A new antibody capture enzyme linked immunoassay specific for transforming growth factor beta

• Published in: The international journal of biochemistry & cell biology 1995-02, Vol.27 (2), p.207-213
• Main Authors: Phillips, A.O., Steadman, R., Donovan, K.D., Williams, J.D.
• Format: Article
• Language: English
• Subjects: ELISA; Enzyme linked immunoassay; Transforming growth factor beta
• ISSN: 1357-2725; 1878-5875
• DOI: 10.1016/1357-2725(94)00077-O

Previous studies which examined Transforming Growth Factor beta 1 (TGF-β 1) generation have relied on the identification of TGF-β 1 mRNA or measurement of TGF-β 1 by bioassay. Quantitation of TGF-β 1 message alone however is inadequate since the regulation of TGF-β 1 synthesis is often post-transcriptional. TGF-β 1 is poorly immunogenic, and sensitive and specific immunoassays for this peptide have proved difficult to develop. Bioassays depend on stimulation or inhibition of cell proliferation in a TGF-β 1 dependent manner, and are very rigid in their requirements for optimal performance. The aims of this work was therefore to develop a sensitive and reproducible immunoassay for TGF-β 1. Microtitre plates were coated with human recombinant TGF-β 1, unbound protein was discarded from the wells prior to blocking with bovine serum albumin. Chicken anti-human TGF-β 1 antibody was incubated with the test solution overnight at 4°C and then added to the coated wells. Bound antibody was detected with alkaline phosphatase conjugated anti-chicken antibody. The assay is sensitive to 0.2 ng/ml with a range to 100 ng/ml. The assay detects the mature form of human recombinant TGF-β 1, natural platelet extracted TGF-β 1, and TGF-β 1 derived from human monocytes stimulated with Phorbol myristate acetate (PMA). Active TGF-β 1 is measured directly and latent TGF-β 1 can be measured indirectly following acid activation of samples. Inter-assay precision ranged from 4.3 to 9.6%, (coefficient of variation, %CV) and intraassay precision ranged from 2.8 to 8.6% (CV). There was no detectable cross reactivity with a range of other cytokines and growth factors, including TGF-β2 or TGF-β3. We describe the development of a simple, sensitive and reproducible Antibody Capture Enzyme Linked Immunoassay specific for Transforming Growth Factor beta 1 (TGF-β 1). The assay measures natural platelet derived TGF-β 1, recombinant TGF-β 1, and TGF-β 1 produced by PMA stimulated monocytes, and has been fully validated in terms of antibody specificity for TGF-β 1, assay precision and accuracy.