LABORATORY TECHNIQUES IN IMMUNOLOGY AND SEROLOGY Flashcards
memorization
1
Q
The clumping and sedimentation of particulate or insoluble antigen/antibody complexes
A
Agglutination
2
Q
Insoluble particle is reacted with an Ab which results in aggregation of particles
A
Direct agglutination
3
Q
The reaction is due to an Ag-Ab reaction wherein the Ag is naturally found on the cell
A
Direct immune agglutination
Note: Direct means the Ag is inherited to the cell/ daan na nga ada ha cell an antigen
4
Q
Aggregation of indicator red cells are not due to Ag-Ab reactions
A
Direct non-immune agglutination
5
Q
Example of Direct immune agglutination:
A
ABO blood grouping
6
Q
Example of Direct non-immune agglutination:
A
Viral hemagglutination tests
7
Q
Reactions where the antigen is attached to the carrier particle:
A
Indirect or passive agglutination
8
Q
Indirect or passive agglutination is used to detect ______ in a biologic sample
A
Antibody
Note: Indirect means antibody ang hinahanap
9
Q
Example of an Indirect or passive agglutination
A
Latex agglutination for Rheumatoid factor
10
Q
Antibody is attached to a carrier particle; detects antigen on a biologic sample:
A
Reverse passive agglutination
11
Q
Example of a Reverse passive agglutination:
A
CRP assay
12
Q
Based on competition between particulate and soluble antigens for limited antibody-combining sites
A
Agglutination inhibition
13
Q
In agglutination inhibition, a lack of agglutination is an indicator of a _______ reaction
A
positive (+) reaction
14
Q
Example of an agglutination inhibition test:
A
Pregnancy test/ HCG
15
Q
In viral hemagglutination inhibition, if the result has no agglutination, it means:
A
positive (+)
note: if there is no hemagglutination, the patient’s serum is positive/has antiviral antibodies. The antiviral antibodies neutralized the virus and hemagglutination was inhibited
16
Q
Detects the presence of non-agglutinating antibodies on red blood cells by adding a second antibody
A
Antiglobulin test
17
Q
Detects in vivo sensitization of red cells by IgG or complements such as C3b or C3d
A
Direct Antiglobulin Test
18
Q
Diseases detected by DAT:
A
- HDN (eg. Rh)
- HTR
- AIHA
- Drug IHA
19
Q
An antiglobulin test used in crossmatching, Ab identification and detection; it also used for RBC phenotyping 9Weak D test)
A
Indirect Antiglobulin Test
20
Q
Uses bacteria as the inert particle to which antibodies are attached
A
Coagglutination
note: in coagglutination, antigen ang hinahanap dito
21
Q
In coagglutination, the bacteria that is frequently used as the inert particle:
A
Staphylococcus aureus
22
Q
Why is S. aureus the most frequently used inert particle for coagglutination?
A
because S. aureus possess the PROTEIN A (it absorbs Fc region of antibody)
23
Q
An immunoassay that uses gold as an inert particle
A
Sol Particle Immunoassay
24
Q
An immunoassay that uses dye as an inert particle
A
Disperse Dye immunoassay
25
An immunoassay that uses latex particles and is the most sensitive
IMPACT/ Immunoassay by particle counting
26
Many tiny agglutinates
Many free cells
May not be visible without a microscope
Grade: ?
W+ (weak positive)
27
Many medium-sized agglutinates
Moderate number of free cells
Grade: ?
2+
28
Many small-sized granulates
Many free cells
Grade: ?
1+
29
Several large agglutinates
Few free cells
Grade: ?
3+
30
One large, solid agglutinate
No free cells
Grade: ?
4+
31
25% cells are agglutinated
Grade: ?
1+
32
75% cells are agglutinated
Grade: ?
3+
33
100% cells are agglutinated
Grade: ?
4+
34
50% cells are agglutinated
Grade: ?
2+
35
The combination of soluble antigen with an antibody to produce an insoluble complex
Precipitation
36
Insufficient Ag to form large immune complexes (Ag
a. Prozone
note: prozone or zone of Ab excess
37
What is the solution for Ab excess?
Dilute the sample
38
Excess Ag leads to decreased cross-linking
a. Prozone
b. Postzone
c. ZoE
d. Avidity
b. Postzone
39
What is the solution for Ag excess?
Repeat the test a week later
Note: Patient will have more time to synthesize antibodies
40
Maximum precipitation occurs (Ag is equal to Ab)
Zone of equivalence
41
Prozone and Postzone causes ______ test results
FALSE-NEGATIVE TEST RESULTS
42
Strength of primary interaction between an epitope and Ab binding site
Affinity
43
The sum of all attractive forces between the Ag and Ab
Avidity
44
Soluble Ag and/or Ab can diffuse through the pores of the gel until they reach optimal concentration to form a stable precipitate
Precipitation in a gel medium
45
Only one reactant is moving (usually Ag)
a. Single diffusion test
b. Single dimension
a. Single diffusion test
46
Both Ag and Ab are moving through the medium
a. Double diffusion test
b. Double dimension
a. Double diffusion test
47
Reaction in tubes; Antigen and antibody migrate up and down
a. Single dimension
b. Double dimension
a. Single dimension
48
Reaction in petri dish; antigen and antibody diffuse radially
a. Double diffusion
b. Double dimension
b. Double dimension
49
What test has a combination of Single diffusion-single dimension test principle?
