A non-radioactive complement-dependent cytotoxicity assay for anti-CD20 monoclonal antibody
| Autor: | Hélène Gazzano-Santoro, Anthony Chen, Thomas C Ryskamp, Venkat R. Mukku, Peter Ralph |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
medicine.drug_class
Recombinant Fusion Proteins Immunology Monoclonal antibody Antibody Isotype Oxazines medicine Immunology and Allergy Potency Humans Viability assay Cytotoxicity Coloring Agents B-Lymphocytes biology Chemistry Antibodies Monoclonal Complement System Proteins Antigens CD20 Cytotoxicity Tests Immunologic Complement-dependent cytotoxicity Antibodies Anti-Idiotypic Biochemistry Relative fluorescence units Xanthenes biology.protein Antibody |
| Zdroj: | Journal of immunological methods. 202(2) |
| ISSN: | 0022-1759 |
| Popis: | A simple and non-radioactive complement-dependent cytotoxicity assay was developed to determine the relative potency of an anti-CD20 mAb, IDEC-C2B8. The assay measures the relative number of viable cells based on the uptake and metabolism of the redox dye, Alamar blue. A linear relationship between the relative fluorescence unit generated and the number of viable cells was demonstrated. The assay is simple, has high throughput (performed in 96-well microtiter plates), and shows reproducible dose–response curves in the concentration range of 0.02–3.3 μg/ml. With intra-assay variability of 5–12%, interassay variability of 6–10% and spike recoveries of 101–109%, the assay has high precision and accuracy. Specificity was demonstrated by the lack of activity of immunoglobulins that do not bind CD20, or anti-CD20 antibody isotype (gamma 4) which does not bind complement. The assay is able to detect degradative changes in the molecule caused by heat, light and proteolytic treatments, suggesting its use as a stability-indicating method. Finally, the Alamar blue method compared favorably with other more conventional methods used to assess cell viability. The assay has the desired properties for use as a potency assay for quality control testing of anti-CD20 mAb. |
| Databáze: | OpenAIRE |
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