Hello,
has someone done chessboard or checkerboard experiments for an indirect sandwich ELISA?
Is it possible to titrate every antibody as it were used for an indirect ELISA??, I mean adding the antigen and then titrate every reagent??
Thanks on advance.
Best,
Carolina
Yes, these experiments are very useful. However, it is not trivial to choose, which concentration is "optimal". Not necessarily the highest signal is the best. Sometimes, this leads to higher background, non-selectivity or a waste of expensive reagents.
Yes I know, but one has to begin with something. There are to many variables in an indirect Elisa that contribute with the final result. It is a difficult approach to achieve just to standardize the conditions in order to make an asset that will last one day and will allow to measure a lot of samples...
thanks Michael!
Yes. To get proper results you have to optimally titrate each immuno-reagents. The antigen to be serially diluted in coating buffer say from 500ng/ml, 250ng/ml, 100ng/ml, and 50ng/ml, and 200 microlitre to be added into the wells (one dilution per eight well strip); for non-specific binding (NSB), 200 microlitre of coating buffer to be added in a separate eight-well strip and incubated for 1 h at 37 degree centigrade or overnight at 4 degree centigrade.
Decanting and washing followed by blocking with blocking protein solution.
Add 100 microlitre of antibody diluted serially in assay buffer, 1:500, 1:1000, 1:2000, and 1:4000 in wells (one dilution per two wells in vertical fashion) and incubated for 1 h at 37 degree centigrade.
Decanting and wash.
In order to estimate the amount of primary antibody immobilized on
microtiter wells add secondary antibody dilution that do not show NSB and incubate for 1 h at 37 degree centigrade.
After incubation, the contents of the wells to be decanted and washed
by filling, decanting, and tapping. Finally, for measuring bound enzyme activity (which is a direct function of primary antibody), 100 microtiter of substrate solution to be added to all the wells and incubated for 15 min at 37 degree centigrade. The reaction to be stopped by adding 100 microtiter of stopping solution and the color intensity to be measured in ELISA plate reader.
If you require any more information please get back to me.
With best wishes
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