Rabbit TNF-Alpha ELISA Protocol

1. Coat plate with 100ul of 0.5ug/ml solution of polyclonal goat anti-rabbit TNF-alpha antibody in 50mM sodium bicarbonate buffer (pH 9.6)
2. Incubate 24 hours at 4^oC
3. Wash 5 times in PBST
4. Add 250ul of 1% BSA in ELISA coating buffer
5. Incubate 30 minutes at room temperature
6. Wash 5 times in PBST
7. Aliquot standards from 1.0 ng/ml to 0.05 ng/ml in 1%BSA, 5mM EDTA in TBST.
8. Add 100ul of samples and standards to ELISA plate
9. Incubate overnight at 4^oC
10. Wash 5 times with PBST
11. Add 100ul to each well of a 0.5ug/ml solution of polyclonal biotinylated rabbit-anti-TNF-alpha antibody in 1% BSA, 5mM EDTA in TBST
12. Incubate one hour at room temperature
13. Wash 5 times with PBST
14. Add 100ul to each well of streptavidin labeled horseradish peroxidase in 1% BSA, 5mM EDTA in TBST
15. Incubate for 30 minutes at room temperature
16. Wash 5 times with PBST
17. Add 100ul of TMB substrate with H₂O₂ to each well
18. Incubate 20 minutes at room temperature
19. Add 50ul of stop solution (1M H₂SO₄)
20. Read absorbance at 450nm
21. Read absorbance at 570nm (value at 570nm is subtracted from 450nm value to control for optical abnormalities in the ELISA plates)

Note: Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.