GM-CSF ELISA Protocol

1. Coat plate with 50ul of a 0.5ug/ml of monoclonal rat anti-murine GM-CSF 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 blcok)
5. Incubate 2 hours at room temperature
6. Wash five times with PBST.
7. Make dilutions from 50 - 1 ng/ml of murine GM-CSF enzyme 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 five times with PBST
11. Add 100ul of 0.5ug/ml of biotinylated rat anti-mouse-GM-CSF antibody in 2% BSA in PBST
12. Incubate 1 hour at room temperature
13. Wash five times with PBST
14. Add 100ul of rabbit anti-mouse IgG anitibody labeled with horseradish peroxidase in 2% BSA in PBST
15. Incubate 60 minutes at room temperature
16. Wash five 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.