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This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti-NGF polyclonal antibody was pre-coated onto 96-well plates. And the biotin conjugated anti-NGF polyclonal antibody was used as detection antibodies. The standards test samples and biotin conjugated detection antibody were added - the wells subsequently and wash with wash buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with wash buffer. TMB substrates were used - visualize HRP enzymatic reaction. TMB was catalyzed by HRP - produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional - the NGF amount of sample captured in plate. Read the O.D. absorbance at 450 nm in a microplate reader and then the concentration of NGF can be calculated.
Preparation of sample and reagents 1. Sample Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20 °C for long term. Avoid multiple freeze-thaw cycles. Body fluids, tissue lysates and cell culture supernatants: Centrifuge to remove precipitate, analyze immediately or aliquot and store at -20 °C . Serum: Coagulate the serum at room temperature (about 4 hours). Centrifuge at approximately 2000 × g for 20 min. Analyze the serum immediately or aliquot and store at -20 °C . Note: 1. Coagulate blood samples completely, then, centrifuge, and avoid hemolysis and particle. 2. NaN3 can not be used as test sample preservative, since it is the inhibitor for HRP.