OUDIN
Single diffusion-Single dimension: OUDIN
50
What test has a combination of single diffusion-double dimension test principle?
Radial Immunodiffusion (RID)/ Mancini
Single diffusion-Double dimension: RID/Mancini
51
What test has a combination of double diffusion-single dimension test principle?
Oakley and Fulthrope
Double diffusion-Single dimension: Oakley and Fulthrope
52
What test has a combination of double diffusion and double dimension test principles?
Ouchterlony and Elek
Double diffusion-Double dimension: Ouchterlony and Elek
53
Antibody is uniformly distributed in a support gel in a petri dish; circular wells are cut into the gel and Ag is loaded; Ag diffuses producing a ring of precipitate
Radial Immunodiffusion or Mancini test
54
Two (2) method of reading RID test:
1. Kinetic (Fahey) Method
2. Mancini/Endpoint
55
RID reading method where measurements are taken while the disc is still expanding (usually 18 hours)
Kinetic (Fahey) Method
56
RID reading method where measurements taken when the ring has stopped expanding, allow maximal precipitation
Mancini/Endpoint method
57
Between kinetic (Fahey) method and Mancini/Endpoint method, which is more sensitive?
Mancini/Endpoint RID reading method
58
In Mancini/Endpoint RID reading method, measurements are taken usually after _____ hours:
24-72 hours
59
Antigen and antibodies diffuse through a semisolid medium in two dimensions
Ouchterlony (double diffusion-double dimension)
60
Ouchterlony; fused band of precipitate around the Ab well, antibody is precipitating identical antigens
Serological identity
61
Ouchterlony; lines of precipitation cross one another, antigens are serologically distinct (nothing in common)
Non-identity
62
Ouchterlony; Spur formation, Ag are not identical but possess a common determinant (common epitope)
Single partial identity
63
Ouchterlony; double spurring formation
Double partial identity
64
Molecules with a net charge are separated when an electric field is applied to the system
Electrophoresis
65
Electrophoresis technique; Single reactant moving in one direction
Rocket Immunoelectrophoresis/
Technique of Laurell
66
Diffusion counterpart of Rocket Immunoelectrophoresis:
OUDIN
67
In Rocket immunoelectrophoresis, the height if the rocket (shape of precipitate) is _______ to Ag concentration
proportional (directly proportional)
68
Electrophoresis technique; single reactant moving in two dimensions; involves two electrophoretic separations
Crossed IE/
Ressler's Method/
Double-crossed IE
69
Diffusion counterpart of Crossed IE/ Ressler's Method/ Double-crossed IE:
RID/Mancini
70
Electrophoresis technique; double reactants moving in one dimension:
Counter Immunoelectrophoresis/
Countercurrent Electrophoresis/
Double Electro-immunodiffusion
71
Electrophoresis technique; double reactants moving in two dimensions; used for identifying myeloma proteins (BJP)
Classic immunoelectrophoresis or IEP/ Grabar and Williams
72
Diffusion counterpart of Classic immunoelectrophoresis or IEP:
Ouchterlony
73
All reactants are mixed together simultaneously then labeled antigens compete with unlabeled patient antigens for a limited number of antibody-binding sites
Competitive assays
74
The amount of bound label is ________ proportional to the concentration of the antigen present
INVERSELY
75
A capture antibody is first passively absorbed into a solid phase. The patient antigen is allowed to react with the antibody. A second antibody is added to the reaction
Noncompetitive assays
76
The label measured is ________ proportional to the amount of patient antigen
DIRECTLY
77
Who discovered Radioimmunoassay (RIA)?
Rosalyn Yalow
78
The most commonly used enzyme in enzyme immunoassay
Horseradish peroxidase
79
An enzyme immunoassay where the bound enzyme is catalytically active
Heterogenous EIA
80
Two (2) types of Heterogenous EIA:
1. Competitive Enzyme-Linked Immunosorbent Assays (ELISA)
2. Non-competitive ELISA
81
Enzyme-labeled antigen competes with unlabeled patient antigen for a limited number of antibody binding sites; the system is washed; enzyme activity is measured
Competitive ELISA
82
The enzyme activity in competitive assay is ______________ proportional to the concentration of the test substance
inversely proportional
83
The enzyme-labeled reagent does not participate in the initial antigen-antibody reactions; the patient antibody is incubated with solid-phase antigen; after a wash step, an enzyme-labeled AHG is added; a second wash step is performed to remove unbound AHG, then substrate us added
Non-competitive ELISA
84
The amount of color fluorescence or luminescence detected is _____________ proportional to the amount of antibody in the specimen
directly proportional
85
If antibody, rather than antigen, is bound to the solid phase, these assays are often called:
SANDWICH IMMUNOASSAYS or CAPTURE ASSAYS
- Patient antigen is incubated with solid-phase antibody
- After washing, a second antibody with an enzyme label is added
- After a second wash, the substrate for the enzyme is added
86
An enzyme immunoassay where the free enzymes are catalytically active; Antigen in patient's sample competes for limited ab-binding sites with an enzyme labeled Ag.
