SERUM ELECTROPHORESIS

1. NARENDRA YADAV
PAPER 4
SERUM ELECTROPHORESIS

2. INTRODUCTION
 Serum is the component that is neither a blood cell nor a
clotting factor; it is the blood plasma not including the
fibrinogens.
 Serum includes all the electrolytes, antibodies, antigens,
hormones, and any exogenous substances (e.g., drugs
and microorganisms).
 Serum is used in numerous diagnostic tests, as well as
blood typing.

3. COMPONENT OF SERUM
 Serum contains two major protein groups: albumin and
globulin.
 Using protein electrophoresis, these two groups can be
separated into five smaller groups (fractions):
1. albumin fraction
2. alpha 1 fraction
3. alpha 2 fraction
4. beta fraction
5.gamma fraction.

4. ALBUMIN
 Produce by liver.
 Largest protein component of serum.
 Moves toward anode durning electrophoresis.
 Usually a single band is seen.
 Heterozygous individuals may produce bisalbuminemia

5. ALPHA 1 FRACTION &
ALPHA 2 FRACTION
 Alpha 1 fraction component:
1.anti-trypsin=protease inhibitor.
2.thyroid binding protein= binding to thyroid hormones.
3.transcortin=Transcortin, transporting cortisol hormones.
 Alpha 2 fraction component:
1.ceruloplamin=copper-carrying protein.
2. haptoglobins= binds free hemoglobin released with
high affinity and thereby inhibits its oxidative activity.

6. BETA AND GAMMA FRACTION
 Beta fractions consist of:
1. transferrin=iron-binding glycoproteins.
2.lipoproteins= LDL& HDL.
 Gamma fraction cosist of:
 1. immunoglobulins.

7. ELECTROPHORESIS
 Electrophoresis is a laboratory technique in which the
sample is placed into a gel, and exposed to an electrical
current to separate the sample on basis of their charge or
size of the sample.
 Serum protein electrophoresis is a laboratory test that
examines specific proteins in the blood called globulins.
 Serum electrophoresis is used separate the serum protein
components into five major fractions by size and electrical
charge: i.e. serum albumin, alpha-1 globulins, alpha-2
globulins, beta globulins, and gamma globulins.

8. PRINCIPLE
 A charged particle placed in an electrical field migrates towards
the anode or cathode depending on the net charge carried by the
particle.
 Serum proteins carry a negative charge at pH 8.6 and move
toward the anode.
 Serum is placed into a sample within low EEO agarose gel in an
alkaline buffer.
 A standardized voltage is applied and upon staining, five distinct
protein bands (albumin, α1, α2, β, and γ) are seen.
 The membrane is then run through a densitometer and the light
absorbance by the protein bands is recorded.
 The absorbance of light is proportional to the relative protein
concentration. The protein concentration of each band is then
calculated.

9. PROTOCOL
 Preparation of agarose gel.
 Sample preparations.
 Electrophoresis.
 Staining.
 Destaining.
 Visualizing.

10. INTERPRETATION
 The densest band are of albumin.
Which run towards the anode durning
the electrophoresis.
 Alpha 1 fraction
 Alpha 2 fraction
 Beta fraction
 Gamma fraction which move towards
the cathode durning the
electrophoresis. The band is
observed back of the origin.

11. Normal Graph after interpretation

12. PATHOLOGICAL CONDITION ASSOCIATED WITH A CHARACTERISTIC
SERUM PROTEIN ELECTROPHORESIS PATTERN.

13. ALBUMIN DISORDER
(HEPATIC CIRRHOSIS)
 Abnormal function of
liver.
 Protein synthesis is
compromised and the
concentration of albumin
and protein in the alpha
and band are decreased.
 Additional common
finding is beta-gamma
bridging due to
increased IgA.

14. NEPHROTIC SYNDROME
 Nephrotic syndrome is a
kidney disorder.
 Loss of protein through the
kidneys(proteinuria) leads
to low protein levels in the
blood.
 Synthesis cause its
accumulation.
 Large proteinuria is due to
an increase in permeability
of the "filtering membrane"
of the kidney, which
selectively filter only
albumin and other smaller
molecular.
 Alpha2 is sufficiently large
so that it is not filtered and
increased Synthesis cause
its accumulation.

15. ALPHA 1
ANTITRYPSIN -DEFICIENCY
 Alpha 1-antitrypsin
deficiency (α1-antitrypsin
deficiency, A1AD) is a
genetic disorder that
causes defective production
of alpha 1-antitrypsin
(A1AT), leading to
decreased A1AT activity in
the blood and lungs.

16. ALPHA1 & ALPHA2 DISORDER
ACUTE INFLAMATION
 There is ncreased alpha 1
and alpha2 band durning the
inflammatory response from
increased hepatic synthesis
of acute phase reactant
protein.

17. GAMA FRACTION DISORDER
CHRONIC INFLAMATION
 Immunoglobulin synthesis
by the activated B
lymphocytes transformed to
plasma cells is
demonstrated by the
increased polyclonal gamma
band.

18. IMMUNOGLOBULIN DEFICIENCY
 Deficient
immunoglobulins
synthesis is
demonstrated by
deminsihed gamma
band.

19. MONOCLONAL GAMMOPATHY
 Usually sharp band in the
gamma region suggest the
presence of a homogenous
immunoglobulins.
 Homogenous
immunoglobulins are also
found in waldenstrom’s
macroglobulinemia

20. Serum Protein Fraction Increased Decreased
Albumin Severe dehydration
Malnutrition, cachexia, liver
disease, nephrotic syndrome,
protein-losing enteropathies,
severe burns
Alpha-1 Inflammatory states, pregnancy Alpha-1 antitrypsin deficiency
Alpha-2
Inflammatory states, nephrotic
syndrome, oral contraceptive use,
steroid use, hyperthyroidism
Hemolysis, liver disease
Beta
Hyperlipidemia, iron-deficiency
anemia
Hypo-B-lipoproteinemia,
malnutrition
Gamma
Polyclonal and Monoclonal
Gammopathies
Agammaglobulinemia,
hypogammaglobulinemia
Various disease states or conditions alter the pattern of proteins in electrophoresis

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Thank you

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REFERENCE
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