Primary Immunologic Reaction - Ag immobilized in assay plate then add labelled Ab
- Initial reaction that happen, initial phase fluorescent Ab/ enzyme labelled Ab direct - Moment of recognition & binding of Ag & Ab detection method - Purpose: usually carried out in clinical lab if conc. Of - Usually detects Ag analyte is too low/ too small - Can be direct, indirect, sandwich method, don’t have Indirect Detection inhibition reaction - Usually detects Ab - 2nd Ab (‘detector Ab) that will bind to 1st Ab in order 4 Types of Primary Reaction to be recognized – for the detection of the complex 1. Radioimmunoassay - Serves as detector Ab 2. Enzyme Immunoassay - Tagged with label 3. Immunofluorescence assay – there is inhibition, - Specific for primary commonly sandwich method 4. Chemiluminescence assay Sandwich assay - Capture procedures - Primary test: has the most sensitive and specific lab - 2 Antigenic sides are occupied with 2 types of Ab test to determine analytes - 2 types of Ab detection Ab - tagged with indicator label to Components of Primary Test (Ag - Ab rxn) establish identity of complex 1. Labelled & unlabeled analyte capture Ab – capture the complex, take up 2. Receptor molecule – known molecule for complex qualitative - Non competitive 3. Std./calibrator soln. – for proper monitoring, conc close to an analyte Radioimmunoassay 4. means to separate the free from the bound - Analyte – tagged with I125 components - Unlabeled analyte – comes from patients’ Ab 5. Means to measure the reporter molecule Whoever has most no of molecules = ↑ activity of (labelled product) binding capacity - Indicator label – used to probe Ag – Ab complex, - Competitive facilitate visual detection of complex - Measured in Ab bound to radiolabel - AKA ‘labelled immunoassay’ procedure - Conc of unknown analyte is Inversely proportional ligand analyte (subs to be detected) in relation to the amt of radioactivity emitted receptor captures analyte (known moleclues - conc of unknown/unlabelled analyte = radioact determines/establish identity to quantitative and emitted qualitative assay of anaylte) A. RIST Assay - AKA receptor – ligand assay - Radioimmunosorbent assay Heterogenous assay – mostly carried out by these - Purpose: Measure total IgE assay - Paper disk makes use of separation method that will Purpose: Separation medium discriminate free from bound Heterogenous Assays Indicator Label Means of Form: Solid phase medium Measurement of Where anti-IgE is attached labelled Paper disk + Anti IgE = solid phase Ab product/analyte RadioIA Radioisotope (I125, Gamma counter/ Paper disk + pt IgE = solid phase+radiolabelled H+3, C12) scintillation anti-IgE (to detect the complex ‘detector Ab’, counter bound to IgE) - Sandwich assay: unknown directly proportional to Enzyme IA Enzyme (*Horse ELISA reader radiolabelled produced Radish - Principle of B. RAST Assay Peroxidase/ measurement - Radioallergosorbent assay Alkaline of spectrophotometry - Purpose: Measure specific IgE Phosphatase) - Indirect assay Immunofluor Fluorescence dye Microscopy, spectrophotometry - Directly proportional (FITC, Rhodamine, - Non competitive auramine) - Ragweed pollen attached to solid phase medium Chemiluminesc Chemiluminescent Luminometer (solid phase Ag) probe (luminal) (same principle Enzyme Immunoassay with spectro) - For any ELISA procedures, follows basic platform Basic Platform Direct Detection 1. Solid phase Ag/Ab - Uses primary Ab – Ab specific for analyte, directly reacting to unlabeled Analyte/ Ag CMYS Immuno Lab 2. Control sera, calibrator/std soln./unknown serves as procedural control monitors sample efficiency of test procedure & test device 3. Enzyme conjugate Assay buffer/ The running buffer – facilitate 4. Substrate chromagen movement of Sx 5. Stop soln. - Microtiter well = solid phase medium, heterogenous, put the Ag - Microtiter well + recombinant protein of Dengue virus = solid phase Ag Each plate is coated - Procedure 1. Put control, calibrator, std soln, sample 2. Incubate (1hr) Purpose: if it has Ab against virus then it will coat allowing rxn of Ag and Ab 3. Wash Purpose: eliminate untreated component, to focus on specific components in order not to interfere 4. Add enzyme conjugate (2nd Ab complexed with indicator label which is an enzyme label e.g. HRP) Purpose: to know if rxn has formed 5. Incubate 6. Wash 7. Add substrate chromogen If substrate is HRP, usually preoxides acts on hydrogen peroxidase Chromogen – Tetramethylbenzidine 8. Incubate 9. When Ab that is conjugated with enzyme, enzyme acts on substrate, if enzyme acts on substrate = (+) blue coloration 10. Incubation of substrate chromogen 11. Add stop soln – bc kinetic rxn 12. Measure analyte – spectrophotometry / ELISA reader Intensity of the colored product with the conc of analyte = directly proportional
sx/ whole blood Reaction port – test area, control area Conjugate pad – holds detector rgt, conjugate to analyte, usually have indicator label colloidal gold – ‘solid phase conjugate IA’ - All are membrane bound - Principle: Lateral flow – most of the assay, straight only Transverse flow – upon addition, flow stops Control area – has anti-detector agent excess goes here