I would guess that the inactivation method has blocked access of your antibody to its epitope. I'm not sure what 'GD inactivated' means, unless it is from a company.

Check for total protein on your plates.

http://www.labome.com/method/Protein-Quantitation.html

If you bought your hemaglutinin antigen from a company, you could ask them what antibody they recommend for use with it.

What is your final goal? What is the big picture experiment here? Is this for a vaccine? Is it for use as a positive control?

Perhaps the antigen you bond to Elisa plate is too few to recognize the specific antibody. You can Increase the antigen by concentration and purification, and may have a better results.

Did you use Nunc plate ELISA Plates?

As Brian said we need more information to figure out a solution. we use Nunc plates and carbonate buffer for coating the plates.

Overall I'm trying to make a passive vaccine.

I'm using Nunc plates and I would like to use plate coated with inactivated HI antigen to catch specific antibodies againts it.

GD is a name of company.

We usually use heat inactivated/ UV inactivated whole PR8 virus as vaccine and get a good response. Serum from mice vaccinated with formalin inactivated vaccine (PR8 virus) works pretty well with plates coated with whole inactivated or live virus (PR8) too. The HA on whole virus should be able to react with the Ab against HA in the serum. We usually look for total IgG and IgG1 and IgG2 isotype response. If you think coating is an issue, you could try using Carbonate buffer instead of PBS (Recipe: http://protocolsonline.com/recipes/buffers/carbonate-buffer/). Hope that helps

Thank you I'll try this for sure.

I think that coating might be an issue cause control serum tested on very similar HA display low optical density and there was no difference with changing dilutions of antigen.

I'm afraid that it may be a non-specific result despite it's 2-fold stronger than blank with antigen.