I did an ELISA experiment using a kit for MCH in rat CSF. The kit recommends using 50 μL sample for each well but since I will also dose orexin, I had to decrease the amount of pipetted sample. Analyzing the results, I saw that many samples were outside the standard range of the curve (the less peptide in the sample the color intensity is higher), which means that I have little peptide in my samples. The other ones were near the limit of the curve (less concentration). I want to know if increasing the pipetted amount of sample could improve the results or if it is really necessary to make an extraction of the peptides and how to do it. I would also like to know if I can use those values that were not very concentrated, but inside the curve for statistical analysis. thanks
Kits are standarized to work with fixed volumes. It is not recommended to change the volumes as it will impact in the reproducibility of the assay. Instead of reducing the volume, you should make dilutions of the samples in order to achieve the proper volume.
I agree with Luis - a kit's overall function, sensitivity and limit of detection are calibrated to using a certain sample volume. The best place for your sample concentration to come out is around 50% binding/linear portion of the curve. That is where you're going to have the most accurate results. Outside of that (too concentrated or not concentrated enough), your data will not be as trustworthy.
That is why it is critical to do validations like parallelism and accuracy, so that you know that appropriate dilution for your sample, whether the kit is actually binding what you think you're binding, and that there is little to no matrix effect. I've attached a great guide on ELISAs put out by the National Zoo - there's all kinds of info on validations, among other things.
Hi!
If you have so low content of the peptide in the each sample you can do
1. use special filters for incereasing peptide concentration in solutions.
2. if properties of this peptide allows this step do cold evaporation under decreased air pressure.
3. if you have not so low concentration you can add double volume of each sample. But! Add 1 volume of buffer into aech calibration wall. In this case (I did this) you can increase time of incubation (empiricaly)
Good luck!
Tatiana
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