ELISA is the most common technique used for detecting and quantifying analytes in biological samples. Generally, ELISA captures specific antigens on a solid surface and then complex them with antibodies linked to an enzyme. The enzyme conjugated with a substrate produces a signal corresponding to the amount of analytes present in the study sample. There are numerous ELISA assays used in clinical and pharmaceutical research. These assays include in vitro/in vivo assays, pharmacodynamic (PD), and pharmacokinetic (PK) ELISA assays.
ELISA assay development consists of choosing an ideal assay format, accumulating all assay components, and developing a working protocol. ELISA validation involves testing and modulating several assay components and parameters to generate robust and reliable results. In this current article, we share the five crucial requirements for ELISA development.
Assay format
Deciding on an appropriate ELISA format is the first consideration for an ELISA assay development. Direct, indirect, and sandwich ELISA are the most common ELISA formats. These formats differ in the strategy used for capturing and detecting target antigens in study samples. Direct and indirect ELISA formats captures an antigen on the ELISA plates. Direct ELISA uses a primary antibody to detect the antigen, whereas indirect ELISA employs labeled secondary antibodies for the same. Sandwich ELISA is the most widely used assay format. It technically sandwiches an antigen between two primary antibodies and then introduces a secondary labeled antibody for detection.
Coating of ELISA plates
The next step in ELISA development is determining and optimizing the immobilization strategy. Although Polyvinyl and Polystyrene surfaces are predominantly used as the solid phase, several other options are available for the solid phase material. These surfaces include nitrocellulose, nylon, and bead-based surfaces. The accuracy of an ELISA assay depends on its immobilization strategy. However, efforts are needed to ensure the complete biological activity of the protein of interest.
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Antigen and antibody
Antigen and antibody determine the specificity and sensitivity of an ELISA assay. Pure and stable antigens can influence the performance of an ELISA assay. Highly pure antigens increase assay sensitivity by enhancing the capturing capability of ELISA antibodies. The antibody pairs that do not interfere with each other and can bind the antigen simultaneously are the most suitable antibody pairs for sandwich ELISAs. The antibody pairs that do not interfere and bind the antigen simultaneously are the most suitable antibody pairs for sandwich ELISAs.
Conjugate strategy
Enzyme conjugated antibodies are a vital component in elisa assay development service. Conjugation of enzymes and antibodies requires the formation of stable covalent bonds between the enzyme and the antigen-specific antibody. The crucial aspect of conjugation strategy is that it should not functionally alter both the active site of an antibody and enzyme. Moreover, alkaline phosphatase and horseradish peroxidase are the two most commonly attached enzymes in ELISA assays.
Enzyme and chromogen systems
Detection is the final step of an ELISA method. The reliability of ELISA assays depends on the accurate detection of analytes in biological matrices. As briefly mentioned in this article, detecting analytes is associated with the signal generated during the enzyme-substrate reaction. The intensity of these generated signals is directly proportional to the amount of analyte captured and bound to the ELISA plate. Understanding the detection system can help researchers troubleshoot and cover common assay errors associated with ELISA assays.
