We know that you put plenty of blood, sweat, and tears work into your research and when it comes time to quantify your protein of interest, you take the time to identify a ready-to-use ELISA kit for the quantification and hope that it works. The kit was developed so that all the components work together, so everything should perform perfectly, right? Each lot should work the same, right? And if it doesn’t…that’s wasted precious time, money, and sample.
ELISA suppliers provide many different ELISA kit quality and validation parameters, but what does it all mean? Better yet, how can you use that information to make sure you purchase the right products for your research? We’re glad you asked!
Let’s review the most important parameters and explain them in terms easier-to-understand terminology:
- Analytical Sensitivity: It is industry standard for ELISA manufacturers to report the analytical sensitivity, more commonly known as the limit of detection (LOD). Analytical sensitivity is the lowest analyte concentration that can be detected from the background. The concentration is calculated as the background +/- 2 standard deviations. It’s important to not confuse analytical sensitivity with functional sensitivity. Functional sensitivity, or the limit of lowest quantitation (LLOQ), is the lowest concentration at which the analyte can be reliably detected. While functional sensitivity might be more useful to researchers, analytical sensitivity is what is reported most of the time.
- Intra-assay precision: Intra-assay precision tests multiple samples in replicate on the same plate. Each well should give comparable results for each sample. The %CV across the replicates should be less than 10%.
- Inter-assay precision: Inter-assay precision tests multiple samples on different days and different plates. This test tends to be more variable then the intra-assay precision, so %CV values may be higher. Inter-assay precision ensures that results obtained will be consistent over time and between kits.
- Spike and Recovery: Spike and recovery testing determines if you are getting the true concentration when testing samples. Some samples may be in matrices such as plasma or serum. These matrices can cause issues with antibody capture and binding. To test if you have captured all the protein you “spike” a known concentration of protein and see how much of it you “recover” upon measurement. Ideally, you should get as close to 100% recovery as possible.
Each of these parameters gives researchers confidence in their chosen pre-coated ELISA kit; no matter if it’s days, months, or even years, the kit should provide you with the same consistent results every time.
To learn more about Invitrogen™ ELISA kit validation and quality testing check out ELISA Validation and Quality Testing.
Invitrogen offers a broad menu of over 1,000 ready-to-use pre-coated ELISA kits that provide accurate, consistent results. To see all the ELISA kits offered, please visit the ELISA Selection Tool.
Jamie Boden, Technical Writer, IMS: Science has been her passion since childhood; she knew at an early age that she wanted to pursue a career in science where she could learn about how humans were connected, down to the cellular level. Jamie is a biologist turned technical writer with experience ranging from the development of handbooks to webpages and everywhere in between. Jamie strongly believes that science should be available to everyone, because it affects every person, animal, and plant on earth.
Stay tuned for more articles from Jamie in the near future!