5 Tips To Ensure An Effective Immunoassay Development And Validation

The ability to develop sensitive, specific, and robust immunoassays is crucial for all biomedical research and drug development pathways. Basic clinical research may need an ELISA assay method to determine protein levels in biological matrices. On the other hand, pharmaceutical studies will need a robust ELISA development and validation protocol to generate reliable results for regulatory approvals.

However, irrespective of applications, numerous factors must be considered while developing and validating an immunoassay. Researchers must not only focus on the immunoassay itself but also consider factors influencing assay workflow. Hence, the current article highlights five tips to ensure effective immunoassay development and validation.

These tips are practical regardless of the type of immunoassay developed for bioanalysis.

Not focusing on a single approach

The immunoassay design depends on a spectrum of assay characteristics, including the abundance and nature of the target analyte, available antibodies, required assay specificity, and sensitivity and complexity of sample matrices. There is a misconception that all ELISA assays are essentially the same. However, this is not the case. The conditions for one target analyte may not be the same for another. Even small requirements, such as wash buffers, affect the assay signal. Hence, each assay component must be considered carefully during ELISA validation and development.

Blaming antibodies for poor results

Researchers generally blame antibodies for poor assay results. However, developing an immunoassay is a stepwise process. So researchers may have the best available antibody for their immunoassay, but they have to focus on each component to generate reliable results. Although a lot depends on antibody performance, other factors must also be considered and controlled in experiments. Moreover, understanding the complex interactions between different immunoglobulins is a must when developing an immunoassay. It is wrong to assume that the primary antibody will bind to its analyte and the secondary antibody to the primary antibody. Anything may go wrong in an immunoassay.

Initial assay parameters

The starting point for developing an immunoassay must always be clear. Especially the parameter to be measured by the immunoassay. This practice may sound obvious. However, understanding this fundamental significance dictates the working of an entire immunoassay.

Taking care of antibodies

Often, poor assay performance of antibodies is due to the mishandling of antibodies during bioanalysis. The most common reasons include storing at the wrong temperatures and unnecessary freeze-thaw cycles. If an antibody needs to be stored at -20℃, one must make aliquots and thaw them on ice before analysis. Besides, conjugated antibodies also need special care. For example, FITC-labeled antibodies left open will photo-bleach rapidly, while freezing HRP will denature it.

Batch-to-batch variability is another factor affecting assay results. Antibodies from separate batches are bound to have different characteristics. Hence, researchers must perform bridging studies to control lot-variability and ensure reliable results.

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Rigorous validation

Only a thoroughly validated immunoassay will generate reliable and accurate results. However, an ideal validation strategy must be able to fulfill the intended purpose of the assay. Different factors must be considered during immunoassay validation, including real study samples, cross-reacting substances, study matrices, spiking experiments, and much more. Although designing ELISA assay methods has come a long way, successful development and validation still rely on the same basic principles.