Presentation on theme: "Part Three Basic Test Methods"— Presentation transcript:
1 Part Three Basic Test Methods Immunologic MethodsPart ThreeBasic Test MethodsCLS 420Clinical Immunology and Molecular DiagnosticsKathy Trudell MLS SBB(ASCP)CM
2 ObjectivesExplain 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 resultsDiscuss general reasons for performing immunologic tests.
3 Precipitation Based Methods Soluble antigen combines with antibody to form aggregates which precipitate out of solution.
4 Nephelometry Y 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.YYYMay use known antigen as a reagent to look for antibodies in a patient’s specimen.
5 NephelometryWhen 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 sourceLight scattered at 90 degrees in commonly measured.Endpoint – reaction is allowed to go to completion. Problems with precipitate settling out, reducing the amount of scatter.Rate – measures rate that scatter increases following addition of reagent.Nephelometry can be used to quantitate serum protein levels.Detector
6 Uses fine particles of antigen to detect antibody in patient’s serum. FlocculationUses fine particles of antigen to detect antibody in patient’s serum.YYYMost common application is testing for syphilis, detecting antibodies [reagin].Patient’s serum is placed within a ring on a slide or card.A measured volume of reagent containing antigen is placed in the ring.The slide or card is rotated to mix the sample and reagent.The reaction is examined macroscopically for fine precipitates, which indicate a positive test.POS NEG
7 Immunofixation Electrophoresis Proteins separated by electrophoresisAntiserum (antibody) is applied to the gel.Ag/Ab complexes form in the gel.The gel is stained to reveal precipitin bands.Electrophoresis used to separate proteins on a gel according to size and electrical charge.If the antibody applied to the gel is directed against a particular protein, precipitin bands form where Ag/Ab complexes have been trapped in the gel.The gel is washed to remove any unprecipitated proteins, then stained to reveal the bands.Application pointAnode Patient serum Cathode(+ electrode) (- electrode)
8 IFE stained gels = Serum application point Example of an IgG monoclonal antibody with kappa light chainsSPE = Serum Protein ElectrophoresisSPE Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-Lambda
9 Western Blot Negative Patient Positive Control specimen Control p24 gp 41gp120/160Negative Patient PositiveControl specimen ControlNo bands Patient bands compared to Pos ControlModification of IFEKnown antigens are electrophoresed to separate them.The separated components are transferred to nitrocellulose paper by blotting the gel.The patient’s serum is applied to the paper.If the patient has antibodies to any of the antigens on the paper, it will form a precipitate.Paper is washed and stained.If antibody to more than one antigen of an organism is detected in the patient’s serum, infection with that particular organism is highly likely.
10 Agglutination Based Methods Antibodies cause the cross-linking of particulate antigens, usually found on a cell.Bacteria and red blood cells (RBCs) are common antigen sources.
11 Y Y Y Direct Agglutination The antigen is a natural part of the solid’s surface.Can be used to detect antigen or antibodyYYYOften performed at room temperature.May use centrifugation to bring antigen and antibody into closer proximity.Useful in detecting bacterial antigens/antibodies and RBC antigens/antibodies (hemagglutination)Watched Infectious mono video in Hematology
13 Passive Agglutination 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.YYYLatex is often used as the carrier molecule.Useful for detecting antibodies to bacteria and viruses.
14 Reverse Passive Agglutination Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen.YYYYYYYYYUses include ID of bacteria, measuring hormone and drug levels, and measuring levels of some proteins.YYYYYYY
15 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.YYYYYY
16 “Labeled” MethodsAttaches a “tag” to either the antigen or antibody. This “tag” can be detected and measured.Tests are fast, sensitive and specific.
17 Elements of a Labeled Assay Analyte (labeled and unlabeled)Specific antibodySeparation of bound and free componentsDetection of labelStandards/calibratorsRadioactivity, fluorescence, chemiluminous materials and enzymes have all been used as tags.
19 ClassificationHeterogeneous: Method that requires a step that separates bound analyte from unbound analyte.Homogeneous: Method that does not require a separation step.Separation methods include : wash step; adsorption coupled with centrifugation or filtration; magnets; chemically modifying the test medium
20 Competitive EIAEnzyme-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 Y YAntibody may be bound to the wells of a microtiter plate or to beads that are placed in a reaction well. The enzyme-labeled antigen is reagent that is added to the well along with the patient’s specimen.
21 Enzyme-multiplied Immunoassay Technique (EMIT) Y Y Y YThe more patient antigen that binds to the antibody, the more enzyme-labeled antigen remains free to react with the chromogen.Level of antigen in patient is directly proportional to the level of color development.Commonly used to test for drugs.This is a homogeneous competitive binding assay.When antibody binds the labeled antigen, it blocks enzymatic activity, reducing the amount of color development.
22 ELISA Y Y Y Y Y Y Y Y Y Y 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 measuredYYYYYYYYYYNoncompetitive EIAMultiple wash steps used to separate bound from unbound.Amount of patient antibody is directly proportional to level of enzyme activityVersatile assay commonly used in the clinical labVery sensitive test (<1pg/mL); not as specific as competitive assaysEasy and inexpensive to performUsed to screen for antibodies to viruses such as HIV, Hepatitis A and Hepatitis C, EBV and infectious mononucleosis
23 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”.YYY Y Y YLevel of color development is DIRECTLY proportional to the amount of antigen “captured”.Used to detect microorganisms such as parasites, fungus, rotavirus and RSV; quanitate levels of immunoglobulins, hormones and look for tumor markers.
24 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.YYYYYYYYMicrobeads may be made of polysaccharides, polyacrylamide or magnetizable cellulose.The test mixture may be centrifuged or magnetized to collect the beads.Used to quantitate hormone levels, viral antibodies, and serum proteins.
25 Direct Fluorescence Y Y Y Y Y Y Y Y Fluorescently labeled antibody is used to detect antigen fixed to a slide.YYYYYThis test has been used for detection of Chlamydia, Legionella, RSV, and other antigens.YYY
26 Indirect Fluorescence YYYYYYPositive Test Negative TestKnown antigen fixed to slidePatient’s serum added (unknown antibody)Incubation & washFluorescently labeled anti-human globulin reagent 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.
27 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.YFree antigen can rotate when hit by polarized light whereas Ag/Ab complexes are too large to turn that quickly.The degree of polarization is inversely proportional to the level of patient antigen.Used to measure hormones and therapeutic drugs.
28 ChemiluminescenceUses 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.Common label materials include:LuminolAcridium estersPeroxyoxalatesRequires sophisticated instrumentation that is specific to the chemical being used.This labeling technique can be applied to heterogeneous or homogenous assays, and may detect antigens or antibodies.Clinical applications include detection of drugs, hormones, and viral antigens.
30 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.Twofold dilutions are the most common, however other dilutions may be used.
32 1 2 3 4 5 6 7 8 9 10 11 12 Tube Saline 0.2 ml Serum 0.2 ml of tube 2 0.2 mlSerum0.2 ml of tube 20.2 ml of tube 30.2 ml of tube 40.2 ml of tube 50.2 ml of tube 60.2 ml of tube 70.2 ml of tube 80.2 ml of tube 90.2 ml of tube 10N/AAntigen0.1 mlFinalDilution1:11:21:41:81:161:321:641:1281:2561:5121:1024control
33 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.Testing in parallel to “control” variation in technique and antigen strength.Change of 2 tubes = 4-fold dilution
34 Reasons to perform a titer Confirm vaccinationVerify past infectionPrenatalAcute and convalescent