Immunology and Serology Lab - 03 Labeled Immunoassays

o IMMUNOLOGY AND SEROLOGY LABORATORY LABELED IMMUNOASSAYS OUTLINE • • • • • • • • Introduction Labeled Immunoassays Constituents of Labeled Assays Radioimmunoassay Techniques Enzyme Immunoassay Fluorescent Immunoassay Chemiluminescent Immunoassay Summary • • LABELED IMMUNOASSAYS INTRODUCTION • • While precipitation and agglutination reactions are techniques that detect secondary phenomenon, labeled immunoassays are serologic techniques that detect primary phenomenon. These techniques are relatively more expensive and are composed of more complex procedures but are generally more sensitive and specific as they are less subjective due to the use of indicators. Used when there is a need for rapid, specific, sensitive assays (Since agglutination and precipitation are laborious and takes a lot of time to yield results). LABELED IMMUNOASSAYS • • • • • If there is no antigen in the sample then there will be a high level of radiation since the radiolabeled antigen can bind to all antibody sites. o If antigen is present in the patient sample then radioactivity will be decreased proportionally to the amount of antigen present. o Standards are run to create a standard curve. o Controls are run to ensure proper technique and reagent reactivity. RAST and RIST – RIA tests o Employed for studying allergic reactions. o RAST – Used to identify which allergen is causing the allergy by detecting the specific antibody (IgE) that binds to it. o RIST – Used to quantitate the amount of IgE generated from the allergic reaction • Some antigen/antibody reactions not detected by precipitation or agglutination. Measured indirectly using a labeled reactant (ie. Enzyme labeled antibody). Referred to as receptor-ligand assays. Ligand is the substance to be measured and is defined as a molecule that binds to another molecule of a complementary configuration, usually it binds to the substance the test is trying to detect. The receptor is what binds the specific target molecule (ie. Paratope/Fc, epitope). RIA Result o Results: ▪ Non-reactive = High radioactivity ▪ Reactive = Low radioactivity o Summary: Relationship is inversely proportional CONSTITUENTS OF LABELED ASSAYS • • Labels which may be used include: fluorescent, radioactive, chemiluminescent and enzymes. The assay includes the use of: o Labeled and non-labeled ligands/reactant o Specific antibody o Standards and calibrators o Means of separating bound from free components o Means of label detection. ENZYME IMMUNOASSAY (EIA) • RADIOIMMUNOASSAY (RIA) TECHNIQUES • • Competitive Binding Assays o Uses radioactive substance as a label, usually I125 (Iodine 125). o Antibody is bound to the walls of the tube or well. o A measured amount of patient sample is added to a measured amount of radiolabeled analyte or ligand. o The antigen in the patient sample and the radiolabeled antigen compete for the binding to the antibody. Advantages of enzyme immunoassay: o labels cheap and plentiful. o labels have a long shelf life o easily adapted to automation o reaction measured using inexpensive equipment o very sensitive o no health hazards associated with reagents o can be used for qualitative or quantitative procedures. Enzymes chosen for us as labels according to the following: o number of substrate molecules converted per molecule of enzyme o purity o sensitivity o ease and speed of detection o stability o absence of interfering substances o availability o cost • • • Typical enzymes used include: o Horseradish peroxidase - cheap, very popular, reacts with a number of chromogens o Glucose oxidase o Glucose-6-phosphate dehydrogenase - uses fluorimetric means o Alkaline phosphatase - expensive o β-D-galactosidase Source Method for enzyme labelling Peroxidase Horseradish Periodate oxidation (Nakane method) B-galactosidase E.coli Dimaleimide method Alkaline Bovine Glutaraldehyde method Phosphatase intestine The enzyme label is linked to antibody or ligand. Two classification of enzyme assays: o Heterogenous requires a step to physically separate bound ligand from free. (washing) o Homogeneous assays require no separation step. (no washing) o o o ADVANTAGES & DISADVANTAGES OF ENZYME IMMUNOASSAY • HETEROGENOUS ENZYME IMMUNOASSAYS • • • Competitive Enzyme Linked Immunosorbent Assays (ELISA) o Enzyme labeled ligand competes with unlabeled patient ligand for binding sites on antibody molecules attached to solid phase. (WELL, WALLS OF A TUBE) o After reacting, a washing step is performed. o Enzyme activity is determined. (ELL-UPL- inverse) o Enzyme activity is inversely proportional the concentration of the test ligand. o Sensitivity in nanograms (10 g/mL) can be achieved. o (Same principle as with the competitive RIA but instead of radioisotopes, we use enzymes as labels) Noncompetitive ELISA o Referred to as indirect ELISA test. o Antigen is bound to solid phase, unlabeled patient antibody is added. o After incubation a wash step is performed and an enzyme labeled substrate is added. o The amount of enzyme label detected is directly proportional to the amount of antibody in the specimen. o Indirect ELISAs are very sensitive. o Disadvantage is there is more manipulation of test. Sandwich or Capture Assays (SANDWICH ELISA) o Used with antigens having multiple epitopes. o Excess antibody attached to solid phase, allowed to combine with test sample. o After incubation, enzyme labeled antibody is added. o Second antibody may recognize same or different epitope than solid phase antibody. o Enzyme activity directly related to