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IMMUNE 1 LEC > CHAPTER 6 – IMMUNOASSAYS > Flashcards

CHAPTER 6 – IMMUNOASSAYS Flashcards

1
Q

Designed for antigens and antibodies that may be small in size or present in very low concentrations.

A

INDICATOR LABELLED IMMUNOASSAYS

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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.

A

labeled reactant

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3
Q

Labels:

A

Fluorescent
Radionuclide (Radioisotopes)
Enzymes
Free radical
Ferritin

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4
Q

Utilzes fluor or fluorochrome

A

FLUORESCENCE IMMUNOASSAYS

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5
Q

Have the ability to absorb light at shorter wavelenghts (infrared light) and emit lgiht waves at a visible spectrum/longer wavelengths (UV light)

A

FLUORESCENCE IMMUNOASSAYS

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6
Q

Covalently linked to Ig or to antiglobin to detect the presence of Ag or Ab.

A

FLUORESCENCE IMMUNOASSAYS

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7
Q

Examples:

Fluoroscein Isothiocyanate (FITC) with fluorescence of color
Tetramethyl rhodamine(TRITC) with fluorescence of color

A

green

red

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8
Q

(direct = Ag+Ab)

A

Ig

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9
Q

(indirect = Ag+Ab+carrier)

A

antiglobin

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10
Q

RADIOIMMUNOASSAYS (RIA) General Uses

A
  1. Monitoring levels of hormone
  2. Detection of vitamins
  3. Detection of viral antigens
  4. Detection of therapeutic and abused drugs such as digoxin, opiates, barbiturates and amphetamine.
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11
Q

RIA DISADVANTAGE

A
  1. Exposure to radiation
  2. Use of sophisticated machine
  3. Disposal of radioisotope waste (burial underground)
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12
Q

RIA ADVANTAGE

A
  1. Extremely sensitive
  2. Relative ease of development
  3. Relatively low cost (FALSE: it is expensive)
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13
Q

most sensitive of all immunoassays

A

RIA

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14
Q

oldest immunoassay (discovered by Rosalyn Yalow)

A

RIA

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15
Q

Radioactive Labels

A
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16
Q

– more automated tests

A

Immunoassays

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17
Q

Emit gamma radiation

A
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18
Q

Emit beta radiation

A

Beta Emitters

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19
Q

Iodine25I and Iodine 311

A

Gamma Emitters

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20
Q

3H (Tritium)

A

Beta Emitters

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21
Q

Uses solid/crystal scintillation counter for measurement

A

Gamma Emitters

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22
Q

Measured with liquid scintillation counter

A

Beta Emitters

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23
Q

Most commonly used

A

Gamma Emitters

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24
Q

• Naturally occurring molecules

A

ENZYME

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25
Q

Catalyze certain biochemical reactions; React with suitable substrates to produce products

A

ENZYME

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26
Q

ENZYME EXAMPLES

A

Chromogenic (change in color), fluorogenic or luminescent

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27
Q

More on measurement of absorbance of the color emitted

A

Chromogenic

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28
Q

• Cheap, readily available and has long shelf life

A

ENZYME LABELS

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29
Q

• Can be used quantitatively (can detect the actual concentration of the analyte) and qualitatively (detect the presence or absence of antibody)

A

ENZYME LABELS

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30
Q

TYPICAL ENZYMES USED

A

• Horseradish peroxidase
• Glusoce-6-Phosphate-Dehydrogenase
• Alkaline phosphatase
• Beta-D-Galactosidase

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31
Q
  • Has the highest turnover rate
A

Horseradish peroxidase
Alkaline phosphatase

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32
Q

Easy to detect

A

Horseradish peroxidase

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33
Q

Often used in EIA together w/ ALP

A

Horseradish peroxidase

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34
Q

Horseradish peroxidase AKA

A

Malunggay

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35
Q

Enzyme Assays

A
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36
Q

Requires steps to physically separate free from bound analyte (washing required)