Homogenous EIA
87
In competitive homogenous enzyme immunoassay, the enzyme activity is ______________ proportional to the concentration of the test substance
Directly proportional
Note: The more Ag in the sample, the more enzyme will remain unbound and catalytically active, therefore there is formation of more colored products
88
Immunoassay that uses fluorochrome; has the ability to absorb light at shorter wavelengths and emit waves at a visible spectrum/ longer wavelengths
Immunofluorescent immunoassay
89
A fluorochrome that emits green color:
Fluorescein isothiocyanate
90
A fluorochrome that emits red light:
Tetramethyl rhodamine
91
Solid-phase antigen fixed to a slide is incubated directly with a fluorescent-labeled antibody; if the specific antigen is present in the patient's sample, fluorescence will be observed
Direct Immunofluorescent Assay
TIP: Patient ANTIGEN (Ag) ang hinahanap dito!
92
Example of a direct immunofluorescent assay test:
Fluorescent Antibody Dark Field Technique for T. pallidum (FTA FTP)
positive result: Fluorescence (+)
93
Patient antibody is reacted with speicific antigen fixed to a microscopic slide; a wash step is performed, and fluorescent-labeled AHG is added; after a wash step, fluorescence is determined
Indirect Immunofluorescent assay
TIP: Patient ANTIBODY (Ab) and hinahanap dito!
94
Example of an indirect immunofluorescent assay test:
FTA ABS (Fluorescent Treponemal Antibody Absorption)
FANA (Fluorescent Antinuclear Antibody)
95
In a precipitation reaction, how can the ideal antibody be
characterized?
a. Low affinity and low avidity
b. High affinity and low avidity
c. High affinity and high avidity
d. Low affinity and high avidity
Option c: High affinity and high avidity
Characteristics of an ideal antibody for precipitation reactions:
1. High affinity: Strong binding between antibody and antigen, ensuring efficient precipitation.
2. High avidity: Overall binding strength of the antibody-antigen complex, promoting stable immune complexes.
High affinity and avidity enable:
1. Efficient antigen recognition
2. Effective precipitation
3. Sensitive detection
96
Precipitation differs from agglutination in which way?
a. Precipitation can only be measured by an automated instrument.
b. Precipitation occurs with univalent antigen, whereas agglutination requires multivalent antigen.
c. Precipitation does not readily occur because few antibodies can form aggregates with antigen.
d. Precipitation involves a soluble antigen, whereas agglutination involves a particulate antigen.
Option d: Precipitation involves a soluble antigen, whereas agglutination involves a particulate antigen.
Key differences:
Precipitation
1. Soluble antigens (e.g., proteins, toxins)
2. Formation of insoluble immune complexes
3. Typically occurs in gel or liquid media (e.g., Ouchterlony double diffusion)
Agglutination
1. Particulate antigens (e.g., bacteria, cells, latex particles)
2. Visible clumping of particles
3. Occurs in liquid media
97
When soluble antigens diffuse in a gel that contains antibody, in which zone does optimal precipitation occur?
a. Prozone
b. Zone of equivalence
c. Postzone
d. Prezone
Option b: Zone of equivalence
Explanation:
In radial immunodiffusion (Ouchterlony technique), optimal precipitation occurs in the:
Zone of Equivalence
1. Antigen and antibody concentrations are optimal.
2. Lattice formation is maximal.
3. Precipitation lines are sharp and clear.
Other zones:
1. Prozone: Excess antibody; inadequate precipitation.
2. Postzone: Excess antigen; weak, diffuse precipitation.
3. Prezone: Insufficient antigen; no precipitation.
98
Which of the following statements applies to rate nephelometry?
a. Readings are taken before equivalence is reached.
b. It is more sensitive than immunoturbidimetry.
c. Measurements are time dependent.
d. All of the above apply.
Option d: All of the above apply.
Rate nephelometry characteristics:
1. Readings taken before equivalence (prozone).
2. More sensitive than immunoturbidimetry.
3. Measurements are time-dependent, monitoring rate of immune complex formation.
Rate nephelometry advantages:
1. High sensitivity
2. Rapid results
3. Suitable for low-concentration analytes
99
Which of the following is characteristic of the endpoint method of RID?
a. Readings are taken before equivalence.
b. The antigen concentration is directly in proportion to the square of the ring diameter.
c. The ring diameter is plotted against the log of the concentration.
d. It is primarily a qualitative rather than a quantitative method.
Option b: The antigen concentration is directly proportional to the square of the ring diameter.
Endpoint method characteristics (Radial Immunodiffusion, RID):
Key Features
1. Antigen diffuses from wells into antibody-containing gel.
2. Precipitation rings form.
3. Ring diameter measured.
Quantitative Relationship
1. Antigen concentration ∝ ring diameter squared.
2. Linear relationship between concentration and diameter squared.
Other Options
1. Option a: Incorrect; readings taken after equivalence.
2. Option c: Incorrect; diameter vs. log(concentration) plots used in some assays, but not endpoint RID.
3. Option d: Incorrect; RID is quantitative.
100
In which zone might an antibody-screening test be false negative?
a. Prozone
b. Zone of equivalence
c. Postzone
d. None of the above
Option a: Prozone
Explanation:
In the prozone, excess antibodies can prevent lattice formation, leading to:
Causes of False Negatives
1. Inhibited precipitation
2. Weak or absent reactions
3. False-negative results
Prozone effects are more common in:
1. Automated antibody screening
2. Gel testing
3. Low-antigen concentrations
To minimize false negatives:
1. Dilute samples
2. Use excess antigen
3. Utilize alternative testing methods
NOTE: the postzone can also cause false-negative results in antibody-screening tests.
Characteristics of Postzone:
1. Excess antigen
2. Antibody saturation
3. Weak or absent precipitation
4. False-negative results
In the postzone, excess antigen binds available antibody sites, preventing lattice formation and precipitation.
101
How does immunoturbidimetry differ from nephelometry?
a. Immunoturbidimetry measures the increase in light after it passes through a solution.
b. Nephelometry measures light that is scattered at an angle.
c. Immunoturbidimetry deals with univalent antigens only.
d. Nephelometry is not affected by large particles falling out of solution.
The correct answer is:
Option b: Nephelometry measures light that is scattered at an angle.
Key differences:
1. Measurement principle:
- Immunoturbidimetry: Measures decreased light transmission (absorbance) due to immune complex formation.
- Nephelometry: Measures scattered light at an angle (90° or 360°).
2. Detection sensitivity:
- Nephelometry is more sensitive than immunoturbidimetry.
3. Particle size:
- Nephelometry detects smaller particles.
Instrumentation:
1. Immunoturbidimetry: Spectrophotometers
2. Nephelometry: Dedicated nephelometers
Applications:
1. Immunoturbidimetry: Quantitative antigen measurement
2. Nephelometry: Quantitative antigen measurement, particularly for low-concentration analytes
102
Which of the following refers to the force of attraction between an antibody and a single antigenic determinant?
a. Affinity
b. Avidity
c. Van der Waals attraction
d. Covalence
Option a: Affinity
Affinity refers to:
1. The binding strength between an antibody's antigen-binding site (paratope) and a single antigenic determinant (epitope).
2. Measures the antibody's ability to bind specifically to an antigen.
Contrast with Avidity
1. Avidity: Measures the overall binding strength of an antibody to a multivalent antigen.
2. Includes contributions from multiple antigen-antibody interactions.
Exclude Options
1. Van der Waals attraction: Weak intermolecular forces, not specific to antigen-antibody interactions.
2. Covalence: Refers to chemical bonding, not relevant to antigen-antibody binding.
103
If crossed lines result in an Ouchterlony immunodiffusion reaction with antigens 1 and 2, what does this indicate?
a. Antigens 1 and 2 are identical.
b. Antigen 2 is simpler than antigen 1.
c. Antigen 2 is more complex than antigen 1.
d. The two antigens are unrelated
Option d: The two antigens are unrelated.
Crossed lines in Ouchterlony immunodiffusion indicate:
Key Points
1. No fusion or partial fusion of precipitation lines.
2. Distinct, intersecting lines.
3. Lack of immune complex formation between antigens.
Interpretation:
1. Antigens 1 and 2 have no common epitopes.
2. Antibodies against Antigen 1 do not recognize Antigen 2, and vice versa.
3. Unrelated antigens.
Other options:
1. Option a: Identical antigens would show fusion or partial fusion.
2. Option b/c: Complexity differences wouldn't necessarily result in crossed lines.
104
Which technique represents a single-diffusion reaction?
a. Radial immunodiffusion
b. Ouchterlony diffusion
c. Counter immunoelectrophoresis
d. Immunofixation electrophoresis
Option a: Radial immunodiffusion (Mancini technique)
Characteristics:
Single-diffusion reaction
1. Antigen diffuses outward from a well.
2. Antibody is uniformly distributed in the gel.
3. Precipitation ring forms around the well.
Other options:
1. Option b: Ouchterlony diffusion - Double-diffusion reaction (antigen and antibody diffuse toward each other).
2. Option c: Counter immunoelectrophoresis - Combines electrophoresis and double-diffusion.
3. Option d: Immunofixation electrophoresis - Combines electrophoresis and specific antibody fixation.
105
Which best describes the law of mass action?
a. Once antigen–antibody binding takes place, it is irreversible.
b. The equilibrium constant depends only on the forward reaction.
c. The equilibrium constant is related to the strength of antigen–antibody binding.
d. If an antibody has a high avidity, it will dissociate from antigen easily
Option c: The equilibrium constant is related to the strength of antigen-antibody binding.
