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

Abstract 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.