Dear Allaa Tariq,

to coate plates with primary antibodies use the following coating buffer:Coating buffer: 1,59 g Na2CO3 + 2,93 g NaHCO3 complete to 1l with demineralized (deionized) water (pH 9,6), for conservation add 0,2 g NaN3 (Sodium azide).The buffer can be kept at room temperature. Incubate the plate with the appropriate dilution of IgG (1:1000?), for 4-12 h at room temperature. Then wash and add antigen.

Best regards

Joachim Hamacher

Coating the plate with primary antibody is a sandwich type ELISA, which generally is used for very sensitive reactions.

1.For it you need to coat with your plate with diluted (dilution generally depends on how much purified the primary antibody is. A purified antibody is coated at nearly 1µg/ml concentration whereas non purified antibodies will need a concentration of around 10µg/ml ) primary antibody in carbonate/ bicarbonate buffer at pH 9.6. 

2. incubating the plate at 4°C for overnight.

3. Decanting the coating solution

4. washing with washing buffer (1X PBS containing 0.05% to 0.1% Tween20, pH 7.4) and Blocking the plate with Blocking buffer (1XPBS,2% to 5% BSA pH 7.4).

5. incubating the plate at 37°C for 1 hr.

6. Again washing with washing buffer.

7. adding the antigen solution. and incubating at 37°C for 1 hr.

8. Washing and then again blocking.

9. Washing and then adding primary antibody.

10. incubating the plate at 37°C for 1 hr.

11. washing.

12. adding secondary antibody (prepared in blocking buffer) tagged with enzyme.

13. incubation for 1 hr.

14. washing and addition of TMB substrate (addition of TMB to be done in minimal lights.). incubation in dark at rt. for 30  minutes.

15. Adding stop solution (2M H2SO4) and reading the plate at 450 nm  immediately.

thank you dear Gaurav sharma  for your answer ....its a very important information