1. Immunological Reactions and Techniques
W. Robert Fleischmann, Ph.D.
Department of Urologic Surgery
University of Minnesota Medical School
(612)

2. Objectives
Become familiar with the various types of immunological techniques that are used for clinical diagnosis.
Understand how each of these immunological techniques work.

3. Shirley Abbott arrives at the clinic
Shirley Abbott arrives at the clinic. She indicates that she has just learned that her best friend has been diagnosed as having an HIV infection.
She indicates that she has been taking i.v. drugs and sharing needles with her best friend for the past several months. She asks to be tested for an HIV infection.
What test would you perform?

4. A Rapid HIV Test is used to test Shirley’s blood.
As they wait for the results, the physician explains that this test is an ELISA test.
The test gives a positive result.
Does this mean that Shirley has an HIV infection?

5. The purposes of immunological tests are to aid in the determination or monitoring of diseases or conditions that may be present in a patients.

6. Immunologic Techniques
Agglutination reactions
Direct antiglobulin test
Indirect antiglobulin test
Electrophoresis
Immunofixation
Immunofluorescence
Enzyme-linked immunosorbent assay
Western blot
Flow cytometry

7. 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

8. 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).

9. 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.

10. Results of an Agglutination Test
Wikipedia

11. 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)

12. Direct Antiglobulin Test (Cont.)
Agent detected:
Antibody
Complement
Indicator:
Agglutinated (clumped) RBCs

13. Direct Antiglobulin Test: Method
Wikipedia

14. 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

15. Indirect Antiglobulin Test (Cont.)
Agent detected:
Antibody to RBCs in a recipient’s blood
Indicator:
Agglutinated (clumped) RBCs in a donor’s blood

16. Indirect Antiglobulin Test: Method
Wikipedia

17. Sample Results for Indirect or Direct Antiglobulin
Kuby

18. Electrophoresis Purpose: Examples:
To detect the levels of various proteins
Examples:
Detect monoclonal IgG produced by myeloma patients
Measuring amount of albumin and other blood constituents

19. 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

20. 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

21. From: Wikipedia

22. 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.
Densitometer Scan
Albumin
Globulins   
Migration distance
Absorbance
IgX from
Myeloma
patients

23. Use of Electrophoresis to Diagnosis Immunodeficiencies
From: Parslow, Medical
Immunology

24. Immunofixation Purpose: Examples:
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)

25. Immunofixation (Cont.)
Agent detected:
Specific heavy and light chain of monoclonal antibody
Indicator:
Antibody against heavy and light chains

26. From: Kristine Krafts

27. Note presence of smear
Note absence of smear
IgG monoclonal antibody composed
of specific gamma and kappa chains
Monoclonal antibody fragment
Composed of specific kappa chain
IgA antibody composed of
a specific alpha and lambda chains
From: Kristine Krafts

28. Immunofluorescence Purpose: Examples:
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

29. 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

30. Immunofluorescence: Method
Direct
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

31. Immunofluorescence Negative for the presence of antigen in the cells
Nuclear Staining:
dark blue (DAPI)
Negative for the presence of antigen in the cells
Cytoplasmic Staining
bright green
Nuclear Staining
pale blue
Positive for the presence of antigen in the cells

32. 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

33. Kinds of ELISA
Indirect ELISA
Sandwich ELISA
Radioimmunoassay

34. ELISA (Cont.) Agent detected: Indicator: Antibody Antigen
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

35. Indirect ELISA Sandwich ELISA Y Y Y Add 1° Ab Add Antigen Y Y Y
Add Reagent
Add Reagent Y
Incubate
Incubate Y

36. 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.

37. Shirley Abbott
The physician explains to Shirley that the results of the ELISA test indicates that she has an antibody in her blood that recognizes at least one HIV protein. He explains that this test suggests that she may have HIV. However, it is not a definitive test.
What test would you run next?

38. Shirley Abbott
The physician explains that he will run a Western blot to determine whether Shirley has antibodies that recognize many HIV proteins.

39. Western Blot Purpose: Examples:
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

40. Western Blot (Cont.) Agent detected: Indicator: Antibody Antigen
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

41. Detection of HIV Infection- Western Blotting
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 Pt Stnd

42. Shirley Abbott
The result of the Western blot become available. The result shows that Shirley’s antibodies recognize only one HIV protein. The physician explains that she is considered to be negative for HIV.
Why did Shirley’s antibody recognize an HIV protein if she is not infected with HIV?
What advice would you give Shirley?
She may have cross-reactive antibody. Shirley should be tested again in another few weeks, as it often takes a prolonged period of time for seroconversion to HIV.

43. Flow Cytometry Purpose: Examples:
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

44. Flow Cytometry (Cont.) Agent detected Indicator
Cells bearing a specific antigen
Indicator
Antibody tagged with fluorescent molecule

45. Flow Cytometry Separation of CD8+ and CD3+ cells from total WBCs.
Rerun of CD8+/CD3+ T cells.