Development and Purification of Polyclonal Antibody Against Glargine in Rabbits
Year: 2026
Type: article
Abstract: Immunogenicity assessment requires the detection of anti-drug antibodies (ADAs) towards therapeutic insulin analogues like insulin glargine. The absence of well-characterized positive control antibodies is however a significant limitation in developing the immunogenicity assays. Rabbit-derived polyclonal antibodies against insulin glargine were successfully generated and affinity-purified for use as positive control antibodies in bridge ELISA-based immunogenicity assays.Insulin glargine was conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehyde-mediated crosslinking followed by sodium borohydride stabilization. Rabbits were immunized subcutaneously with the conjugate using Freund’s adjuvants according to a scheduled booster regimen. Antibody titers were determined by indirect ELISA using BSA-glargine–coated plates to avoid anti-KLH interference. Serum IgG was purified through Protein A affinity chromatography followed by glargine-conjugated Affi-Gel affinity purification. Purity was evaluated by SDS-PAGE. Five out of six immunized rabbits developed strong immune responses with endpoint titers reaching 1:32,000–1:40,000. One animal showed no significant response. Affinity purification yielded highly enriched IgG with improved specificity and reduced non-specific reactivity. SDS-PAGE confirmed antibody purity, and ELISA demonstrated high sensitivity towards glargine with minimal cross-reactivity. A robust and reproducible strategy was established for generating high-titer, affinity-purified polyclonal antibodies against glargine, suitable for immunoassay development and bioanalytical applications.
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W7204043830