Immunologic Techniques (Part 2)

IMMUNOLOGIC TECHNIQUES (PART 2) Prepared by: jmaRMT Labeled Immunoassay ▪ Labeled immunoassays 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. Characteristics of Labeled Assay ▪ Techniques include the use of enzymes, radioactive substances, Fluorochromes, and chemicals as labels. ▪ Competitive versus non-competitive immunoassay ▪ Heterogenous versus Homogenous assays ▪ Currently, most immunoassays use a solid-phase vehicle for separation such as polystyrene test tubes, microtiter plates, glass or polystyrene beads, magnetic beads, plastic beads, and cellulose membranes. ▪ The bound and unbound fractions are usually separated by physical means, including decanting, centrifugation, or filtration. This is followed by a washing step to remove any remaining unbound analyte. Competitive versus non-competitive immunoassay ▪ In a competitive immunoassay, all the reactants are mixed simultaneously; labeled antigen competes with unlabeled patient antigen for a limited number of antibody-binding sites. ▪ The activity of the labeled immunoassay is INVERSELY PROPORTIONAL to the concentration of the unknown. ▪ In a typical noncompetitive immunoassay, an antibody, often called a capture antibody, is first passively absorbed into 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. An 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. ▪ The activity of the labeled immunoassay is DIRECTLY PROPORTIONAL to the concentration of the unknown. Heterogenous versus Homogenous assays Homogenous An immunoassay in which no separation step is Immunoassay necessary. It is based on the principle of a decrease in enzyme activity when a specific antigen-antibody combination occurs. They are less sensitive than heterogeneous assays Heterogenous An immunoassay in which enzyme is used as a label Immunoassay and which requires a separation step such as washing to separate free from bound analyte. DIFFERENT TYPES OF LABELED IMMUNOASSAY ▪ RADIOIMMUNOASSAY (RIA) ▪ ENZYME IMMUNOASSAY (EIA) ▪ FLUORESCENT IMMUNOASSAY ▪ CHEMILUMINESCENCE IMMUNOASSAY ▪ IMMUNOCHROMATOGRAPHIC ASSAY / RAPID IMMUNOASSAY RADIOIMMUNOASSAY (RIA) RADIOIMMUNOASSAY (RIA) ▪ Developed by Rosalyn Yallow ▪ Uses a radioactive substance as a label such as 131I; 125I (most commonly used, 60days halflife); & tritiated hydrogen (3H) ▪ Radioactivity is measured by a Scintillation Counter (Gamma Counter) ▪ The Chief disadvantage are the health hazard involved in working with radioactive substances, disposal problems, short shelf life, and the need for expensive equipment ▪ APPLICATION: ▪ RIST / RADIOIMMUNOSORBENT TEST = measure Total IgE ▪ RAST / RADIOALLERGOSORBENT TEST = measure ALLERGEN-SPECIFIC IgE COMPETITIVE RIA ▪ Radiolabeled antigen competes with unlabeled patient antigen for a limited number of antibody-binding sites. ▪ Radioactivity is inversely proportional to the antigen concentration. NON-COMPETITIVE RIA ▪ Analyte measured is sandwiched between two antibodies. ▪ Radioactivity is directly proportional to the analyte concentration. Coated to beads / wells Radiolabeled component Relationship of Radioactivity of Label to Unk. Ag Conc. Competitive RIA Solid-phase Antibody Antigen Reagent Increase Radioactivity = Decrease Antigen Concentration Inversely Proportional Noncompetitive RIA Antibody Reagent Increase Radioactivity = Increase Antigen Concentration Directly Proportional Solid-phase Antibody ENZYME IMMUNOASSAY ENZYME IMMUNOASSAY ▪ Label: ENZYME ▪ Alkaline phosphatase and horseradish peroxidase have the highest turnover (conversion of substrate) rates, high sensitivity, and are easy to detect, so they are most often used in such assays. ▪ Change in absorbance due to enzymatic activity is measured using SPECTROPHOTOMETER. HETEROGENOUS ENZYME IMMUNOASSAY 1. Competitive EIA ▪ 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. ▪ Enzyme activity is inversely proportional to the concentration of the test substance. HETEROGENOUS ENZYME