Immunological Reactions and Techniques
W. Robert Fleischmann, Ph.D.Department of Urologic SurgeryUniversity of Minnesota Medical [email protected](612) 626-5034
Objectives• Become familiar with the various types
of immunological techniques that are used for clinical diagnosis.
• Understand how each of these immunological techniques work.
The purposes of immunological tests are to aid in the determination or monitoring of diseases or conditions that may be present in a patients.
Immunologic Techniques• Agglutination reactions• Direct antiglobulin test• Indirect antiglobulin test• Electrophoresis• Immunofixation• Immunofluorescence• Enzyme-linked immunosorbent assay• Western blot• Flow cytometry
Agglutination Reactions• Purpose:
– To detect antibodies in a patient’s serum– To detect antigens in a patient’s serum
• Examples:– Detect whether or not a patient has previously
been exposed to an agent such as German measles (rubella) either by vaccination or by contracting the disease
– Testing for the presence of Hemophilus influenzae type B capsular antigens in cerebral spinal fluid
Agglutination Reactions (Cont.)• Agent detected:
– Specific antibody– Specific antigen
• Indicator:– Agglutinated particles.
• Use particles coated with antigen or with antibody.
• These particles could be colored or white latex beads
• The particles could even be RBCs (hemagglutination).
Agglutination Reaction: Method
Harvest a patient specimen.
Clarify the specimen by centrifugation.
Mix specimen with white or colored latex beads that have Bound antibodies on their surface.
Allow binding, if any, to occur.
Read the test as agglutination or no agglutination of the beads.
Results of an Agglutination Test
Wikipedia
Direct Antiglobulin Test
Aka: Coombs Test• Purpose:
– Detect antibody on a patient’s RBCs• Examples:
– Performed in patients with hemolytic anemia caused by autoimmune disease
– Diagnosis of a transfusion reaction– Diagnosis of hemolytic disease of the newborn
(blue baby syndrome, Rh factor incompatibility)
Direct Antiglobulin Test (Cont.)
• Agent detected:– Antibody– Complement
• Indicator:– Agglutinated (clumped) RBCs
Direct Antiglobulin Test: Method
Wikipedia
Indirect Antiglobulin Test
Aka: Indirect Coombs Test• Purpose:
– Detect antibodies that are directed against RBC antigens
• Examples:– Performed in patients about to undergo a blood
transfusion to detect the presence of antibodies to the RBCs about to be transfused (mismatch)
– During pregnancy or at delivery for a woman who is Rh negative
Indirect Antiglobulin Test (Cont.)
• Agent detected:– Antibody to RBCs in a recipient’s blood
• Indicator:– Agglutinated (clumped) RBCs in a donor’s
blood
Indirect Antiglobulin Test: Method
Wikipedia
Sample Results for Indirect or Direct Antiglobulin
Kuby
Electrophoresis
• Purpose:– To detect the levels of various proteins
• Examples:– Detect monoclonal IgG produced by
myeloma patients– Measuring amount of albumin and other
blood constituents
Electrophoresis (Cont.)• Agent detected:
– Antibody– Antigen– Any protein
• Indicator:– Visible band of agent on a gel– Stained band of an agent on a gel
• Coomassie blue or silver stain for protein• Ethidium bromide for nucleic acid
Electrophoresis: Method• Prepare a polyacrylamide or agarose
gel• Load sample on the gel• Apply an electric current across the gel
for a period of time• Visualize the band on the gel
– With the naked eye – By exposure of the band to UV-light– By staining the band with a dye.
• Quantify the amount of material in the band by densitometer reading
From: Wikipedia
Multiple Myeloma• Antibodies are produced by B
cells and plasma cells.• When a single plasma cell
becomes transformed into a cancerous cell, it causes myeloma.– Myeloma patients over-produce a
homogeneous Ig produced by a single plasma cell. This can be observed as a heightened peak of Ig by electrophoresis of blood proteins.
– Myeloma patients also have some immunoglobulin proteins that spill over into their urine. These Bence-Jones proteins are dimers of kappa or lambda light chains.
AlbuminGlobulins
Migration distanceA
bsor
banc
e
IgX from Myeloma patients
Densitometer Scan
From: Parslow, Medical Immunology
Use of Electrophoresis to
Diagnosis Immunodeficiencies
Immunofixation
• Purpose:– Identification of composition of monoclonal
antibody• Examples:
– Detection of monoclonal antibody type in myeloma patients (plasma cell lymphoma giving an overproduction of IgG, IgA, IgE)
– Detection of monoclonal antibody in Waldenstom’s macroglobulinemia (B cell lymphoma giving an overproduction of IgM)
Immunofixation (Cont.)
