Hi Nocolai,

I believe what you need here is; optimization of ELISA generally refereed as Chequerboard Titrations. Depending on the type of ELISA you are testing, you can plan your experiment with diluted Ag vs Diluted Primary Ab. Following reference might help you. http://www-naweb.iaea.org/nafa/aph/public/ras-ai-elisaii.pdf

This will definitely answer your LOD and LOQ queries. And after you have data, and well plotted graph you can determine Accuracy and precision too. However Pipetting will contribute a lot as variable here.

hope this helps.

good luck.

Hi,

I don't know if my answer would be not too late and would help you now but regarding the validation of analytical methhods in biological samples, the EMEA (European Medicines Agency) guidelines say: "Precision should be demonstrated for the LLOQ, low, medium and high QC samples, within a single run and between different runs, i.e. using the same runs and data as for the demonstration of accuracy".  Here enclosed is the file with the complete guidelines if it helps. Its scope is to provide recommendations for validation of bioanalytical method applied to measure drug concentrations in biological samples, not biomarkers... So the recommendations can vary depending on the analyte you want to measure...

I hope my answer could still help you. Good luck!

Hello Cecile,

Thank you for this information. No it's not too late, so i really appreciate your answer. Thank you!

The idea of spike-and-recovery is that you add a certain amount from standard stock solution into the wells containing the solution to be tested (e.g., sample buffer or samples) and measure them, to see whether you can recover that amount again, and how much you can recover from it in %. If, for any reason, you couldn't recover that amount in comparison with a control, where that same amount was added into assay buffer, then this means that something in the test solution is not in favor of the assay.