Samples assayed on kits from two manufacturers may sometimes result in different reported concentrations, preventing the results from being directly comparable. Some of the most common reasons for this are:
Referencing is a term for defining the concentration of a kit standard(s) using one or more outside standards as a metric. The process of assigning standard values is often performed by assay manufacturers before, during, and after producing a lot of kits to ensure the defined concentration of the standard is accurate. Reported sample values may vary from one manufacture’s kit to another based on the metric and or method of determining the concentrations of the kit standards. At Quansys Biosciences, we reference many of our multiplex products using R&D Systems ELISAs. Other companies may reference their antigens using different standards, or may not reference at all.
Antibodies sold for a given biomarker can have a myriad of differences which affect their affinity and avidity. These differences depend on factors such as which epitopes the antibodies target, the host species, the method of purification, and whether the antibodies are monoclonal or polyclonal. Also, antibodies that bind slightly different conformations of the same biomarker may be sold under similar labels. For example, the Quansys Biosciences Human TNFα assay recognizes both soluble TNFα receptor-bound and unbound TNFα, while some TNFα assays only recognize unbound TNFα.
Assays with different sample and/or assay diluents may report discrepant biomarker values because diluents influence such things as antibody affinity, protein stability, and biomarker availability in endogenous matrices. Additionally, the failure of a diluent to prevent non-specific binding may lead to reported false positives. Quansys diluents are each carefully validated for performance, and human kit diluents always contain reagents to help block the non-specific binding of rheumatoid factor and heterophilic antibodies such as HAMA (human anti-mouse antibodies).
To Address These Differences
First, confirm that the two assays are binding the same biomarker conformation. If they are, perform referencing or correlation tests, such as those explained below, to establish equivalency between the two products:
Example Protocol to Reference Kit A Standard to Kit B Standard:
- Prepare the standard curve for Kit B following the Kit B protocol.
- Prepare the standard from Kit A as if it were a sample (using the diluents from Kit B). Consider running duplicates of several dilutions, to ensure that some replicates fall within the quantifiable range of Kit B.
- Calculate concentrations for the Kit A “samples” based on the Kit B standard curve. Average replicates and apply dilution factors to the Kit A samples that fall within the quantifiable range of standard curve B, and discard replicates that do not. The average concentration of the Kit A samples is the new referenced value.
Example Sample Correlation Protocol
- Run a set of the same samples on kits from both manufacturers following the kit protocols.
- Calculate the R² value between the sample sets in order to evaluate the correlation. An R² value of 0.8 or higher is typically considered acceptable correlation.
Contact the Quansys Sales team if you would like additional Q-Plex reagents for such testing.