Differences Between ELISA Kits Explained
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 manufacturer’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 singleplex products from vendors that have been trusted in the industry for years. 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, confirm that the two assays are binding the same biomarker confirmation. 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 curve 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 to evaluate the correlation. An R² value of 0.8 or higher is typically considered an acceptable correlation.