Immuno Lab

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
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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

Immunochromatographic assay
2 phases
1. Stationary phase
2. Mobile phase
 Running buffer / assay diluent
Drawing

 Sample port/sample pad – where you add serum

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

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