9.2.3.2. ELISA

1. Mix coating buffer (CB):
1.1.  0.159% Na2CO3,
1.2.  0.293% NaHCO3,
1.3.  Adjust to pH 9.6.
2. Seed each well with 180µl of CB.
3. Add 5-20 µl sample.
4. Incubate 3hrs at ambient temperature, or overnight at 4oC.  
5. Tip out and wash the wells 3X with PBS-T (= PBS containing 0.05% Tween20 detergent), shaking the ELISA plate dry each time.
6. Prepare a 1/2,000 - 1/5,000 dilution of the primary antibody in PBS-TPO:  
6.1.  2% Polyvynylpyrrolidone (PVP) mw 440000,
    6.2.  0.2% Bovine serum albumin (BSA),  
6.3.  in PBS-T (fresh daily).
7. Add 200 µl of antibody/PBS-TPO to each well.
8. Incubate 3hrs at ambient temperature, or overnight at 4oC.
9. Tip out fluid and wash the wells 3X with PBS-T, shaking the ELISA plate dry each time.
10. Prepare a ProteinA-HorseRadishPeroxidase (PrA-HRP) conjugate stock solution at 100 µg/ml.
11. Make a 1/2,000 – 1/5,000 dilution of PrA-HRP stock solution in PBS-TPO.
12. Add 200 µl to each well.  
13. Incubate 3hrs at ambient temperature, or overnight at 4oC.  
14. Tip out fluid and wash the wells 3X with PBS-T, shaking the ELISA plate dry each time.