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 dilutions. 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 were calibrated and the technique was the same in all. so rest all technological problems were excluded.
what would be the reason for this pattern?
There is not enough information about your assay to really make useful comments. Have you sufficient number of negative samples at similar dilutions to check cut off point validity? Are you testing human sera? If so is the kit has provisions for the problem of HAMA (human anti-mouse igg antibodies)? if all is right then you maybe have very high titre and need further dilution
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