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Chapter 3 Laboratory Diagnosis by Immunologic Methods

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1 Chapter 3 Laboratory Diagnosis by Immunologic Methods

2 Copyright © 2017 Wolters Kluwer • All Rights Reserved
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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

3 Copyright © 2017 Wolters Kluwer • All Rights Reserved
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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

4 Copyright © 2017 Wolters Kluwer • All Rights Reserved
Classes of Human Igs Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

8 Copyright © 2017 Wolters Kluwer • All Rights Reserved
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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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. Copyright © 2017 Wolters Kluwer • All Rights Reserved

10 Complement Fixation (CF)
Copyright © 2017 Wolters Kluwer • All Rights Reserved

11 Hemagglutination Inhibition (HAI)
Inactivated hemagglutinating virus Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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. Copyright © 2017 Wolters Kluwer • All Rights Reserved

14 Principles of EIA (Rubella Virus)
Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

16 Solid-Phase Immunoassay Methods (cont.)
Antibody capture is a preferred method for IgM detection Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

19 Lateral Flow Immunoassay for Detection of Antigen
Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

21 Immunofluorescence Techniques for Antigen Detection
Direct immunofluorescence, also called direct fluorescent antibody (DFA) Copyright © 2017 Wolters Kluwer • All Rights Reserved

22 Immunofluorescence Techniques for Antigen Detection (cont.)
Indirect immunofluorescence, also called indirect fluorescent antibody (IFA) Copyright © 2017 Wolters Kluwer • All Rights Reserved

23 Immunofluorescence Techniques for Antibody Detection (cont.)
Retrospective diagnosis of viral infections by detecting seroconversion or change in titers Example: CMV Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved

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 Copyright © 2017 Wolters Kluwer • All Rights Reserved


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