1. ELISA Capture: Purified A75-2 antibody is useful as the capture antibody in a sandwich ELISA for measuring mouse, human or pig TGF-β1 along with biotinylated anti-TGF-β1 clone A75-3 (Cat. No. 555053) as the detection antibody. The purified antibody should be titrated from 1.0 - 4.0 µg/ml to determine optimal concentration for ELISA capture. For specific methodology, visit the protocols section or the chapter on ELISA in the Immune Function Handbook, both of which can be found at www.bdbiosciences.com. This ELISA pair is recommended for measuring cytokine from experimental cell culture systems and is not recommended for testing serum or plasma samples. For measuring TGF-β1 in serum or plasma the BD OptEIA™ Human TGF-β1 ELISA Set (Cat. No. 559119) is specially 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 1:5 (20 µl serum + 80 µl PBS), then add 1 N HCl in 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 in 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 the 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 any of the cytokines tested (e.g., mouse IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12 p70, IL-15, GM-CSF, IFN-γ, MCP-1, TCA-3, TNF; human IL-1α, IL-1β , IL-2 , IL-3 , IL-4, IL-6, IL-7 , IL-8, IL9 , IL-10, IL-11 , IL-12 p70, IL-12 p40, IL-13, IL-15, G-CSF, GM-CSF, IFN-γ, lymphotactin, MCP-1, MCP-2, MIP-1α, MIP-1β, NT-3, PDGF-AA, sCD23, SCF, TNF, LT-α, VEGF; rat IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF).