I am currently developing an antigen-down assay for Troponin I. I have already optimized the surface deposition of Troponin I, but am looking for ways to increase the kinetic binding parameters of the tracer antibody to enhance the DL of IgG on the surface. I have seen in literature that many researchers compare the effects of pH, temperature, and salt concentration during antibody association to increase the Kon of the reaction. My setup also uses a competitive format, where the sample (standard Troponin) is mixed and applied with the tracer antibody during the same step. Ultimately, we wish to lower the detection limit of IgG (anti-Troponin) on our assay to reach the molar limits required for competitive detection in real samples.
If you would like to influence of the detection limits of your ELISA you should be focused on the antibody affinity instead of the concentration. According to the law of mass action, this parameter will determine the detection limit much more than any other parameter adjustment. So you need to try several antibodies ...
In a competitive format is important both the concentration of the antibody in solution and the concentration of the immobilized antigen. Usually for the same antigen concentration a smaller amount of antibody, if it has a priori a high affinity for the target in solution, can improve the limit of detection. So I would suggest you to try several antibody concentrations for the same immobilized amount of antigen. But as you have been suggested, physico-chemical parameters such as pH and ionic strength are many times crucial to improve the assay features. You can also try to preincubate your antibody with the target in solution before putting in contact with the surface (you favor the initial formation of the Ab-target complex before competing with the immobilized one)...
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