HenryGÇÖs Chapter 43

8/6/2019 HenryGs Chapter 43

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Henrys Chapter 43 Outline:Immunoassay and Immunochemistry


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General Characteristics of Antigen

Antibody Reaction

Biological ligands:

Enzyme and substrate

Hormone and receptor

Antigen and antibody Immunoassays

Optimized to detect less than 0.1 pg/mL of antigenpresent in blood

Detection of: Haptens as small molecules

Proteins and protein complexes as macromolecules

Any antibody to allergens, infectious agents, and autologous

antigens


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General Characteristics of Antigen

Antibody Reaction Antigens

Haptens and hormones

Proteins, glycoproteins, glycolipids, and other naturalproducts

Antibodies/Immunoglobulin

5 classes (isotypes): IgG, IgM, IgA, IgD, and IgE.

IgG: 4 subclasses

Both IgA and IgM: 2 subgroups

Heavy chain: either a or a light chain

Variable regions

Polyclonal or monoclonal


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Polyclonal Antibodies

Obtained through immunization with an

antigen which has various epitopes


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Monoclonal Antibodies

Uniform homogeneous antibodies directed to

specific epitopes

Developed using:

Somatic cell fusion

Selection of the resulting hybridoma

Limiting dilution to obtain monoclonals

Use:

Tumor marker

Identification of isoenzymes, subtypes, isotypes of

protein, and conformational changes of molecules


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Immunoassays

Precipitation immunoassays

Particle immunoassays

Radioimmunoassays Enzyme immunoassays

Fluorescent immunoassays

Chemiluminescent immunoassays Table 43-1.


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Precipitin and Nephelometric

Immunoassays


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Precipitin and Nephelometric

Immunoassays

Large complexes of antigen and antibodycombine to form an insoluble lattice.

Sensitivity limitation (0.1-0.5 mg/dL) of these

assays is a major consideration. Factors affecting the precipitin:

Relative proportions of reactants

Temperature

pH

Ionic strength of the medium

Antibody characteristics of avidity and affinity


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Formats:

1. Qualitative Precipitant Assay Methods

2. Semiquantitative Precipitant Assay Methods

3. Nephelometric immunoassays


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1. Qualitative Precipitant Assay

Methods

Single immunodiffusion

Double immunodiffusion

Double immunodiffusion in two dimensions Electroimmunodiffusion reaction

Immunoelectrophoresis


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2. Semiquantitative Precipitant Assay

Methods Single radial immunodiffusion Single dimension electroimmunodiffusion

(rocket electrophoresis)

3. Nephelometric immunoassays

Immunoprecipitin analysis that use light-scattering devices

Turbidimetry or nephelometry


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Particle Immunoassay


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Particle Immunoassay

Based on agglutination reaction

Does not require separation of bound and freereactants.

Fomats: Hemagglutination tests

Gelatin Particle Agglutination

Latex Agglutination

Particle-Counting Immunoassay

Quasi-elastic scattering


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Hemagglutination tests

Simple to perform and do not require special equipment Detection of antibody to Treponema pallidum

Recommended by WHO

Superior specificity and sensitivity

Detection of antibodies to hepatitis B virus (HBV), hepatitistype C (HCV), human immunodeficiency virus (HIV-1/2),thyroglobulin, thyroid microsome, and other substances

Reverse hemagglutination tests are used for thedetection of HBV surface antigen, -fetoprotein (AFP),

human hemoglobin in stool specimens, and so forth

Sensitivity: 50 ng/mL for antigen detection


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Special gelatin particle

Highly hydrophilic surface

Able to prevent nonspecific binding of materials

present in a specimen 3 m in size

Preparation:

Phase separation and 3-D cross-linkage at 40Cand optimum pH

Fixation with formaldehyde or glutaraldehyde


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Advantages over erythrocytes:

No antigenicity and is therefore free from problemsassociated with heterophilic antibodies

Requires much less serum dilution to avoidnonspecific binding and guarantees more sensitivedetection

Strict temperature control is not required to performthe tests

Detection of antibodies to human T-cell lymphotropicvirus type I (HTLV-1), HIV, HBV, and HCV

High sensitivity and specificity


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Latex Agglutination Latex as particles

Detection of various analytes, such as hCG forqualitative pregnancy tests, and the quantitativedetection of other plasma proteins with orwithout instrumentation

Susceptible to interference from unknown factorspresent in specimens (remedied by addition ofabsorbents)

Sensitivity: less than 0.5 g/mL

Adapted for quantitative assays using lightdetection methods

Turbidimetry (light absorption)

Nephelometry (light scattering)

Enhanced sensitivity of subnanograms per milliliter


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Particle-Counting Immunoassay

Uses optical cell counting to assess thedecrease in the number of unagglutinatedparticles after an immunoreaction.