A

HETEROGENOUS ENZYME IMMUNOASSAY

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37
Q

No separation step is necessary (no washing required)

A

HOMOGENOUS ENZYME IMMUNOASSAY

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38
Q

Enzyme activity diminishes when binding of an antibody and antigen occurs

A

HOMOGENOUS ENZYME IMMUNOASSAY

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39
Q

TYPES OF FIA

A
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40
Q

TYPES OF RIA

A
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41
Q

TYPES OF EIA

A
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42
Q

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

A
  1. Direct Immunofluorescent Assays
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43
Q

Examples: Fluorescent Ab Dark Field Technique for Treponema pallidum

A
  1. Direct Immunofluorescent Assays
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44
Q

Antihuman globulin is combined with patient cells that have become coated with antibody in vivo.

A
  1. Direct Immunofluorescent Assays
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45
Q

Reagent cells are reacted with patient antibody. These are washed, and then antihuman globulin is added to enhance agglutination.

A
  1. Indirect Immunofluorescent Assay
46
Q

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.

A
  1. Indirect Immunofluorescent Assay
47
Q

Example: FTA-Absorption test

A
  1. Indirect Immunofluorescent Assay
48
Q

– one of the confirmatory test for syphilis

A

FTA-Absorption test

49
Q

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

A

Solid-phase Antigen

50
Q

(Fluorescent Antibody Inhibition Test)

A

Inhibition Immunofluorescence Test

51
Q

Px Ab and labelled Ab competes for the Ag binding site

A
  1. Inhibition Immunofluorescence Test
52
Q

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

A
  1. Inhibition Immunofluorescence Test
53
Q

Addition of Ag: free Ag (incapable of binding)

A

(+) due to presence of Ab

54
Q

Addition of labelled Ab: free Ag (capable of binding)

A

(-) due to absence of Ab

55
Q

Antibody is capable of binding to complement due to the Fc portion

A
  1. Complement staining IF Test
56
Q

Complement attaches to the Fc portion of the Ab

A
  1. Complement staining IF Test
57
Q

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

A
  1. Complement staining IF Test
58
Q

(+) due to presence of Ab and complement

A
  1. Complement staining IF Test
59
Q

Measures polarization of light

A
  1. Fluorescence Polarization Immunoassay
60
Q

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

A
  1. Fluorescence Polarization Immunoassay
61
Q

Uses gel particles to show fluorescence

A
  1. Solid Phase Fluorescence Immunoassay
62
Q

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

A
  1. Solid Phase Fluorescence Immunoassay
63
Q

Gel particles can fluoresce

A
  1. Solid Phase Fluorescence Immunoassay
64
Q

Principle: competitive binding assay

A
  1. Classic RIA Competitive Analysis
65
Q

AKA: Displacement/Radioligand Inhibition

A
  1. Classic RIA Competitive Analysis
66
Q

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

A
  1. Classic RIA Competitive Analysis
67
Q

High or reduced?

Very little patient antigen is present, making radioactivity of the solid phase [?].

68
Q

High or reduced?

69
Q

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.

70
Q

to remove the unbound Ag - to prevent false negative results

A

Wash with saline

71
Q

A or B?

More labelled Ag: occupies more binding sites

72
Q

A or B?

Equal labelled and unlabelled antigen

73
Q

Addition of Ag: free Ag (capable of binding)

A
  1. Non-competitive Assays
74
Q

Addition of labelled Ab (doesn’t compete w/ Ag, only attaches)

A
  1. Non-competitive Assays
75
Q

Examples: IRMA: Radioimmunometric assay

A
  1. Non-competitive Assays
76
Q

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

A
  1. Non-competitive Assays
77
Q

Principle: Competitive Binding Assay

A
  1. Indirect Radioimmunosorbent Assay
78
Q

Anti-IgE (Reagent) +Patient IgE + Radiolabeled IgE → More Anti IgE+Patient IgE complex hence, higher Patient IgE = No radioactivity (vice versa)

