CHAPTER 6 – IMMUNOASSAYS Flashcards
(111 cards)
1
Q
Designed for antigens and antibodies that may be small in size or present in very low concentrations.
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INDICATOR LABELLED IMMUNOASSAYS
2
Q
The presence of such antigens or antibodies is determined indirectly by using a [?] to detect whether or not specific binding has taken place.
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labeled reactant
3
Q
Labels:
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Fluorescent
Radionuclide (Radioisotopes)
Enzymes
Free radical
Ferritin
4
Q
Utilzes fluor or fluorochrome
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FLUORESCENCE IMMUNOASSAYS
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Q
Have the ability to absorb light at shorter wavelenghts (infrared light) and emit lgiht waves at a visible spectrum/longer wavelengths (UV light)
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FLUORESCENCE IMMUNOASSAYS
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Q
Covalently linked to Ig or to antiglobin to detect the presence of Ag or Ab.
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FLUORESCENCE IMMUNOASSAYS
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Q
Examples:
Fluoroscein Isothiocyanate (FITC) with fluorescence of color
Tetramethyl rhodamine(TRITC) with fluorescence of color
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green
red
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Q
(direct = Ag+Ab)
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Ig
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Q
(indirect = Ag+Ab+carrier)
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antiglobin
10
Q
RADIOIMMUNOASSAYS (RIA) General Uses
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- Monitoring levels of hormone
- Detection of vitamins
- Detection of viral antigens
- Detection of therapeutic and abused drugs such as digoxin, opiates, barbiturates and amphetamine.
11
Q
RIA DISADVANTAGE
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- Exposure to radiation
- Use of sophisticated machine
- Disposal of radioisotope waste (burial underground)
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Q
RIA ADVANTAGE
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- Extremely sensitive
- Relative ease of development
- Relatively low cost (FALSE: it is expensive)
13
Q
most sensitive of all immunoassays
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RIA
14
Q
oldest immunoassay (discovered by Rosalyn Yalow)
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RIA
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Q
Radioactive Labels
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16
Q
– more automated tests
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Immunoassays
17
Q
Emit gamma radiation
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18
Q
Emit beta radiation
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Beta Emitters
19
Q
Iodine25I and Iodine 311
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Gamma Emitters
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Q
3H (Tritium)
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Beta Emitters
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Q
Uses solid/crystal scintillation counter for measurement
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Gamma Emitters
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Q
Measured with liquid scintillation counter
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Beta Emitters
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Q
Most commonly used
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Gamma Emitters
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Q
• Naturally occurring molecules
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ENZYME
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Catalyze certain biochemical reactions; React with suitable substrates to produce products
ENZYME
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ENZYME EXAMPLES
Chromogenic (change in color), fluorogenic or luminescent
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More on measurement of absorbance of the color emitted
Chromogenic
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• Cheap, readily available and has long shelf life
ENZYME LABELS
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• Can be used quantitatively (can detect the actual concentration of the analyte) and qualitatively (detect the presence or absence of antibody)
ENZYME LABELS
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TYPICAL ENZYMES USED
• Horseradish peroxidase
• Glusoce-6-Phosphate-Dehydrogenase
• Alkaline phosphatase
• Beta-D-Galactosidase
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- Has the highest turnover rate
Horseradish peroxidase
Alkaline phosphatase
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Easy to detect
Horseradish peroxidase
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Often used in EIA together w/ ALP
Horseradish peroxidase
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Horseradish peroxidase AKA
Malunggay
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Enzyme Assays
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Requires steps to physically separate free from bound analyte (washing required)
HETEROGENOUS ENZYME IMMUNOASSAY
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No separation step is necessary (no washing required)
HOMOGENOUS ENZYME IMMUNOASSAY
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Enzyme activity diminishes when binding of an antibody and antigen occurs
HOMOGENOUS ENZYME IMMUNOASSAY
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TYPES OF FIA
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TYPES OF RIA
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TYPES OF EIA
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Procedure:
1. Ag from patient sample fixed to a microscopic slide
2. Fluorescent labelled Ab added
3. Ab binds to Ag and becomes fixed to slide
4. Washing to remove unattached Ab
5. Positive test: Fluorescence
1. Direct Immunofluorescent Assays
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Examples: Fluorescent Ab Dark Field Technique for Treponema pallidum
1. Direct Immunofluorescent Assays
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Antihuman globulin is combined with patient cells that have become coated with antibody in vivo.
