Biomedical research requires a substantial effort to identify and quantify the analyte of interest. Researchers explore several immunoassays and LC-MS approaches to identify the most suitable technique for analysis. ELISA technique is one such approach that has stood the test of time. Researchers often develop an ELISA assay from scratch or procure a ready-to-use kit for quantifying the analyte for interest.

However, developing or buying an ELISA assay does not guarantee its use in a biomedical and pharmaceutical environment. The assay still has to undergo qualification and validation. The primary objective of immunoassay validation and qualification is to generate accurate, consistent, and reliable assay results.

But what is immunoassay validation, and what is assay qualification?

. Immunoassay validation is related to specific processes such as development and manufacturing processes. Assay qualification is a part of the validation strategy. It is a systematic approach to ensure that the processes generate reliable and consistent results. The validation process  focuses on assay equipment and  supplementary structures such as staff and software systems.

Assay qualification strives to optimize systems to meet industry standards, regulatory requirements, and expected performance. On the other hand, qualification is one such section under immunoassay validation that introduces different systems to assay processes.. Hence, the current article highlights the three most crucial steps ELISA service providers must focus on while developing an ELISA method for qualification.

Analytical sensitivity

Regulatory bodies require ELISA developers to demonstrate analytical sensitivity, also known as the   detection limit. Analytical sensitivity  measures of the lowest detectable analyte concentration from the background. Researchers can estimate analytical sensitivity as +/- 2 standard deviation. However, researchers must not confuse functional sensitivity with analytical sensitivity.  Functional sensitivity is the lowest analyte concentration that scientists can detect reliably. Though functional sensitivity might appeal more to scientists, analytical sensitivity is often reported in new drug discovery and development studies.

Intra-assay and inter-assay precision

Intra-assay precision relates to testing samples in the same ELISA plate. Each replicate must have comparable results for every well for that sample. Generally, the coefficient of variance must be less than 10%.

Inter-assay precision means testing the samples on different plates and separate run days. Hence, they can have a higher coefficient of variance compared to intra-assay precision. However, ensuring inter-assay precision instills confidence in results obtained over a longer course of time.

Spike and recovery

Spike and recovery experiments are vital for identifying valid concentrations while testing study samples. Biological samples are usually in matrices such as serum or plasma. These matrices have inherent interferences that may affect antibody capture and binding. Hence, researchers perform spike and recovery testing,  spike a protein with a known concentration, and then perform a recovery experiment to estimate the amount of recovered protein. Ideally, recovery must be close to 100%.

Each of these above factors is crucial to ensure that a developed ELISA technique is ready for qualification. Such timely interventions further give researchers the confidence to use the developed ELISA assay through the entire drug discovery life cycle.

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