Figure 3-23. Antibody and immunogen.
monoclonal - arising from one cell line hybridoma - a fused lymphocyte and myeloma cell used for making specific antibodies clones - genetically identical cells noncompetitive immunoassay - immunoassay that does not contain reagent antigen competing with patient antigen tagged - labeled with some component that allows detection or visualization epitopes - specific antibody binding sites found on an antigen of antigens. Monoclonal antibodies are produced when mice are injected with the antigen, and antibody-producing lymphocyte cells are taken from the animals and fused with cells that grow continuously in culture to form hybridomas. A single hybridoma produces only one type of antibody by dividing to produce a large population of clones all making the same monoclonal antibody. Living hybridomas are frozen indefinitely in liquid nitrogen to produce an indefinite supply of uniform and consistent reagent antibodies of one type. Monoclonal antibodies have many advantages over polyclonal antibodies, including consistency between lots, indefinite supply, high specificity, and longer lead times, but have higher initial costs when compared with polyclonal antibodies.
Immunoassays are named by the acronym that describes the antibody-antigen response, such as ELISA (enzyme-linked immunosorbent assay). To organize the many methodologies that are used in immunoassay, assays are categorized by separation methodology, antibody-antigen reaction, or the label that is used to detect a reaction. Many formats have been used for immunoassay techniques. The choice of format is dependent upon the physical properties of the analyte, the need for quantitative versus qualitative result, the complexity of the sample matrix, sample preparation requirements, and simplicity of tests.
Type I is a single-site, noncompetitive immunoassay. In this format, an antibody of known antigenicity is tagged with label. The label is not part of the antigen-antibody reaction but is used to detect binding. Enzymes and fluorescent or chemi-luminescent molecules are common labels for immunoassay reactions. Radioactive isotopes have historical significance in immunoassay, but are no longer routinely used to detect reactions. In type I immunoassays, the antibody is mixed with patient antigen. If the antigen contains specific epitopes for the antibody, binding will occur. Unbound antibody is then washed away and the label is measured. The amount of label present is directly proportional to the amount of the antigen.
A type II immunoassay, also known as a two-site immunoassay, is also noncompetitive but uses two antibody-binding reactions. Antibodies of known antigenicity are attached to a solid phase, such as plastic wells, tubes, capillaries, membranes, latex particles, or magnetic particles. Patient antigen is then added to the test system. Patient antigen with epitopes to the known antibody will bind to the fixed antibody. A second antibody that is tagged with a label is then introduced into the test system. If the patient antigen has an epitope for this second antibody, binding will occur. Excess labeled antibody is washed away. The remaining label is measured. The amount of label is directly proportional to the amount of antigen present in the patient sample.
Type III refers to an immunoassay that is both competitive and homogeneous. Patient antigen and reagent antigen that has been labeled are introduced to the testing system, which contains antibody in limited numbers. Patient and reagent antigen compete for the limited number of binding sites on antibody that are available. Based on the law of mass action, the ratio of antibody binding of patient to reagent antigen will be proportional to their concentrations. Binding of reagent antigen inactivates the label. Only free reagent antigen label will be measured. The amount of free reagent label measured is directly proportional to the amount of patient antigen that has bound to antibody. Figure 3-24 illustrates competitive immunoassays.
Type IV is a competitive heterogeneous immunoassay. This methodology is similar to type III methodology in that patient antigen and labeled reagent antigen compete for a limited number of antibody binding sites. However, the label is not inactivated by binding. Free and bound reagent antigen must be separated before the label is measured. Label may be measured on either the free or bound reagent antigen and is related to the amount of patient antigen in the sample.
competitive immunoassay -
immunoassay in which patient antigen and labeled reagent antigen compete for the same binding site on the antibody homogeneous assay -
immunoassay in which bound and free antibody need not be separated before label is measured heterogeneous immuno-assay - immunoassay in which bound and free antibody must be separated before label is measured
An enzyme is a common label that is used to detect an immunoassay reaction. After the antibody-antigen reaction has occurred, the product is determined by measuring the enzyme label. A procedure for measuring the product of the enzyme-catalyzed reaction must follow the immunoassay reaction. In most instances, substrate for the enzyme-activated reaction is added to the test system and the product of the reaction is measured by spectrophotometry. Examples of enzyme-labeled immunoassay include enzyme-linked (ELISA) and enzyme-multiplied immunoassays.