1. Direct Immunofluorescent Assays
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Reagent cells are reacted with patient antibody. These are washed, and then antihuman globulin is added to enhance agglutination.
2. Indirect Immunofluorescent Assay
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Procedure
1. Ag spread on slide and patient serum (Ab) added and incubated and washed
2. Fluorescent antihuman globulin added incubated and washed
3. If the patient serum contained the corresponding Ab it would be fixed to the Ag on the slide
4. Tagged reagent reacts with the Ab fixed to the Ag.
2. Indirect Immunofluorescent Assay
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Example: FTA-Absorption test
2. Indirect Immunofluorescent Assay
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– one of the confirmatory test for syphilis
FTA-Absorption test
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Which reactant of Indirect Immunofluorescent Assay is being asked?
attached on a slide or in a wall/microtiter plate; no fluorescence yet; no competition; 2nd rgt only participates in the reaction
Solid-phase Antigen
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(Fluorescent Antibody Inhibition Test)
Inhibition Immunofluorescence Test
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Px Ab and labelled Ab competes for the Ag binding site
3. Inhibition Immunofluorescence Test
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Procedure:
1. The Ag is fixed to the slide and then flooded with patient serum
2. If corresponding Ab is present in the serum, it will attach/fix to the Ag
3. When specific fluorescent Ab reagent is added it cannot become fixed because all of the antigenic sites are already occupied by the unlabeled patient Ab.
4. Labelled reagent washed off
5. Positive test: No fluorescence
3. Inhibition Immunofluorescence Test
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Addition of Ag: free Ag (incapable of binding)
(+) due to presence of Ab
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Addition of labelled Ab: free Ag (capable of binding)
(-) due to absence of Ab
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Antibody is capable of binding to complement due to the Fc portion
4. Complement staining IF Test
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Complement attaches to the Fc portion of the Ab
4. Complement staining IF Test
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Procedure:
1. Patient serum and complement mixed with reagent Ag fixed on the slide
2. Incubate and wash
3. Fluorescent labelled anticomplement is added
4. Positive: Fluorescence
4. Complement staining IF Test
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(+) due to presence of Ab and complement
4. Complement staining IF Test
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Measures polarization of light
5. Fluorescence Polarization Immunoassay
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Procedure:
1. Patient serum and complement mixed with reagent Ag fixed on the slide
2. Incubate and wash
3. Fluorescent labelled anticomplement is added
4. Positive: Fluorescence
5. Fluorescence Polarization Immunoassay
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Uses gel particles to show fluorescence
6. Solid Phase Fluorescence Immunoassay
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Procedure:
1. Patient serum and complement mixed with reagent Ag fixed on the slide
2. Incubate and wash
3. Fluorescent labelled anticomplement is added
4. Positive: Fluorescence
6. Solid Phase Fluorescence Immunoassay
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Gel particles can fluoresce
6. Solid Phase Fluorescence Immunoassay
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Principle: competitive binding assay
1. Classic RIA Competitive Analysis
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AKA: Displacement/Radioligand Inhibition
1. Classic RIA Competitive Analysis
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Procedure:
1. Antigen in patient serum compete with antigen labelled isotope for an Ab binding site
2. Free antigen is separated
3. Bound phase and free phase in quantitated
4. Positive: No radioactivity
1. Classic RIA Competitive Analysis
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High or reduced?
Very little patient antigen is present, making radioactivity of the solid phase [?].
high
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High or reduced?
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High or reduced?
More patient antigen is present, and the radioactivity of the solid phase is [?] in proportion to the amount of patient antigen bound.
reduced
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to remove the unbound Ag - to prevent false negative results
Wash with saline
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A or B?
More labelled Ag: occupies more binding sites
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A or B?
