CLS 420 Clinical Immunology & Molecular Diagnostics Immunologic Methods Part 2 Basic Methods

Objectives Explain the principle of each method presented, and give a clinical use for each. Contrast precipitation, agglutination and flocculation, including: Reaction time and conditions Antigen state Immunoglobulin class Lattice formation Describe heat inactivation of patient serum, including method and purpose. Discuss general reasons for performing immunologic tests.

Precipitation Based Methods Soluble antigen combines with antibody to form aggregates which precipitate out of solution.

Nephelometry Y Y Antibody reagent is combined with patient sample. Y If antigen is present in the patient’s sample, Ag/Ab complexes will form and precipitate out of solution. Y May use known antigen as a reagent to look for antibodies in a patient’s specimen. Click on image at right wait for animation to begin.

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 corresponds to the level 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. Nephelometry can be used to detect serum proteins. Detector

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. Most common application is testing for syphilis, detecting antibodies to 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 for fine precipitates, which indicate a positive test. Negative test Positive test Click on images above wait for animation to begin.

Double Immunodiffusion Ouchterlony Method Testing performed in agar gel. Antigen is placed in one well. Antibody is place in other well. Each diffuses through gel. If antibody is specific to that antigen, a precipitin line will form where the 2 meet. AG AB

Double Immunodiffusion Identity Known antibody with multiple specificities is placed in center well. Known antigen is placed in an outside well. Patient specimen containing antigen is placed in other outside well. The pattern of precipitin lines is interpreted. This test may be used to identify fungal antigens and antibodies to nuclear antigens. Click on image above wait for animation to begin.

Double Immunodiffusion Nonidentity Click on image above wait for animation to begin.

Double Immunodiffusion Partial Identity D antigen Da Db Da AG D AG Dc Dd Anti-D

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)

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

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.

Agglutination Based Methods Antibodies cause the cross-linking of particulate antigen, usually found on a cell.

Direct Agglutination The antigen is a natural part of the solid’s surface. Often performed at room temperature. May use centrifugation to bring antigen and antibody into closer proximity. Can be used to detect antigen or antibody Bacterial antigens/antibodies RBC antigens/antibodies (hemagglutination) Click on image at right wait for animation to begin.

Passive Agglutination Antigens on a carrier molecule, such as latex, combine with patient’s sample for antibody detection. Click on image above wait for animation to begin.

Reverse Passive Agglutination Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen. Uses include ID of bacteria, measuring hormone and drug levels, and measuring levels of some proteins. Click on image above wait for animation to begin.

Inhibition of Agglutination Antibody reagent is combined with patient’s specimen. If patient’s specimen contains the antigen for that antibody, they 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 antigen. Y Y Y Y Y Y Click on images at right wait for animation to begin.

Other Methods

Neutralization Positive Test Negative Test Applications include ASO titers and anti-DNase B titers. The presence of an antibody prevents the antigen from functioning correctly. Click on images above wait for animation to begin.

Complement Fixation The patient’s serum is heated at 56oC for 30 minutes to inactivate any complement present. Patient’s treated serum is incubated with known antigen and a known quantity of guinea pig complement. If the patient has an antibody to the antigen, they will react and the complement will bind to the Fc pieces of the antibodies. Sheep RBCs that are coated with hemolysin are added. The test is incubated, centrifuged and read for hemolysis. In a positive test, the complement will have been used up by the patient’s antibody, and no hemolysis will be present. Hemolysin = antibodies known to activate complement and cause hemolysis. This test is not used frequently, but has been used to detect antibodies to viruses, fungus and rickettsia.

Complement Fixation Negative test Positive test Click on images above wait for animation to begin.

“Labeled” Methods Attaches a “tag” to either the antigen or antibody. This “tag” can be detected and measured. Tests are fast, sensitive and specific.

Parts 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.

Classification Heterogeneous: Method that requires a step that separates bound analyte from unbound analyte. Homogeneous: Method that does not require a separation step. Often a wash step removes the unbound analyte. Other separation methods include adsorption coupled with centrifugation or filtration, magnets, or chemically modifying the test medium so that bound analytes remain in solution while Ag/Ab complexes fall out of solution.

Competitive EIA Enzyme labeled antigen competes with unlabeled patient antigen for binding sites on fixed antibodies. A chromogen is added that reacts with the enzyme. The level of color development is inversely proportional to the level of patient antigen. Click on image at right wait for animation to begin.

Capture (Sandwich) EIA Patient’s sample is incubated with bound antibody. Following a wash, a second antibody that is labeled with a chromogen is added. The level of color development corresponds with the amount of antigen “captured”. Click on image at right wait for animation to begin.

Enzyme-multiplied Immunoassay Technique (EMIT) Y Y Y Y When antibody binds to the labeled antigen, it blocks enzymatic activity, reducing the amount of color development. The more patient antigen that binds to the antibody, the more enzyme-tagged antigen remains free to react with the chromogen. Commonly used to test for drugs. This is a homogeneous competitive binding assay. Color development is _______proportional to the concentration of antigen.

Direct Fluorescence Negative test Positive test This test has been used for detection of Chlamydia, Legionella, RSV, and other antigens. Fluorescently labeled antibody is used to detect antigen fixed to a slide. Click on images above wait for animation to begin.

Indirect Fluorescence 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. Click on images above wait for animation to begin.

Microparticle Capture Uses microbeads coated with known antigen or antibody. The beads are incubated with a fluorescently labeled analyte and the patient’s sample. The test mixture is centrifuged (or magnetized) to collect the beads, which are then analyzed for fluorescence. Microbeads may be made of polysaccharides, polyacrylamide or magnetizable cellulose.

Fluorescent Polarization Free labeled antigen excited by polarized light emits unpolarized light. Labeled antigen/antibody complexes excited by polarized light emit polarized light. FPIA is a competitive binding assay in which 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.

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.

Comparing Antibody Quantities Antibody titers

Antibody Titer An antibody titration can help determine antibody concentration levels. Twofold serial dilutions of serum containing an antibody are made, then tested against cells possessing the target antigen. The titer is the reciprocal of the greatest dilution in which agglutination is observed. Twofold dilutions are the most common, however other dilutions may be used.

Two-fold Serial Dilutions

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 0.2 ml of 6% BSA RBC Suspension 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

Results Titers provide more valuable information when tested in parallel with a previous titer specimen. A comparison of the current specimen’s results and previous specimen’s current results should be made. A change in titer of 2 or more tubes is considered to be significant. Change of 2 tubes = 4-fold dilution

Reasons to perform a titer Acute and convalescent Prenatal Verify past infection Confirm vaccination

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

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