May 24, 2024

10 Tips For Successful ELISA Method Validation

ELISA assay method is the most common antigen-antibody interaction method used in bioanalytical analysis. ELISA lab employ immunoassay to identify and quantify  various molecules such as antibodies, hormones, proteins, and peptides. ELISA assays can not only detect antigens or antibodies; they can determine the immune response intensity, type of response induced, and much more. Such innate capabilities make custom ELISA assay development a possibility for bioanalysis. Despite being first invented almost 50 years ago, ELISA and its different variants are still developed and expanded for a wide range of bioanalysis.

Top 10 tips for a robust ELISA method validation

A validated ELISA method is crucial in the success of relevant clinical and nonclinical studies. A validated method ensures that the generated results are reliable in providing essential insights into the safety and efficacy of pharmaceutical drug products. An ELISA validation incorporates validating the assay against some critical characteristics, including linearity, specificity, precision, accuracy, sensitivity, and robustness. Following are ten tips for successful ELISA method validation.

  1. One size fits all approach does not apply to ELISA assays. All ELISA assays are not the same. Not only does the nature and amount of target analyte dictate the design of an ELISA assay, assay specificity and sensitivity, availability of antibodies, and sample complexity are other crucial aspects in ELISA method validation.
  1. Do not entirely focus on antibodies alone. Antibodies play a crucial part in ELISA assay validation. But focusing only on antibodies and not on other antibody reagents and assay factors will pose a challenge in assay development and validation.
  1. Ensure careful handling of assay antibodies. Most antibodies are poorly handled during assay validation, most commonly during freeze-thaw cycles and storage at wrong temperatures.
  1. Avoid mixing components between separate kit lots. ELISA assays are lot-specific, and they need to be used before their expiry date. Often to conserve reagents, researchers use reagents from different lots during assay protocols.
  1. Many assay reagents are temperature-dependent. Ensure all reagents are at room temperature before 30 mins of analysis, providing enough time for consistent binding kinetics.
  1. Have duplicates for all standard and study samples. Running ELISA in replicates helps identify points that are outliers and help achieve assay accuracy.
  1. Avoiding contamination is the golden rule in all bioanalysis. Use new tips to  prevent  cross-contamination of samples, which will lower the coefficient of variability and background values and provide reliable results.
  1. Focus on reliable standard curves. Keep the serial dilution within the indicated assay range. Adding points beyond the curve will not help increase assay sensitivity as the characteristics of the antibodies limit the assay..
  1. As biomarker levels and matrix interference effects may vary, optimizing  the serial dilution according to the study sample is very crucial.
  1. Before initiating an ELISA assay, ensure all suggested background and control well are appropriately included.


Without an appropriate demonstration of ELISA validation, it is impossible to generate reliable immunoassay results. An ideal method validation should be able to meet a broad range of technician expectations. Although newer ELISA-based techniques are still being developed, a successful immunoassay still runs on basic principles of assay development and optimization.