I am performing a (partial) validation of commercially available ELISA kit. I am using the kit on a previously untested matrix, so I wish to validate the assays Accuracy, intra-assay Precision and Spike-recovery.
To my understanding: Accuracy (AC) is defined as: closeness of mean test results to the true concentration. Precision (intra-assay) (PR) is defined as: closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogenous sample. Spike recovery (SR): used to determine if the assay is affected by the difference between the diluent used to prepare the standard curve and the sample matrix.
Since all these parameters are determined by performing multiple measurements of different concentrations, I am wondering if one can combine these into a single series of samples and use the data to determine the 3 parameters above?
i.e I would perform a series with 4 different concentrations of the analyte spiked into sample matrix, and do 5 determinations for each concentration. Is there any reason that AC, PR and SR determination should be divided into separate series on the validation plate?
I have attached is my planned plate-layout including the other parameters (LOD, LOQ, linearity etc). Am I on the right track here?
I appreciate any thoughts/insight you may have on this
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!
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