title: Comparison of GD2 binding capture ELISA assays for anti-GD2-antibodies using GD2-coated plates and a GD2-expressing cell-based ELISA.
abstract: Two assay methods for quantification of the disialoganglioside (GD2)-specific binding activities of anti-GD2 monoclonal antibodies and antibody immunofusion proteins, such as ch14.18 and hu14.18-IL2, were developed. The methods differed in the use of either microtiter plates coated with purified GD2 or plates seeded with GD2-expressing cell lines to bind the anti-GD2 molecules. The bound antibodies were subsequently detected using the reactivity of the antibodies to an HRP-labeled anti-IgG Fc or antibodies recognizing the conjugate IL-2 part of the Hu 14.18IL-2 fusion protein. The bound HRP was detected using reagents such as orthophenylene diamine, 2, 2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] or tetramethylbenzidine. The capture ELISA using GD2-coated plates was developed earlier in assay development and used to demonstrate assay specificity and to compare lot-to-lot consistency and stability of ch14.18, and Hu14.18 IL-2 in clinical development. During this study, we found a number of issues related to plate-to-plate variability, GD2 lot variability, and variations due to GD2 storage stability, etc., that frequently lead to assay failure in plates coated with purified GD2. The cell-based ELISA (CbELISA) using the GD2 expressing melanoma cell line, M21/P6, was developed as an alternative to the GD2-coated plate ELISA. The results on the comparability of the capture ELISA on GD2-coated plates and the cell-based assay show that both assays give comparable results. However, the cell-based assay is more consistent and reproducible. Subsequently, the anti-GD2 capture ELISA using the GD2-coated plate was replaced with the CbELISA for product lot release testing and stability assessment.
authors: Biopharmaceutical Development Program, SAIC-Frederick Inc., National Cancer Institute, MD 21702, USA. somang@mail.nih.gov
authors: Gopalan
authors: G
authors: Soman
authors: Xiaoyi
authors: X
authors: Yang
authors: Hengguang
authors: H
authors: Jiang
authors: Steve
authors: S
authors: Giardina
authors: Gautam
authors: G
authors: Mitra
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005732:Gangliosides / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology; D008545:Melanoma / Q000276:immunology / Q000473:pathology; D015203:Reproducibility of Results
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D005732:Gangliosides; D007376:Interleukin-2; C048844:anti-IgG; C019403:ganglioside, GD2; C112746:dinutuximab; C493743:lorukafusp alfa