IMS- Finals PDF

Preview

Summary

SEROLOGIC TECHNIQUES: SECONDARY REACTIONS PHASES OF IMMUNOLOGIC REACTIONS: 1. PRIMARY binding of Ag and Ab not easily detected because it is not visible to the eye Ex: Labelled immunoassays such as RIA, EIA, Chemiluminescence, Immunofluorescence 2. SECONDARY in Amino Acid chain no need for labels be...

Description

SEROLOGIC TECHNIQUES: SECONDARY REACTIONS PHASES OF IMMUNOLOGIC REACTIONS: 1. PRIMARY - binding of Ag and Ab - not easily detected because it is not visible to the eye * Ex: Labelled immunoassays such as RIA, EIA, Chemiluminescence, & Immunofluorescence 2. SECONDARY - conformation/change in Amino Acid chain - no need for labels because it is visible to the eye * Ex: Agglutination, Precipitation, Floculation, Neutralization, C-fixation test 3. TERTIARY - folding of Polypeptide chain - binding of hydrogen & hydrophobic bonds - takes place in vivo * Ex: Phagocytosis, Chemotaxis, Opsonization TERMS USED IN Ag-Ab BINDING: * AFFINITY - initial force of attraction between univalent Ag & univalent Ab * AVIDITY - sum forces of attraction between multivalent Ag & multivalent Ab - has stronger force & more stable bond * SPECIFICITY - follows the lock & key model INTRAMOLECULAR FORCES THAT BINDS TO Ag & Ab: 1. IONIC BONDS - exist between two opposite charge particles 2. HYDROGEN BONDS - exist between two polar molecules 3. HYDROPHOBIC BONDS - exist between two non-polar molecules 4. VAN DE WAELS FORCES - exist between electrons of Ag & Ab * LAW OF MASS ACTION - states that “Ag & Ab reaction are reversible” Ag + Ab ↔ Ag-Ab complex - there should be equilibrium so we can compute for K ↑ K1 = ↑ [Ag-Ab] = ↓[Ag] = ↓ [Ab] = ↓ K2 = ↑K

K = K1 . = [ Ag-Ab ] K2 [Ag] [Ab] * AGGLUTINATION / PRECIPITATION CURVE

* LATTICE FORMATION - by Marrack - network / crosslink of immune complexes - can be formed in Equivalent Zone but can’t be found in Pro Zone & Post Zone TYPES OF SECONDARY REACTIONS: I. AGGLUTINATION - binding of particulates to Ag to form large complexes seen as “Agglutinates” > STEPS: 1. Sensitization = Ag & Ab binding 2. Lattice formation > FACTORS AFFECTING LATTICE FORMATION: 1. pH (6.7-7.2) 2. Temperature 3. Ionic strength of medium > WAYS TO ENHANCE LATTICE FORMATION: 1. Add Low Ionic Strength Saline (LISS) or 22% Bovine Serum Albumin (BSA) 2. Add Enzymes such as Bromelin, Papain, & Ficin 3. ↑ Viscosity (PEG, Polyvinyl, Pyrolidone) 4. by Centrifugation or Agitation > KINDS OF AGGLUTINATION: 1. DIRECT AGGLUTINATION - clumping of Ag that is normally present in the particle * Ex: ABO, Widal, Weil-felix * Positive (+) Result: w/ Agglutination 2. INDIRECT / PASSIVE AGGLUTINATION - clumping of Ag that is attached to a carrier * Ex: Latex, Charcoal, RBC - Ag is not normally present in the particle * Unknown: Ab * Ex: Slide-Agglutination, ASO, RF, SLE * Positive (+) Result: w/ Agglutination

