I have researched and understand the basic principle and approach of a checkerboard titration.
In a recent discussion with my manager, several questions have come up:
These relate specifically to sandwich ELISAs
1. Why would the primary Ab be optimized rather than coating as much of the plate as possible to enhance antigen capture?
My thought is cost and finding the level that capture the antigen at the high end of the linear range, but still conserving Ab. I wonder if non-specific binding affects play a role as well.
2. Why would the secondary Ab be optimized?
Here, I'm pretty sure this is a cost consideration, but confirmation would be appreciated. Or correction if needed.
3. If the antigen of interest is in a complex matrix (such as whole blood or serum), how are non-specific interactions characterized? When developing the ELISA, is a buffer used, or is antigen negative sera spiked with a known amount of antigen?
4. Can anyone explain why the usual starting dilutions series is 1:2? Is there a dilution factor that will likely encompass the dynamic range of most Ab-Ag pairs?
Thank you for any insights into the reasons behind the conventions of checkerboard titrations.
Hi Becker
1-Yes, it is very important to optimise the coating Ab concentration. No more quantity means more signal. Frequently Hook effect is present when coating protein is in excess.On the other hand, steric hindrance may occur and non functional Ab are present in coating.Typical concentration curve for coating Ab goes from 1 - 15µg/mL. Frequently 5µg/mL result the best result.
2- Here there are 2 possibilities: A- Using the same Ab as secondary Ab. In this case this Ab must to be conjugated with the enzyme because the use of an anti specie conjugate will react with the coating Ab. It is mandatory that the specific epitope ensure both capture and secondary detection. Be careful if you using MAbs.
B- Using a Ab from other specie. In this case the optimal dilution is very important in order to minimize cross reactivity with coating Ab. This Ab may be either directly conjugated or not. If It is non conjugated you have to use an anti specie Ab conjugated and you have to select correctly this one and optimize the working dilution. In both cases (A or B) excess of reactants will lead to a background in the ELISA, of course a cost consideration is a issue to be in consideration. As rule take the max dilution that offer the max signal.
3- Well this is a complicated question. Former, I think that depend on the specificity of the Abs that you are using. Some ELISA needs some dilution for eliminating interferences when negative sera are analyzed. If you are talking about of a quantitation ELISA you may spike the sample with Ag in order to check the recuperation percent. I think that you have to do it using the same matrix that you perform the assay.
4- Two fold serial dilution is the most common way to assay the dynamic range but it is no a rigid rule. You can test any dilution rate to cover the range of your assay.
Anyway, you can review specialized literature related with ELISA. Best regards, Oscar
Thank you Oscar Otero Alfaro for your explanations. They are super helpful to my understanding.
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