• Agent detected:– Specific heavy and light chain of
monoclonal antibody• Indicator:
– Antibody against heavy and light chains
From: Kristine Krafts
From: Kristine Krafts
IgG monoclonal antibody composedof a specific gamma and kappa chains
Monoclonal antibody fragmentComposed of specific kappa chain
IgA antibody composed ofa specific alpha and lambda chains
Note absence of smear
Note presence of smear
Immunofluorescence• Purpose:
– Detection of an antigen in a specimen• Examples:
– Detection of specific proteins in cells, such as a tumor antigen or a viral antigen
– Detection of bacterial organisms– Detection of antigen-antibody complexes
that have been deposited on cell membrane or basement membrane surfaces
Immunofluorescence (Cont.)
• Agent detected:– Antigen that is precipitated on a cell– Antigen that is part of the cell membrane– Agent that is within the cell (must
permeablize the cell to detect it)• Indicator:
– Antibody that has a fluorescent tag
Immunofluorescence: MethodDirect
• Fix cells to a slide• Add antibody (IgG) specific to
the target antigen that is tagged with fluorescent compound
• Visualize fluorescence by looking through a fluorescence microscope
Indirect• Fix cells to a slide• Add primary antibody (IgG) specific
to the target antigen• Add secondary anti-antibody (anti-
IgG) that is tagged with fluorescent compound
• Visualize fluorescence
Kuby
Immunofluorescence
Negative for the presence of antigen in the cells
Positive for the presence of antigen in the cells
Cytoplasmic Staining bright greenNuclear Staining pale blue
Nuclear Staining: dark blue (DAPI)
Enzyme-Linked Immunosorbent Assay (ELISA)
• Purpose:– Detection of antibodies or antigens in a patient
specimen• Examples:
– Home pregnancy test– Detection of antibody to a virus, bacterium or other
microorganism• HIV test
– Detection of antibody to a foreign antigen– Detection of a viral antigen or bacterial antigen
Kinds of ELISA
• Indirect ELISA• Sandwich ELISA• Radioimmunoassay
ELISA (Cont.)• Agent detected:
– Antibody– Antigen
• Indicator:– Antibody with a bound enzyme that can
catalyze conversion of a colorless molecule to a colored one• Alkaline phosphatase• Horseradish peroxidase
– Antibody with a bound radioisotope
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Add Antigen
Add 2° Ab
Add Reagent
Incubate
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Incubate
Indirect ELISA Sandwich ELISA
Elispot• This is a variation of the ELISA test.• A petri plate is coated with specific
capture antibody.• Cells are added to the plate and
allowed to settle for a period of time.• The cells produce a specific cytokine
that binds to the specific antibody in the area where the cell settled.
• The cells are washed away.• Detection antibody bearing an enzyme
is added and unbound excess antibody is washed away.
• An appropriate substrate is added.• An area of the petri plate where the
cytokine was produced turns color.
Western Blot
• Purpose:– Detection of antibodies or proteins in a
patient specimen• Examples:
– Detection of antibodies to HIV in a patient’s blood
– Detection of HIV proteins in a patient’s blood
Western Blot (Cont.)• Agent detected:
– Antibody– Antigen
• Indicator:– Antibody tagged with fluorescent molecule– Antibody tagged with enzyme to convert non-
colored compound to a colored compound– Antibody tagged with chemiluminescence enzyme
(luciferase)– Antibody tagged with radiolabel
Detection of HIV Infection- Western Blotting
• Western blot– is the confirming test for HIV infection.– detects the presence of antibodies to the
various protein components of HIV (e.g., anti-p18, anti-p24, anti-gp41).
– consists of four steps:1. Electrophoresis of HIV proteins on cellulose
acetate2. Reacting putative serum antibodies of patient with
HIV proteins3. Reacting conjugated (enzyme or radiolabeled)
anti-antibody with the serum antibodies and the HIV proteins
4. Reading color change or radioactivity for positive result
Pt 1 Pt 2 Stnd
Flow Cytometry
• Purpose:– Determine the number or percentage of
cells that express a given antigen• Examples:
– Monitoring CD4+ T cell levels in HIV-infected patients
– Diagnosis of leukemia and lymphoma
Flow Cytometry (Cont.)
• Agent detected– Cells bearing a specific antigen
• Indicator– Antibody tagged with fluorescent molecule
Flow Cytometry
a. Separation of CD8+ and CD3+ cells from total WBCs.
b. Rerun of CD8+/CD3+ T cells.