ELISA assays are a robust analytical tool to detect analytes in complex biological matrices. Their applications in biomedical and clinical research have significantly impacted the approach to analyzing numerous analytes such as proteins, antibodies, peptides, and hormones in multiple biological samples, including blood, plasma, serum, urine, saliva, etc. Several variants of ELISA assays are available to detect antibodies or antigens, determine immune response type and intensity, and much more. These properties, along with lower cost and higher sensitivity, make ELISA the primary tool in biomedical and clinical applications.
During ELISA assay development, accurate and precise assay performance parameters are necessary for individual assay steps. This approach ensures entire assay protocol is optimized for reliable and reproducible results. Moreover, before implementing assay protocols for routine applications, scientists should prove that the assay is reproducible, accurate, and precise. Hence, ELISA assay validation is necessary to generate reproducible, specific, and reliable results. The current article discusses the need for ELISA validation and shares the best ELISA validation practices for laboratories such as PK labs.
The need for ELISA assay validation in biomedical and clinical research
Validated ELISA methods for quantitating analytes such as drugs and biomarkers in complex study matrices are necessary for successful biomedical and clinical studies. Validated methods provide crucial safety and efficacy data. A comprehensive ELISA validation addresses key aspects of drug testing, such as understanding the target analyte concentrations, estimating assay variability, and evaluating the impact of storage and handling on reliability and accuracy.
Today, multiple ELISA assay formats are available. Moreover, regulatory bodies, such as EMA and the US FDA have clear guidelines for validating ELISA assays. These suggested recommendations are crucial for generating reproducible results. Besides, they are necessary for data that are acceptable worldwide. ELISA assay validation includes assessing assay sensitivity, linearity, accuracy, precision, specificity, and robustness. Critically, the acceptance criteria for each of these parameters depend on the intended use of that ELISA assay.
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Best practices for ELISA assay validation
Researchers should develop the ELISA assay to fulfill the intended applications of the assay. The assay should be reproducible, sensitive, and specific. The assay method should mention the ELISA format, the antigen/antibody pair, conjugation molecules, incubation period, temperature, and data analysis. Moreover, each ELISA assay must be optimized for specificity, sensitivity, and reproducibility. Assay optimization includes testing different parameters, such as incubation time and antibody and antigen concentration, to determine optimal conditions for reliable and accurate results. Researchers can evaluate the biochemical properties of therapeutic antibodies by recovery ELISA.
ELISA assay validation includes protocol testing through samples that are analogs of the intended applications. All steps involved in the design, validation, and optimization of ELISA assays should be documented. This documentation should contain the protocol, validation results, data deviation, and optimization approaches. Besides, this document should be secured and made available whenever necessary. After validating the ELISA protocol, researchers should monitor and maintain it to produce reliable and accurate results. This approach includes regular evaluation of control samples, equipment calibration, and monitoring of tools and reagents.
The US FDA recommends inter-laboratory studies for ELISA assays to demonstrate the reliability and reproducibility of the assay among different laboratories. This comparison contains the ELISA assay results across different laboratories following the same protocol and blinded samples.
Addressing common errors during ELISA assay validation
Even after a thorough ELISA Assay Validation approach, other issues may impact assay results. Let us troubleshoot some of the errors observed during ELISA validation.
Prepare standards through serial dilution, beginning with the highest concentration and ending with the lowest. Errors during standard preparation may impact the entire study. Hence, it is critical to mix solutions during dilution. Besides, formulate separate controls for each study.
Binding-solution pH is critical. Wrong pH impacts the binding kinetics. Hence, the binding solution should be adjusted at the appropriate pH.
Washing is one of the most critical steps in ELISA assays. Researchers should use PBS for all washing steps. Ideally, use automated washing devices. Thorough washing eliminates non-specific binding and reduces false positives.
Delaying the reaction process or failing to stop ELISA reactions can lead to non-specific changes in the substrate and generate inaccurate results. Hence, evaluate the incubation period carefully.
The typical wavelengths for ELISA assays are 405, 450, and 630 nanometers. Reading at wavelengths other than the ones recommended by developers may lead to inaccurate results. Smaller wavelengths can cause high absorbance, while larger wavelengths may undermine target molecule estimation. Also, read the samples just after adding the stop solution for accurate readings.
Modern reading systems can save data. This system is advantageous for ensuring data accuracy. Other factors such as stability and conformation of the target antigen, concentration and type of enzyme conjugate, potential reader or human error, and substrate quality may also impact ELISA results. Background value errors can be reduced by increasing blocking solution concentration or adding non-ionic detergent. On the other hand, an extended blocking incubation period and plate shaker can minimize background readings.
