Sandwich ELISA
Definition
An ELISA format in which a capture antibody immobilised on the plate binds one epitope of the target antigen, and a separate detection antibody binds a different epitope on the same antigen. The antigen is held between two antibodies, giving rise to the name. This format is highly specific and is widely used for detecting large antigens with multiple epitopes.
- Format
- Capture antibody plus separate detection antibody
- Requires
- Antigen with at least two distinct epitopes
- Specificity
- High, due to dual antibody binding
- Forensic use
- Detecting large proteins such as haemoglobin
- Sub-field
- Forensic serology, immunoassay methods
Common questions
Why can't sandwich ELISA be used for very small target molecules?+
Small molecules often present only one accessible epitope, leaving no room for a capture and a detection antibody to bind simultaneously without steric interference, so sandwich format is generally reserved for larger multi-epitope antigens.
How does sandwich ELISA reduce false positives compared to a single-antibody format?+
A signal only appears when both the capture and detection antibodies bind the same antigen molecule, so a substance that cross-reacts with just one of the two antibodies will not produce a positive result, adding a built-in specificity check.
What forensic body fluid or protein targets commonly use this format?+
Sandwich ELISA formats are used for antigens such as human haemoglobin in blood identification, where the larger, multi-epitope structure of the target protein suits the two-antibody approach.
Related terms
- Competitive ELISA
- An ELISA format in which sample antigen competes with a fixed quantity of labelled antigen for a limited number of antibody binding...
- Cross-Reactivity
- The capacity of an antibody raised against one analyte to bind structurally related compounds. In RIA, cross-reactivity is the main driver of...
- Cross-Reactivity Matrix
- A table documenting the assay's optical density response to blood from multiple non-target species, expressed as a percentage of the human blood...
- Enzyme Conjugate
- An antibody (or antigen) chemically linked to a reporter enzyme, most commonly horseradish peroxidase (HRP) or alkaline phosphatase (AP). The enzyme catalyses...
- Horseradish Peroxidase (HRP)
- The most common enzyme label in immunoassays. When the substrate TMB (3,3'-5,5'-tetramethylbenzidine) is added, HRP converts it to a blue product that...
- Limit of Detection (LOD)
- The lowest concentration of analyte that produces a signal reliably distinguishable from the instrument noise, conventionally three times the standard deviation of...
- Optical Density (OD)
- The absorbance reading from a microplate reader that measures how much light is absorbed by the coloured product. Higher OD means more...
- Solid Phase
- The inert surface to which an antigen or antibody is adsorbed at the start of an ELISA. Usually the well walls of...
- Standard Curve
- A series of known antigen concentrations run on the same ELISA plate, used to convert the OD reading for an unknown sample...
- Substrate
- The compound converted by the reporter enzyme into a coloured product. For HRP, 3,3',5,5'-tetramethylbenzidine (TMB) is the standard substrate, producing a blue...
Explained in these topics
- Anti-Human Haemoglobin ELISA and Modern ImmunoassaysA two-antibody ELISA format in which a capture antibody immobilised on a microplate well binds the target antigen from the sample, and a second detection antib...
- ELISA: Principles, Formats, and Forensic Applications