3. REVERSE INDIRECT / PASSIVE AGGLUTINATION - clumping of Ab that is attached to a carrier - Ab is not normally present in the particle * Ex: C-Reactive Protein (CR-P) test * Positive (+) Result: w/ Agglutination 4. AGGLUTINATION-INHIBITION 1st step: Patient’s soluble Ag + Ab 2nd step: Add particulate Ag * Positive (+) Result: w/o Agglutination 5. CO-AGGLUTINATION - the carrier used is a bacteria * Ex: Protein-A of S.aureus * Positive (+) Result: w/ Agglutination 6. ANTIGLOBULIN-MEDIATED AGGLUTINATION / COOMB’S TEST a. DIRECT ANTIGLOBULIN TEST (DAT) - in vivo sensitization of RBC b. INDIRECT ANTIGLOBULIN TEST (IAT) - in vitro sensitization of RBC II. PRECIPITATION - consist of soluble Ag & soluble Ab to form insoluble complex > PRECIPITATION IN LIQUID: 1. TURBIDIMETRY - amount of cloudiness in a solution - results to ↓ light intensity due to: light scattering, light reflection, & light absorption 2. NEPHELOMETRY - amount of the light scattered when it passes to a solution - usually @ 10-70 degrees * Ex: IgG, IgM, IgD, IgE, C4, C2, C1 inhibitor, RF a. KINETIC - measures light scattering before the equivalent zone b. ENDPOINT - wait for the equivalent zone first before measuring the light scattering > PRECIPITATION IN GEL: - we used Agar or Agarose because it’s matrix is neutral - with 0.3-1.58% > FACTORS AFFECTING PRECIPITATION IN GEL: 1. Size of particles 2. Temperature 3. Gel viscosity

4. Amount of Hydration 5. Interaction between matrix & reactants * STANDARD CURVE - used to measure the exact amount of Ag a. ENDPOINT / MANCINI METHOD - read the diameter of the ring using vernier caliper after 48hrs, then square it - plot the curve in a graphing paper b. KINETIC / FAHEY & MCKELVEY METHOD - measure the diameter of the ring using vernier caliper after 18hrs, but you don’t have to square it - plot the curve in a semilog paper > KINDS OF PRECIPITATION IN GEL: 1. PASSIVE IMMUNODIFFUSION - you just let Ag & Ab across the gel on their unknown > SOURCES OF ERRORS: - Underfilling or Overfilling of the holes/wells - Incorrect incubation of time & temperature - Spilage of sample over the gel - Specific contamination a. SINGLE DIFFUSION - by: Oudin - only one diffuses the gel (Ag) - establishes relationship with Ag & Ab b. RADIAL IMMUNIDIFFUSION (RID) - modified single diffusion test * Reagent: Ab (incorporated to the gel) * Unknown: Ag * Process: 1. Add the px’s sample in the holes called “wells” 2. Incubate 3. Once the Ag & Ab meet @ the equivalent zone, they will form a precipitin ring * Positive (+) result: Precipitin ring c. DOUBLE DIFFUSION IN 1 DIMENSION - by: Oakley & Fulthorpe - determines the relationship of single Ag & single Ab - both Ag & Ab diffuses d. DOUBLE DIFFUSION IN 2 DIMENSIONS / OUCHTERLONY TECHNIQUE - by: Ouchterlony - determines the relationship of two or more Ag * Process: 1. Add the px’s sample w/ Ag on the small wells (the Ab is placed on the central well) 2. Incubate for 12-48hrs

3. When Ag & Ab meet at the Equivalent Zone, it will form Precipitin line * Positive (+) Results: Precipitin lines > Line of Identity: Smooth arch > Line of Partial Identity: Spur > Line of Non-Identity: Cross line 2. ROCKET IMMUNOELECTROPHORESIS / LAUREL IMMUNOELECTROPHORESIS - by: Laurel - known as “One Dimension Electroimmunodiffusion” - radial immunodiffusion (RID) but w/ applied electric current * Application: used for measurement of Ab & complement in other fluids * Reagent: Ab * Unknown: Ag * Process: 1. Add the Px’s sample 2. Run a standard curve in different concentration 3. Buffer ph: 8.6 4. When Ag & Ab meet at the equivalent zone, they form a rocket-shaped line * Positive (+) result: Rocket-shaped lines 3. IMMUNOELECTROPHORESIS - by: Grabar & Williams - double diffusion but w/ applied electric current * Application: Used to detect Immunodeficiency & immunoproliferative diseases * Reagent: Ab * Unknown: Ag * Process: 1. Add the Px’s sample to the holes called “Through” 2. Incubate for 18-24hrs 3. When the Ag & Ab meet at the equivalent zone, it will form Precipitin Arch 4. Compare the standard & px’s sample in terms of location, intensity & Shape * Positive (+) result: Precipitin arch 4. COUNTERCURRENT IMMUNOELECTROPHORESIS - double diffusion but w/ applied voltage * pH: 8.6 * Application: Used to detect Autoantibodies to infectious agents

