The Matrix: more than just a futuristic sci-fi movie, the matrix can have a confounding effect on your ELISA results. Interactions between your protein of interest and other components in the sample, called matrix effects, can result in erroneous readings.
Matrix effects, or the effect that the other substances in your sample might have on the ability to detect your specific target protein, are most commonly observed when using plasma and serum samples. Matrix components can affect the binding of antibody to protein or alter the signal-to-noise ratio.
Most matrix effects can be attributed to:
- Other substances in the sample (phospholipids, carbohydrates and metabolites)
- pH of the sample
- High viscosity
- Direct interaction between the protein of interest and other proteins in the sample
- Salt concentrations
How to determine if you are seeing matrix effects
To determine if you have a matrix problem, spike your samples: add a known amount of standard protein to a sample. Try to keep the volume of the added standard to a minimum so you don’t disturb the matrix. Compare the reading obtained to one in which the standard is diluted in standard dilution buffer. If the readings are identical, then the matrix is not your problem.
But if the readings don’t match, then the matrix is the most likely culprit.
Once you have determined you have a problem, there are several things you can do to limit matrix effects.
Dilution is the solution
Dilute your samples by 2-5 fold in sample dilution buffer. Hopefully this will dilute out the matrix enough to allow you to accurately quantitate your protein of interest. Just make sure:
- you use the same buffer when diluting samples for your standard curve
- you take the dilution factor into account when calculating the final protein concentration
- that your readings still fall within the linear range of the assay
Use the matrix to calibrate
Dilute your standards and your samples in sample matrix. For example, you could dilute both in normal serum. This will compensate for any effects the matrix is having on the assay.
Take your best guess
If you don’t have a matrix sample that you can use to dilute your standard curve, you can try and approximate the composition of the matrix in your samples and make a dilution buffer that matches it. Some companies even sell diluent buffers for different sample compositions.
Use the standard addition method
A more rigorous approach to compensate for matrix effects is the standard addition method. You can find in-depth explanations of the method on-line, for example at http://www.tau.ac.il/~advanal/StandardAdditionsMethod.htm. In brief, you take the reading of your unknown sample, and your unknown sample that has been spiked with increasing amounts of known standard. Using linear regression, you can then calculate the amount of protein in the unknown sample.
Hopefully your matrix problems won’t be as severe as Neo’s are in the movie. But these tricks should help sort things out.
Photo courtesy of kris krüg.
Leave a Reply