Enzyme-linked immunosorbent assays (ELISAs) are widely used for quantifying protein biomarkers, cytokines, and other molecules in biological matrices. For accurate and consistent results, it is essential to validate ELISA performance based on stringent acceptance criteria and quality control standards. This article delves into some of those factors.
Quantitative ELISAs typically have a sandwich configuration, whereby one analyte-specific antibody is bound to the microplate wells for target capture and another analyte-specific antibody, that recognizes a different epitope, is used for detection. By including a serially diluted standard on each microplate, the concentrations of the test samples can be calculated. However, many different factors can influence ELISA performance, making rigorous validation essential.
The types of validation tests that should be carried out include the following:
• Precision
The International Organization for Standardization (ISO) defines precision as “the closeness of agreement between independent test results obtained under stipulated conditions”.1 To evaluate precision, the International Council for Harmonization (ICH) recommends preparing samples at a minimum of 4 different concentrations, including the lower limit of quantification (LLOQ), 30–50% of the calibration curve range, and a minimum of 75% of the upper limit of quantification (ULOQ).2 After running each of these concentrations multiple times*, the coefficient of variation (% CV) can be calculated with the formula % CV = (standard deviation / sample mean) x 100. For an ELISA to pass precision testing, the ICH specifies that the % CV should not exceed 15%, except at the LLOQ, where a 20% threshold is deemed acceptable.
* The ICH advises a minimum of 5 replicates for determining intra-assay precision and a minimum of 3 analytical runs performed over at least two days for evaluating inter-assay precision.
• Dilution linearity
When running ELISAs, the test samples must often be diluted for their concentration to fall within the linear range of the assay. However, because the sample dilution process can potentially affect the measured concentration, dilution linearity testing should be carried out to confirm that the expected results are produced. This involves spiking the sample matrix with an analyte concentration above the ULOQ and preparing a series of dilutions from which the sample concentration can be calculated by multiplying the measured concentration by the dilution factor. According to the ICH, the calculated mean concentration for each dilution should be within ± 20% of the nominal concentration.
• Matrix effects
The types of samples that are analyzed by ELISA are often complex biological matrices, such as serum, urine, and culture supernatants. Besides the analyte of interest, these contain various proteins, lipids, and other biomolecules that can influence assay performance. To test for these so-called matrix effects, researchers are advised to analyze replicates of low and high concentration samples, using matrix from at least 6 different lots.2 If the measured concentration is within ±15% of the nominal concentration and the % CV is no greater than 15%, matrix effects can be ruled out. Critically, when running these types of tests, the matrix should always match that of the test samples to avoid any confusion later on.
Additional factors to consider during ELISA development include which reference standards to use and how to ensure lot-to-lot consistency. Reference standards should come from an authentic, traceable source and be supplied with a certificate of analysis containing details including the batch number, purity, and expiration date. They should also be identical to the analyte, if possible. Meanwhile to ensure comparability of protein concentration values across experiments conducted with different ELISA kits, the reference standards should be calibrated against NIBSC/WHO International Standards.3,4 One way of assessing lot-to-lot consistency is to run quality control (QC) samples on plates from both the current lot and the new lot, before calculating the inter-assay variance. This should be below a certain threshold (typically 15%) for the new lot to be released.
When purchasing an off-the-shelf ELISA kit, identifying the right product can sometimes seem overwhelming. A good place to start is by looking for established manufacturers with a transparent validation process that covers the tests described here, since that will help to safeguard the accuracy and reproducibility of your results. And, of course, Biocompare lets you narrow down your search by selecting from a broad array of ELISA criteria.
Included in the Biocompare product directory, ClinMax™ ELISA kits from ACROBiosystems are fully validated, ready-to-use immunoassays that are manufactured under strict adherence to an ISO13485:2016 / ISO9001:2015 quality management system and calibrated using NIBSC/WHO International Standards. To learn more about ClinMax ELISA kits and how they can benefit your research, visit acrobiosystems.com/clinmax
1. https://www.iso.org/obp/ui/#iso:std:iso:5725:-1:ed-1:v1:en
2. https://www.ema.europa.eu/en/documents/scientific-guideline/ich-guideline-m10-bioanalytical-method-validation-step-5_en.pdf
3. https://nibsc.org/standardisation/international_standards.aspx
4. https://www.who.int/publications/m/item/annex2-trs932