Hi everyone, I have some questions about the ELISA test in stool sample.
Objective: ELISA assay to detect MMP-9 and Fibrinogen in human stool.
It is known that these two proteins are abundantly expressed in the feces of diseased people, but little or no expression in healthy people.
Result 1: There is no linear relationship between the dilution factor of the target protein in the diseased sample and the final concentration, but the standard curve does. The detected concentrations of proteins were all within the standard curve range.
Result 2: Detecting the healthy sample (A1) and the spike in gradient standard in the healthy sample(A2), it can be found that (A2-A1) has no linear relationship with the original concentration of the standard.
Do these results indicate that there is non-specific binding in the stool sample itself? How should it be solved?
I would not attribute your observations to non-specific binding. Stool is a very complex and heterogeneous matrix, there are many bacteria (and kinds of), and many components which may influence the interaction of a certain protein with the components of an ELISA (capture antibodies, reagents, etc) or simply digest or denature your proteins of interest.
So you might need some standardization/extraction procedure. I'd try optimizing the extraction buffer and removing the lipids from the samples (e.g with petrol ether) first.
I agree with Wolfgang Schechinger concern. The presence of heterogeneous matrix and several interacting compenents that could interact with Fibrinogen as well as other solutions that are used to conduct the ELISA may limit the sensitively of the assay.
As you know that Blank and nonspecific binding (NSB) are always run in parallel in each time of ELISA set up, role of NSB is eliminated by deducting the OD of NSB from all other OD. Hence, you may have to focus on the extraction and normalization of samples prepared for the estimation of MMP-9 and Fibrinogen in human stool using ELISA kit. A brief extraction procedure is given below:
Stool extraction
Stool samples were weighed and added to an extraction buffer, vortexed for 1-minute alternating with a 5 min ice bath incubation until no fecal granules were visible. Following two rounds of centrifugation, the supernatant fraction was collected, assayed for protein content, and frozen in aliquots at −80 °C until the assay.
For more detail for sample extraction procedure and conduction of ELISA, see the following link:
Predicting disease course in ulcerative colitis using stool proteins identified through an aptamer-based screen. Nature Communications volume 12, Article number: 3989 (2021).
https://www.nature.com/articles/s41467-021-24235-0
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