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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 • 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. 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 Light angles. source • The light that is scattered at a particular angle is measured. This corresponds to the level of antigen in the sample. Detector Flocculation Uses fine particles of antigen to detect antibody in patient’s serum. 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 AG other well. • Each diffuses through gel. • If antibody is specific AB to that antigen, a precipitin line will form where the 2 meet. Double Immunodiffusion Identity 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 D Da AG 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. Application point Anode Patient serum Cathode (+ electrode) (- electrode) IFE stained gels = Serum application point SPE Anti-IgG Anti-IgA Anti-IgM Anti-Kappa Anti-Lambda Western Blot p24 gp 41 gp120/160 Negative Patient Positive Control specimen Control No bands Patient bands compared to Pos Control 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 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. 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, Y they will react. Y • Reagent antigen is added. Y • A positive reaction will show Y 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. Click on images at right wait for animation to begin. Other Methods Neutralization Positive Test Negative Test 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. 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. Parts 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 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 •This is a homogeneous competitive binding assay. •Color development is _______proportional to the concentration of antigen. Direct Fluorescence Negative test Positive test 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. 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. Fluorescent Polarization • Free labeled antigen excited by polarized light emits unpolarized light. • Labeled antigen/antibody Y 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. 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 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. Two-fold Serial Dilutions Tube 1 2 3 4 5 6 7 8 9 10 11 12 0 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Saline ml ml ml ml ml ml ml ml ml ml ml 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Serum ml ml ml of ml of ml of ml of ml of ml of ml of ml of ml of ml of tube tube tube tube tube tube tube tube tube 6% 2 3 4 5 6 7 8 9 10 BSA RBC 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Suspen ml ml ml ml ml ml ml ml ml ml ml ml sion Final 1:1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 control Dilution 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. Reasons to perform a titer • Acute and convalescent • Prenatal • Verify past infection • Confirm vaccination Primary vs. Secondary Humoral Response IgG IgM IgM IgG First Second exposure exposure Congratulations You have finished Immunology Student Lab Lectures! Good Luck on your exam!
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