In my case, during one experiment, I manually wash my plate three turns: 4 times per turn. And I experienced big differences in Absorbance (at 450nm) among triplicates.
For example:
Optical Density (Absorbance at 450nm) of the triplicates of one sample is:
0.633; 0.621; 0.569
=> % Coefficient Variance = 60.8%
I doubt the reliability of the assay when the variance is huge.
Hello Nhu Nguyen,
This is how you have to calculate the "Coefficient of Variation" (CV) also called "Relative Standard Deviation" (RSD).
Average of OD of the sample triplicates = 0.608
Standard Deviation of the sample triplicates = 0.034
Coefficient of Variation (CV) = (STDEV / AVERAGE)*100 = 5.60%
So you have made error while calculating the CV of your sample triplicates. Generally a CV of 15% is allowed for immunoassays such as ELISA.
Don't worry, your assay is perfectly working.
Flag this if it is really worthful.
I would suggest you to check with your substrate. If it is old, it might not give reliable results.Also, ensure it is not exposed to sunlight. Sometimes, it might be photo-sensitive.
I believe that is the cause for the reduction in OD here, unless it is giving consistent results with other samples.
http://www.rndsystems.com/resources/images/5660.pdf
you might find this link useful, It's written for an ELISA kit but it has some good points that might help.
Good Luck
Hello Nhu Nguyen,
This is how you have to calculate the "Coefficient of Variation" (CV) also called "Relative Standard Deviation" (RSD).
Average of OD of the sample triplicates = 0.608
Standard Deviation of the sample triplicates = 0.034
Coefficient of Variation (CV) = (STDEV / AVERAGE)*100 = 5.60%
So you have made error while calculating the CV of your sample triplicates. Generally a CV of 15% is allowed for immunoassays such as ELISA.
Don't worry, your assay is perfectly working.
Flag this if it is really worthful.
Another thing you might want to consider is variation not only in OD but also in your actual results calculated based on these ODs. Sometimes you can peacefully ignore relatively large variance in OD if they are within the shallow or even plateau region of your standard curve.
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