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Immunology and Serology [LAB] > Laboratory Activity 3c – Principles of Serologic Reactions > Flashcards

Laboratory Activity 3c – Principles of Serologic Reactions Flashcards

1
Q

Antibodies that aggregate cellular antigens

A

Agglutinins

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2
Q

Antibodies that form precipitates with soluble antigens

A

Precipitins

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3
Q

Antibodies that neutralize toxins

A

Antitoxins

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4
Q

Antibodies that cause dissolution of cell membrane

A

Lysins

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5
Q

Cause the destruction of RBC in the presence of the complement

A

Hemolysins

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6
Q

Cause destruction of cells of gram-negative bacteria under certain conditions

A

Bacteriolysins

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7
Q

Cause destruction of other cell types under appropriate conditions in the presence of the complement

A

Cytolysins

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8
Q

– assays involving antibody-antigen reactions are called immunoassays

A

IMMUNOASSAYS

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9
Q

soluble antibody reacts with insoluble antigen or soluble antigen reacts with an insoluble antibody

A

Agglutination reaction

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10
Q

are made soluble by combining with latex particles, RBCs, dyes or liposomes

A

Reactants

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11
Q

occurs when particles in suspension clump together due to an antibody-antigen reaction

A

Agglutination

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12
Q

Involves the interaction of antibody with a multivalent antigen (particulate): results in the cross-linking of various antigen particles by the antibody

A

Agglutination

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13
Q

Demonstrate the presence of antigen-antibody reactions by the visible aggregation of antigen-antibody complexes

A

Agglutination

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14
Q

These tests are simple to perform and are often the most sensitive test method

A

AGGLUTINATION METHODS

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15
Q

Performed with slide, tube or microtiter technique

A

AGGLUTINATION METHODS

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16
Q

Particulate antigen + antibody ® clumping

A

AGGLUTINATION

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17
Q

antigen binds with Fab sites of 2 antibodies forming bridges between antigens

A

Lattice formation

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18
Q

The process by which particulate antigens, such as cells, aggregate to form large complexes when specific antibody is present

A

Direct agglutination

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19
Q

Febrile agglutinins, Salmonella and Shigella serotyping

A

Examples of Direct agglutination

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20
Q

Antibodies + surface antigens of bacteria in suspension -> visible agglutination

A

Direct Bacterial Agglutination

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21
Q

Antibody bound to latex beads + antigen -> visible agglutination results when antigen binds to latex-bound antibody

A

Latex Agglutination

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22
Q

An antigen-antibody reaction that results in the clumping of red blood cells

A

Hemagglutination

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23
Q

One solid aggregate, clear background

A

4+

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24
Q

Several large aggregates, clear background

A

3+

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25
Q

Medium-sized agglutinates, clear background

A

2+

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26
Q

Small agglutinates, turbid background

A

1+

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27
Q

Tiny agglutinates, turbid background

A

w+

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28
Q

No agglutination or hemolysis

A

0

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29
Q

ABO typing

A

Examples of Hemagglutination

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30
Q

A reaction in which soluble antigens are bound to latex beads, bentonite, or charcoal -> the particles are agglutinated by the corresponding antibody

A

Passive agglutination

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31
Q

Rheumatoid factor

A

Examples of Passive agglutination

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32
Q

A reaction in which soluble antigens are adsorbed onto RBCs (i.e., proteins coupled to RBCs using bisdiazotized benzidine) -> RBCs are agglutinated by the corresponding antibody

A

Passive hemagglutination

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33
Q

Cold agglutinins

A

Examples of Passive hemagglutination

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34
Q

A reaction in which carrier particles coated with antibody clump together due to combination with antigen

A

Reverse passive agglutination

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35
Q

Rapid tests for identification of bacteria

A

Examples of Reverse passive agglutination

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36
Q

An agglutination reaction based on competition between particulate antigen (reagent) and soluble antigen (specimen) for limited sites on a reagent antibody

A

Agglutination inhibition

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37
Q

Detection of illicit drugs

A

Examples of Agglutination inhibition

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38
Q

A test for detecting antibodies to certain viruses that agglutinate RBCs -> in the presence of antibody, the virus is neutralized and hemagglutination does not occur

A

Hemagglutination inhibition

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39
Q

Rubella antibody

A

Examples of Hemagglutination inhibition

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40
Q

An agglutination reaction in which bacteria are used as the carrier for the antibody

A

Coagglutination

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41
Q

Rapid tests for identification of bacteria

A

Examples of Coagglutination

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42
Q

Detection of non-agglutinating antibody by coupling with 2nd antibody (antihuman globulin [AHG])

A

Antiglobulin-mediated agglutination

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43
Q

Direct and indirect antiglobulin test

A

Examples of Antiglobulin-mediated agglutination

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44
Q

Principle: Soluble antigen combines with soluble antibody to produce visible insoluble complexes

A

Precipitation

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45
Q

Clumping together of particles to form visible masses over a narrow range of antigen concentration

A

Flocculation

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46
Q

Similar with precipitation except that the precipitin appears as a fleecy mass or clump

A

Flocculation

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47
Q

Principle: Soluble antigens react with specific antibody to form precipitate of fine particles

A

Flocculation

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48
Q

Application: Venereal Disease Research Laboratory (VDRL) tests, Rapid Plasma Reagin (RPR)

A

Flocculation

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49
Q

Principle: Light scattering by immune complexes is measured -> scattering of light is proportional to the size and amount of immune complexes formed

A

Nephelometry

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50
Q

Application: Immunoglobulins, complement, C-reactive protein

A

Nephelometry

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51
Q

Measures the decrease in light intensity in a solution containing immune complexes

A

Turbidimetry

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52
Q

Principle: Measurement of light transmitted through a suspension of particles -> the formation of immune complexes decreases the amount of light passing through a suspension -> the more immune complexes formed and the larger they are, the greater the decrease in light able to pass through

A

Turbidimetry

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53
Q

Type of Diffusion

A

Single
Double

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54
Q

if only one reactant (usually antigen) is moving

A

Single

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55
Q

if both antigen and antibody are moving through the medium

A

Double

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56
Q

Type of Dimension

A

Single
Double

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57
Q

if the reaction in a medium have only one effective dimension for antigen and antibody migration (i.e., up and down)

A

Single

58
Q

if the reaction in a circular holes (i.e., wells) cut in a gel on a flat surface diffuses from the wells radially

A

Double

59
Q

Principle: Known Antibody fixed in agar + Unknown Antigen (overlaid) -> Precipitin lines

A

Single linear diffusion (SLD) or Oudin technique

60
Q

Application: Used to detect multiple antigen-antibody reactions

A

Single linear diffusion (SLD) or Oudin technique

61
Q

Principle: Known Antibody fixed in agar + Unknown Antigen (well cut in agar plate) ® Precipitin ring

A

Single radial diffusion (SRD)/ Fahey or Mancini method

62
Q

diameter of precipitin ring at 24 hours (Read before it reaches the maximum at 6-12 hours)

A

Fahey method

63
Q

48

A

Mancini method

64
Q

Principle: Antigen diffuses out of well in gel containing antigen -> Precipitin ring forms -> Diameter proportional to concentration of antigen

A

Radial immunodiffusion (RID)

65
Q

Application: Immunoglobulins, complement No longer commonly performed except for low-volume testing of IgD and IgG

A

Radial immunodiffusion (RID)

66
Q

Principle: Antigens and antibodies diffuse out from wells cut in gel and precipitin lines where they meet

A

Ouchterlony technique/Double Immunediffusion

67
Q

Ouchterlony technique/Double Immunediffusion
Three basic reaction patterns:

A

a) Identity
b) Non-identity
c) Partial identity

68
Q

a single smooth arc of precipitation forms between the antigens and antibodies

A

Identity

69
Q

two separate lines of precipitation cross each other

A

Non-identity

70
Q

two precipitating lines meet, forming a spur

A

Partial identity

71
Q

Application: Fungal antigens, extractable nuclear antigens

A

Ouchterlony technique/Double Immunediffusion

72
Q

Common errors include overfilling of wells, irregular well punching, unlevel incubation area, gel drying, increased room temperature, and antigen or antibody contamination by bacteria or fungi

A

Ouchterlony technique/Double Immunediffusion

73
Q

Principle: antigens and antibodies are placed in wells that are directly opposite one another in a gel -> an electrophoretic charge is applied to drive the reactants toward each other -> precipitin band forms where they meet

A

Countercurrent immunoelectrophoresis (CIE)

74
Q

Application: Bacterial antigens

A

Countercurrent immunoelectrophoresis (CIE)

