Laboratory Activity 3c – Principles of Serologic Reactions Flashcards
1
Q
Antibodies that aggregate cellular antigens
A
Agglutinins
2
Q
Antibodies that form precipitates with soluble antigens
A
Precipitins
3
Q
Antibodies that neutralize toxins
A
Antitoxins
4
Q
Antibodies that cause dissolution of cell membrane
A
Lysins
5
Q
Cause the destruction of RBC in the presence of the complement
A
Hemolysins
6
Q
Cause destruction of cells of gram-negative bacteria under certain conditions
A
Bacteriolysins
7
Q
Cause destruction of other cell types under appropriate conditions in the presence of the complement
A
Cytolysins
8
Q
– assays involving antibody-antigen reactions are called immunoassays
A
IMMUNOASSAYS
9
Q
soluble antibody reacts with insoluble antigen or soluble antigen reacts with an insoluble antibody
A
Agglutination reaction
10
Q
are made soluble by combining with latex particles, RBCs, dyes or liposomes
A
Reactants
11
Q
occurs when particles in suspension clump together due to an antibody-antigen reaction
A
Agglutination
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
13
Q
Demonstrate the presence of antigen-antibody reactions by the visible aggregation of antigen-antibody complexes
A
Agglutination
14
Q
These tests are simple to perform and are often the most sensitive test method
A
AGGLUTINATION METHODS
15
Q
Performed with slide, tube or microtiter technique
A
AGGLUTINATION METHODS
16
Q
Particulate antigen + antibody ® clumping
A
AGGLUTINATION
17
Q
antigen binds with Fab sites of 2 antibodies forming bridges between antigens
A
Lattice formation
18
Q
The process by which particulate antigens, such as cells, aggregate to form large complexes when specific antibody is present
A
Direct agglutination
19
Q
Febrile agglutinins, Salmonella and Shigella serotyping
A
Examples of Direct agglutination
20
Q
Antibodies + surface antigens of bacteria in suspension -> visible agglutination
A
Direct Bacterial Agglutination
21
Q
Antibody bound to latex beads + antigen -> visible agglutination results when antigen binds to latex-bound antibody
A
Latex Agglutination
22
Q
An antigen-antibody reaction that results in the clumping of red blood cells
A
Hemagglutination
23
Q
One solid aggregate, clear background
A
4+
24
Q
Several large aggregates, clear background
A
3+
25
Medium-sized agglutinates, clear background
2+
26
Small agglutinates, turbid background
1+
27
Tiny agglutinates, turbid background
w+
28
No agglutination or hemolysis
0
29
ABO typing
Examples of Hemagglutination
30
A reaction in which soluble antigens are bound to latex beads, bentonite, or charcoal -> the particles are agglutinated by the corresponding antibody
Passive agglutination
31
Rheumatoid factor
Examples of Passive agglutination
32
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
Passive hemagglutination
33
Cold agglutinins
Examples of Passive hemagglutination
34
A reaction in which carrier particles coated with antibody clump together due to combination with antigen
Reverse passive agglutination
35
Rapid tests for identification of bacteria
Examples of Reverse passive agglutination
36
An agglutination reaction based on competition between particulate antigen (reagent) and soluble antigen (specimen) for limited sites on a reagent antibody
Agglutination inhibition
37
Detection of illicit drugs
Examples of Agglutination inhibition
38
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
Hemagglutination inhibition
39
Rubella antibody
Examples of Hemagglutination inhibition
40
An agglutination reaction in which bacteria are used as the carrier for the antibody
Coagglutination
41
Rapid tests for identification of bacteria
Examples of Coagglutination
42
Detection of non-agglutinating antibody by coupling with 2nd antibody (antihuman globulin [AHG])
Antiglobulin-mediated agglutination
43
Direct and indirect antiglobulin test
Examples of Antiglobulin-mediated agglutination
44
Principle: Soluble antigen combines with soluble antibody to produce visible insoluble complexes
Precipitation
45
Clumping together of particles to form visible masses over a narrow range of antigen concentration
Flocculation
46
Similar with precipitation except that the precipitin appears as a fleecy mass or clump
Flocculation
47
Principle: Soluble antigens react with specific antibody to form precipitate of fine particles
Flocculation
48
Application: Venereal Disease Research Laboratory (VDRL) tests, Rapid Plasma Reagin (RPR)
Flocculation
49
Principle: Light scattering by immune complexes is measured -> scattering of light is proportional to the size and amount of immune complexes formed
Nephelometry
50
Application: Immunoglobulins, complement, C-reactive protein
Nephelometry
51
Measures the decrease in light intensity in a solution containing immune complexes
Turbidimetry
52
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
Turbidimetry
53
Type of Diffusion
Single
Double
54
if only one reactant (usually antigen) is moving
Single
55
if both antigen and antibody are moving through the medium
Double
56
Type of Dimension
Single
Double
57
if the reaction in a medium have only one effective dimension for antigen and antibody migration (i.e., up and down)
Single
58
if the reaction in a circular holes (i.e., wells) cut in a gel on a flat surface diffuses from the wells radially
Double
59
Principle: Known Antibody fixed in agar + Unknown Antigen (overlaid) -> Precipitin lines
Single linear diffusion (SLD) or Oudin technique
60
Application: Used to detect multiple antigen-antibody reactions
Single linear diffusion (SLD) or Oudin technique
61
Principle: Known Antibody fixed in agar + Unknown Antigen (well cut in agar plate) ® Precipitin ring
Single radial diffusion (SRD)/ Fahey or Mancini method
62
diameter of precipitin ring at 24 hours (Read before it reaches the maximum at 6-12 hours)
Fahey method
63
48
Mancini method
64
Principle: Antigen diffuses out of well in gel containing antigen -> Precipitin ring forms -> Diameter proportional to concentration of antigen
Radial immunodiffusion (RID)
65
Application: Immunoglobulins, complement No longer commonly performed except for low-volume testing of IgD and IgG
Radial immunodiffusion (RID)
66
Principle: Antigens and antibodies diffuse out from wells cut in gel and precipitin lines where they meet
Ouchterlony technique/Double Immunediffusion
67
Ouchterlony technique/Double Immunediffusion
Three basic reaction patterns:
a) Identity
b) Non-identity
c) Partial identity
68
a single smooth arc of precipitation forms between the antigens and antibodies
Identity
69
two separate lines of precipitation cross each other
Non-identity
70
two precipitating lines meet, forming a spur
Partial identity
71
Application: Fungal antigens, extractable nuclear antigens
Ouchterlony technique/Double Immunediffusion
72
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
Ouchterlony technique/Double Immunediffusion
73
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
Countercurrent immunoelectrophoresis (CIE)
74
Application: Bacterial antigens
Countercurrent immunoelectrophoresis (CIE)
75
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
Immunoelectrophoresis (IEP)
76
Application: Serum proteins including immunoglobulins
Immunoelectrophoresis (IEP)
77
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
Immunofixation electrophoresis (IFE)
78
Application: Identification of immunoglobulins in monoclonal gammopathies, Bence-Jones proteins
Immunofixation electrophoresis (IFE)
79
Principle: An electrical charge is applied to an RID assay -> height of the rocket-shaped precipitin band is proportional to the concentration of antigen
Rocket electrophoresis
80
Application: Immunoglobulins, complement, alpha-fetoprotein
Rocket electrophoresis
81
The substance being measured in an immunoassay
Ligand
82
An immunoassay that uses radioisotope as the label
Isotopic
83
An immunoassay that uses something other than a radioisotope as the label
Nonisotopic
84
An immunoassay in which the patient ligand and the labeled reagent ligand compete for a limited number of binding sites on a reagent antibody
Competitive
85
An immunoassay in which the reaction does not involve competition for binding sites
Noncompetitive
86
An immunoassay in which the reaction does not involve competition for binding sites
Noncompetitive
87
An immunoassay in which a separation step is required to remove free reactant from bound reactant
Heterogenous
88
An immunoassay in which a separation step is not required
Homogenous
89
Horseradish peroxidase (HRP)
Fluorescein
Luminol
125I
90
b-D-galactosidase
Rhodamine
Acridium ester
3H
91
Alkaline phosphatase (ALP)
Dioxetane phosphate
14C
92
Labels: 125I, 131I, 3H, 14C
Radioimmunoassay (RIA)
93
Detection: Radioisotopes emit radioactivity
Radioimmunoassay (RIA)
94
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
Radioimmunoassay (RIA)
95
Results: CPM are proportional to the concentration of the ligand in the specimen
Radioimmunoassay (RIA)
96
Employs a sorbent for allergen insolubilization. Homologous antibodies of all immunoglobulin classes may be bound to this allergen-sorbent.