5. IMMUNOFIXATION ELECTROPHORESIS - by: Alfer & Johnson - allows the reagent Ag to be separated in the gel through electrophoresis - modification of this process is “Westernblot” * Process: 1. Add Px’s sample directly on the surface of the gel 2. When the Ag & Ab meet at the equivalent zone, Immunoprecipitates will be formed * Positive (+) result: Precipitin bond > SOURCES OF ERRORS: - Application of current in the wrong direction - Incorrect pH of the buffer - Excess Ag or Ab - Amount of current is weak or too strong

SEROLOGIC TECHNIQUES: PRIMARY REACTIONS * LABELLED IMMUNOASSAYS - also known as “Receptor Ligand Assay” - tests used to detect 1° infection * RECEPTOR: Ab used in test * LIGAND: substance being measured CONSTITUENTS OF LABELLED IMMUNOASSAY: 1. Labelled & Unlabelled Analyte 2. Receptor 3. Standards & Calibrators 4. Means of separating bound from free analyte 5. ↑ of detecting a label 2 GROUPS OF IMMUNOASSAY: 1. HOMOGENOUS - no separation method (no washing) - easier to perform but less sensitive 2. HETEROGENOUS - separates bound from free reactant - there is separation method (washing) SEPERATION METHOD: 1. Centrifugation / Filtration 2. Absorption into particles like talc, silica cellulose, & dextran-coated charcoal 3. Precipitation of immune complexes using (Nh4)2SO4, Ethanol, PEG 4. Use of Precipitating Ab/2nd/Sandwich - Ex: Anti-human globulin (AHG) 5. Use of solid-phase vehicles - Ex: Microtiter plate Nitrocellulose membrane Glass/Magnetic/Polystyrene tube or bead QUALITY CONTROL: * CONTROL - to check for quality of rgt - to check for procedural errors a. Negative control b. Positive control * BLANK - to check for non-specific absorption - to check for unadequate washing

* BACKGROUND - any readings indicative of the presence of label in blank * CUT-OFF VALUE - will serve as comparison between control & sample TYPES OF LABELLED IMMUNOASSAY: I. ENZYME IMMUNOASSAY (EIA) - label: Enzymes * Ex: HRP, ALP, G6-PD, β-Galactoside * Advantages: cheap, readily available, long shelf life - uses K. gluteraldehyde + Maleimide derivative > KINDS OF ENZYME IMMUNOASSAY: ** HETEROGENOUS EIA ** 1. INDIRECT/NON-COMPETITIVE ELISA - uses solid phase Ag coated with Ag * Unknown: Ab * Procedure: 1. Add Px’s sample 2. Incubate then wash 3. Add Ab conjugate (Enzyme labelled AHG) 4. Incubate then wash 5. Add substrate chromogen 6. Add stop solution (0.1 N Sulfuric Acid) 7. Measure absorbance/optical density using ELISA reader “The amount of color produced is directly proportional to the amount of Ag.” 2. DIRECT / SANDWICH ELISA - uses solid phase Ab - detects Ag of—Giardia, Cryptosporidium, Rota virus in stool, & Respiratory Synctial Virus * Unknown: Ag * Disadvantage: Hook effect * Procedure: 1. Add px’s sample (Ag) 2. Incubate then wash 3. Add Ab conjugate (enzyme labeled Ab) 4. Sandwich is formed 5. Add substrate chromogen 6. Add Stop solution (0.1 N Sulfuric Acid) 7. Measure absorbance using ELISA reader “The amount of color produced is directly proportional to the amount of Ag.”