Equal labelled and unlabelled antigen
B
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Addition of Ag: free Ag (capable of binding)
2. Non-competitive Assays
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Addition of labelled Ab (doesn’t compete w/ Ag, only attaches)
2. Non-competitive Assays
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Examples: IRMA: Radioimmunometric assay
2. Non-competitive Assays
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Procedure:
1. Patient serum and complement mixed with reagent Ag fixed on the slide
2. Incubate and wash
3. Fluorescent labelled anticomplement is added
4. Positive: Fluorescence
2. Non-competitive Assays
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Principle: Competitive Binding Assay
3. Indirect Radioimmunosorbent Assay
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Anti-IgE (Reagent) +Patient IgE + Radiolabeled IgE → More Anti IgE+Patient IgE complex hence, higher Patient IgE = No radioactivity (vice versa)
3. Indirect Radioimmunosorbent Assay
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Competition between px IgE and labelled IgE for the binding sites on the solid Ab
3. Indirect Radioimmunosorbent Assay
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↑Px IgE = ↓ Radioacivity
3. Indirect Radioimmunosorbent Assay
5. Radioprecipitation Test (RIP)
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Procedure:
1. Anti-IgE is covalently coupled to a solid phase
2. A constant amount of radiolabled IgE is added and allowed to compete with IgE in patient’s sample
3. The higher the concentration of IgE in the patient’s serum. The lower the number of counts. (Inversely proportional)
3. Indirect Radioimmunosorbent Assay
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Directly proportional
4. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
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Measures total IgE
4. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
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Px IgE binds to solid-phase anti-IgE
4. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
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Binding sites are already occupied by Px IgE
4. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
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Addition of labelled IgE (2nd rgt) will not compete w/ Px IgE, instead attaches itself to the Ag-Ab complex
4. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
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↑Px IgE = ↑Radioacivity
4. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
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Principle: Competitive binding assay
5. Radioprecipitation Test (RIP)
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Competition between px IgE and labelled IgE for the binding sites on the rabbit anti-human IgE
5. Radioprecipitation Test (RIP)
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Procedure:
1. Patient IgE is mixed with rabbit anti-human IgE
2. Followed by addition of radiolabeled IgE
3. The soluble IgE-anti-IgE complex is precipitated by antirabbit globulin
4. Centrifugation washing
5. Measure radioactivity
5. Radioprecipitation Test (RIP)
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HETEROGENOUS ENZYME IMMUNOASSAY
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Principle: competitive binding assay
1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
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Enzyme activity if inversely proportional to the concentration of test substance
1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
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AKA: Displacement/Radioligand Inhibition
1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
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Typically used for measuring relatively small antigens (insulin, estrogen)
1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
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Procedure:
1. Antigen in patient serum compete with antigen labelled isotope for an Ab binding site
2. Free antigen is separated
3. Bound phase and free phase in quantitated
4. Positive: No radioactivity
1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
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Mostly used EIA because of its sensitivity, specificity, and low cost
2. Noncompetitive Enzyme Immunoassay
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Often referred to as indirect enzyme-linked immunosorbent assays (ELISA)
2. Noncompetitive Enzyme Immunoassay
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Enzyme-labelled reagent does not participate in the initial antigen–antibody binding reaction
2. Noncompetitive Enzyme Immunoassay
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Measure antibody production to infectious agents that are difficult to isolate
2. Noncompetitive Enzyme Immunoassay
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Preferred screening test for detecting antibody to HIV, Hepatitis A and Hepatitis C
2. Noncompetitive Enzyme Immunoassay
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Antigen in solid-phase
2. Noncompetitive Enzyme Immunoassay
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Patient antibody is incubated with solid-phase antigen.
2. Noncompetitive Enzyme Immunoassay
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After a wash step, to prevent false (-), enzyme-labeled antiimmunoglobulin is added.
2. Noncompetitive Enzyme Immunoassay
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Does not compete with the binding site of antigen; only participates in the reaction
2. Noncompetitive Enzyme Immunoassay
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This will bind to the patient antibody on solid phase.
2. Noncompetitive Enzyme Immunoassay
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Binding to the Ag-Ab reaction on the fc-portion of the Ab
2. Noncompetitive Enzyme Immunoassay
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A second wash step is performed to remove any unbound anti immunoglobulin, and substrate for the enzyme is added.
2. Noncompetitive Enzyme Immunoassay
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Color development is directly proportional to the amount of patient antibody present.
2. Noncompetitive Enzyme Immunoassay
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Procedure:
a. Excess amount of enzyme labelled Ab is added to patient Ag (Ag-Ab binding)
b. Formation of Ag-Ab complex
c. Next a solid phase Ag is added and absorbs unbound enzyme labelled Ab
d. Centrifuge to settle all immunosorbent particles
e. Substrate of the enzyme is added to the supernatant
f. Measure spectrophotometrically
3. Immunoenzymometric Test
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Used to stoped the reaction from further activation
SUBSTRATE
