IMMUNOELECTROPHORESIS V. MAGENDIRA MANI ASSISTANT PROFESSOR OF BIOCHEMISTRY ISLAMIAH COLLEGE (AUTONOMOUS) VANIYAMBADI VELLORE DIST Email Id:
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IMMUNO ELECTROPHORESIS Immuno-electrophoresis technique is based on the electrophoretic mobility and immuno precipitin reaction. The term electrophoresis is defined as the migration of charged particle under the influence of an electric field. In precipitin reaction, an antigen combines with its specific antibody to form an antigen –antibody complex. Since most of the Ag-Ab complexes are insoluble, they can be seen with the naked eye. In immuno-electrophoresis the antigens are first separated based on their electrical charge and then visualized by the precipitin reaction. There are different types of immuno-electrophoresis techniques are used for various analytical purposes. 1. Qualitative immuno- electrophoresis 2. Cross - over immuno-electrophoresis 3. Quantitative (rocket) immuno-electrophoresis 4. Two - dimensional immuno-electrophoresis QUALITATIVE IMMUNO ELECTROPHORESIS
This analysis is carried out in an agarose gel containing barbitone buffer on a microscopic slide.
A suitable pattern as shown in the figure about 100μg of specific antigen (Human serum albumin-HSA) and mixture of antigens (Human serum- HS) are placed in two different wells. Gel is cut with gel punch.
The slides are connected by thick wetfilter paper wicks to the electrode wells, and a direct electric current of about 8 mA per slide is passed for 1 - 2 hours.
Charged molecules will have been separated electrophoretically.
When electrophoresis over, immediately after the voltage supply has been disconnected, the troughs are filled with appropriate anti-sera and incubated over right at room temperature in a humid chamber.
The antigens diffuse radically and the antibodies diffuse laterally as shown the figure, resulting in the antigen - antibody precipitation areas are formed.
Magendira Mani Vinayagam/ Academia.edu/ Asst. Prof., IC., VNB
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Agar gel wells are made by gel punch, and then wells are filled with antigens, human serum albumin (HAS) and human serum (HS) respectively.
Electrophoresis starts with applied electric field, antigens are migrated
Rabbit anti - HS The troughs are filled with appropriate antisera and incubated over right
Rabbit anti - HSA
The antigen - antibody precipitation areas are formed.
Magendira Mani Vinayagam/ Academia.edu/ Asst. Prof., IC., VNB
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Application: Qualitative immuno- electrophoresis may be used to investigate the purity or to detect the particular antigens in sera, tissue culture filtrates, tissue or cell extracts or subcellular fractions.
CROSS OVER IMMUNO- ELECTROPHORESIS
This is modified method of qualitative immuno- electrophoresis. This method allows precise measurements of each antigen present in the mixture.
In the first step an antigen mixture is placed in a slot in agarose gen and electrophoresed. Bands of each antigen are present, but they are not visible at this time. A narrow longitudinal strip is cut out along the middle of the gel. The strip contains bands of each antigen.
The strip is then placed on a second gel which contains a low concentration of antiserum the antiserum contain antibodies to the antigens present in the mixture applied to the first gel. The second gel is now electrophoresed, but at 90 angle with respect to the movement of the antigens. During this electrophoresis, both the antigens in the strip and antibodies in the lower plate migrate in the electric field. Precipitation zones form for each antigen. This zones move as long narrow peaks rather than small curves. Since the antigens are forced to migrate by the electric field. This results in less overlapping of the components of the mixture and allows a clear count of the number of the components.
Most proteins show anodic migration at pH 8.0 but globulins isexceptional apparently migrating towards cathode, due to electro-osmosis.
Cross - over immuno- electrophoresis as shown in the figure takes advantage of this by moving IgG antibodies (γ-globulins) and antigens towards each other and resulting in this formation of precipitation lines.
Advantages:
More rapid (15 - 20 min) than the Ouchterlong method which may take several days to produce a clear result.More sensitive because all of the molecules migrate towards each other rather than diffusing radically.
Up to 12 samples may be tested on one agar - covered microscopic slide, saving time valuable reagents band samples.
Magendira Mani Vinayagam/ Academia.edu/ Asst. Prof., IC., VNB
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Applications Cross - over immuno- electrophoresis is particularly useful in forensic science for establishing the species of origin of body fluids such as blood, semen and saliva.
Principles of cross – over immune electrophoresis QUANTITATIVE (OR) ROCKET IMMUNO ELECTROPHORESIS: Laurells 'rocket' electrophoresis is related single radical immuno diffusion.
This method allows the determination of the concentration of a particular antigen in mixture.
The antigen sample is placed in wells cut in agar containing specific antiserum to the antigen to be assayed.
When a directelectric current in applied, most antigens migrate towards the anode and the IgG antibodies migrates towards the cathode, initially soluble (Ag-Ab) antigen-antibody complexes are formed in the presence of excess antigen.
Magendira Mani Vinayagam/ Academia.edu/ Asst. Prof., IC., VNB
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When all antigens have migrated into the gel, equivalence
is
reached
and
antigen antibody complexes precipitated.
The area under the rocket shape is directly proportional to the antigen concentration. When the precipitation areas have become stationary (110
hours)
plot
of
rocket
height
against
concentration will be linear. Thus by the use of standards and the preparation of a calibration curve, the concentration of antigen solutions may be determined. TWO DIMENSIONAL IMMUNO - ELECTROPHORESIS i.
Two dimensional immuno - electrophoresis combines the electrophoretic separation in a molecular sieving medium with specificity and speed of rocket electrophoresis.
ii. Initially antigens are separated by agar electrophoresis
one
dimension
(as
in
qualitative immuno- electrophoresis) iii. A suitable slice of gel is transferred into a square glass plate and a layer of agar containing a suitable anti serum is solidified against it over the rest of plate. iv. After rocket electrophoresis in the second dimensions, precipitin areas are formed as shown in the figure. v. By comparison of the area under the axes with those of standard system, a semi - quantitative estimate of the amount of individual antigens present may be made. Magendira Mani Vinayagam/ Academia.edu/ Asst. Prof., IC., VNB
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