Hyaluronic Acid Binding Protein 1 (HABP1) - I need some insight to prevent binding of this molecule to itself (Aug/12/2009 )
I have an ELISA plate format with the Hyaluronic acid binding protein1 (HABP), also known as C1QBP, bound to the microtiter plate. Hyaluronic acid (HA) is added to the wells and then addition of HABP conjugated to HRP tag. What we have observed is a high background of just the HABP binding to itself on the surface of the microtiter plate wells.
We know that adequate blocking is taking place because uncoated wells are NOT showing any binding.
We know that it needs a lower pH of 6.0 and high salt concentration of 0.1M to achieve adequate binding to HA. Even with those conditions, high background is still noted.
As with any large protein, does anyone have any recommendations of what inhibitor I could add to the conjugate (HABP-HRP) solution to prevent the molecule from binding to itself?
I was thinking of using some monoclonal and polyclonal antibodies to HABP or C1Q antigen to the conjugate diluent. But I have to be careful or it will also inhibit the binding of HA.
Any experience in this area?
Hi tokorok -- welcome to the BioForums!
I don't have experience with this protocol, but perhaps you're using too much HRP-conjugated antibody? What is your dilution?
Have you thought about trying a bit of non-ionic detergent (like Tween) in your binding buffer?
Or perhaps a blocking reagent (gelatin, skim milk) or irrelevant protein (like BSA)?
HomeBrew on Aug 12 2009, 06:07 PM said:
I don't have experience with this protocol, but perhaps you're using too much HRP-conjugated antibody? What is your dilution?
Have you thought about trying a bit of non-ionic detergent (like Tween) in your binding buffer?
Or perhaps a blocking reagent (gelatin, skim milk) or irrelevant protein (like BSA)?
We know that the amount of conjugated antibody is appropriate because the upper limit of the standard curve is still within the limits of our spectrophotometer instrument and correlates with the results on a kit manufactured assay.
We will test the addition of Tween, but we have already tested all kinds of blocking reagents such as milk and BSA and some commercial blocking buffers.
Gelatin could be a possible solution. We will try gelatin and tween and see if those reagents help, but we are convinced that the solution lies in an inhibitory additive that is specific for this protein as the solution.