5 Real-World Tips to Optimize an ELISA Development and Validation Experiment

No clinical and life science research can be conducted without using ELISA assays. ELISA assays employ antigen-antibody reactions to capture and detect analytes in biological samples. With the ongoing Covid-19 pandemic, antigen testing has become a common term in regular conversations. Only experts understand how delicate the development and validation of ELISA assays can be.

Today we highlight five real-world tips to optimize ELISA development and validation experiments.

Assay Design

Designing a robust ELISA assay depends on the availability and properties of the target antigen. It also depends on the sensitivity and specificity requirements, availability of antibodies, and properties of the sample matrices. It is commonly misunderstood that ELISA assays are typically the same formats where any target antigen can be coated with.

One target analyte may not hold the same assay condition, and even a minor modification in buffer type can influence the ELISA signal. A rigorous ELISA assay validation is necessary for its utility in routine clinical applications.

Focusing On the Washing Step

Background noise is often the reason for low ELISA output. Background noise is a result of inadequate blocking and washing of ELISA plates. The washing step is particularly crucial as thorough washing of wells ensures the removal of the wash buffer. A traditional ELISA assay consists of several washing steps.

Using advanced solutions such as SPARCL and ELISAONE technology help reduce the majority of wash steps. More advanced options such as bead-based assays eliminate the wash step, reducing human errors.

Understanding Antibody Kinetics

Antibodies are always considered as an issue during the failure of an immunoassay. Several other factors play a critical part in the working of an ELISA assay. Robust antibodies are crucial for the success of ELISA development and validation. Understanding the interplay between all assay components is necessary for the success of immunoassays. These components include the assessment of controls and other elements that are necessary during ELISA method development.

Protecting Antibodies for Optimal Performance

Poor assay performance is often blamed on antibodies used in ELISA assays. In many cases, poor performance is due to improper handling of antibodies.

The two most common reasons for poor assay performance are storage at the wrong temperature and repeated freezing and thawing cycles. Researchers can aliquot samples and thaw them on ice just before use to avoid repeated freeze-thaw cycles. Batch-to-batch inconsistencies are another reason for poor assay performance. In this situation, researchers can perform bridging studies to control lot-to-lot variation, which saves valuable time and resources.

Rigorous ELISA Assay Validation

An adequate ELISA assay validation entrusts confidence in the generated results. An assay validation strategy should consider all aspects of the intended use in a wide range of study samples. Assay cross-reactivity, different matrices, actual study samples, and spiking experiments are some parameters that need to be assessed in MSD ELISA assay validation.

For achieving an ideal binding reaction, acquiring the right assay components and tackling limitations as early as possible will be crucial factors in ELISA development and validation.