Labelled Immunoassays PDF

 CO Are designed for antigens and antibodies that may be small in size or present in very low concentrations. The presence of such antigens or antibodies is determined indirectly by using a labeled reactant to detect whether or not specific b...

 CO Are designed for antigens and antibodies that may be small in size or present in very low concentrations. The presence of such antigens or antibodies is determined indirectly by using a labeled reactant to detect whether or not specific binding has taken place.  CO COMPETITIVE IMMUNOASSAYS all the reactants are mixed together simultaneously; labeled antigen competes with unlabeled patient antigen for a limited number of antibody- binding sites. ,The amount of bound label is inversely proportional to the concentration of the labeled antigen, which means that the more labeled antigen that is detected, the less there is of patient antigen. CO  CO NON-COMPETITIVE IMMUNOASSAYS An antibody, often called capture antibody, is first passively absorbed to a solid phase such as microtiter plates, nitrocellulose membranes, or plastic beads. Excess antibody is present so that any patient antigen present can be captured. Unknown patient antigen is then allowed to react with and be captured by the solid-phase antibody. After washing to remove unbound antigen, a second antibody with a label is added to the reaction  CO NON-COMPETITIVE IMMUNOASSAYS In this case, the amount of label measured is directly proportional to the amount of patient antigen. This type of assay is more sensitive than competitive immunoassays. In both types of assays, the label must not alter the reactivity of the molecule and should remain stable for the reagent’s shelf life CO  CO Immunoassays can also be categorized according to whether or not it is necessary to separate the bound reactants from the free ones. Heterogeneous enzyme immunoassays require a step to physically separate free from bound analyte. Homogeneous enzyme immunoassays, on the other hand, do not need a separation step. LESS SENSITIVE  CO 1ST developed immunoassay, Pioneered by Yalow and Berson (1950’s) Useful for hormones, vitamins and serum proteins The assay uses a radioactive substance as a label. Radioactive elements have nuclei that decay spontaneously, emitting matter and energy. Several radioactive labels have been used, but 125I has been the most popular. CO  CO Enzymes are catalysts that react with substrates to produce breakdown products that may be chromogenic, fluorogenic, or luminescent. Some type of spectroscopy can then be used to measure the changes involved. As labels for immunoassay, enzymes are cheap and readily available, have a long shelf life, are easily adapted to automation, and cause changes that can be measured using inexpensive equipment. Most commonly used: Horseradish Peroxidase, Alkaline phosphatase, and β-D-galactosidase  CO HETEROGENOUS ENZYME IMMUNOASSAYS 1. COMPETITIVE EIA The first enzyme immunoassays were competitive assays based on the principles of RIA. Enzyme-labeled antigen competes with unlabeled patient antigen for a limited number of binding sites on antibody molecules that are attached to a solid phase. This method is typically used for measuring small antigens that are relatively pure, such as drugs and hormones  CO HETEROGENOUS ENZYME IMMUNOASSAYS 2. NON-COMPETITIVE EIA Most noncompetitive assays are indirect immunoassays, or so-called indirect enzyme-linked immunosorbent assays (ELISA), because the enzyme-labeled reagent does not participate in the initial antigen– antibody binding reaction. The enzyme-labeled reagent only binds after the initial antigen–antibody reaction has taken place. This type of assay is one of the most frequently used immunoassays in the clinical laboratory because of its sensitivity, specificity, simplicity, and low cost.  CO HETEROGENOUS ENZYME IMMUNOASSAYS 2. NON-COMPETITIVE EIA Antigen is typically bound to solid phase. A variety of solid-phase supports are used, including microtiter plates, nitrocellulose membranes, and magnetic latex or plastic beads When antigen is bound to solid phase, patient serum with unknown antibody is added and given time to react. After a wash step, an enzyme-labeled antiglobulin, or secondary antibody, is added.  CO HETEROGENOUS ENZYME IMMUNOASSAYS 2. NON-COMPETITIVE EIA This second antibody reacts with any patient antibody that is bound to solid phase. If no patient antibody is bound to the solid phase, the second labeled antibody will not be bound. After a second wash step, the enzyme substrate is added The amount of color, fluorescence, or luminescence detected is directly proportional to the amount of antibody in the specimen HIV, HEPATITIS B AND C SCREENING  CO HETEROGENOUS ENZYME IMMUNOASSAYS 3. CAPTURE ASSAYS / SANDWICH ASSAY If antibody, rather than antigen, is bound to the solid phase, these assays are often called sandwich immunoassays or capture assays. Antigens captured in these assays must have multiple epitopes. Excess antibody attached to solid phase is allowed to combine with the test sample to capture any antigen present. After an appropriate incubation period, enzyme labeled antibody is added. This second antibody recognizes a different epitope or binding site than the solid-phase antibody and completes the “sandwich.”  CO HETEROGENOUS ENZYME IMMUNOASSAYS 3. CAPTURE ASSAYS / SANDWICH ASSAY Enzymatic activity is directly proportional to the amount of antigen in the test sample. Capture assays are best suited to antigens that have multiple determinants, such as antibodies, cytokines, proteins, tumor markers, and microorganisms, especially viruses.  