concentration of antigen. o Detects antigens present in low concentrations. o Suited to antigens with multiple epitopes. o Epitope must be unique to the organism and present in all strains. o Major use of this technique is for measurement of immunoglobulins, the immunoglobulin and the antigen and an anti-antibody is added to the test system. Disadvantage: linear relationship does not exist, must prepare standard curve, test conditions must be carefully controlled. Results: ▪ Reactive: Change in color ▪ Non-reactive: No change in color Sandwich ELISA • Advantages of Enzyme Immunoassay o Sensitivity similar to RIA w/o health hazards (radiation) and reagent disposal problems. (radioactive isotopes) o Expensive equipment not necessary, spectrophotometers or notation of color change. o Most are fairly simple Disadvantages o Some samples may have natural inhibitors. (w/c will inhibit ab-ag reaction) o Size of enzyme label limiting factor in designing some assays. o Nonspecific protein binding may occur. (some proteins might have an affinity towards the ab) o Enzyme reactions very sensitive to temperature. FLUORESCENT IMMUNOASSAY (FIA) • • • • • Use fluorescent compounds called fluorophores or fluorochromes as markers. Markers have the ability to absorb energy and convert it into light. Fluorescent probe must exhibit high intensity to distinguish from background. Two compounds most frequently used are fluorescein and rhodamine. o Fluorescein isothiocyanate emits green color, it has high intensity and good photostablility. (absorbs 490495nm and emits green color at 517 to 520nm) o Tetramethylrhodamine emits red light. (absorbs at 550nm and emits red light at 580 to 585nm) o Newer compounds include algae, porphyrins and chlorophylls. (plants) Fluorescent tags first used for localization of antigen in tissues. o Immunofluorescent assay (IFA) is qualitative observation of fluorescent reaction using fluorescent scope for visualization. o Can detect antigens in fixed cells or live cell suspensions. CHEMILUMINESCENT IMMUNOASSAYS • • FLUORESCENT MICROSCOPY • • • Uses light source that emits light of the appropriate wavelength necessary to excite fluorochrome used. The presence of a specific antigen is determined by the appearance of localized color against a black background. Fluorescent staining o Direct immunofluorescent assay the antibody has a fluorescent tags, added directly to unknown antigen fixed to slide, after incubation and washing observe for fluorescence. o Indirect immunofluorescent assay reacts with patient serum with known antigen attached to solid phase, wash, add anti-human antibody attached to flourescent tag, wash, observe for fluorescence. • • • • • It 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. Most common substances used are: luminol, acridium esters and dioxetane phosphate. The substance is oxidized, usually with hydrogen peroxide and an enzyme for a catalyst. Energy given off in the form of light. Heterogeneous and homogenous assays available. Disadvantages: o False results may be obtained if lack of precision in addition of hydrogen peroxide. o Some biologic materials cause quenching. o Needs dedicated instrumentation. Advantages: o reactive stability of label o speed of detection o sensitivity comparable to RIA and EIA o low cost HETEROGENEOUS FLUORESCENT IMMUNOASSAYS • • • • • • • • Fluorescent immunoassays (FIA) are classified as heterogeneous or homogeneous. Heterogeneous assays require a separation step and include: o indirect o competitive o sandwich o capture Same principle as EIA except fluorescent label is used. *not enzymes Can detect either antigen or antibody. Solid phase uses antigen or antibody attached to beads which may be magnetized. When beads are utilized the reaction occurs, the tube centrifuged, supernatant discarded, and the beads (sediment) observed for fluorescence. Magnetized beads are placed on a magnetic surface to attract beads to the bottom of the tube. Solid phase can also employ a dipstick coated with antigen or antibody, react with patient sample then add a labeled antibody. SUMMARY • • • • • ADVANTAGES & DISADVANTAGES OF FLUORESCENT TECHNIQUES • • Advantages: o Methods fairly simple. o No hazardous reagents to use or discard. o Increased sensitivity over radiolabeled and enzyme reactions. Disadvantages include: o Fluorescent compounds are sensitive to environmental changes. o Stability of labels o Nonspecific binding can diminish signal. (there will be no light) o Bilirubin (photosensitivity) or hemoglobin can absorb the excitation or emission energy. o Requires expensive dedicated instrumentation • • • Labeled assays are used to measure antigen or antibody. Different labels can be used. Two classification of enzyme assays: o Heterogenous requires a step to physically separate bound ligand from free. o Homogeneous assays require no separation step. Immunoassay labels must be very specific and have high affinity. (prevent false change in reaction) Radioactive labels o RIA is COMPETITIVE - known antibody added in specific amount to unknown sample, competes with patient antibody to attach. Indirect measurement. Enzymes can be used as labels, results in a color reaction. (e-z REACTION) o Most assays today are non-competitive. o Sandwich technique very popular o Homogeneous enzyme assays - no separation necessary, antibody binding causes antigen labeled with enzyme to lose activity. Fluorescence o Direct immunofluorescence detects antigen directly by using labeled antibody. o Indirect immunofluorescence utilizes sandwich technique.