Law of Mass Action:
1. Dynamic equilibrium between antigen-antibody binding and dissociation.
2. Equilibrium constant (K) reflects binding strength.
Key Points
1. K = [Antigen-Antibody complex] / ([Antigen] × [Antibody])
2. High K: Strong binding (high affinity)
3. Low K: Weak binding (low affinity)
Exclude Options:
1. Option a: Binding is reversible.
2. Option b: Equilibrium constant considers both forward and reverse reactions.
3. Option d: High avidity indicates strong binding, not easy dissociation.
106
Agglutination of dyed bacterial cells represents which type of reaction?
a. Direct agglutination
b. Passive agglutination
c. Reverse passive agglutination
d. Agglutination inhibition
Option a: Direct agglutination
Direct Agglutination:
1. Immediate antigen-antibody reaction.
2. Antibodies bind directly to antigens on bacterial cell surfaces.
3. Agglutination occurs.
Examples:
1. Slide agglutination tests
2. Tube agglutination tests
3. Direct bacterial agglutination assays
Incorrect Options
1. Passive Agglutination (B): Indirect reaction using coated particles, not direct bacterial interaction.
2. Reverse Passive Agglutination (C): Detects antigens using antibody-coated particles, opposite of direct agglutination.
3. Agglutination Inhibition (D): Measures inhibition of agglutination, not direct bacterial agglutination.
107
If a single IgM molecule can bind many more antigens than a molecule of IgG, which of the following is higher?
a. Affinity
b. Initial force of attraction
c. Avidity
d. Initial sensitization
Option c: Avidity
Avidity refers to the overall binding strength of an antibody to a multivalent antigen, considering multiple binding sites.
IgM's higher avidity:
1. Pentameric structure (5 subunits)
2. Multiple antigen-binding sites (10)
3. Increased binding strength
In contrast:
1. Affinity (Option a): Measures binding strength per antigen-binding site.
2. Initial force of attraction (Option b): Similar for IgM and IgG.
3. Initial sensitization (Option d): Unrelated to antibody-antigen binding strength.
108
Agglutination inhibition could best be used for which of the
following types of antigens?
a. Large cellular antigens, such as erythrocytes
b. Soluble haptens
c. Bacterial cells
d. Coated latex particles
Option b: Soluble haptens
Agglutination inhibition is ideal for detecting soluble haptens (small antigens) because:
Characteristics
1. Small size: Haptens cannot agglutinate on their own.
2. Solubility: Haptens inhibit agglutination reactions.
3. Specificity: Inhibition assays detect specific antibody-hapten interactions.
Applications
1. Toxicology screening (e.g., detecting drugs or toxins)
2. Infectious disease serology (e.g., detecting viral antigens)
3. Allergy testing
Other options:
1. Option a: Large cellular antigens (erythrocytes) are better suited for direct agglutination tests.
2. Option c: Bacterial cells are often used in direct agglutination or indirect fluorescent antibody tests.
3. Option d: Coated latex particles are used in passive agglutination assays.
109
Which of the following correctly describes reverse passive
agglutination?
a. It is a negative test.
b. It can be used to detect autoantibodies.
c. It is used for identification of antigens.
d. It is used to detect sensitization of RBCs
Option c: It is used for identification of antigens.
Reverse Passive Agglutination (RPA) characteristics:
Key Features
1. Antibody-coated particles (e.g., latex, erythrocytes) agglutinate in presence of specific antigens.
2. Detects soluble antigens.
3. High sensitivity and specificity.
Applications
1. Bacterial antigen detection (e.g., Streptococcus, Salmonella).
2. Viral antigen detection (e.g., HIV, Hepatitis).
3. Fungal antigen detection.
Exclude options:
1. Option a: RPA is a positive test detecting antigen presence.
2. Option b: Primarily detects antibodies in patient serum.
3. Option d: Direct antiglobulin test (DAT) detects RBC sensitization.
110
Reactions involving IgG may need to be enhanced for which reason?
a. IgG is only active at 25°C.
b. IgG may be too small to produce lattice formation.
c. IgG has only one antigen-binding site.
d. IgG is only able to produce visible precipitation reactions.
Option b: IgG may be too small to produce lattice formation.
IgG characteristics:
1. Monomeric structure (single unit)
2. Limited cross-linking capability
3. Difficulty forming visible precipitates/lattices
Enhancement methods:
1. Aggregation techniques (e.g., heat, enzymes)
2. Adding carrier molecules (e.g., latex particles)
3. Increasing antibody concentration
4. Using secondary antibodies (e.g., anti-IgG)
Exclude options:
1. Option a: IgG functions optimally at 37°C.
2. Option c: IgG has two antigen-binding sites (Fab regions).
3. Option d: IgG can produce precipitation reactions, but may require enhancement.
111
For which of the following tests is a lack of agglutination a positive reaction?
