ELISA assessment of recombinant salivary proteins

BUFFERS:
• Coating Buffer: carbonate/bicarbonate buffer
   o 0.8 g Na₂CO₃ (disodium carbonate)
   o 1.46 g NaHCO₃ (sodium bicarbonate)
   o Adjust pH= 9.6 (with HCl)
   o In a total Volume= 500 ml ddH₂O
• Wash Buffer: 0.025% Tween-20-PBS
• Blocking Buffer: 3% BSA-PBS
• Serum-antibody Buffer: 1% BSA-PBS
• Secondary-antibody Buffer: 1% BSA-0.05% Tween-PBS
• TMB: to develop a conjugated HRP-2^o Ab
• PNPP: to develop a conjugated Phosphatase Alkaline-2^o Ab

Procedure:
1. Coat plates with antigen (250 ng/well; 100 μl/well) diluted in coating buffer. Seal and incubate Over Night /4 °C
2. Wash 3X in wash buffer (250 μl/well)
3. Add blocking buffer (200 μl/well) and incubate 1h at 37 °C
4. Wash 3X in wash buffer
5. Add serum antibody dilutions in serum-antibody buffer: i.e. 1:500, 1:5K or 1:50K (100 μl/well) and incubate for 2h at room temperature.
6. Wash 3X in wash buffer
7. Add 2o Antibody diluted at 1:2000 in secondary-antibody buffer (100 μl/well) and incubate for 1h at 37 °C.
8. Wash 3-5 times in wash buffer
9. Add 100 μl/well of TMB (KPL:52-00-02); incubate for a maximum of 30 min at RT, then add 100 μl of stop solution (KPL:50-85 05).
10. Read plate at 450nm (for HRP)
11. For alkaline phosphatase, use 100 μl of PNPP (SIGMA: N-2770) incubate 20-30 min at RT and read at 405