ELISA Detection: For a sandwich ELISA to measure mouse, human, or pig TGF-β1 protein levels, the biotinylated monoclonal anti-mouse, human, or pig TGF-β1 antibody A75-3 (Cat. No. 555053) can be used as detector in conjunction with the purified A75-2 antibody (Cat. No. 555052) as the capture antibody, and TGF-β1 derived from human as the standard. The biotinylated A75-3 antibody should be titrated from 0.5 - 2.0 µg/ml to determine the optimal concentration for ELISA detection. To obtain standard curves, doubling dilution of TGF-β1 standard ranging from ~4000 to 30 pg/ml are recommended for inclusion in each ELISA plate. For specific methodology, please see Chapter 7 on ELISA in the Techniques for Immune Function Analysis Application Handbook, 1st Edition or visit the protocols section of our website, www.bdbiosciences.com.
Note: This ELISA pair is recommended primarily for measuing cytokine from experimental cell culture systems. These ELISA reagents are not recommended for assay of serum or plasma samples. For measuring human TGF-β1 in serum or plasma our human TGF-β1 BD OptEIA Set (Cat. No. 559119) is especially formulated and recommended.
Important: Prior to ELISA assay for TGF-β1 protein levels, samples of biological fluids must be treated by acidification. The protocol is described below:
For serum samples, first dilute serum in PBS in 1:5 (i.e., 20 µl serum + 80 µl PBS), then add 1 N HCl 1:25 to adjust to pH3 (i.e., 4 µl 1 N HCl to 100 µl diluted serum). Cell supernatants can be treated undiluted with 1 N HCl 1:25 to adjust to pH3 (i.e., 4 µl 1 N HCl to 100 µl supernatant). Incubate acidified samples for 60 minutes at 4°C (alternatively, samples can be treated at room temperature for 15 minutes). After incubation neutralize samples by treating each sample with 1 N NaOH 1:25 and test immediately or store at -70°C until testing.
Note: This ELISA pair shows no cross-reactivity with other cytokines or chemokines (e.g., human IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, GM-CSF, IFN-γ, TNF, LT-α, MCP-1, MCP-2, MIP-1α, MIP-1β, SCF, lymphotactin, VEGF, G-CSF, NT-3, PDGF, CD23, IP-10, GRO, NAP-2, PF4).
Western blot: The purified A75-3 antibody is useful for Western blotting. Please note that this application is not routinely tested at BD Biosciences Pharmingen.