How Immunoassays Work
What is an immunoassay?
Immunoassays are based on the principles that specific antigens will stimulate very specific (unique) immune responses and that the proteins produced by the immune response, called antibodies, can be used to signal the presence of a target compound in a sample.
There are two types of immunoassays: sandwich and competitive.
Sandwich assays use two antibodies to bind a specific target. One antibody is typically immobilized to a solid support such as nitrocellulose (as is the case for lateral flow test strips) or a microtiter plate (as is the case for ELISA tests). The antibody attached to the solid support specifically binds a large substance (known as an antigen) and then another antibody (which is soluble or suspended in solution) binds the other side of the antigen. The soluble antibody is attached to a reporter system (e.g., a gold particle for a lateral flow test or an enzyme for an ELISA test) which is used to visualize the binding event. Gold particles create a red colored line at the zone of detection on a test strip whereas the enzyme catalyzes a reaction that turns the solution a different color in the microtiter plate. In a sandwich assay, the amount signal produced (i.e., line intensity or optical density on a strip or in a plate, respectively) that is produced is directly proportional to the amount of antibody sandwich formation that occurs. Thus, by adding known amounts of a specific sample to a detection system and measuring the amount of signal produced, a standard curve can be produced. When a sample containing an unknown amount of analyte is tested, the result can be compared to the standard curve and the amount of analyte in the unknown sample can be quantitated. The QuickScan system performs quantitation for EnviroLogix test strips. A plate reader performs quantitation of ELISA tests.
Competitive assays use only one antibody to detect the presence of a specific antigen. Competitive assays are designed for analytes that are too small to be sandwiched. In competitive assays, analyte in the sample competes with analyte attached to a solid support (test strip) or enzyme (ELISA) for the antibody binding site. In competitive assays, the darker the line (test strip) or greater the optical density (ELISA) the lesser the concentration of analyte in the sample.
What is a Lateral Flow Device?
Lateral flow devices, also known as test strips or LFDs, are immunoassays that work by applying a fluid sample to one end of the strip (called the sample pad) and allowing the sample to flow to the other end of the strip (called the absorbent pad). During flow, either a sandwich assay (GMO detection) or competitive assay (mycotoxin detection) occurs. The strip components are engineered with various pads containing buffer salts, antibodies, or competitive analytes which bind or compete to bind with the analyte in the sample as the fluid migrates from the sample pad to the absorbent pad. Certain areas of the strip are functionalized to provide zones of detection. These zones are known as the test line or the control line. The control line is always the last line that the fluid encounters and is an indicator that the assay is working properly. Thus, if no control line appears, the test result is considered invalid. For GMO detection, sandwich assays are used, which means a very dark test line indicates a high concentration of GMO protein while no visible test line indicates the absence of GMO protein in the sample. For mycotoxin detection, a competitive assay is used, and therefore if no test line is present than the sample contains a high concentration of the specific mycotoxin the test was designed for. EnviroLogix develops and manufactures test strips for both qualitative and quantitative analyte detection. Qualitative results are interpreted by eye and provide a yes or no test result. Quantitative results are obtained using the QuickScan system.
Interpretation of results with QuickStix LFDs
If no “control line”, the test has not run properly and should be repeated. As with the plate and tube formats, certain tests are “competitive assays” and others are so-called “sandwich assays”. For example, EnviroLogix’ pesticide assays, whether plate, tube or QuickStix, are all competitive assays, while the Bt endotoxin assays are all sandwich assays.
In competitive LFD assays, a positive result is indicated by the absence of a line in the test result zone.
In sandwich LFD assays, a positive result is indicated by the presence of a line in the test result zone.
Interpreting ELISA Immunoassays
EnviroLogix plate and tube kits are supplied with Calibrators (known concentrations of the target analyte in solution) and a negative Control (known to be free of the target analyte) for both visual and instrumented interpretation of the test results. These Standards (Calibrators and Control) exhibit distinctly different shades of blue color at the different concentrations provided (for example: zero, 0.5 ppb (parts per billion), 1 ppb, 5 ppb, 10 ppb). By comparing the color of the sample against the Standards, the person testing may visually determine the concentration range of the sample, for example, “between 1 and 5 ppb”. This interpretation is semi-quantitative.
Quantitative interpretation can be performed by inserting the microwells in a “plate reader” which precisely measures the optical density of all samples and all Standards at the same time. Using software provided with the reader the user then calculates the sample concentration from the Standard.
For field use, a “strip reader” or “mini-photometer” can be used to process one eight-well or twelve-well strip at a time. This type of device is much more portable than a plate reader, and less costly, but it does require more manual manipulation.
Plate and Tube Format Assays
Antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) methods have been widely used in medical applications since the early-70’s. Countless studies have demonstrated the correlation between ELISA results and traditional methodologies such as gas chromatography (GC) and high performance liquid chromatography (HPLC).
Most EnviroLogix 96-well format (Plate) kits and tube kits (including all the pesticide ELISA assays) are competitive assays. As with all competitive immunoassays, sample concentration is inversely proportional to color development:
Darker color = Lower concentration of the suspected analyte
Lighter color = Higher concentration of the suspected analyte
In these tests, pesticide residues in the sample compete with known amounts of pesticide analogue that has been enzyme labeled (typically with horseradish peroxidase) for a limited number of antibody binding sites on the inside surface of the test wells.
After a simple wash step, the outcome of the competition is visualized with a color development step.
Note: Certain non-pesticide tests use a different format for displaying immune response called a “sandwich” assay. In such assays, darker color means higher concentration of the analyte. Be careful to read the product insert that accompanies each test to be certain of the proper interpretation.