CLS_A_Bottomline_Approach_5e IMMUNOSERO.pdf

33 IMMUNOLOGY AND SEROLOGY by Patsy Jarreau...

33 IMMUNOLOGY AND SEROLOGY by Patsy Jarreau TYPES OF IMMUNITY Active vs. Passive -l- ACTIVE PA!:>!:>IVE Individual Produces Antibody Antibody Transferred to Individual Follows Immunization or Infection Example: Gamma Globulin Injections, Memory (lasting) placental transfer No Memory (temporary) What type of immunity is associated with rubella immunization?-------,- Active Immunity What type of immunity is associated with neonatal ( 12 hours to develop Leprosy Graft vs. Host Disease (GVHD) Precipitin Curve Tests for Allergy: Equivalence Skin Tests, RIST, RAST Prozone (antibody excess) Postzone ELISA for total lgE and (antigen excess) lgE to specific antigens Antigen Association of EOtJUJophilB Concentration and°lgEwith Hypersensiti:vity Type I Principles of Antigen-Antibody Reactions Used in Serologic Testing REMEMBER! PRECIPITATION Pros Have 1. Principle a. Soluble antigen + antibody (in Good Bodies proper proportions) b. Lattice formation (antigen binds with Fab sites of two antihodies) ➔ visible precipitate 2. Examples a. Double diffusion ( Ouchterlony) PROZONE = b. Single diffusion (radial immunodiffusio11) Anti~ c. lmmunoelectrophoresis Excess d. lmmunofixation 38 COMPLEMENT FIXATION (CF) l. Step 1: Antibody and antigen allowed to combine in presence of complement 2. Step 2: Indicator is added (SRBC EUSA for total lgE and coa ted with hemolysin) IgE specific antigens 3. Positive test (No hemolysis): If complement is fixed in step 1, it will not be available to combine with indicator AGGLUTINATION ➔ no h,e molysis occurs 1. Principle 4. Negative test (Hemolysis): complement a. Particulate antigen + antibody was not bound in step 1 and is available b. Lattice formation (anti.gen binds to r eact with indicator ➔ hemolysis with Fah sites of two antibodies occurs forming bridges between antigens) ➔ clumping 5. Limitati,o ns a. Serum must be heat inactivated 2. Examples b. Stored serum becomes a. Direct agglutination (Blood Bank) anticomplementary b. Passive hemagglutination (antigen c. Elaborate QC and standardization reagent produced by treating RBCs required with tannic acid to allow adsorption d. Only used for IgM antibodies of protein antigens) c. Passive latex agglutination (antigen in reagent is attached to latex Complement (C') Fixation particle) Serum with Ab Serum without Ab AGGLUTINATION INHIBITION 1. Step 1: Patient serum (antigen) is reacted with limited amount of ~ + Ag v + antibody r eagent C' 2. Step 2: Indicator is added (same V ~ anti.gen for which you are testing bound Ag-Ab-C' Ag C' to RBC or latex carrier particle) 3. Positive test (No agglutination): If ~ + ABC-Ab v (Indicator) patient has antigen for which you are testing, the reagent antibody will be V ~ bound in step 1 and unavailable to NOC' Available: C' Available: react with the indicator NO Hemolysis Hemolysis Positive Test Negative Test 4. Negative test (Agglutination): If patient does not have the antigen, reagent RADIAL IMMUNODIFFUSION (RID) antibody is not bound in step 1 and is 1. Also called single immmunodiffusion available to react with indicator 2. Principle: 5. Examples of inhibition reactions : a. Unlimited antibody incorporated a. Hemagglutination inhibition test for into agar in plate rubella b. Serum and standards are added to b. Latex agglutination inhibition test circular wells precut in agar for other viruses c. Incubate d. Diffusion occurs and ring of precipitate forms e. Measure diameter (d ) of ring  39 2. Methods 3. Fill trough in agar with known a. Fahey (kinetic) antibody ❖ Read before ring reaches maximal size (6-12 hours) 4. Antigen and antibody diffuse through ❖.Logarithmic relationship between agar diameter (d) ofprecipitin ring and a. In equivalence zone, precipitation antigen concentration ~ read from arc appears plotted standard curve b. Size of arc determined by antigen b. Mancini (end-point) concentration ❖ Read at maximal size (24-48 c. Abnormal contour of arc may hours) indicate monoclonal gammopathy ❖.Linear relationship between diameter squared (d2) of 5. Most commonly used to determine heavy and light chains involved precipitin ring and a. Serum IEP: monoclonal antigen concentration~ gammopathies read from plotted standard curve b. Urine IEP: Bence Jones protein DOUBLE DIFFUSION (OUCHTERLONY) 1. Used to determine relationships + Monoclonal Gammopathy between antigens and antibodies SPE 2. Antibody is added to precut wells in center of agar plate (Agar contains no antigen or antibody) 3. Patient sera and standards are alternated in wells surrounding the center well (antibody well) Normal Serum '-..__../ 4. Incubate IEP Anti-lgG in Trough 5. Diffusion occurs and results in visible Monoclonal lgG in Patient Serum bands of precipitation + Polyclonal Gammopathy 6. Patient wells are read in relation to standards in adjacent wells (see SPE patterns below) 7. Location of bands depends on concentration and rate of diffusion 8. Used to identify antibodies associated with autoimmune disorders Double Diffusion Patterns Normal Serum '-..__../ IEP Anti-lgG in Trough @IDEITTITT Polyclonal lgG in Patient Serum IMMUNOFIXATION 1. Protein electrophoresis + :ONSOEmlN@ immunoprecipitation 2. Procedure a. Apply specimen to 6 positions on @PARTIAL IDENTITY agarose plate b. Electrophorese to separate proteins IMMUNOELECTROPHORESIS (IEP) c. Apply monospecific antisera to 5 1. Gel diffusion + electrophoresis lanes using a 6th for reference d. If antigen present, band of antigen- 2. Electrophorese serum proteins on agar antibody complexes form and gel precipitate; wash, stain 40 3. Highly sensitive method, easy to read RADIOIMMUNOASSAY (RIA) 1. Very sen sitive and sp ecific 4. Used to classify monoclonal gammopathies (determine heavy and 2. Can be u sed for detecting antigen or light chains involved) antibody (explanation b elow detects antigen, lmt using known antigen to lmmunofixation + detect serum antibody is also possible) 3. Comp etitive binding assay a. Patient antigen and labeled antigen are incuba ted with known amount of sp ecific antibody (unlabeled and labeled antigen compete for binding with antibody) b. Wash to remove unbound antigen c. Radioactivity counted on a gamma counter; compare to standard curve d. Results ❖ The lower the radioactive count, the JJigher the concentration of SPE lgG lgA lgM K ?,. (ref.) unlabeled antigen (patient) 4. Examples a. Tests for hepatitis antigens and antibodies ROCKET IMMUNOELECTROPHORESIS (LAUREL) b. R adioimmunosorhent Test (RIST) - 1. Similar to RID but electrophoresis is measures total IgE c. Radioallergosorhent Test (RAS]) - used to speed formation of precipitate measures l gE to sp ecific allergens 2. Procedure ENlYME IMMUNOASSAY (EIA/ELISA) a. Gel contains antibody 1. " Sandwich technique" b. Add patient sera (antigen ) to weUs a. Monoclonal or polyclonal antibody cut in gel adsorbed on solid surface (h ead or c. Add assayed standards to other microtiter well) wells b. Add patient serum ; if antigen is d. Apply electric current to rapidly present in serum, it hinds to migrate antigen through gel antibody-coated head or well e. A cone-shaped area of precipitation c. Add excess enzyme-labeled antibody forms (looks lilrn a rock et) (antibody conjugate); forms f. Measure height of rocket and antigen-antibody-labeled antibody compare to standa rd curve "sandwich" (antibody in conjugate COUNTERCURRENT IMMUNOaECTROPHORESIS is directed agai.nst anotlier epitope 1. Procedure of antigen b eing assayed) a. Two rows of wells are cut in gel d. Add en zyme substrate, incubate h. Add antigen to well in one row; add and read absorbance antibody to corresponding well in e. Important: Washing required other row ( one known, the other between each step. Improper iwlrnown) washing leads to false positive c. Apply electric current to gel results d. Antigen and antibody