A
  1. Indirect Radioimmunosorbent Assay
79
Q

Competition between px IgE and labelled IgE for the binding sites on the solid Ab

A
  1. Indirect Radioimmunosorbent Assay
80
Q

↑Px IgE = ↓ Radioacivity

A
  1. Indirect Radioimmunosorbent Assay
  2. Radioprecipitation Test (RIP)
81
Q

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)

A
  1. Indirect Radioimmunosorbent Assay
82
Q

Directly proportional

A
  1. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
83
Q

Measures total IgE

A
  1. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
84
Q

Px IgE binds to solid-phase anti-IgE

A
  1. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
85
Q

Binding sites are already occupied by Px IgE

A
  1. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
86
Q

Addition of labelled IgE (2nd rgt) will not compete w/ Px IgE, instead attaches itself to the Ag-Ab complex

A
  1. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
87
Q

↑Px IgE = ↑Radioacivity

A
  1. Direct Noncompetitive Binding Radioimmunosorbet Test (RIST)
88
Q

Principle: Competitive binding assay

A
  1. Radioprecipitation Test (RIP)
89
Q

Competition between px IgE and labelled IgE for the binding sites on the rabbit anti-human IgE

A
  1. Radioprecipitation Test (RIP)
90
Q

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

A
  1. Radioprecipitation Test (RIP)
91
Q

HETEROGENOUS ENZYME IMMUNOASSAY

92
Q

Principle: competitive binding assay

A
  1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
93
Q

Enzyme activity if inversely proportional to the concentration of test substance

A
  1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
94
Q

AKA: Displacement/Radioligand Inhibition

A
  1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
95
Q

Typically used for measuring relatively small antigens (insulin, estrogen)

A
  1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
96
Q

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

A
  1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
97
Q

Mostly used EIA because of its sensitivity, specificity, and low cost

A
  1. Noncompetitive Enzyme Immunoassay
98
Q

Often referred to as indirect enzyme-linked immunosorbent assays (ELISA)

A
  1. Noncompetitive Enzyme Immunoassay
99
Q

Enzyme-labelled reagent does not participate in the initial antigen–antibody binding reaction

A
  1. Noncompetitive Enzyme Immunoassay
100
Q

Measure antibody production to infectious agents that are difficult to isolate

A
  1. Noncompetitive Enzyme Immunoassay
101
Q

Preferred screening test for detecting antibody to HIV, Hepatitis A and Hepatitis C

A
  1. Noncompetitive Enzyme Immunoassay
102
Q

Antigen in solid-phase

A
  1. Noncompetitive Enzyme Immunoassay
103
Q

Patient antibody is incubated with solid-phase antigen.

A
  1. Noncompetitive Enzyme Immunoassay
104
Q

After a wash step, to prevent false (-), enzyme-labeled antiimmunoglobulin is added.

A
  1. Noncompetitive Enzyme Immunoassay
105
Q

Does not compete with the binding site of antigen; only participates in the reaction

A
  1. Noncompetitive Enzyme Immunoassay
106
Q

This will bind to the patient antibody on solid phase.

A
  1. Noncompetitive Enzyme Immunoassay
107
Q

Binding to the Ag-Ab reaction on the fc-portion of the Ab

A
  1. Noncompetitive Enzyme Immunoassay
108
Q

A second wash step is performed to remove any unbound anti immunoglobulin, and substrate for the enzyme is added.

A
  1. Noncompetitive Enzyme Immunoassay
109
Q

Color development is directly proportional to the amount of patient antibody present.

A
  1. Noncompetitive Enzyme Immunoassay
110
Q

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

A
  1. Immunoenzymometric Test
111
Q

Used to stoped the reaction from further activation

IMMUNE 1 LEC (28 decks)

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