How do best show my ELISA results if I have tested two new peptide antigens (say, A& B) by immunizing mice and collecting sera. I coated the plates with the antigens, added sera at three dilutions (in triplicates) and did the rest of ELISA process and have absorbance values. Sera from one set (mice immunized with antigen A) has significantly high absorbance at all dilutions over other (mouse immunized with antigen B). Pre-immunization sera from both set was my negative control. Can I just use the absorbance values to show results?
Or should I also compare the IgG concentration from each sera to determine if antigen A or B is a more potent antigen? Should I report antigen A is more immunogenic than B or A generates IgG with high specificity than B?
You can and you must use only absorbance values to show your results. The antisera you produced in both cases have polyclonal antibodies to each of your peptides. You don't know anything about affinities of each monoclonal Ab in their mixtures, as well as if one of your peptide is more immunogenic than another for this host animal. You also don't know anything about their specificities to each particular epitope on your peptides. Therefore, any determination of total IgG concentration will not be very helpful.
I do agree with Viktor, in addition you might do a titration of your sera to identify the EC50
Hello, since you measured the absorbance, you will have to report it in absorbance. If however you are interested in concentration, you will have to prepare a concentration curve, with known concentrations of the standard antigen, then plot a curve and convert all the absorbance values to concentration. From there, you can now use concentrations which is a better way to really determine which reaction is better and higher.
I think this link is very useful,
http://www.instanano.com/characterization/theoretical/concentration-calculation-from-uv-vis-absorbance/
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