ELISA method is a plate-based technique used for detecting and quantifying analytes such as proteins, antibodies, hormones, and peptides in complex biological matrices. The primary principle of ELISA assays is based on antigen-antibody interactions. The target analyte is immobilized on the microplate surface. This analyte is then complexed with a conjugated antibody that generates a measurable signal in the presence of its substrate.
ELISA assays comprise several segments. ELISA services and ELISA lab heavily focus on individual components as they are crucial for analytical method transfer in pharma projects. The current article discusses various assay components that are a specialty of robust ELISA testing services.
Generally, ELISA assay plates are 96-well plates made from polyvinyl chloride or polystyrene. Today there are different plate variants with additional wells. However, a higher number of wells will require automated handling to reduce pipetting errors. Besides, opaque plates are needed for chemiluminescent or fluorescent signals.
Ideally, researchers must use assay plates designed for ELISA analysis. This initiative reduces edge effects and provides optimal data. Moreover, ELISA assays for diagnostic, commercial, and quality control applications must test plates from different manufacturers to assess plate-to-plate and batch-to-batch variability.
There are three assay buffers used primarily in the ELISA method. They include coating, washing, and blocking buffers.
Coating buffers are the first step of an ELISA assay. Antigens and antibodies are diluted in coating buffers and then adsorbed to assay well surfaces. Both the concentrations of the coating agent and the pH of the coating buffer control the hydrophobic interactions between the antigen and the plastic plate surface.
Blocking buffers is crucial to prevent non-specific binding. Detergents and proteins are the two primary blocking buffers. Proteins are permanent blockers. Hence, they are added after the capture antibody. On the other hand, detergents are temporary blockers and may disappear after the washing step.
Finally, washing buffers remove unbound materials in between each step. They are vital in reducing unwanted signals. Usually, PBS with non-ionic detergent is used as a washing buffer.
Researchers use different types of antibodies in ELISA assays. These antibodies include monoclonal, polyclonal, or both these antibodies combined. Each antibody has distinct advantages. Hence, one must choose an antibody based on individual assay needs.
Moreover, antibody selection also depends on antigen-antibody interactions. These interactions can be described as specificity, avidity, and affinity. All these factors determine the level of assay optimization during ELISA development.
Monoclonal antibodies are homogeneous antibodies specific to a small region or a single epitope. Hence, they don’t generally interact with other proteins to generate non-specific signals. On the other hand, polyclonal antibodies are complex. They have specificity to various epitopes of a single antigen. Therefore, polyclonal antibodies must undergo tests and validation to reduce batch-to-batch variability.
A combination of monoclonal and polyclonal antibodies is used widely in sandwich ELISA formats. This combination works such that each antibody will act as a capture and detection antibody against a single antigen. However, these combinations can consist of two polyclonal antibodies, two monoclonal antibodies, or a blend of both.