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.
PROCEDURE
1. Specimen Collection and Handling: Specimens should be clear, non-hemolyzed and non-lipemic. In the case of cell-culture, remove any particulate material by centrifugation and assay immediately or store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Where as in the case of patient blood, use a serum separator tube and allow samples to clot for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum and assay immediately or store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles.