Immunoelectrophoresis (IEP) Serum Protein Electrophoresis (SPE) Immunofixation (IFX

)

Immunoelectropho  resis (IEP) 

1. Definition
also called gamma globulin electrophoresis, or immunoglobulin electrophoresis

developed by French immunologist Petr Nikolaevich Grabar in 1950s

qualitative method that combines electrophoresis and immunodiffusion technique Both identification and approximate quantitation may be accomplished for individual proteins present in serum, urine, or other biological fluids method of determining the blood levels of 3 major immunoglobulins: IgM, IgG, and IgA

2. Purpose
powerful analytical technique with high resolving power as it combines separation of antigens by electrophoresis with immunodiffusion against an antiserum aids in the diagnosis and evaluation of the therapeutic response in many disease states affecting the immune system used frequently to diagnose multiple myeloma, a disease affecting the bone marrow

Multiple Myeloma
most common of the monoclonal gammopathies Characterization - neoplastic proliferation of plasma cells or morphologically abnormal plasma cells (myeloma cells), primarily occurring in the bone marrow. Common symptoms – bone pain, with the presence of bone lesions and frequent bone fractures. Indication  - an increase in one of the Ig, excluding an increase of IgM.

3. Preparation of Agarose gel : prepared with alternating well and IEP two side troughs.
Antiserum A Mixed antigens

 

Antiserum B

Well : antigen mixture (usually a serum sample) Troughs : antibody/mixture of antibodies Electrophoresis : to separate proteins in the sample
-antigens and antibodies migrate toward 1 another

precipitin arcs at the region of optimal concentration

4. Description
When serum applied to the agarose plate subjected to normal zone electrophoresis Channels that are filled with specific antiserum can be directed against all proteins or a specific proteins The antiserum and sample proteins are then allowed to diffuse (immunodiffusion) forming precipitin lines at the equivalence boundaries

After diffusion the gel is washed to remove all unprecipitated proteins, then stained. Proteins that have reacted with the antiserum used will show as a precipitin arc.

4. Principle of IEP
Electrophoresis of antigens and

immunodiffusionof antigens with a polyspecific antiserum to form precipitin bands

Electrophoresis:

- antigen molecules acquire charge and move towards appropriate electrode. - mobility depends on : a) size/shape of molecules b) pH c) heat generated by ionic strength buffer d) charge particles

Immunodiffusion:

- resolved antigen subjected to immunodiffusion with antiserum - immunodiffusion is due to the diffusion coefficient, density, and gradient of Ag and Ab which formed Ag-Ab complex precipitin lines at the zone of equivalence.

Point of equivalence
0bserved by maximum Ag-Ab complex

formation

A sample of Ig profile

5. APPLICATIONS
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clinical and research laboratories - diagnostic tool to probe the protein composition of serum. to detect a particular antigenic site following the transfer of the proteins from a gel to a special support, such as nitrocellulose capillary immunoelectrophoresis : - a sample can be simultaneously sly drawn up into many capillary tubes. - a sample can be subdivided into very many sub volumes (the very diameter of the tubes = little sample is required to fill a tube). - each volume can be tested against a different antibody preparation. IEP is used to examine alterations in the content of serum, especially changes concerned with Igs.

6. ABNORMAL RESULTS
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IgM levels – malignancy, chronic infections, such as hepatitis or mononucleosis and autoimmune diseases, like rheumatoid arthritis IgM levels - AIDS, immunosuppression caused by certain drugs like steroids or dextran, or leukemia IgG levels - chronic liver disease, autoimmune diseases, hyperimmunization reactions, or certain chronic infections, such as tuberculosis or sarcoidosis. IgG levels - Wiskott-Aldrich syndrome, AIDS and leukemia IgA levels - chronic liver disease, chronic infections, or inflammatory bowel disease. IgA levels - ataxia, a condition affecting balance and gait, limb or eye movements, speech, and telangiectasia, an increase in the size and number of the small blood vessels in an area of skin, causing redness. IgA levels are

7. NORMAL RESULTS
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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 1mg = approximately 0.000035 oz. 1dL = approximately 0.33 oz.

8. PRECAUTIONS
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Drugs that may cause increased Ig levels should be avoided :- 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 - lead to the increased immunoglobulin levels resulting in false positive results.

