Labeled Immunoassays PDF - Chapter 11

6/27/2024 CHAPTER 11 LABELED IMMUNOASSAYS PREAMBLE PowerPoints are a general overview and are provided to help students take notes over the video lect...

6/27/2024 CHAPTER 11 LABELED IMMUNOASSAYS PREAMBLE PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/27/2024 CHAPTER OVERVIEW Labeled immunoassays Heterogeneous vs. homogenous assays Competitive vs. noncompetitive immunoassays Radioimmunoassay Enzyme immunoassays (EIAs) Interferences in EIAs Chemiluminescent assays Fluorescent immunoassays Rapid immunoassays LABELED IMMUNOASSAYS Are designed for antigens and antibodies that may be small in size or present in very low concentrations Use a reactant labeled with a detection molecule to monitor the amount of specific binding that has taken place Detect analytes such as microbial antigens, hormones, drugs, tumor markers, and specific immunoglobulins Animation of EIA (Enzyme Immunoassays) https://www.youtube.com/watch?v=70TPrfL_8-M 2  6/27/2024 IMMUNOASSAY LABELS A variety of labels can be used for Immunoassays Enzyme/colorimetric substrate Chemiluminescent molecule/trigger solution Fluorescent compound (fluorophore) Radioactive isotope (older methods) Heterogeneous immunoassays Involve physical separation of bound and free components Most commonly involve binding to a solid phase (polystyrene reaction HETEROGENEOUS wells, microparticle beads, latex VS. beads, plastic tubes) HOMOGENOUS Magnetic separation or IMMUNOASSAYS centrifugation may be used. Homogenous immunoassays Do not require a physical separation step 3  6/27/2024 COMPETITIVE IMMUNOASSAYS All the reactants are mixed together simultaneously. Labeled and unlabeled antigen compete for a limited number of binding sites on reagent antibody. The amount of bound label is inversely proportional to the concentration of the labeled antigen. Highly specific assays measure small antigens that are relatively pure (e.g., drugs and hormones). Have high specificity COMPETITIVE IMMUNOASSAY PRINCIPLE 4  6/27/2024 NONCOMPETITIVE IMMUNOASSAYS Also known as capture, sandwich, or immunometric assays Patient antigen is captured by antibody bound to a solid phase. After washing to remove unbound antigen, a second antibody with a label is added to the reaction. The amount of label is directly proportional to the amount of antigen in the patient sample. NONCOMPETITIVE IMMUNOASSAY PRINCIPLE 5  6/27/2024 RADIOIMMUNOASSAY (RIA) The first immunoassay developed Uses radioactive labels – 125 I is most popular. Emits gamma radiation, which is detected by a gamma counter. Is extremely sensitive and precise. Measures trace amounts of analytes (such as hormones, serum proteins, and drugs) that are small in size. RADIOIMMUNOASSAY (RIA) (CONTINUED) Competitive assay—the amount of label in the bound phase is Disadvantages: indirectly proportional to the amount of patient antigen present. Working with radioactive Disposal of low-level Short shelf life of some Therefore, testing in substances (health radioactive waste reagents clinical labs is limited. hazard) 6  6/27/2024 ENZYME IMMUNOASSAYS (EIAS) Highly sensitive assays that use enzymes as labels, which react with suitable substrates to produce breakdown products that may be chromogenic, fluorogenic, or luminescent Are available in competitive and noncompetitive formats Commonly used enzymes include Alkaline phosphatase, Horseradish peroxidase, Glucose-6-phosphate dehydrogenase (G6PDH), and β-D-galactosidase. Alkaline phosphatase and horseradish peroxidase Have the highest turnover (conversion of substrate) rates Have high sensitivity and are easy to detect HETEROGENEOUS EIAS Require a step to physically separate free analyte from bound analyte 1. Competitive assays Involve enzyme-labeled antigen competing with unlabeled patient antigen for binding sites on antibody molecules Used for measuring small antigens that are relatively pure (e.g., drugs and hormones) Have high specificity 7  6/27/2024 HETEROGENEOUS EIAS 2. Noncompetitive enzyme immunoassays Offer high sensitivity and specificity, simplicity, and low cost Enzyme-linked immunosorbent assay (ELISA) Used to measure antibody production to infectious agents that are difficult