As we are using approved semi-quantitative/qualitative the indirect ELISA for antibody detection. we ran 10 known positive samples in dilution neat (as per kit insert which is 20 dilution), 16, and 32 dilution. all ran in one batch.
Result ;
10/10 neat positive.
4/10 gives the linear OD value as respective to all dilution.
6/10 gives the OD value approximate twice than the cut off value in all neat, 16, 32 diluted samples.
all materials used was calibrated and technique were the same in all. so rest all technological problm were excluded.
what would be the reason for this pattern?
What is your sample, it look like some kind of intereference. Can you supply results or more information. Sample diluent?
serum samples and dilutions were done with known Antibody negative serum outside of the procedure.
Is it possible to dilute the samples with buffer not in serum?
It seem that there is serum interference IgG, biotin etc.
Ok sure. Can try.
Can u elaborate the machanism of these types of interference, how these work ?
If you use serum as sample and diluent there are components in rhe serum that reacts with the antigen or
antibodies causing false signals. To verify this you have to make serial dilution of the sample or negative serum with buffer and see reduction in signal. If you have biotin streptavidin as detection tried to avoid them or screen other secondary prefered mouse origin
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