Presentation on theme: "Immunologic Methods Part Three Basic Test Methods CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP) CM"— Presentation transcript:
Immunologic Methods Part Three Basic Test Methods CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP) CM
Objectives Explain the principle of each method presented, and give a clinical application of each. Contrast precipitation, agglutination and flocculation. Discuss performing antibody titers and determine the antibody titer when given appropriate laboratory results Discuss general reasons for performing immunologic tests.
Precipitation Based Methods Soluble antigen combines with antibody to form aggregates which precipitate out of solution.
Nephelometry Antibody reagent is combined with patient sample. If antigen is present in the patient’s sample, Ag/Ab complexes will form and precipitate out of solution. Y Y Y
Nephelometry When light is passed through the solution, the precipitates cause the light to scatter at various angles. The light that is scattered at a particular angle is measured. This directly corresponds to the amount of antigen in the sample. Light source Detector
Flocculation Uses fine particles of antigen to detect antibody in patient’s serum. Y Y Y POS NEG
Immunofixation Electrophoresis Proteins separated by electrophoresis Antiserum (antibody) is applied to the gel. Ag/Ab complexes form in the gel. The gel is stained to reveal precipitin bands. Anode Patient serum Cathode (+ electrode) (- electrode) Application point
Western Blot Negative Patient Positive Control specimen Control No bands Patient bands compared to Pos Control p24 gp 41 gp120/160
Agglutination Based Methods Antibodies cause the cross-linking of particulate antigens, usually found on a cell.
Direct Agglutination The antigen is a natural part of the solid’s surface. Can be used to detect antigen or antibody Y Y Y
Direct Agglutination (Hemagglutination)
Passive Agglutination Y Y Y Antigens on a carrier molecule combine with antibodies in the patient’s sample. If the specific antibodies are present, the carrier molecules will clump together.
Reverse Passive Agglutination Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Inhibition of Agglutination Antibody reagent is combined with patient’s specimen. If patient’s specimen contains the target antigen, Ag/Ab will react. Reagent antigen is added. A positive reaction will show no agglutination, because the antibodies were bound to the patient antigen before the reagent antigen was added. A negative reaction shows agglutination between reagent antibodies and reagent antigen. Y Y Y Y Y Y
“Labeled” Methods Attaches a “tag” to either the antigen or antibody. This “tag” can be detected and measured.
Elements of a Labeled Assay Analyte (labeled and unlabeled) Specific antibody Separation of bound and free components Detection of label Standards/calibrators
Classification Heterogeneous: Method that requires a step that separates bound analyte from unbound analyte. Homogeneous: Method that does not require a separation step.
Competitive EIA Enzyme-labeled antigen competes with unlabeled patient antigen for binding sites on fixed antibodies. A wash step removes unbound antigen. A chromogen is added that reacts with the enzyme. The level of color development is inversely proportional to the level of patient antigen. Y Y
Enzyme-multiplied Immunoassay Technique (EMIT) Y Y This is a homogeneous competitive binding assay. When antibody binds the labeled antigen, it blocks enzymatic activity, reducing the amount of color development.
ELISA Known antigen fixed to test platform. Patient serum added; Ab will bind to antigen. Labeled anti-human globulin added; reacts with patient antibody. Substrate added; reacts with label; level of activity measured YYY Y Y Y Y Y Y Y
Capture (Sandwich) EIA Patient’s sample incubated with bound antibody. Following wash, a second chromogen-labeled antibody is added. Chromogen substrate is added and the color is developed and measured. Level of color development is _____ proportional to the amount of antigen “captured”. Y Y Y Y
Microparticle Capture Microbeads coated with known antigen or antibody. Beads incubated with fluorescently labeled analyte and the patient’s sample. Beads are collected and analyzed for fluorescence. Y Y Y Y Y Y Y Y
Direct Fluorescence Fluorescently labeled antibody is used to detect antigen fixed to a slide. Y Y Y Y Y Y Y Y
Indirect Fluorescence Positive TestNegative Test Known antigen fixed to slide Patient’s serum added (unknown antibody) Incubation & wash Fluorescently labeled anti-human globulin reagent added. Y Y Y Y Y Y
Fluorescent Polarization Free labeled antigen excited by polarized light emits unpolarized light. Labeled antigen/antibody complexes excited by polarized light emit polarized light. Labeled antigen competes with unlabeled (patient) antigen for antibody binding sites. The more labeled antigen that is bound to antibody, the more polarized light is emitted. Y
Chemiluminescence Uses chemical labels that, when oxidized, produce a substance of a higher energy level. When this substance decays to its original state, it emits energy in the form of light.
Comparing Antibody Quantities Antibody titers
Antibody Titer Helps determine antibody concentration levels. Twofold serial dilutions of serum are made, then tested against the target antigen. The titer is the reciprocal of the greatest dilution in which a positive reaction is observed.
Two-fold Serial Dilutions
Tube Saline ml Serum 0.2 ml 0.2 ml of tube ml of tube ml of tube ml of tube ml of tube ml of tube ml of tube ml of tube ml of tube 10 N/A Antigen0.1 ml Final Dilution 1:11:21:41:81:161:321:64 1:1281:2561:512 1:1024control
Results Often tested in parallel with a previous specimen. Comparison of current specimen’s results and previous specimen’s current results should be made. A change in titer of 2 or more tubes is considered significant.
Reasons to perform a titer Confirm vaccination Verify past infection Prenatal Acute and convalescent