a. Hemagglutination
b. Passive agglutination
c. Reverse passive agglutination
d. Agglutination inhibition
Option d: Agglutination inhibition
Agglutination Inhibition:
1. Antibodies react with antigens, preventing agglutination.
2. Lack of agglutination indicates antibody presence (positive result).
3. Used for detecting antibodies or antigens in serum.
Other options:
1. Option a: Hemagglutination - agglutination is positive.
2. Option b: Passive Agglutination - agglutination is positive.
3. Option c: Reverse Passive Agglutination - agglutination is positive.
Examples of Agglutination Inhibition:
1. Hemagglutination Inhibition (HAI) assays
2. Viral serology tests
3. Toxicology screening
112
Typing of RBCs with reagent antiserum represents which type of reaction?
a. Direct hemagglutination
b. Passive hemagglutination
c. Hemagglutination inhibition
d. Reverse passive hemagglutination
Option a: Direct hemagglutination
Direct Hemagglutination:
1. Reagent antiserum (antibodies) directly bind to RBC antigens.
2. Immediate agglutination reaction.
3. Used for:
Blood typing (ABO, Rh)
Cross-matching
Red cell antibody screening
Other options:
1. Option b: Passive Hemagglutination - uses coated RBCs.
2. Option c: Hemagglutination Inhibition - measures inhibition.
3. Option d: Reverse Passive Hemagglutination - detects antigens.
113
Which of the following statements accurately describes
heterogeneous competitive binding immunoassays?
a. Excess binding sites for the analyte are provided.
b. Test signal is generated in solution without the need of a solidphase support material.
c. The concentration of patient analyte is inversely proportional to bound label.
d. All the patient analyte is bound in the reaction.
Option c: The concentration of patient analyte is inversely proportional to bound label.
Characteristics of Heterogeneous Competitive Binding Immunoassays:
Key Features
1. Limited binding sites on solid-phase support (e.g., microtiter plate).
2. Labeled analyte (tracer) competes with patient analyte for binding sites.
3. Unbound components removed through washing.
Principles
1. Inverse proportionality: Higher patient analyte concentrations → Lower bound label.
2. Signal generation: Bound label detected (e.g., fluorescence, radioactivity).
Examples
1. Radioimmunoassay (RIA)
2. Enzyme-Linked Immunosorbent Assay (ELISA)
3. Immunoradiometric Assay (IRMA)
Exclude options:
1. Option a: Excess binding sites are not provided.
2. Option b: Solid-phase support material is required.
3. Option d: Not all patient analyte is bound.
114
How do heterogeneous immunoassays differ from homogeneous immunoassays?
a. Heterogeneous immunoassays require a separation step.
b. Heterogeneous immunoassays require less technical skill to perform than homogeneous immunoassays.
c. For noncompetitive immunoassays, the concentration of patient analyte is indirectly proportional to bound label in heterogeneous immunoassays.
d. Homogeneous immunoassays have better analytical sensitivity compared with heterogeneous immunoassays.
Option a: Heterogeneous immunoassays require a separation step.
Key differences:
Heterogeneous Immunoassays
1. Separation step: Unbound components removed through washing.
2. Solid-phase support: Antigen/antibody-coated surface (e.g., microtiter plates).
3. Non-homogeneous: Phases separated (solid/liquid).
Homogeneous Immunoassays
1. No separation step: Reaction occurs in a single phase (solution).
2. No solid-phase support: Reagents and sample mixed together.
3. Homogeneous: Single phase (solution).
Examples:
1. Heterogeneous: ELISA, RIA, IRMA
2. Homogeneous: Fluorescence Polarization Immunoassay (FPIA), Chemiluminescence Immunoassay (CLIA)
Exclude options:
1. Option b: Heterogeneous assays often require more technical skill.
2. Option c: Concentration is directly proportional in non-competitive heterogeneous assays.
3. Option d: Heterogeneous assays often have better sensitivity.
115
Which of the following responses characterizes a capture or
sandwich enzyme immunoassay?
a. Less analytically sensitive than competitive enzyme
immunoassays
b. Labeled antigen attached to a solid phase
c. Best for small antigens with a single epitope determinant
d. Excess number of antibody sites on solid-phase material
Option d: Excess number of antibody sites on solid-phase material.
Characteristics of Capture/Sandwich Enzyme Immunoassay:
1. Excess antibody binding sites on solid-phase.
2. Two antibodies: Capture antibody (solid-phase) and detection antibody (labeled).
3. Sandwich complex formation: Capture antibody-antigen-detection antibody.
4. High analytical sensitivity and specificity.
5. Suitable for large antigens (e.g., proteins).
6. Reduced non-specific binding.
Why other options are incorrect:
1. Option a: Capture EIA is more sensitive than competitive EIA.
2. Option b: Labeled antigen is used in competitive EIA.
3. Option c: Best for large antigens with multiple epitopes.
116
Which of the following is an advantage of enzyme immunoassay over radioimmunoassay?
a. Decrease in hazardous waste
b. Shorter shelf life of kit reagents
c. No interference by biological inhibitors
d. Must be read manually
Option a: Decrease in hazardous waste
Enzyme Immunoassay (EIA) advantages over Radioimmunoassay (RIA):
1. Reduced hazardous waste (no radioactive materials).
2. Improved safety (minimized radiation exposure).
3. Longer shelf life (enzymes more stable).
4. Easier disposal (non-radioactive waste).
5. Cost-effective.
6. Higher sensitivity and specificity.
7. Faster results.
117
Which of the following is characteristic of direct fluorescence immunoassays?
a. The anti-immunoglobulin has the fluorescent tag.
b. Antibody is attached to a solid phase.
c. This method can be used for rapid identification of microbial antigens.
d. The amount of color is inversely proportional to the amount of antigen present.
