Hi all,
I am trying to establish an ELISA method for the quantification of a recombinant protein injected into mice. In the meantime I am trying to make calibration curves with the purified proteins and not serum samples. I am coating a maxisorp plate with 10 ug/ml of an anti cMyc antibody, incubated overnight at 4 degrees in carbonate buffer at pH 9.6. I then block with 1% BSA in PBS pH=7.4 for 1 hr at RT. Wash 4 times (0.05% Tween 20 in PBS, pH=7.4) and then I add the recombinant protein diluted in 0.1% BSA in PBS pH=7.4, incubate for 2 hrs at RT, then wash 4 times. For detection I am using an anti-FLAG antibody conjugated to HRP and incubating 2 hrs at RT and using Sigma's TMB as substrate and reading absorbance after at least 15 min. My lowest detection concentration is 500 ng/ml, I would like to achieve lower detection down to 0.6 ng/ml. Any ideas?
Thanks,
Jason
Have you considered using a pool (mixture) of biotinylated detection antibodies that recognize distinct epitopes on your recombinant protein (if they exist and are available)? You would then use SA-HRP to reveal binding of these biotinylated "secondary" antibodies, and in principle this should yield an assay with improved sensitivity.
Greg A Kirchenbaum I did not try that, it could be a good solution. I will look into how feasible it is. I understand that there is streptavidin with 25 HRP molecules conjugated to it.
Rissin et al. have described a nifty form of ELISA that results in aM sensitivity (Article Single-Molecule enzyme-linked immunosorbent assay detects se...
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