CAT ELISA Protocol

1. Coat plate with 50ul of a 0.5ug/ml of polyclonal rabbit anti-CAT antibody in 50mM sodium bicarobonate buffer (pH 9.4)
2. Incubate plates 24 hours at 4^oC
3. Aspirate wells
4. Add 250ul of 5% non-fat milk or 1% BSA in PBS
5. Incubate 30 minutes at room temperature
6. Wash five times with PBST
7. Make dilutions from 1.0 - 0.05 ng/ml of CAT enzyme in 1% BSA in PBS
8. Add samples to be tested. If necessary, dilute samples as in step 7
9. Incubate 24 hours at 4^oC (This time might be able to be decreased depending on which plates you are using. I routinely use 2 hours in some ELISAs)
10. Wash three to five times with PBST
11. Add 100ul of 0.5ug/ml of monoclonal anti-CAT-Digoxin labeled antibody in 1% BSA in PBS
12. Incubate 2 hours at room temperature
13. Wash three to five times with PBST
14. Add 100ul of 0.5ug/ml monoclonal mouse anti-Digoxin biotinylated antidody in 1% BSA in PBS
15. Incubate 2 hours at room temperature
16. Wash three to five times with PBST
17. Add 100ul streptavidin labeled horseradish peroxidase
18. Incubate 30 minutes at room temperature
19. Add 100ul of TMB substrate with H₂O₂ to each well
20. Incubate 20 minutes at room temperature
21. Add 50ul stop solution (1M H₂SO₄)
22. Read absorbance at 450nm
23. 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.