Option c: This method can be used for rapid identification of microbial antigens.
Direct Fluorescence Immunoassay (DFA) characteristics:
Key Features
1. Fluorescently labeled antibody (directly conjugated).
2. Single-step reaction.
3. Rapid results.
Advantages
1. Rapid identification of microbial antigens.
2. High specificity.
3. Sensitivity.
4. Simple protocol.
Applications
1. Microbial antigen detection (e.g., influenza, HIV).
2. Immunofluorescence microscopy.
3. Diagnostic testing.
Why other options are incorrect
1. Option a: Incorrect; antibody has fluorescent tag in indirect DFA.
2. Option b: Characteristic of indirect/sandwich immunoassays.
3. Option d: Describes competitive immunoassays.
118
Which of the following is true of the enzyme-multiplied
immunoassay technique (EMIT)?
a. It is classified as a heterogeneous method.
b. Nicotinamide adenine dinucleotide (NAD+) is a substrate used in the test reaction.
c. When the patient sample antigen concentration is high, the final test signal will be low.
d. Patient sample antigen blocks the enzyme active site in the test reaction.
Option b: Nicotinamide adenine dinucleotide (NAD+) is a substrate used in the test reaction.
EMIT (Enzyme-Multiplied Immunoassay Technique) often employs enzymes like lactate dehydrogenase or malate dehydrogenase, which utilize NAD+ as a cofactor. When antigen-antibody binding occurs, enzyme activity decreases, reducing NADH production.
119
A fluorescent substance is best described as one in which
a. light energy is absorbed and converted to a longer wavelength.
b. the emitted wavelength can be seen under normal white light.
c. there is a long time between the absorption and emission of light.
d. it spontaneously decays and emits light.
Option a: Light energy is absorbed and converted to a longer wavelength.
Fluorescence characteristics:
Key Features
1. Absorption of light energy (excitation).
2. Energy transfer to a higher energy state.
3. Relaxation and emission of light at a longer wavelength.
4. Short-lived emission (nanoseconds to microseconds).
Properties
1. Excitation wavelength < emission wavelength.
2. Energy loss due to vibrational relaxation.
3. Fluorescence spectrum specific to substance.
Options clarification:
1. Option b: Fluorescence typically requires UV or specific excitation light.
2. Option c: Phosphorescence exhibits delayed emission.
3. Option d: Describes phosphorescence, not fluorescence.
120
In a noncompetitive ELISA, if a negative control shows the presence of signal, which of the following might be a possible explanation?
a. No reagent was added.
b. Washing steps were incomplete.
c. The enzyme was inactivated.
d. No substrate was present.
Option b: Washing steps were incomplete.
Incomplete washing can cause:
1. Residual antibodies/enzyme conjugates.
2. Non-specific binding.
3. High background signal.
Other possible explanations:
1. Cross-reactivity.
2. Contaminated reagents.
3. Instrument calibration issues.
4. Sample contamination.
5. Insufficient blocking.
To resolve:
1. Repeat assay.
2. Optimize washing steps.
3. Verify reagent concentrations.
4. Check instrumentation calibration.
5. Use fresh reagents.
121
Which of the following best characterizes chemiluminescent
immunoassays?
a. Only the antigen can be labeled.
b. Tests can be read manually.
c. These are only homogeneous immunoassays.
d. A chemical is oxidized to produce light.
Option d: A chemical is oxidized to produce light.
Chemiluminescent Immunoassay (CLIA) characteristics:
Key Features
1. Chemical oxidation produces light emission.
2. Luminol or acridinium esters commonly used.
3. Light detection via photomultiplier or CCD camera.
Advantages
1. High sensitivity.
2. Wide dynamic range.
3. Low background signal.
4. Rapid results.
Applications
1. Hormone assays.
2. Tumor marker detection.
3. Infectious disease testing.
4. Drug monitoring.
Options clarification
1. Option a: Both antigen and antibody can be labeled.
2. Option b: Typically automated readers are used.
3. Option c: CLIA can be homogeneous or heterogeneous.
122
Immunofluorescence assays may be difficult to interpret for which reason?
a. Autofluorescence of substances in serum
b. Nonspecific binding to serum proteins
c. Subjectivity in reading results
d. Any of the above
Option d: Any of the above.
