Quantitative measurement of anti-ErbB-2 antibody by flow cytometry and ELISA

Abstract

To establish standard methods for measuring anti-ErbB-2 antibody, a flow cytometric and an enzyme-linked immunosorbent assay (ELISA) were developed and compared. In the flow cytometric assay, the antibody was measured by binding to human breast cancer cell line SKBR3 and the result expressed as mean channel fluorescence (MCF). In ELISA, the antibody was measured by binding to a recombinant, secreted human ErbB-2 containing the N-terminal 505 amino acids of ErbB-2 fused to myc and His tags (secE2/myc/His or secE2) and the result expressed as O.D. 405. A mouse anti-human ErbB-2 mAb 9G6 was used as the standard. Using flow cytometry, MCF of 9G6 binding increased with linearity between 0.6 and 10 μg/ml. Using ELISA, O.D. 405 increased with linearity between 0.015 and 1 μg/ml, indicating greater sensitivity of ELISA. The titer of an immune mouse serum was determined to be 400 and 8000 with flow cytometric and ELISA assay, respectively, consistent with the assay sensitivity. Based on standard curves generated with 9G6, antibody concentration in the same serum sample was calculated to be ∼230 μg/ml by ELISA and ∼270 μg/ml by flow cytometry. Therefore, excellent concordance in antibody concentration was obtained with different assays using linear regression and properly diluted samples. This concordance was verified with multiple serum samples.