Development of an in vitro potency assay for therapeutic TGFbeta antagonists: the A549 cell bioassay

• Published in: Journal of immunological methods Vol. 316; no. 1-2; p. 18
• Main Authors: Rapoza, Martha L, Fu, Daotian, Sendak, Rebecca A
• Format: Journal Article
• Language: English
• Published: Netherlands 20-10-2006
• Subjects: Antibodies, Monoclonal - pharmacology; Biological Assay - methods; Cell Line, Tumor; Humans; Inhibitory Concentration 50; Interleukin-11 - analysis; Interleukin-11 - biosynthesis; Protein Isoforms; Sensitivity and Specificity; Transforming Growth Factor beta - antagonists & inhibitors; Transforming Growth Factor beta - immunology
• ISSN: 0022-1759

A bioassay was developed to assess the potency of TGFbeta antagonists by measuring IL-11 production in TGFbeta-1 treated human lung epithelial cells (A549). The production of IL-11 by A549 cells, measured by ELISA, was shown to be proportional to the TGFbeta-1 concentration. The A549 cells were responsive to all three isoforms of TGFbeta in the range of 3.0 ng/mL to 1.4 pg/mL, with an 18 to 24 h exposure time found to be within the linear portion of the bioassay response range. The Effective Dose at 80% of the maximal response (ED80) of TGFbeta-1 determined for the assay was 0.3 ng/mL. With this level of TGFbeta-1, a human anti-TGFbeta-1 antibody (CAT-192) yielded an approximate median Inhibitory Concentration (IC50) value of 3 microg/mL. To investigate assay specificity, alternate members of the TGFbeta superfamily were evaluated. Recombinant human activin B, inhibin A and BMP-2 (Bone Morphogenetic Protein-2) did not elicit a significant IL-11 response from the A549 cell line. Bioassay qualification was performed to obtain estimates of precision and accuracy, as well as to establish plate validity criteria. Assay precision was estimated at 30%, while the accuracy was +/-13%. Additionally, the ability of the A549 cell potency assay to detect potency differences in structurally modified samples was investigated.