An immunoassay is a biochemical test that measures the presence or concentration of a specific substance, known as an analyte, in a biological sample. These tests are founded on the specific binding between an antibody and its corresponding antigen, an interaction often compared to a lock and key. The term “wash-based” distinguishes tests that use a physical rinsing step to separate bound and unbound molecules. This ensures that only the specific antibody-antigen interactions are measured.
The Heterogeneous Assay Principle
Wash-based immunoassays are also called heterogeneous assays, highlighting the required separation of bound and free components. The process begins with immobilization, where an antibody or antigen is attached to a solid support like a microplate well, membrane, or bead. This step anchors one of the reagents for interaction with the sample.
Once the surface is prepared, the biological sample—such as blood, serum, or urine—is introduced. The target analyte within the sample, if present, binds to the immobilized antibody or antigen. This binding event is the primary recognition step of the assay.
The surface is subjected to one or more wash steps using a buffer solution. This washing procedure physically removes all components from the sample that did not specifically bind to the immobilized molecules. This includes unbound analytes and other interfering substances, and the removal of this background noise allows for a sensitive and specific measurement.
After the wash, a detection antibody is added. This antibody is engineered to recognize the captured analyte and carries a label that can generate a measurable signal. In many formats, this creates a “sandwich” with the analyte positioned between the capture and detection antibodies. The amount of labeled antibody that remains is proportional to the amount of analyte in the original sample.
Key Formats and Variations
The principle of immobilizing a reagent, binding a target, and washing is applied across several formats. The most prevalent is the Enzyme-Linked Immunosorbent Assay, or ELISA. Performed in 96-well microplates, ELISAs are common in diagnostics and research, allowing for simultaneous analysis of multiple samples. Variations include the direct ELISA and the sandwich ELISA, which uses matched antibody pairs to capture and detect the antigen.
Another application is the Western Blot, a technique used to identify a specific protein from a complex mixture like a cell extract. It first involves separating proteins in the sample by size using gel electrophoresis. The separated proteins are then transferred to a solid membrane, which serves as the support for antibody binding, washing, and signal detection.
The principle extends to Immunohistochemistry (IHC), where the immunoassay is performed on a thinly sliced section of biological tissue. An antibody specific to a protein of interest is applied to the tissue. After binding and washing, a labeled secondary antibody generates a signal, allowing researchers to visualize the location of specific proteins within the tissue’s structure.
Signal Generation and Quantification
Once the final wash step is complete, the invisible molecular interactions must be translated into a measurable signal. The method of signal generation is determined by the type of label attached to the detection antibody.
The most common approach is colorimetric detection, where the antibody is linked to an enzyme, such as horseradish peroxidase. When a specific chemical substrate is added, the enzyme catalyzes a reaction that produces a visible color change, and the intensity of this color can be measured with an instrument called a spectrophotometer.
A different method relies on fluorescence. In this format, the detection antibody is conjugated to a fluorophore, a molecule that absorbs light at one wavelength and emits it at a longer one. A specialized instrument excites the fluorophore and measures the emitted light, and the intensity of the fluorescent signal corresponds to the quantity of the analyte captured in the assay.
A third strategy is chemiluminescence, which offers high sensitivity. The antibody is linked to an enzyme that triggers a chemical reaction producing light as a byproduct. Unlike fluorescence, this light is generated without an external light source for excitation. The emitted photons are captured by a luminometer, providing a measurement that can detect low concentrations of the analyte.
Diagnostic and Research Applications
The specificity and sensitivity of wash-based immunoassays make them useful across many fields. In medical diagnostics, they are the basis for numerous tests. Pregnancy tests, for example, detect the hormone human chorionic gonadotropin (hCG) in urine. They are also used for screening blood donations for infectious diseases like HIV and hepatitis, allergy testing, and measuring hormone levels.
In pharmaceutical research and development, these assays are used to screen potential drug candidates and understand how new therapies affect protein levels. Researchers can measure the concentration of biomarkers in response to treatment, providing information on a drug’s efficacy and mechanism of action.
In the food industry, immunoassays are employed to detect common allergens, such as peanuts or gluten, in processed foods. They are also used to identify harmful bacterial toxins or other contaminants, helping to prevent foodborne illness. This testing allows manufacturers to monitor their products and ensure they meet safety standards.
Environmental science also benefits from this technology. Wash-based immunoassays provide a method for detecting and quantifying pollutants, such as pesticides or industrial chemicals, in soil and water samples. Their ability to detect low concentrations makes them a tool for monitoring environmental contamination.