to isolate and for autoantibody testing HIV Hepatitis B and hepatitis C Is easily applied to point-of-care and home testing INDIRECT ELISA Enzyme-linked immunosorbent assay (ELISA) Noncompetitive immunoassay used to detect antibody in patient sample: Antibody to infectious agents such as hepatitis B, rubella virus Autoantibodies (e.g., antinuclear antibodies, thyroglobulin antibody) 8  6/27/2024 INDIRECT ELISA PRINCIPLE Automated immunoassay commonly used because of its high sensitivity and specificity, simplicity of use, and low cost CAPTURE (SANDWICH) IMMUNOASSAYS EIA that detects antigen in patient sample Best suited for antigens that have multiple determinants (e.g., cytokines, proteins, tumor markers, microbial antigens) Can also be used to detect immunoglobulins of a particular class (e.g., IgM for acute infections) 9  6/27/2024 Antigen in the test sample binds to antibody attached to a solid phase. After incubation, enzyme-labeled CAPTURE antibody is added, and binds to the (SANDWICH) antigen to complete the “sandwich.” IMMUNOASSAY After addition of substrate, a colored PRINCIPLE or chemiluminescent reaction product is detected. Enzyme activity is directly proportional to the amount of antigen in the test sample. Vitamin B7 Biotin (Vitamin H) BIOTIN- Bacterial protein AVIDIN Streptavidin (SAv) that has a strong affinity for biotin LABELING Biotin can be complexed to antibody and streptavidin to solid phase material to increase signaling and sensitivity in ELISAs and capture immunoassays 10  6/27/2024 May be caused by properties of the specimen, antigen interference, or antibody interference. INTERFERENCES High dose biotin supplements can WITH cause interference in assays using biotin- IMMUNOASSAYS SAv labeling. False-positive or false-negative results can occur. HIGH-DOSE HOOK EFFECT Excess patient antigen causes falsely decreased detection. Analyte concentration appears to be low or normal when it is actually high. 11  6/27/2024 ANTIBODY INTERFERENCES Heterophile Autoantibodies antibodies e.g., Rheumatoid Usually cause false- factor can cause a positive results false positive. e.g., Human and mouse antibodies (HAMA) HOMOGENOUS EIAS Are less sensitive than heterogeneous assays Rapid and simple to perform Include EMIT and CEDIA Used to determine low-molecular-weight analytes in serum and urine Hormones Therapeutic drugs Drugs of abuse 12  6/27/2024 HOMOGENOUS EIAS (CONTINUED) Does not require a washing or separation step. Enzyme activity is directly proportional to the concentration of patient antigen or hapten present in the test solution. When antibody binds to specific determinant sites on the antigen, the active site on the enzyme is blocked, resulting in a measurable loss of activity. GENERAL PRINCIPLE OF A HOMOGENOUS IMMUNOASSAY 13  6/27/2024 CHEMILUMINESCENT IMMUNOASSAYS Highly sensitive automated immunoassays that can be used to detect antigens (e.g., therapeutic drugs, steroid hormones) or antibodies. Involve 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. Chemiluminescent molecules include acridinium esters, ruthenium derivatives, and nitrophenol oxalates. Chemiluminescent technology can be applied to heterogeneous and homogenous assays. CHEMILUMINESCENT IMMUNOASSAYS Chemiluminescent microparticle immunoassay (CMIA) Heterogeneous assay in which patient antigen competes with chemiluminescent antigen for antibody-coated microparticles; magnets attract the particles for physical separation and washing. Electrochemiluminescence immunoassay (ECLIA) Ruthenium label undergoes a chemical reaction at the surface of an electrode. 14  6/27/2024 FLUORESCENT IMMUNOASSAYS Use a fluorochrome as the label These compounds absorb energy from incident light and convert it to a longer wavelength and lower energy as excited electrons return to the ground state. Examples: Fluorescein—absorbs light at 490– Rhodamine—absorbs light at 550 495 nm and emits green light at 520 nm and emits red light at 585 nm nm. DIRECT IMMUNOFLUORESCENCE ASSAYS Used to identify pathogens in patient samples. Antibody conjugated with a fluorescent tag is added to tissue sections or cells fixed onto a microscope slide. After incubation and a wash step, the slide is read using a fluorescence microscope. Fluorescent-labeled antibodies to CD antigens can be used to identify lymphocytes and other cells by flow cytometry. 