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Part Three Basic Test Methods

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1 Part Three Basic Test Methods
Immunologic Methods Part Three Basic Test Methods CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)CM

2 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.

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. Y Y Y May use known antigen as a reagent to look for antibodies in a patient’s specimen.

5 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 Light 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.
Flocculation Uses fine particles of antigen to detect antibody in patient’s serum. Y Y Y Most 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 electrophoresis Antiserum (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 point Anode Patient serum Cathode (+ electrode) (- electrode)

8 IFE stained gels = Serum application point
Example of an IgG monoclonal antibody with kappa light chains SPE = Serum Protein Electrophoresis SPE Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-Lambda

9 Western Blot Negative Patient Positive Control specimen Control p24
gp 41 gp120/160 Negative Patient Positive Control specimen Control No bands Patient bands compared to Pos Control Modification of IFE Known 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 antibody Y Y Y Often 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

12 Direct Agglutination (Hemagglutination)

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. Y Y Y Latex 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. Y Y Y Y Y Y Y Y Y Uses include ID of bacteria, measuring hormone and drug levels, and measuring levels of some proteins. Y Y Y Y Y Y Y

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. Y Y Y Y Y Y

16 “Labeled” Methods Attaches 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 antibody Separation of bound and free components Detection of label Standards/calibrators Radioactivity, fluorescence, chemiluminous materials and enzymes have all been used as tags.

18 Standard Curve Known concentrations of unlabeled analyte, used to determine relationship between labeled analyte (in this case, Hepatitis B antigen labeled with radioactive iodine) and unlabeled analyte in specimen. Figure 6-9b Kuby Immunology , 6th ed ©2007, WH Freeman & Co. Used with permission

19 Classification Heterogeneous: 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 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 Y Y Antibody 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 Y The 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 measured Y Y Y Y Y Y Y Y Y Y Noncompetitive EIA Multiple wash steps used to separate bound from unbound. Amount of patient antibody is directly proportional to level of enzyme activity Versatile assay commonly used in the clinical lab Very sensitive test (<1pg/mL); not as specific as competitive assays Easy and inexpensive to perform Used 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”. Y Y Y Y Y Y Level 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. Y Y Y Y Y Y Y Y Microbeads 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. Y Y Y Y Y This test has been used for detection of Chlamydia, Legionella, RSV, and other antigens. Y Y Y

26 Indirect Fluorescence
Y Y Y Y Y Y Positive Test Negative Test Known antigen fixed to slide Patient’s serum added (unknown antibody) Incubation & wash Fluorescently 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. Y Free 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 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. Common label materials include: Luminol Acridium esters Peroxyoxalates Requires 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.

29 Comparing Antibody Quantities
Antibody titers

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.

31 Two-fold Serial Dilutions

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 ml Serum 0.2 ml of tube 2 0.2 ml of tube 3 0.2 ml of tube 4 0.2 ml of tube 5 0.2 ml of tube 6 0.2 ml of tube 7 0.2 ml of tube 8 0.2 ml of tube 9 0.2 ml of tube 10 N/A Antigen 0.1 ml Final Dilution 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 control

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 vaccination Verify past infection Prenatal Acute and convalescent

35 Acute Titer (IgG) 1 2 3 4 5 6 7 8 9 10 11 12 + 1:1 1:2 1:4 1:8 1: 16
Tube 1 2 3 4 5 6 7 8 9 10 11 12 Dilution 1:1 1:2 1:4 1:8 1: 16 32 64 128 256 512 1024 Control Results + Titer

36 Convalescent Titer (IgG)
Tube 1 2 3 4 5 6 7 8 9 10 11 12 Dilution 1:1 1:2 1:4 1:8 1: 16 32 64 128 256 512 1024 Control Results + Titer

37 Primary vs. Secondary Humoral Response
IgG IgM IgM IgG First exposure Second exposure

38 Any Questions?

39 You have finished Immunology Student Lab Lectures!
Congratulations You have finished Immunology Student Lab Lectures! Good Luck on your exam!

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