9.2.3.3. Sandwich ELISA

1. Seed each well with 200 µl of a 1/2,000-1/5,000 dilution of the Fab fragment (Harlow and Lane, 1988) of the primary antibody in coating buffer (CB, see section 9.2.3.2.).
2. Incubate 3hrs at ambient temperature, or overnight at 4oC.  
3. Tip out and wash the wells 3X with PBS-T, shaking the ELISA plate dry each time.
4. Add 180 µl PBS-TPO to each well.
5. Add 20 µl sample to each well.
6. Incubate 3hrs at ambient temperature, or overnight at 4oC.  
7. Tip out fluid and wash the wells 3X with PBS-T, shaking the ELISA plate dry each time.  
8. Add 200 µl 1/2,000 – 1/5,000 dilution of PrA-HRP stock solution in PBS-TPO to each well.  
9. Incubate 3hrs at ambient temperature, or overnight at 4oC.  
10. Tip out fluid and wash the wells 3X with PBS-T, shaking the ELISA plate dry each time.