I am trying to do indirect ELISA from human serum samples. The protein mixture we use as antigen is made in our laboratory. I coat with same concentration and in multiple dilutions. The serum samples are frozen in -80 C till used for ELISA.
I coat the antigen over night in carbonate/bicarbonate buffer pH9.6.
Next day, I block at 37 C for 2 hours with 1% BSA in PBS. After washing 5 times with PBST, I incubate it for 2 hours with diluted serum samples. Followed by 5 times washing with PBST, I incubate with Alkaline phosophatase conjugated Antibody and incubate for 1.5 hours. After that washes with PBST, followed by PBS and addition of 1 step PNPP. I let the colour develop for 15 mins and add stop solution. NaOH.
I keep most things constant. Dilutions, Antibodies, timings and so on. Still I get different results from same serum samples. Since the standard is not available, I don't have standard curve. What would be the reason of inconsistent results?
Dear, inconsistent ELISA can be a result of minor things apart from the dilutions, Antibodies, timings and so on as mentioned by you. I feel you should check into things like quality of plate, washes, buffers, key reagent etc. The way in which you explain I think its some problem with low or high signal/background.
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