75
Q

Principle: Proteins are separated by electrophoresis, then subjected to double diffusion with reagent antibodies placed in a trough cut in the agar ® shape, intensity, and location of the precipitin arcs develop are compared with those of a normal control

A

Immunoelectrophoresis (IEP)

76
Q

Application: Serum proteins including immunoglobulins

A

Immunoelectrophoresis (IEP)

77
Q

Principle: Proteins are separated by electrophoresis -> cellulose acetate strip impregnated with antiserum is placed on the separated proteins -> the antiserum diffuses into the gel and antigen-antibody complexes precipitate

A

Immunofixation electrophoresis (IFE)

78
Q

Application: Identification of immunoglobulins in monoclonal gammopathies, Bence-Jones proteins

A

Immunofixation electrophoresis (IFE)

79
Q

Principle: An electrical charge is applied to an RID assay -> height of the rocket-shaped precipitin band is proportional to the concentration of antigen

A

Rocket electrophoresis

80
Q

Application: Immunoglobulins, complement, alpha-fetoprotein

A

Rocket electrophoresis

81
Q

The substance being measured in an immunoassay

A

Ligand

82
Q

An immunoassay that uses radioisotope as the label

A

Isotopic

83
Q

An immunoassay that uses something other than a radioisotope as the label

A

Nonisotopic

84
Q

An immunoassay in which the patient ligand and the labeled reagent ligand compete for a limited number of binding sites on a reagent antibody

A

Competitive

85
Q

An immunoassay in which the reaction does not involve competition for binding sites

A

Noncompetitive

86
Q

An immunoassay in which the reaction does not involve competition for binding sites

A

Noncompetitive

87
Q

An immunoassay in which a separation step is required to remove free reactant from bound reactant

A

Heterogenous

88
Q

An immunoassay in which a separation step is not required

A

Homogenous

89
Q

Horseradish peroxidase (HRP)
Fluorescein
Luminol

A

125I

90
Q

b-D-galactosidase
Rhodamine
Acridium ester

A

3H

91
Q

Alkaline phosphatase (ALP)

Dioxetane phosphate

A

14C

92
Q

Labels: 125I, 131I, 3H, 14C

A

Radioimmunoassay (RIA)

93
Q

Detection: Radioisotopes emit radioactivity

A

Radioimmunoassay (RIA)

94
Q

Principle: Radiolabeled ligand and unlabeled ligand in the specimen compete for binding sites on reagent antibody -> the amount of labeled ligand bound is determined by count per minute (CPM) on a scintillation counter

A

Radioimmunoassay (RIA)

95
Q

Results: CPM are proportional to the concentration of the ligand in the specimen

A

Radioimmunoassay (RIA)

96
Q

Employs a sorbent for allergen insolubilization. Homologous antibodies of all immunoglobulin classes may be bound to this allergen-sorbent.

A

Radioallergosorbent Test (RAST)

97
Q

Principle: Performed by incubating specific allergen-coated particles (i.e., sorbent) with the patient’s serum in a tube ®the tube is centrifuged, and the sorbent is washed to remove all IgE molecules except those specific for the allergen -> a radiolabeled anti-IgE antibody is then allowed to incubate with the complexes followed by centrifugation, washing, and counting

A

Radioallergosorbent Test (RAST)

98
Q

Application: RIA method specifically designed to measure antigen-specific IgE

A

Radioallergosorbent Test (RAST)

99
Q

Indirect RIST
Direct RIST

A

Radioimmunosorbent Test (RIST)

100
Q

(1) Anti-IgE covalently coupled to cross-linked dextran particles (Sephadex), (2) radiolabeled IgE and (3) patient’s serum are all incubated in one tube
(2) The radiolabeled IgE and patient’s IgE compete for the antibody receptor sites of the anti-IgE bound to Sephadex particle
(3) Following incubation, the tube is centrifuged and washed three times with buffer
(4) Decantation after the last wash leaves a pellet of complexes consisting of Sephadex bead + anti-IgE + IgE
(5) Emission of gamma rays per unit time from the radiolabeled IgE is counted