ELISAs are commonly used immunoassay techniques. An ELISA is a type II procedure. An antibody with known antigenicity is attached to a solid phase (usually a fiberglass membrane or multiwell plate). Patient antigen is then added to the test system. Antigen that is specific to the antibody will bind to the antibody. A second enzyme-labeled antibody that is specific to another site on the antigen is then added into the test system. Antigen that contains epitopes for both antibodies will be "sandwiched" between the antibodies. Free enzyme-labeled antibody is washed away. Substrate for the enzyme-catalyzed reaction is added and the product of the reaction is measured. The amount of enzyme activity is directly proportional to the amount of antigen present in the patient sample. Alkaline phosphatase and horseradish peroxidase enzymes are commonly used enzyme labels. Both enzymes catalyze reactions that produce colored compounds that can be measured on a spectrophotometer.
Reagent Antigen with label dFree Reagent antigen label is active
Patient antigen o
Bound Reagent antigen: label is inactive o
More patient antigen means less bound reagent antigen label ane less activity
More patient antigen means less bound reagent antigen label ane less activity
An enzyme-multiplied immunoassay technique (EMIT) is a type III procedure. Patient antigen and enzyme-labeled reagent antigen are mixed with a limited amount of antibody. Patient and reagent antigen compete for the limited number of binding sites that are available. Binding of reagent antigen inactivates the enzyme. Substrate is added to the test system, but only free reagent antigen will catalyze the enzyme reaction. Because more patient antigen will allow more enzyme-labeled reagent antigen to remain free and active, greater enzyme activity is an indication of increased patient antigen in the sample. Glucose-6-phosphate dehydrogenase (G6PD) is commonly used as enzyme label in EMIT procedures. When the antigen binds G6PD-labeled antibody, the active site of the enzyme is blocked. Substrate and a coenzyme (NAD) are added to the test system. The enzyme reaction is measured as change in absorbance as NAD is consumed.
Cloned enzyme donor immunoassays (CEDIAs) use a genetically altered enzyme as label. The enzyme exists in two sections; one section is bound as label to reagent antigen and the other section is introduced into the test system after antigen-antibody binding. The enzyme is active only when the two sections are intact. When reagent antigen is bound to antibody, the second section of the enzyme cannot bind to the first section and the enzyme remains inactive. The immunoassay procedure follows the type III format. Patient antigen and enzyme fragment-labeled reagent antigen compete for a limited number of binding sites on the antibody. The second section of the enzyme is added to the test system, and enzyme fragment-labeled reagent antigen binds to antibody, which results in inactivated enzyme. Increased amounts of patient antigen result in increased free reagent antigen and increased enzyme activity.
Microparticle-capture enzyme immunoassay (MEIA) employs a modified type II immunoassay format. The test cuvet contains antibody that is bound to latex microparticles. Patient antigen is allowed to bind to the antibody. A second antibody that is tagged with enzyme label is then added to the test system. Free labeled antibody is washed away and substrate is added to the test mixture. Enzyme activity is measured as an indication of the amount of antigen that is present in the patient sample. Alkaline phosphatase is commonly used as label, with 4-methylum-belliferyl phosphate as substrate. The fluorescent product of the reaction, methy-lumbelliferone, is measured.
The measurement of fluorescent compounds increases the sensitivity of the measurement of the label reaction. Methods that measure fluorescence must be carefully controlled for false-positive reactions from contaminating substances and false-negative reactions that can occur from fluorescence quenching. A fluorescence polarization immunoassay (FPIA) uses a type III immunoassay format with a fluorophore as label. Patient antigen and fluorescent-labeled reagent antigen compete for a limited number of antibody binding sites. Spectrophotometry, which uses polarized light, is used to excite the fluorophore, and the polarization of the emitted light is then measured. Small, unbound fluorophore-labeled reagent antigen will rotate quickly, emitting unpolarized light. Larger, bound fluorophore-—olarized li ht li ht which- labeled reagent will rotate more slowly, emitting polarized light. Increased patient antigen will result in greater amounts of unpolarized light. vibrates in one plane D D r d
Chemiluminescence is produced by compounds such as luminol and acridinium esters that have the ability to produce light energy by chemical reaction. The light may be seen as a flash or a glow. Immunoassay systems that measure chemi-luminescence reactions often employ a type IV methodology. Patient antigen and chemiluminescent compound-labeled reagent antigen compete for a limited number of antibody binding sites. The label is not inactivated by binding. Upon separation of free and unbound reagent antigen, an enzyme (usually firefly luciferase) is used to produce chemiluminescence, which is measured with a lumi-nometer. See the example of the use of chemiluminescent immunoassay in Test Methodology 3-16.
Particle immunoassays assess the formation or inhibition of a precipitation or aggregation event. Aggregation is measured by nephelometry or turbidimetry. Particle-enhanced turbidimetric immunoinhibition assay (PETINA) is a type IV immunoassay formatted procedure. Patient antigen and particle-labeled reagent antigen compete for a limited number of antibody binding sites. The binding of particle-labeled antigen to antibody produces aggregation and increased turbidity in the test solution, which can be measured by photometry.
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