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Immunoassay Principles and Methodology
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Immunoassay Principles and Methodology
Immunoassay Principles and Methodology
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1
Question
What is the primary immunoglobulin class used as an immunochemical reagent in immunoassays?
Page 159
Answer
Immunoglobulin G (IgG)
2
Question
How do polyclonal antibodies differ from monoclonal antibodies in terms of their origin and specificity?
Page 159
Answer
Polyclonal antibodies are derived from different plasma cell lines or clones, while monoclonal antibodies are derived from one plasma cell line or clone and target a single epitope.
3
Question
What are three advantages of using monoclonal antibodies in immunoassay systems?
Page 159
Answer
They provide a well-defined reagent, yield unlimited quantities of homogeneous reagent with consistent affinity and specificity, and can be prepared using nonpurified antigens.
4
Question
What is an immunogen in the context of immunochemical reactions?
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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?
Page 159
Answer
An antigen is any material capable of reacting with an antibody, but not necessarily inducing antibody formation, whereas an immunogen induces antibody formation.
6
Question
What is a hapten and how does it produce antibodies?
Page 159
Answer
A hapten is a chemical determinant that, when conjugated to an immunogenic carrier, stimulates synthesis of hapten-specific antibodies but cannot do so alone.
7
Question
What are five enzymes commonly used as labels in immunoassays?
Page 160
Answer
Peroxidase, β-galactosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase.
8
Question
What detection method is used for radioisotope labels like iodine-125 in immunoassays?
Page 160
Answer
Scintillation counting
9
Question
Name three chemiluminescent labels used in immunoassays and their detection method.
Page 160
Answer
Acridinium esters, dioxetane, luminol; detected via chemiluminescence.
10
Question
What is the kinetic equation for a reversible antigen-antibody reaction?
Page 160
Answer
\( \text{Ag} + \text{Ab} \underset{k_2}{\overset{k_1}{\rightleftharpoons}} \text{AgAb} \)
11
Question
How does affinity differ from avidity in antigen-antibody binding?
Page 160
Answer
Affinity is the thermodynamic energy of a single binding site interaction; avidity is the overall binding strength summing all individual site affinities.
12
Question
What role does solid-phase material play in heterogeneous immunoassays?
Page 162
Answer
It immobilizes antibodies via covalent bonding or adsorption to enable physical separation of bound from free fractions.
13
Question
How does signal relate to analyte concentration in competitive immunoassays?
Page 162
Answer
Inversely proportional, as labeled antigen competes with sample analyte for limited antibody binding sites.
14
Question
What is the key procedural difference between homogeneous and heterogeneous immunoassays?
Page 162
Answer
Homogeneous assays do not require physical separation of bound and free forms; heterogeneous assays do.
15
Question
Describe the signal-analyte relationship in noncompetitive sandwich immunoassays.
Page 162
Answer
Nonlinear and directly proportional, as analyte concentration determines labeled second antibody binding.
16
Question
What substrate and enzyme are used in microparticulate enzyme immunoassay (MEIA)?
Page 162
Answer
Alkaline phosphatase (ALP) enzyme label with 4-methylumbelliferyl phosphate (4-MUP) substrate producing fluorescent methylumbelliferone (MU).
17
Question
How does chemiluminescent enzyme immunoassay (CL-EIA) produce light at 477 nm?
Page 165
Answer
ALP cleaves AMPPD phosphoester bond, releasing electron-rich dioxetane phenolate (AMP-D) that decomposes to emit light.
18
Question
In the enzyme multiplied immunoassay technique (EMIT), how does enzyme activity relate to sample analyte concentration?
Page 165
Answer
Directly proportional, as sample antigen displaces enzyme-labeled antigen from inhibitory antibody binding.
19
Question
What makes the substrate-antigen conjugate nonfluorescent in substrate-labeled fluorescent immunoassay (SLFIA)?
Page 165
Answer
β-galactosidase cleaves β-galactosylumbelliferone only when not bound by antibody, producing fluorescent umbelliferone.
20
Question
What technology enables cloned enzyme donor immunoassay (CEDIA) to split β-galactosidase into acceptor and donor fragments?
Page 166
Answer
Recombinant DNA technology engineering large polypeptide enzyme acceptor and small enzyme donor.
21
Question
Why does net polarization decrease with higher analyte concentration in fluorescence polarization immunoassay (FPIA)?
Page 167
Answer
High analyte binds antibody, leaving free rotating fluorescein-labeled antigen emitting depolarized fluorescence.
22
Question
What trigger reagents activate acridinium ester in chemiluminescent immunoassay (CLIA)?
Page 170
Answer
Pretrigger and trigger reagents including base/acid and H2O2 producing N-methyl acridone light emission.
23
Question
How does electrochemiluminescent immunoassay (ECLIA) generate 620 nm light using ruthenium?
Page 171
Answer
Ru(bpy)₃²⁺ and TPA oxidize on gold electrode; excited Ru(bpy)₃²⁺ decays emitting photons.
24
Question
What triggers chemiluminescence at 612 nm in luminescent oxygen-channeling immunoassay (LOCI)?
Page 172
Answer
Singlet oxygen (¹ΔgO₂) diffuses from Sensibead to Chemibead in bead immunocomplex after 680 nm light stimulation.
25
Question
How does kinetic interaction of microparticles in solution (KIMS) detect analyte?
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Answer
Analyte inhibits microparticle-analyte conjugate aggregation by antibody, reducing light transmission absorbance.
26
Question
What causes the hook effect in immunoassays and how is it validated?
Page 173
Answer
Excess analyte over antibody flattens or reverses dose-response curve; validated by dilution linearity testing.
27
Question
Why did Yalow and Berson develop radioimmunoassay (RIA) for insulin in 1960?
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Answer
To overcome limitations of prior assays lacking sensitivity, specificity, precision, and accuracy for proteins, steroids, vitamins.
28
Question
What is a label in immunoassay procedures?
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Answer
An element or compound attached to an antibody or antigen for detection.
29
Question
How do cation salts affect antigen-antibody binding?
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Answer
They inhibit binding by altering hydration of charged groups in antibody sites or cationic antigens.
30
Question
What common separation substances are used in heterogeneous immunoassays?
Page 162
Answer
Double antibodies, solid-phase antibodies, polyethylene glycol, talc, solvent or salt precipitates, charcoal.