Download Immunofluorescence Techniques for Antigen Detection

Chapter 3
Laboratory Diagnosis by
Immunologic Methods
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
2
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
3
Classes of Human Igs
Copyright © 2017 Wolters Kluwer • All Rights Reserved
4
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
•
•
•
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
•
Principal antibody on mucosal surfaces and extracellular secretions
•
Found on surfaces of immature B lymphocytes, acting as a cellular antigen receptor
•
•
Binding activates degranulation of mast cells and basophils.
Major role in allergic reactions, may also be elevated during intestinal helminth
infections
• IgM
• IgA
• IgD
• IgE
Copyright © 2017 Wolters Kluwer • All Rights Reserved
5
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
6
Monoclonal Antibodies
• Enable isolation of cloned lines of individual lymphocytes
that produce unique, monospecific antibody molecules
• Procedure
1.
2.
3.
4.
5.
6.
7.
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
7
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
8
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 antibodycoated 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
9
Complement Fixation (CF)
Copyright © 2017 Wolters Kluwer • All Rights Reserved
10
Hemagglutination Inhibition (HAI)
Inactivated hemagglutinating virus
Copyright © 2017 Wolters Kluwer • All Rights Reserved
11
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
12
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
13
Principles of EIA (Rubella Virus)
Copyright © 2017 Wolters Kluwer • All Rights Reserved
14
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
15
Solid-Phase Immunoassay Methods (cont.)
Antibody capture is a preferred
method for IgM detection
Copyright © 2017 Wolters Kluwer • All Rights Reserved
16
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
17
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
18
Lateral Flow Immunoassay for Detection of Antigen
Copyright © 2017 Wolters Kluwer • All Rights Reserved
19
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
20
Immunofluorescence Techniques for Antigen Detection
Direct immunofluorescence,
also called direct fluorescent
antibody (DFA)
Copyright © 2017 Wolters Kluwer • All Rights Reserved
21
Immunofluorescence Techniques for Antigen Detection (cont.)
Indirect immunofluorescence,
also called indirect fluorescent
antibody (IFA)
Copyright © 2017 Wolters Kluwer • All Rights Reserved
22
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
23
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
24
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
25
Immunofluorescence and EIA Approaches
• Immunofluorescence
•
•
DFA techniques are more labor intensive.
Allow direct observation to determine adequacy of specimen
•
EIA methods for antigen detection advantageous in high-volume labs
where many samples are examined for a single determinant
• EIA
Copyright © 2017 Wolters Kluwer • All Rights Reserved
26