Dear students, researchers, and anyone who can help

I'm attempting to optimize and validate an indirect ELISA test with peptides for a parasite, but it's lacking reproducibility. The test works sometimes and then does not at others. I tried changing different variables, but the reproducibility is still poor. I cannot find what variable I need to change.

To standardize, I used a pool of sera of known infected animals as a positive control; individual sera from healthy animals as a healthy control, and individual sera of animals infected with other parasites. The test worked perfectly, differentiating the positive control from the healthy control and animals infected with other parasites.

However, when I performed the ELISA with individual samples of infected animals (not the positive control, but the sera of infected animals with the same parasite), the group results were similar to healthy control animals. The O.D. values of healthy control and blank samples are the only ones constant on every test. Also, I used a native antigen as the reagent control, which worked every time.

I tried several modifications: different antibody dilutions, peptide concentrations, substrate dilution, blocking buffer, washing buffer, the water used on the washing buffer, the type of washing (manual vs automatic), different volumes added to the well, and the microplate brand.

I appreciate your suggestions!

Buffers used:

For coating the wells: carbonate-bicarbonate, pH 9.6

Blocking and diluting samples and substrate: BSA 1%

Washing: PBS-Tween 0,05%