Dear all

I used an RnD system's Duo set ELISA kit for analysing cytokine level in tissue samples. The standards and samples were diluted using the diluent received with the kit (1%BSA in PBS). The absorbance I got for the same sample at different dilutions is mentioned below:

1. Neat dilution: 0.3462 (concentration: 48.89508 pg/ml)

2. 2-fold dilution: 0.4558

3. 4-fold dilution: 0.4384

Since diluting the sample did not alter matrix interference, I tried changing the buffer in which I homogenized the tissues. Separate tissues were homogenised in RIPA buffer, PBS buffer and a combination of RIPA and PBS. I performed spike studies and found that samples homogenized in PBS alone had a better recovery percentage than the other. However the absorbance issue still remains. The absorbance more or less does not change when I dilute the sample. Spike recovery is still around 60%. What could be the reason. What else should I do?