Immunoelectrophoresis - PowerPoint PPT Presentation

1 / 40
About This Presentation
Title:

Immunoelectrophoresis

Description:

A. Aboutalebi Immunoelectrophoresis Outline Introduction Immunoelectrophoresis(IEP) IEP Methods Application Conclusion References Introduction IEP Definition: A ... – PowerPoint PPT presentation

Number of Views:9564
Avg rating:5.0/5.0
Slides: 41
Provided by: ghaziaska
Category:

less

Transcript and Presenter's Notes

Title: Immunoelectrophoresis


1
Immunoelectrophoresis
  • A. Aboutalebi

2
Outline
  • Introduction
  • Immunoelectrophoresis(IEP)
  • IEP Methods
  • Application
  • Conclusion
  • References

3
Introduction
Sample
Immuno- Electrophoresis
4
Introduction
  • IEP DefinitionA separation technique combines
    the principles of agarose gel electrophoresis)AEG
    ) with immuno-precipitation.
  • IEP Purpose
  • IEP is a method of determining the blood
    levels of three major immunoglobulins IgM,IgG,
    and IgA, aids in the diagnosis and evaluation of
    the therapeutic response in many disease states
    affecting the immune system.

5
Electrophoresis
The migration of particles under the influence of
a direct electrical current,
  • and requires
  • Charged particles
  • Medium capable of carrying a current

6
Effective Factors
  • Choice of electrolyte
  • Electrophoretic conditions
  • Choice of support
  • pH
  • Ionic strength
  • Voltage
  • Current
  • Heat

7
Support Characteristics
  • Ease of use
  • Properties affecting separation
  • Suitability for appropriate stains or other
    detection
  • Suitability for post electrophoretic data analysis

8
Agarose Gel
AGE
  • Now increasing popularity as commercially
    prepared gels available
  • Suitable for most stains and immunofixation
  • Dries to permanent record
  • Suitable for densitometry
  • Automated/semiautomated systems now available

9
AGE
10
Immunoprecipitation
  • When the antigen contacts the antibody, in right
    concentration ratio of Ag-Ab, a precipitate can
    form that named precipitin.

11
Antigen
  • A chemical substance that the body perceives as
    foreign, when it is introduced into the body,
    induces an immune response and stimulates the
    production of an antibody.

12
Antibody
  • Proteins produced in blood and tissue to help
    neutralize and destroy foreign bodies (antigens).
  • Monoclonal
  • Polyclonal

13
Antibody Structure
  • Immunoglobulins
  • Light chain (? ? )
  • Heavy chain ?(IgA),?(IgG),
  • µ(IgM),?(IgE),
  • ?(IgD)

14
Ag-Ab Intraction
15
Precipitine Formation
16
Ag-Ab Ration
Ag Ab ltgt AgAb
Ka AgAb/AgAb Liters/mole
17
(No Transcript)
18
Double Immunodiffusion
  • Identification

19
Quantitation
20
IEP
21
Detection Methods
  • Radioisotope
  • Enzyme
  • Fluorescence
  • Staining

22
Instrumentation
23
Instrumentation
24
IEP Methods
  • Rocket IEP.
  • Crossed IEP.
  • Tandem Crossed IEP.
  • Intermediate Gel Crossed IEP.
  • Fused-Rocket Crossed IEP.
  • Countercurrent Immunoelectrophoresis

25
Applications
  • Analysis and purification of proteins, enzymes
    and nucleic acids
  • Identification and characterization of monoclonal
    Igs in serum, urine and cerebrospinal fluid
  • Analysis of drugs and amino acids
  • Diagnose multiple myeloma
  • Visualize the location of antigenic compounds
    from animal and plant tissues, body fluids, cell
    materials and microorganisms
  • Determination of allergens in foods
  • Detection of species-specific muscle proteins in
    heat-processed products

26
Conclusion
  • The sensitivity and precision of IEP can improved
    by modern data analysis techniques such as image
    analysis.
  • This technique use in medicine, pharmacology
    (vaccine and drug preparation), clinical
    laboratory and food industry.

