1. Chapter 3 Laboratory Diagnosis by Immunologic Methods

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Antigens
Antigen
A substance that evokes the formation of antibodies in an animal that is immunized with that particular antigen
Generally immunogenic
Immunodominant antigenic determinants
Also called epitopes
The antigen’s molecular structures that are immunogenic and recognized by antibodies
Cross-reactivity
Different molecules may share antigenic determinants and may be recognized by antibodies directed against these determinants.
Example: certain streptococcal antigens and heart muscle and valve tissue proteins
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Antibodies
Immunoglobulins (Igs)
Antibodies belong to this group of structurally related glycoprotein molecules in blood and extracellular fluid.
Produced by B lymphocytes that secrete antibodies against the target antigen or present the antigen to T lymphocytes to stimulate additional antibody production and cellular immune responses
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Classes of Human Igs
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5. Classes of Human Igs (cont.)
IgG
Actively transported across placenta, providing passive immunity to newborn infant
Appears in serum 4 to 6 weeks after infection, usually persists for life
IgG1: major Ig in serum, fix and activate complement
IgG2 & IgG4: response to polysaccharide antigens, including encapsulated bacteria
IgG3: secondary immune response, viral neutralization
IgM
First Ig class produced by fetus, does not cross placenta
First to appear following immunization or infection, helpful in complement fixation
Presence in blood usually indicates recent infection, may appear during reactivation of latent infection, may persist for weeks to months
IgA
Principal antibody on mucosal surfaces and extracellular secretions
IgD
Found on surfaces of immature B lymphocytes, acting as a cellular antigen receptor
IgE
Binding activates degranulation of mast cells and basophils.
Major role in allergic reactions, may also be elevated during intestinal helminth infections
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6. Serologic Diagnosis of Infections
First specimen collected 5 to 7 days after onset of symptoms
Second specimen collected 2 to 4 weeks later
≥ fourfold rise in antibody titer suggests recent or intercurrent infection
Antibody titer: the reciprocal of the dilution of serum that still gives a positive test for the presence of antibodies
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7. Monoclonal Antibodies
Enable isolation of cloned lines of individual lymphocytes that produce unique, monospecific antibody molecules
Procedure
Selection of antigen
Animal immunization
Fusion of splenic lymphocytes and myeloma cells
Selection of hybrid lymphocyte–myeloma cells
Cloning the hybridoma cells
Screening for desired antibodies
Mass production of monoclonal antibodies
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Precipitin Reactions
Test systems that allow free diffusion of antigen and antibody fronts toward one another
Single diffusion
Tube (Oudin) immunodiffusion method
Radial immunodiffusion
Double diffusion
Countercurrent immunoelectrophoresis (CIE)
Possible false-negative reactions if antibody or antigen is in excess
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9. Complement Fixation and Hemagglutination Inhibition
Older methods for the diagnosis of infectious diseases that can either identify viral antigens or detect antibodies
Largely superseded by other serologic and nucleic acid-based methods
Complement fixation (CF)
In stage 1, antigen, antibody, and complement are mixed. If antigen and antibody bind, complement is fixed and will be unable to act on antibody-coated erythrocytes added in stage 2. The final reaction appears as an absence of hemolysis. If antigen and antibody do not bind in stage 1, complement is not fixed and remains free to act on the antibody-coated erythrocytes added in stage 2.
Hemagglutination inhibition (HAI)
In stage 1, hemagglutinating viruses and antibodies are mixed. In stage 2, erythrocytes are added. If antibodies bind to virus in stage 1, the virus is inhibited and fails to hemagglutinate the erythrocytes. If antibodies do not bind, virus remains active and hemagglutinates erythrocytes in stage 2.