3. COMPETITIVE ELISA - uses solid phase Ab - detects Ag of—Estrogen, Insulin * Unknown: Ag * Procedure: 1. Add Px’s sample (Ag unlabelled) 2. Add Enzyme labelled Ag (part of rgt) 4. Incubate then wash 5. Add substrate chromogen 6. Add stop solution (0.1 N Sulfuric Acid) 7. Measure absorbance/optical density using ELISA reader “The amount of color produced is inversely proportional to the amount of Ag.” 4. MEMBRANE-BASED CASSETTE ASSAY - also known as “Capture Assay” - single used disposable assay in plastic cartilage * Principle: Immunochromatography * Filter paper: Nitrocellulose membrane * Test area: coated with Ab * Control area: coated with Anti-Ig ** HOMOGENOUS EIA ** 5. ENZYME MULTIPLIED IMMUNOASSAY TECHNIQUE - follow competitive form of ELISA - uses solid phase Ab * Unknown: Ag * Procedure: 1. Add Px’s sample (Ag) 2. Add Enzyme labeled Ag 3. Incubate 4. Add substrate chromogen 5. Add stop solution (0.1 N Sulfuric Acid) 6. Read the absorbance/optical density “The amount of color produced is directly proportional to the amount of Ag.” 6. CLONED ENZYME DONOR IMMUNOASSAY - 1st manufactured using recombinant unit * Enzyme: β-galactosidase * Unknown: Ag “The amount of color produced is directly proportional to the amount of Ag.”

II. IMMUNOFLUORESCENCE - label: Fluorescent Dye, Fluorochrome, Fluorophores * Ex: Lucifer yellow, Phycoerythrin, & Europium * FITC: green @ 517-520 nm * TMR: red @580-585 nm > KINDS OF IMMUNOFLUORESCENCE: 1. DIRECT IMMUNOFLUORESCENCE - detects Ag of—Legionella, Pneumosistis, Toxoplasma, & Chlamydia * Specimen: Px’s tissue (via biopsy) * Unknown: Ag * Procedure: 1. Coat/attach px’s tissue (Ag) 2. Add fluorescent-labelled Ab 3. Incubate then wash 4. Read for fluorescence 2. INDIRECT IMMUNOFLUORESCENCE * Unknown: Ab * Procedure: 1. Coat/attach px’s tissue (Ab) 2. Incubate then wash 3. 2. Add fluorescent-labelled AHG 4. Incubate then wash 5. Read for fluorescence 3. SANDWICH IMMUNOFLUORESCENCE * Disadvantage: QUENCHING— decrease in fluorescence due to non-specific absorption Px’s unlabelled + Known Ag coated + Fluorescent Ab on the slide labelled Ab

III. CHEMILUMINESCENCE - label: Chemiluminescent tags/probes * Ex: Luminol, Acridine ester, Dioxthane, Nitrphenyl Phosphate, Rutherium derivatives, Peryoxalates - amount of light produced as a result of chemical reaction IV. RADIOIMMUNOASSAY - label: Radioactive isotopes (emits radiation) > Ex: 3H, 123I, 131I - very specific but test but also toxic > KINDS OF RADIOIMMUNIASSAY: ** HETEROGENOUS RIA ** 1. COMPETITIVE RIA - original RIA discovered by Rosalyn Yalow a. CONTROL SYSTEM - determines radiolabelled Ag - determines the ratio of the bound to the free

b. TEST SYSTEM - determines the unknown in the test - determines the control & the px’s sample 2. RADIOIMMUNOSORBENT TEST (RIST) - a sandwich phase RIA - uses solid phase IgE - measures the total IgE * Procedure: 1. Add Px’s sample (IgE) 2. Add radiolabelled anti-IgE 3. Incubate then wash 4. Measure radioactivity using Scintillation counter 3. RADIOALLERGOSORBENT TEST (RAST) - an indirect RIA - used solid phase Ag of a specific thing - measures Ag-specific IgE ** HOMOGENOUS RIA ** 4. IMMUNORADIOMETRIC ASSAY (IRMA) - a competitive RIA - uses radiolabelled Ab on excess * Unknown: Ag with magnetic bead * Procedure: 1. Add px’s sample (Ag) 2. Add solid phase Ag 3. Incubate 4. Centrifuge 5. Measure radioactivity @ supernatant “The amount of color produced is directly proportional to the amount of Ag.”...