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
IEP
Agarose gel : prepared with alternating well and
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
l clinical and research laboratories - diagnostic tool
to probe the protein composition of serum.
l to detect a particular antigenic site following the
transfer of the proteins from a gel to a special
support, such as nitrocellulose
l 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.
l IEP is used to examine alterations in the content of
serum, especially changes concerned with Igs.
 6. ABNORMAL RESULTS
l IgM levels – malignancy, chronic infections, such as
hepatitis or mononucleosis and autoimmune diseases, like
rheumatoid arthritis
l IgM levels - AIDS, immunosuppression caused by certain
drugs like steroids or dextran, or leukemia
l IgG levels - chronic liver disease, autoimmune diseases,
hyperimmunization reactions, or certain chronic infections,
such as tuberculosis or sarcoidosis.
l IgG levels - Wiskott-Aldrich syndrome, AIDS and
leukemia
l IgA levels - chronic liver disease, chronic infections, or
inflammatory bowel disease.
l 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
l Reference ranges vary from laboratory to
laboratory and depend upon the method
used.
l 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
l 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.

l 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.
SPE

SERUM PROTEIN ELECTROPHORESIS

 Muscle
carry a (+) or a
Enzymes
(-) electrical
PROTEI Amino
Acids
Hormones
charge.
Hemoglobin
Move in fluid
Otherplaced
when body in
tissues
an electrical

Chemical
Separat methods
Electrophore
Electrophore
e sis
sis
Elemen 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 the total amount of protein
specific types of proteins in the blood, and to
called immunoglobulins. establish the levels of other
types of proteins - albumin,
ordered if a SPE test has a
Electrophore
"spike," or rise, at the
alpha1 globulin, alpha2
globulin, beta-globulin &
immunoglobulin level sis Gamma globulin.

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.

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

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

With B-2 Barbitol

buffer
(1 sample:4 buffer)
3-5 

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

toward the
Anode 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
☺Excessive production of a single type of Ig
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
THANK YOU…