Formats: Rate assay

Rate of decrease in the number of unagglutinatedparticles

End point of the assay Sensitivity: nanogram-per-milliliter

Longer incubation time


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Quasi-elastic scattering

Measurement of the change in response to

particle size distribution

Uses a laser light beam to measure the

reduction in the mean diffusion coefficient of

particles as a result of immunoreactions

Other methods measure the change in

angular anisotropy of scattered light with the

increasing average size of the particle


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Radioimmunoassay (RIA)


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Radioimmunoassay (RIA) Radioisotopes as labels (See Table 43-3)

RIA techniques:

Competitive

Noncompetitive heterogeneous formats

Immunoradiometric or sandwich assays

Advantages: Precision and high

sensitivity

Ease of isotope conjugation

Signal detection withoutoptimization

Stability against interferencefrom the assay environment

Disadvantages: Short shelf-life of the

reagentsHazardous radioactivity

May not be applied tohomogeneousimmunoassays becausesignals from isotopes cannotmodulate antigenantibody

reactions


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Enzyme Immunoassay


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Enzyme Immunoassay

Enzymes as labels

Enzyme-linked immunoabsorbent assay

(ELISA), the EIA, and the enzyme-multiplied

immunoassay technique (EMIT) are most

widely used.


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Advantages: Sensitive assays can be

developed by the amplificationeffect of enzymes.

Reagents are relatively cheapand can have a long shelf-life.

Multiple simultaneous assays

can be developed. A wide variety of assay

configurations can bedeveloped.

Equipment can be inexpensiveand is widely available.

No radiation hazards occurduring labeling or disposal ofwastes.

Disadvantages:

Measurement of enzyme activity

can be more complex than

measurement of the activity of

some types of radioisotopes.

Enzyme activity may be affected

by plasma constituents.

Homogeneous assays at the

present time have the sensitivity

of 109 M and are not as

sensitive as radioimmunoassays.

Homogeneous EIAs for large

protein molecules have been

developed but require complex

immunochemical reagents.


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Heterogeneous Enzyme Immunoassays

Commonly used enzymes Horseradish peroxidase

Alkaline phosphatase

-galactosidase

Glucose oxidase

Urease

Catalase

Colorimetric Enzyme Immunoassay

Fluorescent Enzyme Immunoassay

Chemiluminescent Enzyme Immunoassay


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Colorimetric Enzyme Immunoassay

Chromogenic substrates

ABTS (diammonium salt of 2,2-azino-di[3-ethyl-benzothiazoline-6-sulfonate])

Horseradish peroxidase

ABTS + H2O2 green color

Alkaline phosphatase

p-nitrophenyl phosphate yellow color

Fl t E I


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Fluorescent Enzyme Immunoassay Fluorescent substrates

Light excitation

Chemiluminescent EnzymeImmunoassay (CL-EIA)

Chemiluminescent substrates: Luciferin-adenosine triphosphate

Luminol derivatives (peroxidase, glucose oxidase oruricase) with p-iodophenol as an enhancer

Dioxetane derivatives (alkaline phosphatase)

Adamantyl dioxetane derivative: AMPPD (disodium 3-(4-methylspiro-[1,2-dioxetane-3,2-tricyclo-[3.3.1.1]decan]-4-yl) phenyl phosphate


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Homogeneous Enzyme Immunoassays

Competitive or noncompetitive binding assays

Advantages:

Rapid and simple

Adaptable to

conventional

instruments

Disadvantages:

Less sensitive thanheterogeneous EIA

Require compleximmunochemical

reagents


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Competitive

Enzyme-Multiplied Immunoassay Technique(EMIT) Antigen with lysozyme, G6PD

Substrate-Labeled Fluorescent Immunoassay(SLFIA) Antigen with substrate

Apoenzyme Reactivation Immunoassay (ARIS) Antigen with prosthetic group

Cloned Enzyme Donor Immunoassay (CEDIA) Antigen with fragments of enzyme

ARIS f t


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ARIS formats: Enzyme-channeling immunoassay

Antigen with G6PDH and hexokinase

Biotin-enzyme avidin immunoassay

Antigen with avidin

Noncompetitive Hybrid antibody immunoassay

Hybrid antibodies specific to antigen and to inhibitor

Proximal linkage immunoassay Antibody with G6PDH and hexokinase

Enzyme Inhibitory Homogeneous Immunoassay (EIHIA) Antibody with amylase

Enzyme enhancement immunoassay Antibody with -galactosidase and succinyl antibody

AEST Antibody with peroxidase

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HenryGÇÖs Chapter 43