27
References
  • 1 Z.Deyl, ,J.Chromatography Lib.,1979,Elsevier
    Scientific Co.
  • 2 Clifton E Meloan, ChemicalSeparations
    ,1999,John Wiley Sons,Inc.
  • 3 P.F.Cotter et al., Poultry Science
    ,2000,7912761281.
  • 4 C.Canavese et al., Original Investigation,
    JNEPHROL 2000 13352-359.
  • 5 M.Besler et al., Trends in analytical
    chemistry,2001, vol.20, 11, Elsevier Co.
  • 6 L.Necidova et al.,Original Paper,
    Vet.Med.-Czech,47,2002 (5) 143-147.
  • 7 A.Chan et al.,Computers Chemistry 23 (1999)
    577-586, Pergamon Press.
  • 8 A.Heger et al.,Thrombosis Research 106 (2002)
    157-164, Pergamon Press.
  • 9 V.Mosqueira et al., Biomaterials 22 (2001)
    2967-2979, Elsevier Co.
  • 10 V.Rivera et al.,J.Pharmaceutical and
    Biomedical Analysis 29 (2002) 431-441, Elsevier
    Co.
  • W.Grob et al., Analyitical Chimica Acta 383
    (1999) 169-184 , Elsevier Co.
  • www.fao.org.
  • www.webmed.unipv.it.
  • www.savec.com.
  • www.medschool.lsumc.edu.

28
(No Transcript)
29
(No Transcript)
30
Agarose Molecule
31
  • agarosesSevern Biotech's range of high-quality
    agaroses are all prepared to exacting standards
    which ensure that the products can be relied on
    time after time to produce optimal performance.
    By altering critical characteristics, the
    agaroses have been developed to meet the specific
    requirements of a diverse range of applications.
    The main characteristics are
  • Melting / gelling temperatures
  • Gel strength
  • Electroendosmosis (EEO)
  • Gels with low melting temperatures (below the
    temperature at which DNA denatures) allow DNA to
    remain intact on melting, thus enabling easy
    purification of nucleic acid using b-agarase. SB
    Low Gel and Recovery will remain liquid for
    several hours at temperatures compatible with
    many in-gel enzymatic reactions and can be
    performed without having to separate the nucleic
    acids from the media. High gel strength is
    important if the application involves handling
    and mechanical manipulation such as Southern or
    Northern blotting. Finally the EEO (the movement
    of non-charged molecules through a medium towards
    the cathode during electrophoresis) of an agarose
    will determine the mobility of certain
    macromolecules within the gel. Low EEO values
    allow nucleic acids to migrate faster thereby
    improving their resolution since migration is
    determined by size only. The agaroses within
    Severn Biotech's range can be used for a wide
    variety of applications from nucleic acid
    separation to immunoelectrophoresis and from
    tissue cell culture to viral plaque assays.
    Recommended applications are indicated with the
    description of each agarose type.

32
  • Immunofixation electrophoresis (IFE) is a
    two-stage process combining agarose gel
    electrophoresis with immuno-precipitation. It has
    replaced IEP (immunoelectrophoresis) as the
    method of choice for identification and
    characterisation of monoclonal immunoglobulins in
    serum, urine and cerebrospinal fluid. IFE allows
    ease of interpretation, fast results (less than 2
    hours) and has excellent sensitivity to low
    levels of monoclonal immunoglobulin (Ig) as may
    be found in cases of early myelomatosis. Samples
    are normally screened using Serum Protein
    Electrophoresis (see above), those with abnormal
    results are further investigated using Freelite
    and/or IFE.

