Module Two Labeled Test Methods CLS 404 Immunology Immunologic Methods Module Two Labeled Test Methods

Objectives Describe the part of a labeled assay List materials used to label analytes Explain the principle of each test method presented and give an example of a clinical application for each test Differentiate the following types of assays: Competitive vs. non-competitive Homogeneous vs. heterogeneous Direct vs. indirect

“Labeled” Methods Attaches a “tag” to either the antigen or antibody. Antigen and antibody are allowed to react. The degree of the reaction can be determined by detecting and measuring the “tagged” material. Analyte is usually a small molecule or present in small quantities. Tests are fast, sensitive, and specific.

Parts of a Labeled Assay Analyte Specific antibody Separation of bound and free components Detection of label Standards/Calibrators

Analyte The substance being measured Unlabeled in patient specimen As a reagent, is often labeled Common analytes include: Bacterial and viral antigens Hormones Drugs Tumor markers Immunoglobulins

Specific antibody High affinity for antigen Important for test sensitivity Specific to the antigen in the reaction No cross-reactivity Often a monoclonal antibody is used

Separation of bound and free components Many tests include a means of removing any unbound antigen and/or antibody. Heterogeneous: Method that requires a step that separates bound analyte from unbound analyte. Homogeneous: Method that does not require a separation step.

Separation Methods Washing to remove unbound antigen/antibody Adsorption of unbound reactants onto talc, charcoal or cellulose followed by centrifugation or filtration to remove adsorbed materials Chemical modification of the test medium that results in the precipitation of antigen-antibody complexes, leaving unbound analyte in solution

Separation Methods Solid-phase reaction surfaces which bind labeled analyte, including: Microtiter plates Magnetic beads Cellulose membranes Spherical surface preferred, as spheres have a larger surface area

Labels and Their Detection Radioactivity Gamma counter detects gamma radiation as radioactive isotope “tag” decays Fluorescence Fluorochromes are excited by light of a specific wavelength Emit light of a different wavelength Emitted fluorescent light is detected microscopically

Fluorochromes Fluorescein – absorbs blue light, emits yellow-green Rhodamine – absorbs yellow-green light, emits deep red Since these emit light at different wavelengths, they may be combined in one assay to detect different antigens on the same cell Phycoerythrin – emits bright red light

Labels and Their Detection Enzymes Break down a colorless chromogenic substrate Color development is measured using a spectrophotometer Common enzymes include: horseradish peroxidase alkaline phosphatase glucose – 6- phosphate dehydrogenase (fluorometric)

Labels and Their Detection Chemiluminescent materials The “tag” molecule is excited to a state of high energy (oxidation) As the excited molecule returns to its original lower energy state, light is emitted May be a short burst of light May be a longer lasting glow Common “tag” materials include: Luminol Acridium esters Peroxyoxalates

Standards/calibrators Known concentrations of analyte, used to determine relationship between labeled analyte and unlabeled analyte in specimen. Typically used to produce a curve from which analyte concentration in patient’s specimen can be determined.

Standard Curve Figure 6-9b Kuby Immunology , 6th ed ©2007, WH Freeman & Co. Used with permission

Specific Labeled Assays

Competitive ELISA Enzyme labeled antigen competes with unlabeled patient antigen for binding sites on fixed antibodies. A chromogen is added that reacts with the enzyme. The level of color development is inversely proportional to the level of patient antigen. Used to measure small molecules such as insulin or estrogen.

Radioimmunoassay (RIA) Competitive binding Uses a radioactive tag rather than an enzyme 125I common tag This method has been used for measuring hormone levels, IgE levels and other small trace analytes Has been replaced in most laboratories by ELISA

Noncompetitive (Indirect) ELISA Antigen is bound to a solid phase. Patient’s serum is added. If antibodies to the antigen are present in the serum, they react with the antigen bound to the solid phase The patient’s antibody is free to bind as much antigen as possible, since the labeled antibody is not present in the test system at this point A wash step removes unbound patient antibody. Y Y Y Y Y Y

Noncompetitive (Indirect) ELISA Y An anti-human globulin (AHG) antibody that has been enzyme labeled is added. This antibody will react with the patient’s antibody, if present. A second wash removes unbound AHG reagent. The enzyme substrate is added. The degree of color development is directly proportional to the amount of patient antibody in the specimen. Y Y Y Y Y Y Y Y Y Anti-Human Globulin (AHG) is antibody to human antibodies. The Fab portion of AHG is directed at the Fc portion of the human immunoglobulin. Y

Noncompetitive (Indirect) ELISA More sensitive than competitive methods. Used to diagnose viral infections and to detect autoantibodies.

Capture (Sandwich) ELISA Patient’s sample is incubated with bound antibody. Following a wash to remove unbound antigen, a second chromogen-labeled antibody is added. Second antibody is also specific for the antigen (analyte) The substrate is added. The level of color development corresponds with the amount of antigen “captured”. Multiple uses include detecting antibodies, hormones, viruses, proteins and tumor markers.

Enzyme-multiplied Immunoassay Technique (EMIT) This is a homogeneous competitive binding assay. When antibody binds to the labeled antigen, it blocks enzymatic activity, reducing the amount of color development. The more patient antigen that binds to the antibody, the more enzyme-tagged antigen remains free to react with the chromogen. Color development is _______proportional to the concentration of antigen. Directly Commonly used to test for drugs.

Chemiluminescence This labeling technique can be applied to heterogeneous or homogeneous assays. Used to detect antigens or antibodies. Requires sophisticated instrumentation that is specific to the chemical being used. Clinical applications include detection of drugs and hormones.

Direct Immunofluorescence Negative test Positive test Fluorescently labeled antibody is used to detect antigen in tissue fixed to a slide.

Quick Question Which paraprotein disorder uses direct immunofluorescence to detect light chain deposits in tissue? Amyloidosis This test has been used for detection of Chlamydia, Legionella, RSV, and other antigens.

Indirect Immunofluorescence Negative Test Positive Test Known antigen fixed to slide Patient’s serum added (unknown antibody) Incubation & wash Fluorescently labeled anti-human globulin added. Anti-Human Globulin (AHG) is antibody to human antibodies. The Fab portion of AHG is directed at the Fc portion of the human immunoglobulin. Clinical applications of indirect fluorescence include detection of viral, treponemal, and antinuclear antibodies.

Please proceed to Module Three, Antibody Titers. The End Please proceed to Module Three, Antibody Titers.