Hello, I did multi-analyte ELISA to detect the concentrations of several cytokines, but how should I calculate the concentration as each well represents a different sample and a different cytokine. there are rows of negative control and positive controls but each is a different cytokine. I am confused.
From your question, I assume you used the multi-analyte ELISA kit from Qiagen. The kit does not come with a full standard curve; all you have is a single well that corresponds to one cytokine. I used it a long time ago and if I remember correctly, the way you get concentration values from the OD readings when you only have a single standard value (+ve control) is like this: if you get an OD of 2.0 for your positive control which has a concentration of 1000 pg/mL and your sample has an OD of 1.2, then the analyte concentration in you sample would be calculated as 600 pg/mL. This is based on many assumptions including the one that your curve is a straight line and that your OD is in the linear range (the manufacturer does say that values over OD 2.5 are not linear).
The values you get from this kit are meant to be relative, not absolute. The idea is you profile a range of cytokines (whether present/absent and whether more/less depending on what you are comparing it to) to identify the ones which are modulated in your experimental setup. This way, you do not have to run ELISAs for all 12 analytes but only for the ones that are relevant to you. Once you identify the more interesting ones, you are supposed to run single analyte ELISAs with full standard curves for accurate quantification (either buy commercial kits with established standard curves or develop in house ELISAs like Ákos Hornung said). As such, the multi-analyte kit is just a good starting point, not the whole picture.
Dear use the individual cytokine absorbance against the standard curve and call the required concentration of each cytokine in the multiplex assay.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA