G-CSF ELISA Protocol (human)

1. Coat plate with 100ul of a 0.5ug/ml of monoclonal rat anti-human G-CSF antibody (1 ug/ml stock) in 50mM sodium bicarobonate buffer (pH 9.4)
2. Incubate plates 24 hours at 4^oC
3. Aspirate wells
4. Add 250ul of 5% milk in PBS (Blotto blcok)
5. Incubate 30 minutes at room temperature
6. Wash five times with PBST
7. Make 1:2000 dilution of 2 ug/ml G-CSF standard
8. Make dilutions of working stock in step 7 from 1.0 - 0.05 ng/ml in 0.1% BSA in PBS
9. Add samples to be tested. If necessary, dilute samples as in step 8
10. Incubate 24 hours at 4^oC
11. Wash five times with PBST
12. Add 100ul of 0.5ug/ml polyclonal biotinylated-rabbit anti-G-CSF antibody in 0.1% BSA in PBST
13. Incubate 2 hours at room temperature
14. Wash five times with PBST
15. Add 100ul of streptavidin labeled horseradish peroxidase
16. Incubate 30 minutes at room temperature
17. Add 100ul of TMB substrate with H₂O₂ to each well
18. Incubate 20 minutes at room temperature
19. Add 50ul 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.