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Immunoassay Basics
Understanding the basic principles of immunoassays is easy. The essential components of antibody-based immunoassay systems are threefold: an antigen that we would like to detect and perhaps quantitate; a specific antibody to this antigen; and a system to measure the amount of antigen in a given sample. Although it appears to be a very simple system, in many cases a number of other assay materials are necessary to allow for quick and convenient measurement. Not all assays are antibody-based. Some assays utilize simple enzymatic reactions to detect small molecules or perhaps measure the activity of an enzyme sample. The most common immunoassay systems are outlined below.
Competitive Immunoassays
In competitive enzyme immunoassays (EIA) the antigen in a sample competes for limited antibody binding sites with antigen conjugated to a reporter enzyme. This produces an inverse relationship between antigen concentration and substrate turnover. Competitive EIAs typically use a single antibody to a small molecular weight antigen, generally less than 10,000 Daltons. During incubation, samples with high antigen content result in unlabeled antigen being bound in greater amounts than conjugated antigen. When chromogenic substrate is added to the assay to develop color, samples with high antigen concentration generate a lower signal than those containing low antigen concentration, yielding the inverse correlation between antigen concentration in the sample and color development in the assay. This relationship can then be used to extrapolate antigen concentration in an unknown sample from a standard curve. This type of reaction is one of the few methods possible for small molecular weight antigens, such as steroids, drugs, lipids and peptides.
The typical format for one of our competitive EIA Kits is shown above. We use alkaline phosphatase (AP) as the reporter enzyme because of its stability, turnover number and lack of interferences. The antibody-bound conjugate is captured using a secondary antibody that specifically binds to the primary antibody coated onto a microtiter plate.
Immunometric Assays
Immunometric assays, also known as sandwich assays, use two antibodies specific to the antigen to capture or “sandwich” antigen in the well for detection. Immunometric assays exhibit a direct correlation between antigen concentration and substrate response. Immunometric assays typically employ a “capture” antibody coated on the plate to bind the antigen of interest. During a second incubation, the antigen is bound by a second “detection” antibody that is also specific to the antigen. The detection antibody can either be bound by a secondary antibody-enzyme conjugate, or the detection antibody itself is enzyme-conjugated. When chromogenic substrate is added to the assay to develop color, samples with high antigen concentration generate more signal than those with low antigen concentration, producing a signal directly proportional to the amount of antigen in the sample. This correlation can then be used to extrapolate the concentration of antigen in an unknown sample from a standard curve.
Indirect Immunoassays
The enzyme-linked immunosorbent assay (ELISA), also known as an indirect immunoassay, is a third type of immunoassay commonly used to detect the presence of antibodies to specific antigenic sites on viruses. These assays have been used extensively in HIV and hepatitis testing. This “true” ELISA format uses a solid phase coated with antigen. Samples are then applied to the solid phase. Any antibodies in the sample will bind to the antigen. A second antibody, labeled with an enzyme, is added to the reaction. This antibody is specific to IgG or IgM and binds to the sample antibody, which is bound to the target antigen. The presence of enzyme is detected by addition of a colorimetric substrate.
Fluorescence Polarization Immunoassays
Fluorescence polarization immunoassays (FPIA) are quick and efficient homogeneous mix-and-read assays. The principle of the FPIA is similar to that of the competitive EIA. Both assays work on the principle of small molecule competitive binding where quantification is based on antibody equilibrium binding to free antigen and conjugate. As the amount of antigen increases, the amount of conjugate bound to antibody decreases leading to a detection response that is inversely proportional to antigen concentration.
In Enzo Life Sciences' FPIA kits, the conjugate is covalently attached to a fluorescence tag, the aromatic molecule fluorescein. All binding events in an FPIA take place in free motion whereas the EIA utilizes solid phase binding. The units used to quantify FPIA are the milli-polarization unit (mP). When excited by polarized light the conjugate molecule will emit light of a lower energy state. Molecules are in motion which results in the non-specific directional emission of light. The faster the molecule tumbles, the more depolarized the light. Therefore conjugate bound to antibody will tumble more slowly due to its size which will yield a greater amount of polarized light and a larger mP. This is how bound conjugate molecules are differentiated from unbound conjugate which has been displaced by analyte in the standard or sample.
Non-Antibody Based Assays
Activity Assays
Activity assays do not use antibodies as detection reagents. The kits measure the activity of enzymes by measuring the amount of a specific substrate that is turned-over by the enzyme being analyzed. Many activity assays have homogeneous mix-and-read formats that do not require wash steps. They are often adaptable to multiple assay formats including kinetic, end-point and inhibition assays.
Detection Assays
As with activity assays, detection assays do not use antibodies as detection reagents. Very small molecular weight analytes require relatively simple biochemical techniques to measure their concentrations. Assay Designs Nitric Oxide and Hydrogen Peroxide assays are examples of detection kits for very small molecular weight antigens. In these kits, chemically driven color changes are used to measure the concentration of these molecules in samples. Detection assays also have simple homogeneous mix-and-read format.
Acridinium Ester
We supplie an Acridinium Ester Labeling Kit for use with antibodies, proteins, peptides and nucleic acids. Acridinium ester-labeled samples can be used for a number of different applications. They can be added as tracers to samples containing unlabeled materials as an alternative to the use of 125I labeled molecules. They can also be used as labeled antigens for competitive immunoassays or as labeled signal antibodies for immunometric assays. In addition, chemiluminescent-labeled nucleic acids may be used as reporter molecules in homogeneous DNA probe-based systems.
