Factor IX ELISA Protocol

1. Coat plate with 50ul of a 0.5ug/ml of monoclonal rat anti-human factor IX antibody in 50mM sodium bicarobonate buffer (pH 9.4)
2. Incubate plates 24 hours at 4^oC
3. Aspirate wells
4. Add 200ul of 5% milk in PBS (Blotto block)
5. Incubate 1 hour at room temperature
6. Wash three times with PBST
7. Make dilutions from 40 - 0.625 ng/ml of human Factor IX standard in 0.1% BSA in PBS
8. Add samples to be tested. If necessary, dilute samples as in step 7
9. Incubate 24 hours at 4^oC
10. Wash three times with PBST
11. Add 50ul of 0.5ug/ml of Rabbit-anti-human Factor IX antibody in 2% BSA in PBST
12. Incubate 1 hour at room temperature
13. Wash three times with PBST
14. Add 50ul of horseradish peroxidase labeled goat-anti-rabbit antibody in 1% BSA in PBS
15. Incubate 60 minutes at room temperature
16. Wash three times with PBST
17. Add 100ul ABTS substrate to each well
18. Incubate 30-120 minutes at room temperature
19. Read absorbance at 405nm at 30 minutes, 1 hour and 2 hour timepoints
20. Read absorbance at 570nm for each timepoint in 19 and subtract from absorbance at 405nm to control for optical abnormalities in the ELISA plate

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