19. Sero Techniques- Primary Reactions PDF

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IMMUNOLOGY SEROLOGY SEROLOGIC TECHNIQUES: PRIMARY REACTION LABELLED IMMUNOASSAYS also known as Ligand tests used to detect infection RECEPTOR: Ab used in test LIGAND: substance being measured will serve as comparison between control sample CONSTITUENTS OF LABELLED IMMUNOASSAY: 1. Labelled Unlabelled...

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IMMUNOLOGY & SEROLOGY

SEROLOGIC TECHNIQUES: PRIMARY REACTION - will serve as comparison between control & sample

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 reagent - to check for procedural errors BLANK - to check for non-specific absorption - to check for unadequate washing BACKGROUND - readings indicative of the presence of label in blank CUT-OFF VALUE JOYCE ANN S. MAGSAKAY

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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 EIA:  HETEROGENOUS EIA: 1. INDIRECT/NON-COMPETITIVE ELISA - uses solid phase Ag coated with Ag - Unknown: Antibody - 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 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: Antigen - Disadvantage: Hook effect - Procedure: 1. Add Px’s sample 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 BS MEDICAL LABORATORY SCIENCE |

OLFU VALENZUELA

IMMUNOLOGY & SEROLOGY

SEROLOGIC TECHNIQUES: PRIMARY REACTION - uses solid phase Ab - detects Ag of—Estrogen, Insulin - Unknown: Antigen - Procedure: 1. Add Px’s sample 2. Add Enzyme labelled Ag (part of rgt) 3. Incubate then wash 4. Add Substrate Chromogen 5. Add Stop solution (0.1 N Sulfuric Acid) 6. Measure absorbance 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: Antigen - Procedure: 1. Add Px’s sample 2. Add Enzyme labeled Ag 3. Incubate 4. Add Substrate Chromogen 5. Add Stop solution (0.1 N Sulfuric Acid) 6. Read the absorbance using ELISA reader “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: Antigen

 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: Antigen - Procedure: 1. Coat/attach Px’s tissue (Ag) 2. Add Fluorescent-labelled Ab 3. Incubate then wash 4. Read for fluorescence 3. INDIRECT IMMUNOFLUORESCENCE - Unknown: Antibody - Procedure: 1. Coat/attach Px’s tissue 2. Incubate then wash 3. Add fluorescent-labelled AHG 4. Incubate then wash 5. Read for fluorescence 4. 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

“The amount of color produced is directly proportional to the amount of Ag.”

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Kinds of Radioimmunoassay:  HETEROGENOUS RIA: IMMUNOFLUORESCENCE 1. COMPETITIVE RIA - Label: Fluorescent Dye, Fluorochrome, Fluorophores - original RIA discovered by Rosalyn Yalow - Ex: Lucifer yellow, Phycoerythrin, & Europium JOYCE ANN S. MAGSAKAY | BS MEDICAL LABORATORY SCIENCE | OLFU VALENZUELA

IMMUNOLOGY & SEROLOGY

SEROLOGIC TECHNIQUES: PRIMARY REACTION 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.”

JOYCE ANN S. MAGSAKAY

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BS MEDICAL LABORATORY SCIENCE

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OLFU VALENZUELA...