We have performed IHC studies on gastric tissue to identify ghrelin expression before and after treatment and results were clear. Treatment increased immunoreactivity. To assess whether production was similarly affected we performed ELISAs, but the results were the opposite. We are now trying to overcome the problem by measuring ghrelin in plasma, which doesn't require application of detergents but still wonder what interfered with our tests. Any suggestions?
thank you!
"To assess whether production was similarly affetcted we performed ELISAs, but the results were the opposite". Kindly clarify which production. Did you mean concentration of ghrelin in tissue. Then how you processed sample (Tissue) for ghrelin estimation by ELISA to be provided in detail
Dear Tulsidas, tissue was homogenized using Classic RIPA buffer, spun down and surnatant was then quantified using Bradford's method. Equal amounts of protein were finally tested using ELISA on 96 multiwells.
This effect may be due to the antibody preparation you are using. If the antibodies react with conformational epitiopes and you have denatured the target protein then the antibodies will not recognise it.
I totally agree with Simon Worrall comments. Antibody for IHC purpose is for denatured epitope of protein and in ELISA it is for native (undenatured) protein.
Thank you very much Simon and Tulsidas. Based on your comments, it looks like I should try to lyse tissues under non-denaturing conditions and then assay lysates on ELISA, right? Please keep in mind that the Ab used in ELISAs is provided with the kit and is different from the one used for IHC studies!
It depends on:
the detergent (non-ionic doesn't disturb at normal final concentrations 0,1 ... 0.5%, ionic does even at low concentrations below 0.1%)
the sample dilution (this will dilute the detergent as well)
the presence of an inert protein (like BSA, hydrolysed gelatine, ...). The can "neutralize" ionic detergents.
... and as sugguested already above: Use you lysis buffer in the same dilution as your samples as negative control.
Thank you very much Dr Kiessig. I will soon attempt to test ghrelin concentration with the ELISA approach According to yours and others suggestions. I am confident these changes will make it work!
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