help ELISA! - color all over plate - (Jun/12/2008 )

Problem I had from ELISA:

Im using a commercial ELISA kit. I found High absorbance in the empty wells and blanks. Very high O.D in positive sample wells. Entire plate is YELLOW and color development was really fast. Perhaps the washing buffer is off or too much antibody?? I have done this many times but this is the first time that occurs to me.

My Standard is ok. Shows r2=0.97

After substracting from the blank, the negative controls shows negative and samples are very concentrated (over detectable range).

Q1. Can you still trust the sample reading after substraction from the blank?
Q2. Does TBST washing buffer needs to be freshly made each time?


Please, if anyone could enlight me with your answer! Thanks in advance!!

I would NOT trust it

did you wash a lot?

did you block a long time?

are you certain you're blocking with the right agent?

if everything you're using is from the kit, you may wish to contact the manufacturer to troubleshoot the particular reagents with which you are working

Could be the following:

1. Unblocked plate or wrong blocking agent
2. Undiluted conjugate (added very high concentration)
3. Incomplete washing or blotting.

Did the whole plate turn colour in a few seconds? The most likely cause was not washing out the conjugate prior to substrate addition.