title: A novel homogeneous Biotin-digoxigenin based assay for the detection of human anti-therapeutic antibodies in autoimmune serum.
abstract: Electrochemiluminescence (ECL) assays have been widely used for the detection of anti-therapeutic antibodies (ATAs) against biotherapeutics. With the discontinuation of BioVeris (BV) ECL platform, an alternative technology was needed to replace BV assays to ensure continuous support of multi-year clinical studies. After evaluation of several immunoassay platforms, a novel homogeneous Biotin-digoxigenin (DIG) based bridging ELISA format was selected to develop an anti-rhuMAbX antibody screening assay to test serum samples from rheumatoid arthritis (RA) patients. With a homogeneous overnight sample incubation, the Biotin-DIG ELISA achieved comparable relative sensitivity and free drug tolerance to the previous BV ATA assay for rhuMAbX. To abrogate potential auto-antibody interference in RA sera, various assay conditions were thoroughly evaluated and a horseradish peroxidase (HRP)-conjugated chicken anti-DIG antibody was selected as the detection conjugate. Other potential interferences from serum Biotin, naturally occurring anti-avidin antibodies, and concomitant medications such as digoxin and hydrocortisone, which have similar structures to digoxigenin, were also investigated. Under optimized final assay conditions, the Biotin-DIG assay showed a relative sensitivity of approximately 11 ng/mL using a polyclonal anti-complementarity determining region (CDR) enriched positive control; the assay could detect 500 ng/mL of the positive control in the presence of approximately 27 μg/mL of rhuMAbX in RA serum. In addition, a confirmatory step was optimized for the assay based upon pre-incubating serum samples with an excess of free drug. Overall, the Biotin-DIG assay met the performance requirements for an ATA screening assay and had comparable sensitivity and drug tolerance to the BV assay; therefore this assay was a suitable replacement for the BV assay used for previous clinical studies of rhuMAbX. The Biotin-DIG based assay format can be broadly used as an effective screening platform for the detection of anti-therapeutic antibodies.
authors.affiliation: Department of BioAnalytical Research & Development, Genentech, 1 DNA Way, South San Francisco, CA 94080, United States. jqiu@gene.com
authors.forename: Zhihua Julia
authors.initials: ZJ
authors.lastname: Qiu
authors.forename: Yong
authors.initials: Y
authors.lastname: Ying
authors.forename: Michael
authors.initials: M
authors.lastname: Fox
authors.forename: Kun
authors.initials: K
authors.lastname: Peng
authors.forename: Sock-Cheng
authors.initials: SC
authors.lastname: Lewin-Koh
authors.forename: Daniel
authors.initials: D
authors.lastname: Coleman
authors.forename: Jeremy
authors.initials: J
authors.lastname: Good
authors.forename: John
authors.initials: J
authors.lastname: Lowe
authors.forename: Amena
authors.initials: A
authors.lastname: Rahman
authors.forename: Jihong
authors.initials: J
authors.lastname: Yang
authors.forename: Jenny
authors.initials: J
authors.lastname: Jiang
authors.forename: Valerie
authors.initials: V
authors.lastname: Quarmby
authors.forename: An
authors.initials: A
authors.lastname: Song
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000008:administration & dosage / Q000276:immunology*; D000888:Antibodies, Anti-Idiotypic / Q000097:blood* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000097:blood* / Q000188:drug therapy / Q000276:immunology; D001710:Biotin / Q000737:chemistry*; D002986:Clinical Trials as Topic; D004076:Digoxigenin / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D005260:Female; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods* / Q000592:standards; D008297:Male; D012680:Sensitivity and Specificity
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D001710:Biotin; D004076:Digoxigenin