I am trying to optimize a sandwich ELISA with antigen. I think, the higher antigen should give the higher OD. But in my case, the higher condition of antigen give the lower OD and it is increase until and then decrease again.
My question is why it can be happen?
Thank you for your help
Can you supply more information such as: Coating antibody, washing, blocking and detecting steps. Also say what the various colours are. Very difficult to assist if this is not available.
I coat the pfcsp antigent several concentration (x axis).I used bsa in pbs for blocking and used antibody against pfcsp in several concentration (showed in differen color).
My purpose is want to measure the optimum concentration of antigen. I Think, the higher antigent concentration the higher OD until the optimum of Ag concentration, and then the OD will stay straight. But in my case, the OD decrease after optimum concentration. I did 2 times and the result is still the same.
Just to make sure, are you changing coating antigen concentrations in the curve?
Do you use a highly purified antigen for coating? May be preferential binding of a contaminant in antigen solution to plate preventing antigen binding at concentrations > 3 ug/ml. Alternatively could be due to prozone effects. Very common at high concentrations for some ELISA systems. Think it will be best to keep antigen less than 3 ug/ml and use the antibody between 10 and 100. Check if you can inhibit antibody binding by adding antigen to antibody solution. Will also check the specificity of the antibody using Western Blot. Always a good experimental back-up if you end up with problems.
Gertrudis Rojas# Yes, I made several antigen concentration
Edmund Pool# I have purify the antigent but not highly purify. Thank you for your explanation sir
Alternatively, your antigen can be in conditions (pH, ionic strength, presence of additives) that interfere with coating, unless it is diluted enough to minimize the effect. If you use relatively low antigen concentrations for coating, dilution in coating buffer can provide a good coating.
You plate surface has a mox binding capacity of about 400 ng/cm². So 1 - 10 µg/mL coating concentration is sufficient. At higher concentration (you have used) you will have double layers of the antigen at the solid phase. They can be removed dirung the washing steps.
But you are already at the right way. Comparing the Ratio between positve and nagative sample OD is expressed in the P/N-Ratio. Use this ratio to optimize your assay for the coating, the buffers (washing, dilution, conjugate concentration, incubation time ...
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