immunological reactions and techniques n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Immunological Reactions and Techniques PowerPoint Presentation
Download Presentation
Immunological Reactions and Techniques

Loading in 2 Seconds...

play fullscreen
1 / 40

Immunological Reactions and Techniques - PowerPoint PPT Presentation


  • 99 Views
  • Uploaded on

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.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Immunological Reactions and Techniques' - aulani


Download Now An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
immunological reactions and techniques

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
Objectives
  • Become familiar with the various types of immunological techniques that are used for clinical diagnosis.
  • Understand how each of these immunological techniques work.
slide3

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

direct antiglobulin test
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
Direct Antiglobulin Test (Cont.)
  • Agent detected:
    • Antibody
    • Complement
  • Indicator:
    • Agglutinated (clumped) RBCs
indirect antiglobulin test
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
Indirect Antiglobulin Test (Cont.)
  • Agent detected:
    • Antibody to RBCs in a recipient’s blood
  • Indicator:
    • Agglutinated (clumped) RBCs in a donor’s blood
electrophoresis
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
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
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
multiple myeloma

Albumin

Globulins

Absorbance

Migration distance

Multiple Myeloma

Densitometer Scan

  • 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.

IgX from

Myeloma

patients

immunofixation
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
Immunofixation (Cont.)
  • Agent detected:
    • Specific heavy and light chain of monoclonal antibody
  • Indicator:
    • Antibody against heavy and light chains
slide25

Note presence of smear

Note absence of smear

IgG monoclonal antibody composed

of a 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

immunofluorescence
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
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 method
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

immunofluorescence1
Immunofluorescence

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

enzyme linked immunosorbent assay elisa
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
Kinds of ELISA
  • Indirect ELISA
  • Sandwich ELISA
  • Radioimmunoassay
elisa cont
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
slide33

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Indirect ELISA

Sandwich ELISA

Add 1° Ab

Add Antigen

Add 2° Ab

Add 2° Ab

Add Reagent

Add Reagent

Incubate

Incubate

elispot
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
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
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
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
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
Flow Cytometry (Cont.)
  • Agent detected
    • Cells bearing a specific antigen
  • Indicator
    • Antibody tagged with fluorescent molecule
flow cytometry1
Flow Cytometry

Separation of CD8+ and CD3+ cells from total WBCs.

Rerun of CD8+/CD3+ T cells.