Immunofluorescence assay challenges:
Interpretation difficulties
1. Autofluorescence: Endogenous substances (e.g., serum proteins, cells) emit fluorescence, masking specific signals.
2. Nonspecific binding: Antibodies bind non-specifically to serum proteins or cellular components.
3. Subjectivity: Reader variability in interpreting fluorescence intensity and patterns.
Additional challenges:
1. Background fluorescence
2. Photobleaching
3. Fluorophore quenching
4. Sample preparation artifacts
5. Instrument calibration variations
To overcome these challenges:
1. Optimize antibody dilutions
2. Use blocking agents (e.g., BSA, milk)
3. Select appropriate fluorophores
4. Standardize reading protocols
5. Utilize controls (positive/negative)
123
Which statement best describes rapid immunochromatographic assays?
a. Test results are always reported as quantitative values.
b. They are designed primarily for point-of-care testing.
c. Urine is the only acceptable sample type.
d. Formation of a colored line always indicates a positive result.
Option b: They are designed primarily for point-of-care testing.
Rapid Immunochromatographic Assays (RICAs) characteristics:
Key Features
1. Portable, lateral flow design
2. Quick results (5-30 minutes)
3. Simple, user-friendly protocol
4. Qualitative or semi-quantitative results
Applications
1. Point-of-care testing (POCT)
2. Clinical diagnostics (influenza, HIV, pregnancy)
3. Field testing (infectious diseases)
4. Food safety testing
5. Environmental monitoring
Sample Types
1. Urine
2. Blood
3. Saliva
4. Serum
5. Other bodily fluids
Result Interpretation
1. Colored line(s) indicate positive results
2. Intensity may correlate with analyte concentration
3. Negative results typically show no colored line
Options clarification:
1. Option a: Results are often qualitative or semi-quantitative.
2. Option c: Various sample types are acceptable.
3. Option d: False positives/negatives possible; interpret results carefully.
124
Which of the following is characteristic of an indirect enzyme
immunoassay?
a. The first antibody has the enzyme label.
b. Only one antibody is required.
c. Color is directly proportional to the amount of patient antibody present.
d. Enzyme specificity is not essential.
Option c: Color is directly proportional to the amount of patient antibody present.
Indirect Enzyme Immunoassay (EIA) characteristics:
Key Features
1. Two-step process
2. Unlabeled primary antibody (patient sample)
3. Enzyme-labeled secondary antibody (anti-human Ig)
4. Color development proportional to patient antibody
Advantages
1. High sensitivity
2. Flexibility (multiple enzyme conjugates)
3. Cost-effective
Applications
1. Antibody detection (infectious diseases, autoimmune disorders)
2. Serology testing
3. Immunological research
Options Clarification
1. Option a: First antibody is unlabeled.
2. Option b: Two antibodies required (primary and secondary).
3. Option d: Enzyme specificity crucial for accurate results.
125
In an indirect ELISA, what would be the outcome of an improper wash after the antibody–enzyme conjugate is added?
a. Results will be falsely decreased.
b. Results will be falsely increased.
c. Results will be unaffected.
d. No wash step is required in the ELISA procedure.
Option b: Results will be falsely increased.
Improper washing after antibody-enzyme conjugate addition leads to:
Causes
1. Residual conjugate remaining.
2. Non-specific binding.
3. High background signal.
Effects
1. False positives.
2. Elevated optical density (OD) values.
3. Inaccurate results.
Best Practices
1. Multiple wash cycles.
2. Optimize wash buffer composition.
3. Verify washing efficiency.
126
In a heterogeneous enzyme immunoassay, if the patient sample produces more signal than the highest positive control, what action should be taken?
a. Report the results out as determined.
b. Dilute the patient sample.
c. Repeat the immunoassay using one-half the volume of the patient sample.
d. Report the results as falsely positive.
Option b: Dilute the patient sample.
When patient sample signal exceeds highest positive control:
Causes
1. Sample concentration exceeds assay dynamic range.
2. Hook effect or prozone phenomenon.
3. Non-specific binding.
Recommended Actions
1. Dilute patient sample (1:2 to 1:10) and retest.
2. Verify dilution factor and calculate corrected results.
3. Consider alternative assays or confirmatory testing.
Incorrect Options
1. Option a: Reporting inaccurate results.
2. Option c: Insufficient correction; dilution recommended.
3. Option d: Premature conclusion; dilution and retesting necessary.
127
Which of the following can uniquely cause interferences in immunoassays designed with streptavidin bound to the solid-phase surface?
a. Elevated concentration of biotin in the patient test sample can cause falsely increased results.
b. Heterophile antibodies present in the patient sample cause falsely decreased results.
c. Antibody excess in the patient sample leads to a hook effect.
d. Solid-phase particles will dissolve easily if the reaction temperature is increased.
Option a: Elevated concentration of biotin in the patient test sample can cause falsely decreased results.
Biotin interference:
1. Elevated biotin levels (>10 ng/mL) in patient samples.
2. Biotin competes with biotinylated antibodies/antigens.
3. False negatives or decreased signal.
Other interferences:
1. Option b: Heterophile antibodies (HAMA) cause false positives.
2. Option c: Hook effect occurs due to antibody excess.
3. Option d: Temperature increase doesn't specifically affect solid-phase particles.
Prevention strategies:
1. Biotin-free sample collection tubes.
2. Sample dilution.
3. Biotin-blocking agents.
4. Alternative assay designs (e.g., non-avidin/biotin systems).
