ELISA (Enzyme-Linked Immunosorbent Assay) – Ultra Sensitive Detector of Proteins
ELISA is a quantitative and ultra-sensitive plate-based assay designed to detect and quantify substances such as antibodies, hormones, proteins (e.g. cytokines) and peptides, receptors on cells as well as small molecule drugs. The ELISA has been a workhorse method since the 1970s.
The method has a sensitivity/detection level of approaching 1pg/ml for a particular protein, even in a complex matrix such as serum. Specificity and sensitivity are determined by the antibodies being used. Polyclonal antibodies have a high affinity which adds to increased sensitivity.
Example of a very sensitive Gamma Interferon ELISA with sensitivity to <1.3pg/mL. This method was used to measure the secondary immune response, recall response, of human blood lymphocytes immune to cytomegalovirus (CMV) by challenge with CMV peptide in vitro. Interferon (IFN) gamma is one of the cytokines produced during an immune response.
The basic design of an ELISA is to immobilize the antigen or a capture antibody on the plate surface as in a 96 well format. Non-specific sites on the plate are blocked with a protein, such as bovine serum albumin. For the capture antibody format, the antigen is incubated and is captured by the immobilized antibody. The next step includes binding a specific primary antibody directed to the analyte. This primary antibody may be enzyme-linked, and if not, then a secondary enzyme-linked antibody is used to bind the primary antibody. Subsequent development of the substrate will result in a measurable signal. Development of the ELISA includes choice of binding reagents, coat method, assay diluent to reduce nonspecific matrix effects, and readout detection methods. The method is perfect for all types of body fluids, tissue homogenates, etc.
Several applications can be used:
– Direct or competitive measure
– Sandwich method
– ELISpot, which detects a signal (e.g. cytokine) from a single cell
– Membrane-bound proteins
– Intracellular antigens in adherent cells
Some of the numerous ELISAs we perform at Marin Biologic are:
– RIA (RadioImmune Assay)
– Small molecule detection with a competitive ELISA
– Multiplex (measuring many analytes within the same well)
– Phosphorylation of proteins
– Cell-based ELISAs and many more variations.
At Marin Biologic Laboratories, there is an option of running your valuable samples using our Ella platform which may produce results with larger signal ranges and better sensitivity over other methods. Ella utilizes a cartridge, so each immunoassay is run the same way every time, eliminating manual steps, washes or reagent additions. Ella is also volume-independent.
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