15  6/27/2024 The slide is Patient serum is A sandwich is washed, then This technique INDIRECT incubated with an anti-human formed with the can be used to microscope first antibody, IMMUNOFLUORESCENCE slide to which a immunoglobulin which localizes identify patient ASSAYS labeled with a antibody (e.g., known antigen the fluorescent tag ANAs) is attached. fluorescence. is added. DIRECT VS. INDIRECT IMMUNOFLUORESCENCE ASSAYS 16  6/27/2024 Used to determine concentrations of therapeutic drugs and hormones FLUORESCENCE Based on the change in POLARIZATION polarization of fluorescent light IMMUNOASSAY emitted from a labeled molecule when it is bound by antibody (FPIA) If the labeled molecule is bound to antibody, the molecule emits an increased amount of polarized light Fluorescent immunoassay that allows multiple antibodies or antigens to be detected simultaneously To detect antibodies, patient serum is incubated with polystyrene beads MULTIPLEX conjugated to different antigens. IMMUNOASSAY (MIA) Antibody binding is detected by addition of a fluorescent-tagged anti-human immunoglobulin. Beads containing bound antibody are identified by flow cytometry and can be distinguished by their unique shade of red. 17  6/27/2024 RAPID IMMUNOASSAYS Membrane-based, single-use assays based on immunochromatography Easy to perform, with fast turnaround time; ideal for point-of-care testing. Patient sample is added to the test membrane and combines with labeled antigen or antibody conjugated to colored latex or colloidal gold particles. Immune complexes are formed that migrate across the membrane to produce a colored reaction. RAPID IMMUNOASSAYS: IMMUNOCHROMATOGRAPHY 18  6/27/2024 SUMMARY Labeled immunoassays measure small antigens or low concentrations of antigens and antibodies. Antibody or antigen reagents in these techniques are labeled with radioactivity, enzymes, or chemiluminescent or fluorescent compounds. There are two major types of immunoassays: competitive and noncompetitive. Immunoassays may be heterogeneous (requiring separation of bound and free components) or homogenous. SUMMARY.2 Radioimmunoassay (RIA) is based on competition between labeled and unlabeled antigen for a limited number of antibody-binding sites. In noncompetitive indirect enzyme immuno-assays (e.g., ELISA), patient sample binds to antigen on a solid phase, and a second enzyme-labeled antibody is added to detect antibody in the sample. In capture assays, antibody is bound to the solid phase, and any patient antigen is allowed to bind or be captured. A second labeled antibody is added that also binds to patient antigen, creating a “sandwich.” Fluorochromes are fluorescent compounds that absorb energy from an incident light source and convert that energy to light of a longer wavelength. 19  6/27/2024 SUMMARY.3 Direct immunofluorescence assays involve antigen detection through a specific antibody that is labeled with a fluorescent tag. In indirect immunofluorescent assays, the antibody specific to the antigen is unlabeled. Incubation with antigen is followed by addition of a second fluorescent-labeled anti- immunoglobulin that detects the antigen–antibody complexes. Interferences in immunoassays can be caused by a variety of factors, including: * physical properties of the specimen, * antigen excess (high dose hook effect), * presence of autoantibodies or heterophile antibodies, or, * in the case of biotin labeling assays, high dose dietary biotin. These interferences can cause false-positive or false-negative results, depending on the assay. SUMMARY.4 Chemiluminescence is produced by certain compounds when they are oxidized and emit light as they return to their original ground state. Chemiluminescent labels are commonly used in automated immunoassays. Rapid immunoassays involve quick detection of patient antibody or antigen by addition of the sample to a test membrane containing reagents that produce immune complexes and visible color formation. 20  6/27/2024 POSTAMBLE READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 21