A

Indirect RIST – Procedure

101
Q

(1) Anti-IgE is rendered insoluble by being coupled to Sephadex beads and incubated with patient’s serum in a tube
(2) The tube is centrifuged and washed to remove any free antigen
(3) Radiolabeled anti-IgE is added followed by incubation, centrifugation, and washing to remove any unattached labeled reagent
(4) Decantation after the last wash leaves a pellet of complexes consisting of Sephadex bead + anti-IgE + IgE
(5) Emission of gamma rays per unit time from the radiolabeled anti- IgE is counted

A

Direct RIST – Procedure

102
Q

Application: competitive binding technique used to quantitate total IgE

A

Radioimmunosorbent Test (RIST)

103
Q

Principle:
(1) Radiolabeled IgE, IgE in patient’s serum and anti-IgE antibodies are all incubated in one tube
(2) Soluble IgE + anti-IgE complexes are formed from competitive binding
(3) A second anti-antibody directed to the anti-IgE is added (i.e., anti-anti-IgE) to the tube to promote precipitation or insolubilization of the IgE + antiIgE complex
(4) Contents are mixed, incubated, centrifuged and washed
(5) Last wash leaves a complex consisting of anti-anti-IgE + anti-IgE + IgE
(6) Emission of gamma rays per unit time from the radiolabeled IgE is counted

A

Radioimmunoprecipitation (RIP) assay

104
Q

Labels: Alkaline phosphatase, horseradish peroxidase, D-galactosidase, glucose-6phosphate dehydrogenase

A

Enzyme Immunoassay (EIA)

105
Q

Detection: Enzymes react with substrate to produce color change

A

Enzyme Immunoassay (EIA)

106
Q

Principle: Enzyme-labeled ligand and unlabeled patient ligand compete for binding sites on antibody molecules attached to a solid phase

A

Enzyme-linked immunosorbent assay (ELISA)

107
Q

Application: used to detect antibodies to viruses (HIV, HAV, HCV, EBV)

A

Enzyme-linked immunosorbent assay (ELISA)

108
Q

Principle: Antibody in the specimen attaches to a solid-phase antigen ® after incubation and washing to remove unbound antibody, an enzyme-labeled antiglobulin is added ® this second antibody reacts with the Fc portion of the patient antibody bound to the solid phase ® following another wash, substrate is added

A

Indirect or non-competitive ELISA

109
Q

Principle: The antigen in the specimen is sandwiched between an antibody attached to a solid phase and enzyme-labeled antibody

A

Sandwich enzymemultiplied immunoassay or capture assay

110
Q

Application: Antigens must have multiple determinants; used to measure immunoglobulins, hormones, proteins and detect tumor markers, viruses, parasites, fungi

A

Sandwich enzymemultiplied immunoassay or capture assay

111
Q

Principle: The antigen in the specimen and an enzyme-labeled antigen compete for binding sites on reagent antibody -> when the enzyme-labeled antigen binds to antibody, enzyme activity is inhibited

A

Enzyme-multiplied immunoassay technique (EMIT)

112
Q

Application: Used for determination of low molecular weight analytes not readily measured by other methods, e.g., hormones, therapeutic drugs, drugs of abuse

A

Enzyme-multiplied immunoassay technique (EMIT)

113
Q

Labels: Commonly used fluorochromes include fluorescein isothiocyanate (FITC), Rphycoerythrin, quantum red, tetramethyl-rhodamine isothiocyanate, rhodamine B isothiocyanate, Texas red, phycocyanin, acridine orange, and propidium iodide

A

Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)

114
Q

absorbs maximally at 490-495 nm; emits green color at 517 nm

A

Fluorescein isothiocyanate (FITC)

115
Q

absorbs at 550 nm: emits bright red light at 580-585 nm

A

Tetra methyl rhodamine isothiocyanate (TRITC)

116
Q

derived from algae, porphyrins, and chlorophylls: exhibits red fluorescence over 600 nm

A

Phycobiliproteins

117
Q

Detection: Fluorochromes absorb energy from light source; convert to a longer wavelength (lower energy)

A

Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)

118
Q

Common methods include direct and indirect IFA

A

Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)

119
Q

Principle: Conjugated (fluorescent labeled) reagent antibody reacts with an antigen in a clinical sample to form an antigen-antibody complex

A

Direct immunofluorescence

120
Q

Analytes: Bacterial, viral antigens

Pataba ka haaaaa!!! HUHUHU

A

Direct immunofluorescence

121
Q

Principle: Antigen reacts with unlabeled antibody forming an antigen-antibody complex that is then complexed with a labeled antihuman antibody, creating an antibody-antigen-antibody “sandwich”

