Hello,
Can one compare binding (curve, EC50...) of 2 antibodies by ELISA if they require different detection secondary mAbs? Both have the same variable domain and hence the same target but the constant chains are from two different species. I do understand assays such as SPR would be more appropriate but can anything be concluded from an early comparison by ELISA?
Many thanks any thoughts!
I would say that such a comparison would be affected by various uncerteinties (differences between species, binding activity, specificity and efficacy, experimentators' influence etc.).
However, if you know there specificity and efficacy for binding to the target/protein of interest you could possinly compare those results.
The curves cannot be compared because the detection systems are different. You should not directly compare the signals. But as the EC50 is a relative measure calculated taking into account half-maximal signal within each curve, I think you could calculate both of them and compare the values in a reasonable way. You need to make sure that both detection systems are working properly and specifically.
Another alternative is to detect one of the molecules (labelling with biotin or enzyme, for instance) and inhibit its binding with both unlabelled molecules. The concentrations of each competitor able to reduce signal by 50% can be compared without any problem in that format.
Curves cannot be compared because detection systems are different.
In addition to that each of them has a special method and conditions of experience They can be separated and then compared
In your assay the binding of the primary antibodies to the antigen would be compounded by the binding of the secondary antibodies to the primary ones. In other words, if you find two different IC50 values, you'd not know which of the two binding events causes the difference. I'd separate these measurements:
- titrate secondary antibody binding first, using known concentrations of the primary as antigen.
- only if there is little difference in affinity and avidity, you can titrate the binding of the primary antibody to the antigen using the secondary as detection reagent.
- if the primary-secondary interaction is different, try to characterise the primary-antigen interaction by labelling the primary directly (RIA may be better than ELISA for that)
As you mentioned yourself, direct measurement of antibody-antigen interaction by methods like SPR would be more suitable for this problem.
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