What is an ELISA (enzyme-linked immunosorbent assay)?
This plate-based assay is used to detect and quantify soluble macro-molecules “the target antigen” such as peptides, proteins, antibodies, and hormones. Antibodies specific to the target antigen are immobilized on a microplate surface which is complexed with an antibody connected to a reporter enzyme. Detection of the target antigen is made by assessment of reporter enzyme activity during incubation. The target antigen binds to the immobilized antibody linked to an appropriate substrate to produce a product to measure the amount of target antigen bound to the plate.
The concentration of target antigen is determined by signal interference as the target antigen and labeled reference sample compete to bind to the immobilized antibody.
This method is commonly used to detect the concentration of an unknown target antigen in a sample. Plates are coated with an immobilized antibody and a blocking buffer is applied to reduce non-specific binding. The sample containing the target antigen is applied to the plate and binds to the immobilized antibodies. Once the plate is washed, a detection antibody solution is added which binds a different epitope on the target antigen to the immobilized antibody. A chromogenic, chemiluminescent, or fluorescent detection substrate is added to measure the amount of target antigen present in the sample relative to the amount of pigment, light, or fluorescent signal produced from the detection substrate.
A compact version of the Elisa Kits
Clia (Chemiluminescence) Kit
A combination of Chemiluminescence and immunohistochemical reaction techniques, CLIA techniques utilize light-emitting chemical probes to measure the chemical reaction between target antigens and kit antibodies.
The range of target antigen the assay is able to detect.
This is the lowest value of the target antigen in the assay buffer that the assay can statistically differentiate from background “noise”.
With a known amount of target antigen added (spiked), the Elisa is run to assess the recovery of the target antigen compared to an identical spiked standard diluent.
The closeness of agreement between independent test results obtained under stipulated conditions.
- Intra-assay precision: Reproducibility between wells within an assay plate to ensure samples running in different wells give comparable results.
- Inter-assay precision: Reproducibility between assays performed on separate occasions. This is typically less than <10% to ensure consistent results over time and between different kits.
Using serial dilutions of a known amount of target antigen (spiked), the precision of results over a range of dilutions is assessed to ensure the assay works with different concentrations of the target antigen.