“ELISA…is still the most used platform in assay development”
The ELISA (enzyme-linked immunosorbent assay) is a popular method for protein detection and quantification. This type of biochemistry assay uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. Performing an ELISA involves at least one antibody with specificity for a particular antigen. ELISAs are simple to carry out and they are designed to rapidly handle a large number of samples in parallel. The ELISA still is a very popular choice for the evaluation of various research and diagnostic targets.
ELISAs can be quite complex and there are many things to consider with the development of an ELISA. The first is what format to use; direct, indirect, sandwich, Competition or Inhibition ELISA. Formats differ in how the target antigen is captured and detected.
Direct and indirect ELISA both immobilize the antigen on the assay plate and then use either a labeled primary antibody (direct) or primary antibody and labeled secondary antibody (indirect) to detect the antigen. This type of ELISA has two advantages: It is faster, since fewer steps are required and it is less prone to error, since there are fewer steps and reagents.
Sandwich ELISA is considered the most robust format because the antigen is “sandwiched” between two primary antibodies. The first antibody (capture) is coated to the polystyrene plate. Next, the analyte or sample solution is added to the well. A second antibody layer (detection) follows this step in order to measure the concentration of the analyte.
Future Diagnostics…the Life hack for ELISA development
The development of an ELISA involves choosing a format, gathering the right components, and constructing a good working protocol.
ELISA optimization takes systematically adjusting and testing all the components and variables to ensure results are robust and accurate.
The ELISA formats provide different levels of specificity, sensitivity, simplicity, and speed. They also require different numbers and varieties of components. When we develop an ELISA from scratch, it is often because the target of interest is “new” to research, in which case a known pair of antibodies may not be available. Searching and matching for the right antibodies may be required.
When was the last time you looked at optimizing:
… the Concentration of Capture Antibody
… the Blocking Buffer
… the Sample Concentration
… the Concentration of Detection Antibody
… the Concentration of Enzyme Conjugate
… Signal Detection
… the Standard Diluent
It is important to make the right choices when you start on Assay Development. Our experience helps you in making the right choices so you save time, labor and money.