Hello dear all, I am processing serum and plasma (heparinized) samples of guanaco (Lama guanicoe) with competitive ELISA commercial kits for estradiol from DRG. First I made dilutions tests with pooled samples of pregnant and not pregnant females. I obtained the dilution factor for each group (pregnant and not pregnant). After that, I processed the samples with the calculated dilutions but I am obtaining OD values even higher than my zero. Samples were obtained in 2016 and frozen at -20ºC, according to the bibliography, estrogens tend to diminish their concentration during long-term storage at this temperature. I am also performing the measurements on the same samples that were unfroze to perform the pools before. Can these facts be influencing hormone measurements?

I intend to measure progesterone also, any suggestions to take in account for this hormone?

Regards,

Antonela