I developed an ELISA, in which I test the different concentration of coated Ag protein was from 200 vg/ml to 800 vg/ml. However, even at the concentration of 800 vg/ml, the diluted serum with high concentration of antibody easily saturated the Ag protein, which leading to an unsatisfactory calibration curve and a narrow dynamic range. What should I do ?
Only thing you can do is to dilute the serum until you don't have saturation.
Assuming that everything else is correct, and if you can't dilute the serum to a point where you see a difference between the 200 and the 800... that means you've already saturated your coating protein concentration at 200.
Usually, when I start these ELISA development, it a checker board where I have the coated protein diluted by 1/2 for 8 to 10 dilutions, and the serum is also diluted 1/2 by 8 or 10 dilution. At some point, you should start to see a difference that is caused by the serum dilution and a difference that is caused by the coated protein concentration.
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