Development of a quantitative antigen-specific cell-based ELISA for the 7G7/B6 monoclonal antibody directed toward IL-2Ralpha.
Interleukin-2 receptor alpha (IL-2Ralpha, CD25) has been identified as a valuable target for immunotherapy. The 7G7/B6 monoclonal antibody, a mouse IgG2a kappa, recognizes an epitope of the IL-2Ralpha peptide, other than that identified by anti-Tac. This antibody is currently being explored for potential therapeutic and diagnostic applications. Here, we show a cell-based enzyme-linked immunosorbent assay (CbELISA) method for quantitative measurement of the binding activity of the 7G7/B6 antibody to the Kit-225-iG3 cell line expressing IL-2Ralpha antigen on the cell surface. The cell- and antigen-specificity of the assay was established using specific cell lines and irrelevant control antibodies. Satisfactory binding curves were demonstrated with Kit-225-iG3 cells grown between 3 and 25 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The assay shows reproducible dose-response curves in the concentration range of 10-1000 ng/ml. The assay validation data presented here indicate that this CbELISA assay is quantitative, reproducible, robust, precise, and can be used to test the biological activity, lot to lot comparison, and stability of 7G7/B6 monoclonal antibody.
Yang
Xiao Yi
XY
Bioanalytical Development Laboratory, Biopharmaceutical Development Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Building 458, Room 17, P.O. Box B, Frederick, MD 21702, USA.
Jiang
Hengguang
H
Hartmann
Wanda K
WK
Mitra
Gautam
G
Soman
Gopalan
G
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit; D019169:Jurkat Cells; D018123:Receptors, Interleukin / Q000276:immunology*; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; C508017:IL2RA protein, human; C508018:Il2ra protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; D018123:Receptors, Interleukin