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SPE

SERUM PROTEIN ELECTROPHORESIS

PROTEI

Amino Acids

Muscle (+) or a carry a Enzymes (-) electrical Hormones charge. Hemoglobin Move in fluid Other body when placed in tissues an electrical

Separat e Elemen

Chemical methods Electrophore Electrophore sis sis Ultracentrifu ge

A process of separating electrically charged particles in solution Protein by passing Immunoelectrophor an electric current through the solution Electrophores esis is
assess the blood levels of specific types of proteins called immunoglobulins. the total amount of protein in the blood, and to establish the levels of other types of proteins - albumin, alpha1 globulin, alpha2 globulin, beta-globulin & Gamma globulin.

ordered if a SPE test has a "spike," or rise, at the immunoglobulin level

Electrophore sis

Blood SERUM, urine, CSF
sample

SERUM Fluid – separated from clotted blood. Same composition as in plasma but lacks fibrinogen & other substances that are used in coagulation process.

Serum protein electrophoresis uses an electrical field to separate the proteins in the blood serum into groups of similar size, shape & charge.

SP

Albumin & Globulin

albumin, alpha-1 globulin, alpha-2 globulin, beta-globulin & Gamma globulin

PURPOSE
To screen :
H & E stains, 100x

Multiple myeloma

Kidney amyloidosis

Waldenstrom‘s macroglobulinemia

the cause of hypogammaglobulinemia (HGG) (a condition characterized by low levels of gamma globulin antibodies. HGG can make a person more susceptible to infection).

METHODS
1- Bloodsample & stain 3- Immobilize 2- Load collection 5- Visually & 4- Dilute serum & run electrophoresis quantitate

1- Blood collection 2- Dilute serum 3- Load sample & run electrophoresis 4- Immobilize, stain 5- Visually, quantitate

3-5 

With B-2 Barbitol buffer (1 sample:4 buffer)

25 min, 100V

Destain

Ge l E lectro phor esi sii
 -ve charge conferred on molecules  gel is porous  molecules travel through gel according to size when electrical current applied  smaller travel faster
Migration rate -Charge of the molecule -Size & shape of the molecule -Voltage -Support medium -pH & ionic strength of the buffer

PRINCIPLE
 Serum sample is applied closed to the cathode  All major serum protein carry a net –ve charge at pH 8.6 migrate toward the anode after current passed through  Serum protein is arranged into 5 bands Albumin, α1 –globulin,α2-globulin,β-globulin,ɣ-globulin

Anode (+)

toward the anode

Catode (-)

Intrepretation of result

Abnormal result

Continue

Abnormal result

WEAKNESSES

Not be able to have the test or effect the results

High levels of lipids (hyperlipidemia). Iron deficiency anemia. Medicines (e.g. corticosteroids, birth control pills, aspirin, bicarbonates, chlorpromazine, neomycin, isoniazid, and sulfonamides (sulfa). Medicine used to treat cancer (chemotherapy). administration of a contrast dye used in some other tests may falsely elevate protein levels. Haemolysed blood will increase the globin value. Pregnancy.

SUMMARY
• SPE is used to identify a monoclonal protein in the serum of patients with Multiple Myeloma & Monoclonal Gammopathy (e.g Monoclonal Gammopathy of Undetermined Significance, MGUS) beside other particular disease based on the pattern of protein band. • Patients with Multiple Myeloma and MGUS are followed by measuring the concentration of the monoclonal protein using SPE.

IMMUNOFIXATION

Immunofixation Technique
☺is used to identify of monoclonal gammopathy (protein @ antibody) that is not obviously detect by HRE. ☺Quantification of IgG, IgA, IgM, kappa and lambda. ☺2 stage procedure; agarose gel electrophoresis and immunoprecipitation ☺Specimen; serum, urine or CSF

Monoclonal Gammopathies type of Ig ☺Excessive production of a single

molecule by a large number of plasma cell ☺The presence of monoclonal gammopathies indicated: 1) Multiple myeloma 2) Monoclonal Gammopathy of undetermined significance (MGUS) 3) Waldenstrom’s macroglobulinemia ☺Associated with lymphomas, leukemias, amyloidosis, autoimmune disease.

Waldenstrom’s Syndrome
Characterized by uncontrolled proliferation of

lymphoplasmacytic cells in the lymph nodes, spleen, and BM. Large amounts of a pentameric IgM monoclonal protein → clinical symptoms. Condition: anemia, a bleeding tendency, generalized lymphadenopathy, hyperviscosity syndrome. Weakness and fatigue

Methodology
The HRE was perform

on the patient’s serum run on several adjacent tract One tract should be fixed per routine Other tracts are apply with specific antisera against IgG, IgA, IgM, kappa and lambda.

Continue…
Specific antisera

reacted with specific antigen to form antigen-antibody complexes. No umbrella effect The result was straightforward to interpret

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