Radioallergosorbent Test (RAST)
97
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
Radioallergosorbent Test (RAST)
98
Application: RIA method specifically designed to measure antigen-specific IgE
Radioallergosorbent Test (RAST)
99
Indirect RIST
Direct RIST
Radioimmunosorbent Test (RIST)
100
(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
Indirect RIST – Procedure
101
(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
Direct RIST – Procedure
102
Application: competitive binding technique used to quantitate total IgE
Radioimmunosorbent Test (RIST)
103
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
Radioimmunoprecipitation (RIP) assay
104
Labels: Alkaline phosphatase, horseradish peroxidase, D-galactosidase, glucose-6phosphate dehydrogenase
Enzyme Immunoassay (EIA)
105
Detection: Enzymes react with substrate to produce color change
Enzyme Immunoassay (EIA)
106
Principle: Enzyme-labeled ligand and unlabeled patient ligand compete for binding sites on antibody molecules attached to a solid phase
Enzyme-linked immunosorbent assay (ELISA)
107
Application: used to detect antibodies to viruses (HIV, HAV, HCV, EBV)
Enzyme-linked immunosorbent assay (ELISA)
108
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
Indirect or non-competitive ELISA
109
Principle: The antigen in the specimen is sandwiched between an antibody attached to a solid phase and enzyme-labeled antibody
Sandwich enzymemultiplied immunoassay or capture assay
110
Application: Antigens must have multiple determinants; used to measure immunoglobulins, hormones, proteins and detect tumor markers, viruses, parasites, fungi
Sandwich enzymemultiplied immunoassay or capture assay
111
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
Enzyme-multiplied immunoassay technique (EMIT)
112
Application: Used for determination of low molecular weight analytes not readily measured by other methods, e.g., hormones, therapeutic drugs, drugs of abuse
Enzyme-multiplied immunoassay technique (EMIT)
113
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
Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)
114
absorbs maximally at 490-495 nm; emits green color at 517 nm
Fluorescein isothiocyanate (FITC)
115
absorbs at 550 nm: emits bright red light at 580-585 nm
Tetra methyl rhodamine isothiocyanate (TRITC)
116
derived from algae, porphyrins, and chlorophylls: exhibits red fluorescence over 600 nm
Phycobiliproteins
117
Detection: Fluorochromes absorb energy from light source; convert to a longer wavelength (lower energy)
Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)
118
Common methods include direct and indirect IFA
Fluorescence Immunoassays (FIA)/ Immunofluorescence assay (IFA)
119
Principle: Conjugated (fluorescent labeled) reagent antibody reacts with an antigen in a clinical sample to form an antigen-antibody complex
Direct immunofluorescence
120
Analytes: Bacterial, viral antigens
Pataba ka haaaaa!!! HUHUHU
Direct immunofluorescence
121
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”
Indirect immunofluorescent assays
122
Analytes: Fluorescent antinuclear antibody (FANA), fluorescent Treponemal antibody (FTA)
Indirect immunofluorescent assays
123
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
Biotin-avidin immunofluorescence
124
Principle: Based on the change of polarization of fluorescent light emitted from a labeled molecule when it is bound by antibody
Fluorescence Polarization Immunoassay
125
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.
Fluorescence Polarization Immunoassay
126
b. The higher the concentration of bound labeled ligand, the more polarized fluorescence.
Fluorescence Polarization Immunoassay
127
c. The amount of polarized fluorescence is _______________ proportional to the concentration of the ligand in the specimen.
Fluorescence Polarization Immunoassay
128
Analytes: Therapeutic drugs, hormones
Fluorescence Polarization Immunoassay
129
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
Substrate-labeled fluorescent immunoassay (SLFIA)
130
Result: Fluorescence is _______________ proportional to the concentration of the ligand in the specimen
Substrate-labeled fluorescent immunoassay (SLFIA)
131
Label: Luminol, acridium esters, ruthenium derivatives, nitrophenyl oxalates
Chemiluminescence Immunoassay (ChLIA)
132
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
Chemiluminescence Immunoassay (ChLIA)
133
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
Chemiluminescence Immunoassay (ChLIA)
134
Used to detect the presence of complement-fixing antibodies (IgM and IgG) in patient serum against soluble antigens
COMPLEMENT FIXATION
135
Serum sample must be heated at ______ for __________ to inactivate native complement
Di ko alam beh sorry
136
• Bacteriolytic test system
• Hemolytic indicator system
Indicator Requirements
137
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
COMPLEMENT FIXATION
138
If the complement fixation test is positive
no further testing is required
139
If the complement fixation test is negative
Rice test must be performed
140
(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
Rice Test
141
(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.
Rice Test
142
(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
Rice Test
