I am trying to develop an ELISA that is coated with an antigen and specifically binds IgY. What I am seeing is that a control IgY is non-specifically binding as well. Is IgY non-specific? If so how do I overcome the non-specific binding to be able to demonstrate that the IgY I am purifying has specificity for the antigen the chickens are immunized with?
There are two kinds of non-specific binding which normally occurs (1) there is nonspecific binding to the surfaces of ELISA Plate wells. If that is the case the blocking buffer should be optimized. The second reason for non-specific binding a is binding to substances in solution in presence of high concentration of albumin or immunoglobulin. There are buffer in the market that can help you (eg: LowCross-Buffer)
Also You have to kee in mind that non-specific binding of secondary antibodies in immunoassays is a common occurrence, there are several reasons for this:
• Cross-reactivity of labeled secondary antibodies with endogenous immunglobulins on the tissues or cells.
• Re-activity of labeled secondary antibodies with albumin or immunoglobulins in the diluent.
• Cross-species reactivity
The solution can direct labeling of primary antibodies
Her, I recommend you to read the following article about non-specific binding in immunoaassays:
Reduction of non-specifi c binding in immunoassays
requiring long incubations. Scandinavian Journal of Clinical & Laboratory Investigation, 2012; 72: 531–539.
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