Hello Somaye Mazaheri

There could be many reasons. I would suggest you dilute the primary and secondary antibodies. Both the primary and secondary antibodies may be too concentrated and therefore likely to bind to the well. Try coating the negative control well with an unrelated antigen because non-coating of the negative control well will result in the antibodies binding to the well causing higher background.

Also try changing the blocking buffer. You can try using normal serum from the host species of the labelled secondary antibody because BSA used in blocking buffer contains bovine IgG and many secondary antibodies will react with bovine IgG.

I hope this is helpful.

Best Wishes.

The ELISA negative control is indicative than positive control and test samples because the highest negative control value excluding possible false positive.

Malcolm Nobre

I was use another antigen and did not have any respons

i was dilute the primary and secondary antibodies with skimmed bloker and dont have any respons

The use of dual antigen system can eliminate false positive.Thanks

Somaye Mazaheri

You can try changing the ELISA microplate. Sometimes the quality of the plate also plays a role. Try using ELISA microplate from another vendor.

Good Luck.

Titre one by one your steps in the Elisa and use the maximun dilution before the lecture declines for each one reatives in your Elisa sistem