33
  • Immunoelectrophoresis
  • Qualitative procedure used to measure monoclonal
    proteins such as lambda and kappa chains
  • Proteins are separated by electrophoresis
    (albumin, gamma globulins)
  • Then a through is filled with AHG and allowed to
    diffuse for 16 hours ppt. lines.
  • Stain and Read bands (hard to read)
  •  

34
  • Other methods using electricity
  • Countercurrent Immunoelectrophoresis
  • Antigen and Antibody are placed at opposite ends
    of a plate
  • Electrical current causes them to migrate toward
    each other
  • Line forms if there is AG-AB binding
  • Qualitative procedure, good if time is a factor
    (ex meningitis)

35
  • Rocket (Laurell)
  • Used primarily in research labs to quantitate
    antigens other than IgG
  • Anti-serum is incorporated into the agar
  • Unknown antigen is placed in the wells and
    electrophoresised
  • As it migrates AG-AB bind and form rocket shape
  • Total distance migrated prop. to quantity

36
  • DEFINITIONA technique combining protein
    electrophoresis and double immunodiffusion
    proteins are separated by agarose gel
    electrophoresis then specific antisera are
    placed in a trough cut parallel to the protein
    track, and the proteins and antibodies are
    allowed to diffuse through the gel, the proteins
    diffusing radially from their electrophoretic
    placement and the antibodies diffusing
    perpendicularly from the trough, resulting in a
    distinct elliptical precipitin arc for each
    protein detectable by the antisera. Abbreviation
    IEP.

