Hi, I am developing a sandwich ELISA for Rubella viral quantitation. I'm using Rubella Glycoprotein E1 for coating and a polyclonal HRP conjugated Ab for detection. In my last experiments, I designed a standard range 20ug/ml to 0.12ug/ml and using the same material I prepared my unknown 2ug/ml to check if the assay is sensitive enough to correctly quantify it.
However, I'm constantly getting 1ug/ml result for my unknown. I checked my calculation and it seems perfectly fine. I diluted the unknown 1:2, 1:4, 1:8 and got 1.0, 1.02, 1.06 ug/ml conc. results.
I prepared standard by using 0.54 mg/ml of reference (protein conc. was determined by Bradford assay).
Can anyone understand and rectify the problem?
Hi Falk Fish , Thanks for the answer. I tried removing Tween-20 from the diluent and keeping samples on ice, which somehow gave me correct concentration. Nonetheless, I will repeat the experiment and see if I get the same results.
Thank you.
Aakash,
1. I would look for different method to determine the concentration of the Glycoprotein E1 concentration since Bradford or other conventional methods for estimating protein concentration will not give to you the accurate reading in case of modified proteins.
2. Make sure, 1:2, 1:4, and so on are your final dilutions (you are not further diluting the samples and using, for example, 2 ul of the1:2 solution + 8 ul of buffer), - that would've been an additional dilution.
Good luck!
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