CO HOMOGENOUS ENZYME IMMUNOASSAYS Homogeneous enzyme immunoassays are generally less sensitive than heterogeneous assays, but they are rapid, simple to perform, and adapt easily to automation. No washing or separation steps are necessary. Their chief use has been in the determination of low-molecular-weight analytes such as hormones, therapeutic drugs, and drugs of abuse in both serum and urine CO  CO Rapid immunoassays are membrane based, easy to perform, and give reproducible results. Although designed primarily for point-of-care or home testing, many of these have been modified for increased sensitivity and can be made semiquantitative for use in a clinical laboratory. Another type of rapid assay, called immunochromatography, combines all the previously mentioned steps into one. The analyte is applied at one end of the strip and migrates toward the distal end where there is an absorbent pad to maintain a constant capillary flow rate  CO The labeling and detection zones are set between the two ends. As the sample is loaded, it reconstitutes the labeled antigen or antibody and the two form a complex that migrates toward the detection zone. An antigen or antibody immobilized in the detection zone captures the immune complex and forms a colored line for a positive test, which may be in the form of a plus sign CO  CO In 1941, Albert Coons demonstrated that antibodies could be labeled with molecules that fluoresce. These fluorescent compounds, called fluorophores or fluorochromes, can absorb energy from an incident light source and convert that energy into light of a longer wavelength and lower energy as the excited electrons return to the ground state. The two compounds most often used are fluorescein and rhodamine, usually in the form of isothiocyanates, because these can be readily coupled with antigen or antibody.  CO In 1941, Albert Coons demonstrated that antibodies could be labeled with molecules that fluoresce. These fluorescent compounds, called fluorophores or fluorochromes, can absorb energy from an incident light source and convert that energy into light of a longer wavelength and lower energy as the excited electrons return to the ground state. The two compounds most often used are fluorescein and rhodamine, usually in the form of isothiocyanates, because these can be readily coupled with antigen or antibody.  CO Fluorescein absorbs maximally at 490 to 495 nm and emits a green color at 520 nm. It has a high intensity, good photostability, and a high quantum yield. Tetramethylrhodamine absorbs at 550 nm and emits red light at 585 nm  CO IMMUNOFLUORESCENT ASSAY - Fluorescent tags or labels on antibodies were first used for localization of antigen in cells or tissue when bound to antigen in the tissue, the fluorescent probe attached to the antibody is detected under ultraviolet light using a fluorescent microscope. 2 CATEGORIES: 1. DIRECT IMMUNOFLUORESCENCE 2. INDIRECT IMMUNOFLUORESCENCE  CO DIRECT IMMUNOFLUORESCENCE In a direct immunofluorescent assay, antibody that is conjugated with a fluorescent tag is added directly to unknown antigen that is fixed to a microscope slide. After incubation and a wash step, the slide is read using a fluorescence microscope. Antigens are typically visualized as bright apple green or orange-yellow objects against a dark background. Examples of antigens detected by the direct method include Legionella pneumophila and Chlamydia trachomatis.  CO INDIRECT IMMUNOFLUORESCENCE Indirect immunofluorescent assays, which are more commonly used than direct assays, involve two steps. In the first step, patient serum is incubated with a known antigen attached to a solid phase. The slide is then washed and an anti-human immunoglobulin containing a fluorescent tag is added. This immunoglobulin combines with the first antibody to form a sandwich, which localizes the fluorescence. CO  CO FLUORESCENCE POLARIZATION IMMUNOASSAY One of the most popular techniques developed is fluorescence polarization immunoassay (FPIA), which is based on the change in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody. In FPIA, labeled antigens compete with unlabeled antigen in the patient sample for a limited number of antibody-binding sites. The more antigen that is present in the patient sample, the less the fluorescence-labeled antigen is bound and the less the polarization that will be detected. Hence, the degree of fluorescence polarization is inversely proportional to concentration of the analyte  CO FLUORESCENCE POLARIZATION IMMUNOASSAY One of the most popular techniques developed is fluorescence polarization immunoassay (FPIA), which is based on the change in polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody. In FPIA, labeled antigens compete with unlabeled antigen in the patient sample for a limited number of antibody-binding sites.  CO FLUORESCENCE POLARIZATION IMMUNOASSAY Homogeneous FIA, just like the corresponding enzyme immunoassay, requires no separation procedure, so it is rapid and simple to perform. There is only one incubation step and no wash step, competitive binding is usually involved. The more antigen that is present in the patient sample, the less the fluorescence-labeled antigen is bound and the less the polarization that will be detected. Hence, the degree of fluorescence polarization is inversely proportional to concentration of the analyte CO  CO Chemiluminescent immunoassay is another technique employed to visualize antigen–antibody combination. Chemiluminescence is the emission of light caused by a chemical reaction, typically, an oxidation reaction, producing an excited molecule that decays back to its original ground state. A large number of molecules are capable of chemiluminescence, but some of the most common substances used are luminol, acridinium esters, ruthenium derivatives, and nitrophenyl oxalates CO Any questions? Don't hesitate to ask for help