Materials and Instrument:
1. Capture Antibody : anti-Mouse TNF monoclonal antibody. It is supplied as 1 vial (1ml) and a recommended 1:250 dilution in coating buffer is used for coating ELISA plate.
2. Detection Antibody : Biotinylated anti-Mouse TNF monoclonal antibody. It is supplied as 1 vial (0.5ml) and a recommended 1:500 dilution in reaction buffer is used for detecting TNF-α in the sample.
3. Enzyme Reagent : Streptavidin-horseradish peroxidase conjugate (SAv-HRP)
4. Standards : Recombinant mouse TNF, It is supplied as lyophilized powder (30ng) and serial dilution of the stock (30ng/ml) in reaction buffer is used for drawing calibration curve.
5. Enzyme reagent Streptavidin –HRP conjugate: It is supplied as 1 vial (1ml) and a recommended 1:250 dilution in reaction buffer is used for detecting TNF-α in the sample.
6. Coating Buffer - 0.2 M Sodium Phosphate, pH 6.5 : Weight 12.49 g Na2HPO4, 15.47 g NaH2PO4 and make up the volume to 1.0 L. Adjust the pH to 6.5. Prepare freshly and use within 7 days of preparation, stored at 2-8°C.
7. Assay Diluent-PBS with 10% FBS, pH 7.0. Freshly prepare and use within 3 days of preparation, store at 2-8°C.
8. Wash Buffer – PBS with 0.05% Tween-20. Freshly prepare and use within 3 days of preparation, stored at 2-8°C.
9. Substrate Solution - Tetramethylbenzidine (TMB) and Hydrogen Peroxide.
10. Stop Solution -1 M H3PO4 or 2 N H2SO4
11. 96-well ELISA flat bottom plates are recommended
12. Microplate reader capable of measuring absorbance at 450 nm
13. micropipettes
14. Tubes to prepare standard dilutions
15. Plate sealers or parafilm.