migrate f. Absorhance is directly proportional toward each other to antigen concentration e. Precipitate forms if specific antigen for the antibody in the 2. Examples: corresponding well is present in a. HIV testing b. Serum HCG (pregnancy) patient's serum c. Tests for hepatitis antigens and antibodies d. Antibodies to bacteria and viruses  41 3. Examples of IIF a. Testing for Antinuclear Antibodies (ANA) b. Fluorescent Treponemal Antibody Test (FTA-Abs) Principles ofAntigen- FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA).Antibody Reactio~ Used in Serology 1. Principle a. Add reagent antibody and ENlYME MULTIPLIED IMMUNOASSAY (EMITI fluorescent- tagged antigen to patient 1. Used to measure concentrations of serum small molecules such as drugs and b. Positive test ❖ Antigen present in patient serum hormones binds to reagent antibody leaving 2. Principle most tagged antigen unbound a. Add patient serum to an enzyme- ❖ Unbound tagged antigens rotate drug conjugate; then add anti-drug quickl.y reducing amount of antibody (reagent) polarized light produced b. Add enzyme substrate and incubate c. Negative test c. Positive test ❖ If no antigen present in patient ❖ Drug in patient serum combined serum, tagged antigen binds to with anti-drug antibody; sites on reagent antibody the enzyme portion of the ❖ Tagged antigen antibody conjugate remain available to hind complexes rotate slowly givinB· off with substnlte increased polarized ligl1t ❖ Color prnduced d. Negative test FLOW CYTOMETRY ❖ Anti-drug antibody attached to See Lab Operations and Instrumentation the drug in the enzyme dmg Chapter for explana tion conjugate and blocks the active sites on the enzyme; substrate SERIAL DILUTIONS unable t·o bind to enzyme 1. Testing for infectious diseases is ❖ No color produced performed on acute and convalescent NEPHELOMETRY specimen s collected about 2 week s apart 1. Procedure a. Serum substance r eacts with 2. Must see 4-fold or 2 tube rise in titer to specific antisera and forms be clinically significant insoluble complexes TERMS USED IN EVALUATING TEST METHODOLOGY b. Light is passed through susp ension c. Scattered {reflected) light 1. Sensitivity absorbance is proportional to a. Analytical Sensitivity - Ability of a number of insoluble complexes; test to detect very small amounts of compare to standards a substance b. Clinical Sensitivity - Ability of test 2. Examples : to give positive result if patient has a. Complement component the disease - 100% clinical sensitivity concentration gives no false negative results b. Antibody concentration (IgG, IgM, IgA , etc.) N is for Negative IMMUNOFLUORESCENCE seNsitivity (%)= TP/(TP+FN) X100 1. Direct - Add fluorescein-labeled (False negatives yield lower sensitivity) antibody to patient tissue, wash & examine under fluorescent microscope P is for Positive 2. Indirect (IIF)-Add patient serum to sPecificity (%)= TN/(TN+FP) x 100 r eagent (tissue containing known antigen), wash, add fluorescein labeled (False positives yield lower specificiity) antiglobulin, wash & examine under fluorescent microscope 42 Serial Dilution Relative Sensitivities of Calculations Serologic Methods Compare Pre & Post Titers Disease States and Associated to Determine Diagnosis of Measles and Rubella Laboratory Tests INFLAMMATION Definitions for Sensitivity 1. C-reactive protein (acu te ph ase and Specificity protein) a. Released rapidly after inflanunation or tissue damage 2. Specificity. b. Concentration quickly decreases as a. Analytical Specifici ty -Ability of inflammation subsides test to detect substance without c. Used to monitor presence of interference from cross-reacting inflammation &uh.stan ces d. Does not indicate source of b. Clinical Specificity - Ability of test inflammation to give negative result if patient e. High sensitivity CRP (hs-CRP) used does not have disease - high clinical to indicate risk of cor onary artery specificity test gives no false positives disea