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When the TBX2 ELISA kit (ADI-900-002 and ADI-901-002) was developed with pooled samples, 1:100 dilution worked. So, this is a good starting point. The levels of TXB2 depends on age, sex and host of factors. We would not be able to suggest a best dilution that would work with your specific samples. We suggest to run an initial optimization with pooled samples by serially diluting samples starting (e.g. 1:1, 1:10, 1:100 etc). With each dilution, one should see linear drop in OD. You should identify OD values that fall within the range of the standard curve, calculate the returned values of those samples, and look for dilutional ranges in which you achieve a linear response between returned value and dilution (for example, if a diluted sample that returned 200pg/ml is further diluted 1:1 with buffer, you would want to see the subsequent sample read 100pg/ml [before correction for the subsequent dilution]).