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Principles of Immunochemistry and Immunoassay Techniques
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Principles of Immunochemistry and Immunoassay Techniques
Principles of Immunochemistry and Immunoassay Techniques
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1
Question
What are antibodies in the context of immunochemical reactions?
Page 159
Answer
Immunoglobulins that bind specifically to natural and synthetic antigens such as proteins, carbohydrates, nucleic acids, and lipids.
2
Question
How do polyclonal antibodies differ from monoclonal antibodies?
Page 159
Answer
Polyclonal antibodies are derived from different plasma cell lines or clones, while monoclonal antibodies are derived from a single plasma cell line or clone.
3
Question
What are three advantages of using monoclonal antibodies in immunoassays?
Page 159
Answer
They provide well-defined reagents, unlimited quantities of homogeneous material with consistent affinity and specificity, and can be produced from nonpurified antigens.
4
Question
What is an immunogen in immunochemistry?
Page 159
Answer
A chemical substance capable of inducing an immune response and eliciting antibody formation when injected into a host.
5
Question
How does an antigen differ from an immunogen?
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Answer
An antigen reacts with an antibody without necessarily inducing antibody formation, while an immunogen induces antibody production.
6
Question
What is a hapten and how does it produce antibodies?
Page 159
Answer
A chemical determinant that, when conjugated to an immunogenic carrier like a protein, stimulates synthesis of hapten-specific antibodies but cannot alone.
7
Question
What are examples of labels used in enzyme immunoassays and their detection method?
Page 160
Answer
Peroxidase, β-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase; detected by absorbance spectroscopy.
8
Question
What label is used in radioimmunoassays and how is it detected?
Page 160
Answer
Radioisotopes like ¹²⁵I, ¹³¹I, ³H (tritium); detected by scintillation counting.
9
Question
What are common chemiluminescent labels used in immunoassays?
Page 160
Answer
Acridinium esters, dioxetane, luminol; detected by chemiluminescence producing light via chemical reaction.
10
Question
How does affinity differ from avidity in antigen-antibody binding?
Page 160
Answer
Affinity is the thermodynamic energy of a single antibody site-epitope interaction; avidity is the overall binding strength summing all site affinities.
11
Question
In competitive immunoassays, what is the relationship between sample analyte concentration and label signal?
Page 161
Answer
Inverse: higher analyte concentration means less labeled antigen binds antibody, reducing bound label signal.
12
Question
What distinguishes heterogeneous from homogeneous immunoassays?
Page 162
Answer
Heterogeneous require physical separation of bound from free fractions; homogeneous do not.
13
Question
What is the key requirement for antigens in noncompetitive sandwich immunoassays?
Page 162
Answer
Antigen must have at least two binding sites to complex both capture and labeled antibodies.
14
Question
In noncompetitive sandwich immunoassays, what is the relationship between analyte concentration and signal?
Page 162
Answer
Direct: more analyte captures more labeled antibody, increasing signal.
15
Question
What is microparticulate enzyme immunoassay (MEIA)?
Page 163
Answer
Homogeneous immunoassay using latex microparticles coated with antibody and alkaline phosphatase-labeled antibody; fluorescence from 4-MUP substrate measured.
16
Question
How is fluorescence produced in MEIA?
Page 163
Answer
ALP cleaves 4-methylumbelliferyl phosphate (4-MUP) to fluorescent methylumbelliferone (MU); excitation 380 nm, emission 450 nm.
17
Question
What substrate is used in chemiluminescent enzyme immunoassays (CL-EIA)?
Page 164
Answer
Adamantyl 1,2-dioxetane phenyl phosphate (AMPPD); cleaved by ALP to produce light at 477 nm via CIEEL.
18
Question
Why are chemiluminescent immunoassays more sensitive than EIAs or FIAs?
Page 164
Answer
They detect low analyte concentrations via light production without external excitation, offering high efficiency.
19
Question
What occurs in EMIT when no analyte is present in the sample?
Page 165
Answer
Antibody binds enzyme-labeled antigen, inhibiting enzyme activity by steric hindrance or conformational change.
20
Question
In EMIT, which enzyme is commonly conjugated to antigen and what is its substrate?
Page 165
Answer
Glucose-6-phosphate dehydrogenase; substrate glucose-6-phosphate and NAD produce colored product measured colorimetrically.
21
Question
How does substrate-labeled fluorescent immunoassay (SLFIA) generate fluorescence?
Page 166
Answer
β-galactosidase cleaves β-galactosylumbelliferone-antigen conjugate to fluorescent umbelliferone when not bound by antibody.
22
Question
What technology creates the enzyme donor and acceptor in cloned enzyme donor immunoassay (CEDIA)?
Page 167
Answer
Recombinant DNA technology engineers β-galactosidase into large enzyme acceptor and small enzyme donor polypeptides.
23
Question
Why is CEDIA linear with analyte concentration?
Page 167
Answer
Amount of active β-galactosidase tetramer formed is directly proportional to free enzyme donor displaced by analyte.
24
Question
What principle underlies fluorescence polarization immunoassay (FPIA)?
Page 168
Answer
Bound fluorescein-labeled antigen rotates slowly, emitting polarized light; free rotates fast, depolarizing emission.
25
Question
In FPIA, why does high analyte concentration produce low polarized signal?
Page 169
Answer
Analyte displaces most fluorescein-labeled antigen from antibody, leaving small free molecules that depolarize rapidly.
26
Question
What triggers light emission in acridinium ester chemiluminescent immunoassay (CLIA)?
Page 170
Answer
Pretrigger (base/H₂O₂) and trigger (acid) convert acridinium to N-methyl acridone, emitting light.
27
Question
How does electrochemiluminescent immunoassay (ECLIA) generate light at 620 nm?
Page 171
Answer
Ru(bpy)₃²⁺ oxidized to Ru(bpy)₃³⁺ with TPA on gold electrode; de-excitation emits photon.
28
Question
What is the key element in luminescent oxygen-channeling immunoassay (LOCI)?
Page 172
Answer
Singlet oxygen (¹Δg O₂) diffuses from Sensibead to Chemibead, triggering chemiluminescence at 612 nm.
29
Question
What happens in kinetic interaction of microparticles in solution (KIMS) without analyte?
Page 172
Answer
Antibody binds microparticle-analyte conjugate, forming aggregates that increase absorbance.
30
Question
What causes the hook effect in immunoassays and how is it detected?
Page 173
Answer
Excess analyte over antibody saturates both capture and labeled antibodies, flattening/negatively sloping dose-response; detected by dilution linearity test.