I have to measure irisin levels in mouse serum samples and I would like to estimate the dilution of the sample, has anyone else done this before?
The manufacturer recommends 1:8
Hello Dr. Joana Antonela Asensio
Yes, 1:4 dilution. But it may not be the same for you. You may have to determine the optimal sample dilution for your experiment. I would suggest you try with neat (undiluted) sample as well as work with two dilutions i.e., 1:2 and 1:4 dilutions. You may also include 1:8 dilution as recommended by the manufacturer.
If the sample falls outside the range of the assay, a lower or higher dilution may be required.
Regards,
Malcolm Nobre
Diluting serum samples for Irisin measurement by ELISA is a common practice to improve the accuracy and sensitivity of the assay. The recommended dilution ratio by the manufacturer, in this case, 1:8, is a general guideline and may need to be optimized for your specific sample types and concentrations.
Here's a step-by-step guide on how to dilute serum samples for Irisin measurement by ELISA:
1. Start by performing a pilot study to determine the optimal dilution ratio for your serum samples. Prepare a series of dilutions using different ratios, such as 1:4, 1:6, 1:8, 1:10, and 1:12.
2. Measure the absorbance of each dilution using an ELISA reader. Choose the dilution ratio that yields the highest absorbance reading. This will likely be the dilution ratio that maximizes the signal-to-noise ratio and minimizes the background noise.
3. Check the linearity range of the ELISA kit. The linearity range represents the concentration range where the assay exhibits a linear response. Make sure that the diluted samples fall within the linearity range. If the diluted samples fall outside of this range, they may not accurately represent the original sample concentration.
4. Consider the characteristics of your serum samples. Factors such as lipid content, protein concentration, and turbidity can affect the efficiency of the ELISA reaction. You may need to adjust the dilution ratio based on the properties of your samples. For instance, if your serum samples contain high lipids or proteins, a higher dilution ratio may be required to prevent interference.
5. Evaluate intra-assay variability. Calculate the coefficient of variation (CV%) for each dilution ratio used in the pilot study. The CV% measures the relative variability among replicate measurements. Choose the dilution ratio with the lowest CV%. This will help ensure that your results are consistent and reliable.
6. Validate the dilution method. Compare the results obtained with the diluted samples to those obtained with undiluted samples. Ensure that the diluted samples yield comparable results to the undiluted samples, and that the differences in results are not statistically significant.
7. Document and standardize the dilution ratio. Record the optimized dilution ratio for future reference and incorporate it into your laboratory protocols. Use the same dilution ratio for all subsequent experiments to ensure consistency and accurate comparison of results.
8. Periodically revalidate the dilution method. Biological samples can vary in composition over time, so it's essential to revalidate the dilution method every few months to maintain accurate results.
In summary, determining the optimal dilution ratio for Irisin measurement by ELISA requires careful consideration of factors such as sample characteristics, linearity range, and intra-assay variability. By following these steps, you can establish a validated dilution method that ensures accurate and reliable results in your research.
All the best with your assay, Good luck
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