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Absorbance results are low - Is it because of the TMB? (Oct/30/2008 )

Dear all,
I'm new to ELISA. First time when I did it, all absorbance values were low (no samples reached 0.4), including standards (usually the range are from 0.04 to 1.6, but my result are 0.04 to 0.289). Can someone tell me what's wrong?
Coz when I repeat it with the same samples, it gives different result, with standards range from 0.04 to 1.18 and samples absorbance until 1.49.
Is it because of the TMB? Was something happen to it when I put the TMB under the light exposure?
Thanks a lot..


It's very hard to tell what causes can give these results.
To test the TMB ,mix a little bit with the conjugaat and the color should develop to blue.
The plate should be read within 30 minutes after the stopreagent is added because it is light sensitive.
Azide in the reagens can also be a course of low OD's.


Thanks for the reply..
I got more infos from u..

However, i forgot to tell that when I repeated the same samples, I used the same reagents (buffer, TMB, stopping solution containing sulfuric acid, etc) that I had used before (that give low absorbance results). I only used different set of standards.

That's perfectly right bout the 30 min. After adding the stop solution, I've tried to read it for three times. The faster I read, the bigger the absorbance is. Using positive control would help us to know the most accurate concentration of samples.

Nice sharing with u all..


Could be the conjugate. Do you make up fresh each time? (Are you using an established method or developing a new one via checkerboard titrations?) If not fresh, how do you store it?

Looking at your standards, your top standard is less than your top samples. I think you need to raise the top standard or try diluting the samples that lie above the top standard.


Yes I make the conjugate fresh every time, and i follow the procedures given by the kit, not checkerboard titrations.
About ur suggestion, I dilute some samples with absorbance values above than the top standard.
Many thanks..


It is a commercial kit. Reagents appear to be OK. But results vary when you repeat.

May I ask if the kit lot changed? Is it same lot and were components stored properly?

Are you using same (lot) of wells? If wells not coated signals would be low.

Did you dilute conj in correct buffer and use correct buffer to wash plate?

You can ask supplier if they have noticed problems.

You indicated you changed standards. That would account for lot to lot differnce in standard results but not account for low sample results one time and higher results(OD) with corresponding higher ODs with next test.