Immunological Reactions and Techniques

W. Robert Fleischmann, Ph.D.

Department of Urologic Surgery

University of Minnesota Medical School

rfleisch@umn.edu

(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

acetate

2. Reacting putative serum antibodies of patient with HIV proteins

3. 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.