I did a lab designed ELISA for OVA-specific IgE levels measurement in mouse serum. The results are as one would expect, however the standard curve failed completely (except for BLANK); it didn t titrate at all. Can I use the O.D. values to compare my samples in arbitrary O.D. units or even compare them relatively? All samples were diluted similarly.
hey,
sure you can compare them in O.D. You can tell questions like lavel 1 is 4 times higer than level 2 or something. but you do not have any quantitative answer. its all qualitativ. but a comparison is for sure possible if you handled al probes equally!
greetings,
alex
Try to find a high titered serum which is in higher volumes or constantly available. Just define that this serum has 100 arbituary units. So you can use is as a standard for the calculations for the "concentrations" in all your other samples. But, you have to make sure, that differences in the antibody affinity of your samples hasn't to much impact on the titration (your titration curves should be parallel).
It's not that difficult to modify the assay: just use for the incubations and washing a 0.01 mol/L phosphate buffer pH 7.2 and add instead of 0.15 mol/L NaCl the double concentration: 0.3 mol/L. So you can exclude low affinity antibodes (which are mostly irrelevant for the physiological action) from binding on the surface antigen.
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