title: A rapid method for generating and characterizing anti-variable region monoclonal antibodies.
abstract: The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.
authors.affiliation: Centocor Research & Development Inc. 145 King of Prussia Rd., Radnor, PA 19087, USA. KStaquet@cntus.jnj.com
authors.forename: Kimberly
authors.initials: K
authors.lastname: Staquet
authors.forename: Deborah
authors.initials: D
authors.lastname: Kwok
authors.forename: Michael A
authors.initials: MA
authors.lastname: Rycyzyn
authors.forename: Theodore
authors.initials: T
authors.lastname: Petley
authors.forename: Eilyn R
authors.initials: ER
authors.lastname: Lacy
authors.forename: Gordon
authors.initials: G
authors.lastname: Powers
authors.forename: Jill
authors.initials: J
authors.lastname: Giles-Komar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000096:biosynthesis* / Q000276:immunology / Q000493:pharmacokinetics / Q000627:therapeutic use; D001402:B-Lymphocytes; D001709:Biotechnology / Q000379:methods; D019013:CD40 Antigens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D007114:Immunization; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D007370:Interferon Type I; D051379:Mice; D008807:Mice, Inbred BALB C; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007370:Interferon Type I