se 2. Erythrocyte sedimentation rate (ESR ) a. Also used to indicate presen ce of REMEMBER! inflamrna tion b. Not as sensitive to increasing or decreasing inflammation as CRP Test Sensitivities Nonlattice (More Sensitive) SYPHILIS lmmunoassays 1. Caused by Trepor1ema pallidum RIA (Radial Immunoassay) 0.001mg/ml EIA (Enzyme Immunoassay) 2. Course of disease : Primary, FIA (Fluorescent Immunoassay) secondary, latent, tertiary ; also Nepholometry congenital infections may occu r TREPONEMAL TESTS 1. Darkfield microscopy - used to Lattice (Less Sensitive) visualize motile organisms from CIE (Counter Current lmmunoelectrophoresis) primary & secondary lesions (..."tiori" or... "sion'J CF (Complement FixaliQ[]) 2. Fluorescent treponemal antibody Agglutination absorption test (FTA-Abs) a. Indirect immunofluorescence assay Flocculation (PrecipitaliQ[]) b. Remove nonspecific antibodies from Rocket Electrophoresis serum by using sorbent RID (Radial lmmunodiffusion) c. React serum with Nichol 's strain of Ouchterlony (Double immunodiffusion) T. pallidum !FE (lmmunofixalion) cl. Add fluorescein-labeled antihuman IEP (lmmunoelectrophoresis) globulin and wash e. Read for fluorescence 500 mg/ml  43 3. Treponema pallidum Immobilization Test (TPI) Sensitivity of Tests for Syphilis a. Darkfield microscopy b. Add live treponemes to patient PRIMARY STAGE serum c. If antibody is present, treponemes immobilized FrA-ABS } RPR VDRL i Sensitivity d. Expensive, seldom used 4. Microhemagglutination Assay for SECONDARY STAGE All Tests Equally Sensitive T. pallidum (MFIA-TP) a. Add patient serum to red cells sensitized with T. pallidum b. If antibody is present, agglutination LATE STAGE occurs FrA-ABS } Equal Sensitivity MHA-TP NONTREPONEMAL TESTS (REAGIN TESTS) TPI 1. Venereal Disease Research Laboratory Reagin Tests Negative in treated patients (VDRL) a. Microflocculation (microscopic) I FTA-ABS: Most Sensitive in All Stages I b. Antigen = cardiolipin + lecithin c. Antibody (reagin) = lgM or lgG RHEUMATOID ARTHRITIS (RA) directed against damaged tissue or organism 1. Production of IgM or IgG antibodies d. Serum requires heat inactivation directed against lgG e. Flocculation indicates reactive 2. Diagnosis requires radiologic, clinical serum and laboratory findings f. Test of choice for screening CSF g. False positive in malaria (100% 3. Laboratory findings biologic false positive), SLE, RA, a. High titer s of rheumatoid factor h epatitis, pneumonia, aging and (RF) infectious mononucleosis b. Low titers of complement c. Positive anti-cyclic citrullinated 2. Rapid Plasma Reagin Test (RPR) peptide (anti-CCP) a. Microflocculation and coagglutination of charcoal particles 4. Screening test: Rheumatoid factor (macroscopic) (RF) assay b. More sen sitive, less specific than a. Patient serum added to reagent VDRL composed of particulate carrier c. Antigen = cardiolipin + charcoal (latex or RBC) attached to lgG particles b. Run positive and negative controls d. No heat inactivation necessary c. Positive test: visible agglutination e. Black clumps form in reactive test d. Detects serum IgM f. False positives - same as VDRL 5. Confirmatory test: Anti-cyclic citrullinated p eptide assay (anti-CCP) a. Used to confirm positive RF tests REMEMBER! b. ELISA technique c. High sensitivity and specificity Do NOT Refrigerate d. Allows earlier diagnosis and earlier Specimen for Cold treatment which may reduce joint erosion and deformity Agglutinin Assay Antibody will bind to red cells leaving serum free of antibodies and result in a false negative or decreased cold agglutinin titer. Screening and Con.irmatroy Test for RA 44 CELIAC DISEASE (CELIAC SPRUE) EBV Antibodies 1. Hypersensitivity to gliadin (a protein component of gluten) found in grains EBV Antibodies such as wheat, barley, rye C 0 2. Laboratory findings ~.... a. Tissue transglutaminase antibody c , -0 0 b. Endomysial antibody (EMA-IgA).0 Anti-EBNA c. Antigliadin antibody (AGA-IgG,.E