ELISA Method Validation

Know The Basics: ELISA Method Validation And Standardization

ELISA assays are one of the most commonly employed immunoassay methods for assessing analytes in complex biological matrices. The advent of ELISA has significantly upgraded how researchers quantify molecules such as antibodies, antigens, peptides, and hormones in study samples. ELISA assays have numerous assay variants that can detect antibodies or antigens, determine the pathological intensity of an immune response, differentiate between the type of induced immune response, and much more.

Despite existing for almost five decades, scientists still develop newer variants of ELISA assays for detecting and quantifying molecules and compounds. Such multifacet capabilities have made ELISA assays crucial during drug discovery, preclinical drug development  and also during later stages of clinical trials. Besides, ELISA assays are widely used in toxicology, veterinary medicine, the food industry, allergology, etc.. The current article aims at helping ELISA labs understand the basics of ELISA method validation and standardization. We shall first clinical drug development understand method standardization followed by  ELISA method validation.

Fundamentals of ELISA method standardization

Several laboratories develop in-house ELISA assays. However, after the ELISA method development, the results generated must be compared to other standard values to be valid for analysis. Hence, consistency in ELISA results is crucial and will require standardized methodological protocols. Several leading regulatory agencies have harmonized the protocols with international standards. WHO has established its standard procedure for biological reference materials. Several laboratories participate and test representative materials through WHO Collaborating Center. Once the reference material is tested, it can serve as a comparator for results performed in any other laboratory.

Hence, standardization of an ELISA method development involves comparing the values of unknown analytes with a known concentration of the assay standard. However, this process is not that straightforward. If ELISA assays are used to measure the end detectable dilution, a reference standard may not be available. However, a reference standard is essential for reliable ELISA assay results. Hence, in any circumstances, an ideal reference standard must represent the expected immune response in the target organism.

Basics of ELISA method validation

Validated ELISA assays for detecting and quantifying drugs, biomarkers, and biological products in complex sample matrices are vital for successful nonclinical and clinical studies. A validated ELISA assay ensures that the generated data is reliable to support the efficacy and safety profiles of drug products.

Literature is abundant for supporting immunochemical methods. Besides, the US FDA has guided the validation of bioanalytical methods. This guidance also applies to ELISA assays  used in toxicology, diagnostics, and applied or basic research. The methodology to conduct validation studies must align with FDA recommendations for the satisfactory conduct of quality measures. Generally, ELISA validation involves the characterization of the following parameters:

  • Linearity
  • Limits of detection
  • Specificity
  • Accuracy 
  • Sensitivity
  • Robustness
  • Precision

Moreover, the acceptance criteria for these parameters are based on the intended purpose of the ELISA assay. Irrespective of the analyte type used for analysis, researchers must always review WHO and US FDA guidelines for assessing molecules through ELISA assays.