A

Indirect immunofluorescent assays

122
Q

Analytes: Fluorescent antinuclear antibody (FANA), fluorescent Treponemal antibody (FTA)

A

Indirect immunofluorescent assays

123
Q

Principle: This is an indirect assay in which the detection system is modified by using a biotin-labeled antibody followed by avidin-labeled fluorochrome ® This extra step increases the specificity and sensitivity of the assay

A

Biotin-avidin immunofluorescence

124
Q

Principle: Based on the change of polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody

A

Fluorescence Polarization Immunoassay

125
Q

a. Unlabeled ligand in the specimen and fluorogenic ligand compete for sites on reagent antibody.
• Free-labeled ligand rotates _______________ and emits little polarized fluorescence.
• Bound labeled ligand rotates more _______________and emits more polarized fluorescence.

A

Fluorescence Polarization Immunoassay

126
Q

b. The higher the concentration of bound labeled ligand, the more polarized fluorescence.

A

Fluorescence Polarization Immunoassay

127
Q

c. The amount of polarized fluorescence is _______________ proportional to the concentration of the ligand in the specimen.

A

Fluorescence Polarization Immunoassay

128
Q

Analytes: Therapeutic drugs, hormones

A

Fluorescence Polarization Immunoassay

129
Q

Principle: Unlabeled ligand in the specimen and fluorogenic ligand compete for sites on reagent antibody -> free-labeled ligand produces fluorescence; bound-labeled ligand does not produce fluorescence

A

Substrate-labeled fluorescent immunoassay (SLFIA)

130
Q

Result: Fluorescence is _______________ proportional to the concentration of the ligand in the specimen

A

Substrate-labeled fluorescent immunoassay (SLFIA)

131
Q

Label: Luminol, acridium esters, ruthenium derivatives, nitrophenyl oxalates

A

Chemiluminescence Immunoassay (ChLIA)

132
Q

Principle: Chemiluminescent substance -> oxidation using H2O2 or enzyme -> produces intermediates at higher energy -> the intermediates spontaneously return to their original state -> giving off energy in the form of light

A

Chemiluminescence Immunoassay (ChLIA)

133
Q

Detection: Chemiluminescent molecules produce light from chemical reaction
o Emission of light is caused by a chemical reaction producing an excited molecule that decays back to the original ground state measured using an illuminometer

A

Chemiluminescence Immunoassay (ChLIA)

134
Q

Used to detect the presence of complement-fixing antibodies (IgM and IgG) in patient serum against soluble antigens

A

COMPLEMENT FIXATION

135
Q

Serum sample must be heated at ______ for __________ to inactivate native complement

A

Di ko alam beh sorry

136
Q

• Bacteriolytic test system
• Hemolytic indicator system

A

Indicator Requirements

137
Q

Principle:
• Patient serum is incubated with antigen and complement -> if the corresponding antibody is present in the serum, it forms a complex with the antigen and the complement -> when sensitized RBCs are added, there is no free complement to lyse them

A

COMPLEMENT FIXATION

138
Q

If the complement fixation test is positive

A

no further testing is required

139
Q

If the complement fixation test is negative

A

Rice test must be performed

140
Q

(1) The patient’s serum is mixed with 1 unit of antigen and 1 unit of complement and is incubated. If the serum contains a non-complement-fixing antibody, it will bind some or all of the antigen but none of the complement

A

Rice Test

141
Q

(2) A known complement-fixing antibody specific for the antigen is added. Since the antigen was in short supply, to begin with, most or all of it has been bound by antibodies in the patient’s serum. There is little or no antigen with which the complement-fixing antibody can unite; hence the complement remains free.

A

Rice Test

142
Q

(3) Finally, sensitized sheep red cells are added.
o The free complement attaches to the antibody causing lysis of red cells -> indicative of a noncomplement-fixing antibody
o If a patient’s serum contains no antibody, both antigen and complement will remain free -> Addition of known complement-fixing antibody results in an antigen-antibody-complement complex -> when sensitized sheep red cells are added, there is no more free complement with which the hemolysin (i.e., antibody attached on the red cell surface) can combine -> hence, the red cells do not lyse

A

Rice Test

Immunology and Serology [LAB] (16 decks)

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