IMMUNOASSAY 2. Noncompetitive / Indirect EIA ▪ Indirect because enzyme-labeled secondary antibody does not participate in the initial antigen-antibody binding. ▪ Enzyme activity is DIRECTLY proportional to the analyte concentration. HETEROGENOUS ENZYME IMMUNOASSAY 3. Capture Assay/Sandwich EIA ▪ Antigen captured must have multiple epitopes ▪ Enzyme activity is DIRECTLY proportional to antigen concentration ▪ Prone to Hook Effect. (increase analyte / Ag excess → result is lower than the actual measurement.) HOMOGENEOUS ENZYME IMMUNOASSAY 4. Enzyme Multiplied Immunoassay Technique (EMIT) ▪ HOMOGENEOUS ASSAY ▪ Antigen is labeled with an enzyme tag. ▪ When the antibody binds to specific determinant sites on the antigen, the active site on the enzyme is blocked, resulting to loss of enzyme activity. FLUORESCENT IMMUNOASSAY FLUORESCENT IMMUNOASSAY ▪ Label: fluorophores or fluorochromes ▪ TWO most commonly used fluorophores or fluorochromes: ▪ Fluorescein isothiocyanate (FITC)- Emits an apple green color at 520nm ▪ Tetramethyl rhodamine isothiocyanate (TRITC)- emits a red color at 585nm ▪ Other fluorochromes are phycobiliprotein, europium (􀇃-naphthyl trifluoroacetone), and lucifer yellow VS ▪ 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. ▪ One of the major disadvantages of fluorescent immunoassay is subjected to QUENCHING. GRADING FOR FITC FLUORESCENCE INTENSITY 0 No apple green fluorescence. 1+ Faint yet unequivocal apple-green fluorescence 2+ Apple green fluorescence 3+ Bright Apple green fluorescence 4+ Brilliant Apple green fluorescence 1. DIRECT IMMUNOFLUORESCENT ASSAY (DFA) ▪ 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 2. INDIRECT IMMUNOFLUORECENT ASSAY (IFA) ▪ Involves TWO STEPS: ▪ First is incubation of patient serum with a known antigen attached to a solid phase ▪ Second the slide is washed, and then an antihuman immunoglobulin containing a fluorescent tag is added. ▪ It is the principle employed in FANA testing, and FTA-ABS test 3. INHIBITION IMMUNOFLUORESCENT ASSAY ▪ Is a blocking test in which antigen is first exposed to an unlabeled antibody, then to a labeled antibody, and is finally washed and examined. ▪ If the unlabeled and labeled antibodies are both homologous to the antigen, there should be no fluorescence. 4. FLUORESCENT POLARIZATION IMMUNOASSAY (FPIA) ▪ A type of homogenous immunoassay ▪ An immunoassay 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 degree of fluorescence polarization is inversely proportional to concentration of the analyte. ▪ Increase polarization/fluorescence = No patient antigen is present ▪ Decrease polarization/fluorescence = Patient antigen is present FIA DETECTS REAGENT POSITIVE RESULT DIRECT IF Unknown Ag Known Ab + FITC FLUORESCENCE INDIRECT IF Unknown Ab 1. Known Ag 2. AHG + FITC FLUORESCENCE INHIBITION IF Unknown Ab 1. Known Ag 2. Homologous Ab + FITC. NO FLUORESCENCE CHEMILUMINESCENCE IMMUNOASSAY CHEMILUMINESCENCE IMMUNOASSAY ▪ 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. ▪ Chemiluminescence substances are LUMINOL, ACRIDINIUM ESTERS, RUTHENIUM DERIVATIVES, & NITROPHENYL OXALATES. ▪ A newer technique called electrochemiluminescence immunoassay uses electrochemical compounds that generate light when an oxidation-reduction reaction occurs RAPID IMMUNOASSAYS / IMMUNOCHROMATOGRAPHIC ASSAY RAPID IMMUNOASSAYS / IMMUNOCHROMATOGRAPHIC ASSAY • Rapid immunoassays are membrane based, easy to perform, and give reproducible results • Designed primarily for point-of-care or home testing • Typically, these are designed as single-use, disposable assays in a plastic cartridge. The membrane is usually made of microporous nylon, which can easily immobilize proteins • Patient sample is added to a test strip and migrates through the strip. • Labeled antigen or antibody binds and is captured by a second reagent in • the detection zone. • If test is positive, a line or plus sign will form on the test strip where patient antigen or antibody is captured END!