37
  • Immunoelectrophoresis
  • Author/s Janis O. FloresDefinition of
    Immunoelectrophoresis
  • Immunoelectrophoresis, also called gamma globulin
    electrophoresis, or immunoglobulin
    electrophoresis, is a method of determining the
    blood levels of three major immunoglobulins
    immunoglobulin M (IgM), immunoglobulin G (IgG),
    and immunoglobulin A (IgA).Purpose of
    Immunoelectrophoresis
  • Immunoelectrophoresis aids in the diagnosis and
    evaluation of the therapeutic response in many
    disease states affecting the immune system. It is
    usually requested when a different type of
    electrophoresis, called a serum protein
    electrophoresis, has indicated a rise at the
    immunoglobulin level. Immunoelectrophoresis is
    also used frequently to diagnose multiple
    myeloma, a disease affecting the bone
    marrow.Precautions of Immunoelectrophoresis
  • Drugs that may cause increased immunoglobulin
    levels include therapeutic gamma globulin,
    hydralazine, isoniazid, phenytoin (Dilantin),
    procainamide, oral contraceptives, methadone,
    steroids, and tetanus toxoid and antitoxin. The
    laboratory should be notified if the patient has
    received any vaccinations or immunizations in the
    six months before the test.It should be noted
    that, because immunoelectrophoresis is not
    quantitative, it is being replaced by a procedure
    called immunofixation, which is more sensitive
    and easier to interpret.Description
  • Immunoelectrophoresis is performed by placing
    serum on a slide containing a gel designed
    specifically for the test. An electric current is
    then passed through the gel, and immunoglobulins,
    which contain an electric charge, migrate through
    the gel according to the difference in their
    individual electric charges. Antiserum is placed
    alongside the slide to identify the specific type
    of immunoglobulin present. The results are used
    to identify different disease entities, and to
    aid in monitoring the course of the disease and
    the therapeutic response of the patient to such
    conditions as immune deficiencies, autoimmune
    disease, chronic infections, chronic viral
    infections, and intrauterine fetal
    infections.There are five classes of antibodies
    IgM, IgG, IgA, IgE, and IgD, but
    immunoelectrophoresis is ordered primarily to
    test for IgM, IgG, and IgA.IgM is produced upon
    initial exposure to an antigen (for example, when
    a person receives the first tetanus vaccination,
    antitetanus antibodies of the IgM class are
    produced 10 to 14 days later). IgM is abundant in
    the blood but is not normally present in organs
    or tissues. IgM is primarily responsible for ABO
    blood grouping and rheumatoid factor, yet is
    involved in the immunologic reaction to other
    infections, such as hepatitis. Since IgM does not
    cross the placenta, an elevation of this
    immunoglobulin in the newborn indicates
    intrauterine infection such as rubella,
    cytomegalovirus (CMV) or a sexually transmitted
    disease (STD).IgG is the most prevalent type of
    antibody, comprising approximately 75 of the
    serum immunoglobulins. IgG is produced upon
    subsequent exposure to an antigen. As an example,
    after receiving a second tetanus shot, or
    booster, a person produces IgG antibodies in five
    to seven days. IgG is present in both the blood
    and tissues, and is the only antibody to cross
    the placenta from the mother to the fetus.
    Maternal IgG protects the newborn for the first
    months of life, until the infant's immune system
    produces its own antibodies.IgA constitutes
    approximately 15 of the immunoglobulins within
    the body. While it is found to some degree in the
    blood, it is present primarily in the secretions
    of the respiratory and gastrointestinal tract, in
    saliva, colostrum (the yellowish fluid produced
    by the breasts during late pregnancy and the
    first few days after childbirth), and in tears.
    IgA plays an important role in defending the body
    against invasion of germs through the mucous
    membrane-lined organs.IgE is the antibody that
    causes acute allergic reactions it is measured
    to detect allergic conditions. IgD, which
    constitutes the smallest portion of the
    immunoglobulins, is rarely evaluated or detected,
    and its function is not well understood.Preparatio
    n of Immunoelectrophoresis
  • This test requires a blood sample. The patient
    should have nothing to eat or drink for 12 hours
    before the test.Aftercare of Immunoelectrophoresis
  • Since this test is ordered when either very low
    or very high levels of immunoglobulins are
    suspected, the patient should be alert for any
    signs of infection after the test, including
    fever, chills, rash, or skin ulcers. Any bone
    pain or tenderness should also be immediately
    reported to the physician.Risks of
    Immunoelectrophoresis
  • Risks for this test are minimal, but may include
    slight bleeding from the blood-drawing site,
    fainting or feeling lightheaded after
    venipuncture, or bruising.Normal results of
    Immunoelectrophoresis
  • Reference ranges vary from laboratory to
    laboratory and depend upon the method used. For
    adults, normal values are usually found within
    the following ranges
  • IgM 60-290 mg/dL
  • IgG 700-1,800 mg/dL
  • IgA 70-440 mg/dL.
  • Abnormal results of Immunoelectrophoresis
  • Increased IgM levels can indicate Waldenstrom's
    macroglobulinemia, a malignancy caused by
    secretion of IgM at high levels by malignant
    lymphoplasma cells. Increased IgM levels can also
    indicate chromic infections, such as hepatitis or
    mononucleosis and autoimmune diseases, like
    rheumatoid arthritis.Decreased IgM levels can be
    indicative of AIDS, immunosuppression caused by
    certain drugs like steroids or dextran, or
    leukemia.Increased levels of IgG can indicate
    chronic liver disease, autoimmune diseases,
    hyperimmunization reactions, or certain chronic
    infections, such as tuberculosis or
    sarcoidosis.Decreased levels of IgG can indicate
    Wiskott-Aldrich syndrome, a genetic deficiency
    caused by inadequate synthesis of IgG and other
    immunoglobulins. Decreased IgG can also be seen
    with AIDS and leukemia.Increased levels of IgA
    can indicate chronic liver disease, chronic
    infections, or inflammatory bowel
    disease.Decreased levels of IgA can be found in
    ataxia, a condition affecting balance and gait,
    limb or eye movements and/or speech and
    telangiectasia, an increase in the size and
    number of the small blood vessels in an area of
    skin, causing redness. Decreased IgA levels are
    also seen in conditions of low blood protein
    (hypoproteinemia), and drug immunosuppression. 

38
Heat effects
  • evaporation
  • siphoning of electrolytes
  • denaturation of molecules

39
Electrolyte
  • pH
  • Ionic strength

40
  • stains
  • Coomasie Brilliant Blue
  • Ethidium Bromide
Write a Comment
User Comments (0)
About PowerShow.com