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10. Complement Fixation (CF)
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11. Hemagglutination Inhibition (HAI)
Inactivated hemagglutinating virus
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12. Agglutination Reactions
Specific immunochemical aggregation of particles coated with antigen or antibody that can be used to detect either soluble antibodies or antigens
Latex agglutination
Streptococcal grouping from positive cultures and cryptococcal antigen detection
Staphylococcal coagglutination
Serologic grouping of β-hemolytic streptococci
Immune electron microscopy (IEM)
Visual detection of viral agents that are noncultivable
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13. Solid-Phase Immunoassay Methods
EIAs for antibody detection
Binding of an antigen or antibody to a variety of solid materials
Incubation in contact with the solid phase, specific antibody binds to immobilized antigen, mixture is washed, antiglobulin is conjugated with a “tag” and incubated, antiglobulin binds to antibody if initial antigen–antibody reaction has occurred
Able to assess antigen/antibody interactions, glycoproteins, specificities of antibodies against other agents
Western immunoblot: antigen specificities of antisera
Detection of HIV-1 antibodies has undergone several modifications since 1984.
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14. Principles of EIA (Rubella Virus)
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15. Solid-Phase Immunoassay Methods (cont.)
EIA antibody-capture methods for IgM detection
Detect primarily IgG, sometimes in combination with IgM to differentiate recent from past infections
Methods to separate IgM from IgG—none are totally effective
Ion exchange column chromatography and gel filtration—molecular size and charge
Sucrose gradient density centrifugation—molecular size
Serum specimens treated with anti-IgG antibodies
Staphylococcal protein A to bind up IgG molecules
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16. Solid-Phase Immunoassay Methods (cont.)
Antibody capture is a preferred method for IgM detection
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17. Solid-Phase Immunoassay Methods (cont.)
EIAs for antigen detection
Capture antibody fixed to the solid phase allows for detection of antigens rather than antibodies.
Diverse microbial pathogens or virulence factors in stool, urine, and serum
Example: H. pylori
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18. Solid-Phase Immunoassay Methods (cont.)
Immunoconcentration and immunochromatographic immunoassays
Basic technology described in 1960s
First commercial application: home pregnancy test in 1980s
Applications in clinical, veterinary, and industrial applications
Example: Immunocard Mycoplasma to detect Mycoplasma pneumoniae–specific IgM in serum
Immunochromatographic or lateral flow immunoassays allow use in test strip format.
Example: rapid HIV antibody assays allowing efficient detection of infection in developed and developing countries
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19. Lateral Flow Immunoassay for Detection of Antigen
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20. Immunofluorescence Techniques
To detect and localize antigens to diagnose bacterial, fungal, parasitic, and viral diseases
To detect antibodies for retrospective diagnosis of infectious diseases
Fluorescence
Radiation of energy when light of a shorter “excitation” wavelength incites the electrons of a molecule to a higher energy state for a very short time
As electrons return to preexcitation state, energy is released as light of a longer wavelength.
Direct immunofluorescence: antigen detection
Indirect immunofluorescence: antigen and antibody detection
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21. Immunofluorescence Techniques for Antigen Detection
Direct immunofluorescence, also called direct fluorescent antibody (DFA)
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22. Immunofluorescence Techniques for Antigen Detection (cont.)
Indirect immunofluorescence, also called indirect fluorescent antibody (IFA)
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23. Immunofluorescence Techniques for Antibody Detection (cont.)
Retrospective diagnosis of viral infections by detecting seroconversion or change in titers
Example: CMV
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24. Immunofluorescence Techniques for Antibody Detection (cont.)
Anticomplement immunofluorescence (ACIF) test
Example: detecting low levels of Epstein–Barr nuclear antigen (EBNA) in EBV-infected cells
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25. Immunofluorescence Techniques for Antibody Detection (cont.)
Fluorescent antibody to membrane antigen (FAMA) test
Test of choice to determine immune status for certain virus
High sensitivity
Example: VZV
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26. Immunofluorescence and EIA Approaches
DFA techniques are more labor intensive.
Allow direct observation to determine adequacy of specimen
EIA
EIA methods for antigen detection advantageous in high-volume labs where many samples are examined for a single determinant
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