutralizing antibodies in patients with Fabry disease during infusion","volume":"141","author":"Lenders","year":"2018","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"334","DOI":"10.1002\/jimd.12176","article-title":"Neutralising anti-drug antibodies in Fabry disease can inhibit endothelial enzyme uptake and activity","volume":"43","author":"Stappers","year":"2020","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Nakano, S., Tsukimura, T., Togawa, T., Ohashi, T., Kobayashi, M., Takayama, K., Kobayashi, Y., Abiko, H., Satou, M., and Nakahata, T. (2015). Rapid Immunochromatographic Detection of Serum Anti-\u03b1-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0128351"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1007\/s10545-015-9886-9","article-title":"Mannose receptor-mediated delivery of moss-made \u03b1-galactosidase A efficiently corrects enzyme deficiency in Fabry mice","volume":"39","author":"Shen","year":"2016","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1002\/jimd.12052","article-title":"Pharmacokinetics, pharmacodynamics, and safety of moss-aGalactosidase A in patients with Fabry disease","volume":"42","author":"Hennermann","year":"2019","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1180","DOI":"10.1182\/blood-2014-09-598268","article-title":"Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans","volume":"125","author":"Hofbauer","year":"2015","journal-title":"Blood"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Pratt, K.P. (2018). Anti-Drug Antibodies: Emerging approaches to predict, reduce or reverse biotherapeutic immunogenicity. Antibodies, 7.","DOI":"10.3390\/antib7020019"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/j.ymgme.2020.08.005","article-title":"Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant \u03b1-galactosidase A in patients with Fabry disease","volume":"31","author":"Scharnetzki","year":"2020","journal-title":"Mol. Genet. Metab."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1602","DOI":"10.1053\/j.gastro.2020.06.007","article-title":"Effects of orally delivered alpha-Galactosidase A on gastrointestinal symptoms in patients with Fabry disease","volume":"159","author":"Lenders","year":"2020","journal-title":"Gastroenterology"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"948","DOI":"10.1039\/C7CC08758F","article-title":"Dynamic blue light-switchable protein patterns on giant unilamellar vesicles","volume":"54","author":"Bartelt","year":"2018","journal-title":"Chem. Commun."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3307","DOI":"10.1039\/D0SC00518E","article-title":"Recruitment of receptors at supported lipid bilayers promoted by the multivalent binding of ligand-modified unilamellar vesicles","volume":"11","author":"Lu","year":"2020","journal-title":"Chem. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1016\/0009-8981(81)90384-3","article-title":"Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry\u2019s disease","volume":"112","author":"Mayes","year":"1981","journal-title":"Clin. Chim. Acta"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/5\/2680\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:34:15Z","timestamp":1760160855000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/5\/2680"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,6]]},"references-count":22,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,3]]}},"alternative-id":["ijms22052680"],"URL":"https:\/\/doi.org\/10.3390\/ijms22052680","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,6]]}}}