A sandwich immunoassay is a laboratory method used to detect and measure a specific molecule within a liquid sample. Its name comes from its structure, where the target molecule is held between two specific antibodies, similar to filling in a sandwich. This technique is valued for its ability to precisely identify and quantify substances like proteins, hormones, and other biomarkers.
Core Principles and Components
A sandwich immunoassay has three main components. The first is the analyte, or antigen, which is the specific molecule the test aims to detect. This could be a protein indicating a disease, a hormone, or a viral component. The test’s ability to find this substance in a complex sample like blood or urine makes it a valuable diagnostic tool.
The second component is the capture antibody. This antibody is immobilized onto a solid surface, such as a microtiter plate well. Its function is to capture the target analyte from the sample fluid, isolating the molecule of interest from the rest of the sample’s contents.
The final element is the detection antibody. After the analyte is captured, this second antibody is introduced to bind to a different location, or epitope, on the same analyte. The detection antibody is linked to a reporter, such as an enzyme, that generates a measurable signal. Using two distinct antibodies for separate sites on the target molecule is a defining feature of the sandwich assay and provides its high specificity.
The Sandwich Immunoassay Procedure
The procedure for a sandwich immunoassay follows a sequence of timed steps. First, a solid surface, like a multi-well plate, is coated with a layer of capture antibodies. These antibodies adhere to the surface, preparing it to bind with the target analyte from the sample.
After coating, the surface is treated with a blocking buffer, such as bovine serum albumin (BSA). This step prevents non-specific binding, where other molecules in the sample might stick to the plate. By occupying available binding sites, the blocking agent reduces background noise and improves accuracy.
Once the plate is prepared, the test sample is added to the wells. During an incubation period, any analyte present binds to the immobilized capture antibodies. The plate is then washed to remove unbound materials, leaving only the capture antibodies and the bound analyte. This washing step is repeated to purify the components at each stage.
Next, detection antibodies linked to an enzyme are added to the wells. These antibodies bind to the captured analyte, completing the “sandwich” structure. A second washing step removes unbound detection antibodies, and a chemical substrate is introduced. The substrate reacts with the enzyme to produce a visible signal, and its intensity is proportional to the amount of analyte in the sample.
Applications in Diagnostics and Research
The precision of sandwich immunoassays makes them suitable for many applications, particularly in medical diagnostics. Common over-the-counter tests, like home pregnancy tests that detect human chorionic gonadotropin (hCG), use this technology. It is also used in rapid tests for infectious diseases like HIV and Strep A, and for measuring biomarkers that indicate cardiac conditions or cancer.
Beyond clinical settings, these assays help ensure food safety. They are employed to detect allergens, such as peanut or gluten proteins, in food products. The technology is also used to identify contaminants like bacterial toxins.
In environmental science, sandwich immunoassays are used to monitor for pollutants and toxins in water and soil samples. This allows for the rapid detection of harmful substances that could impact ecosystems and human health. Quantifying these substances helps assess environmental quality.
The pharmaceutical industry relies on sandwich immunoassays during drug development and for quality control. These tests can measure the concentration of protein-based drugs or monitor the response to a therapeutic agent by measuring specific biomarkers.
Assay Sensitivity and Specificity
The performance of a sandwich immunoassay is defined by its sensitivity and specificity. Sensitivity is the test’s ability to detect very low concentrations of the target analyte. The signal amplification from the enzymatic reaction allows these assays to detect minute quantities of a substance, making them highly sensitive.
Specificity is the test’s ability to bind exclusively to the intended target molecule without being cross-reactive with similar molecules. The high specificity of the sandwich immunoassay results from using two different antibodies that recognize distinct binding sites on the analyte. This dual-recognition makes it highly unlikely the assay will mistakenly identify a non-target molecule.
A limitation of this method is the “hook effect,” which can occur at extremely high analyte concentrations. In such cases, excess analyte can saturate both the capture and detection antibodies simultaneously, preventing the formation of the sandwich complex. This leads to a weaker signal and an underestimation of the analyte concentration.