IMMUNO ELECTROPHORESIS

IMMUNO ELECTROPHORESIS Immuno electrophoresis combines the principles of both immuno preciptin reaction and electrophoresis. In IE the antigens are first separated based on their electrical charge and then visualized by the precipitin reaction. There are different types of IE; 1. Quantitative immunoelectropphoresis 2. Cross - over immunoelectrophoresis 3. Quantitative (rocket) immunoelectrophoresis 4. Two - dimentional immunoelectrophoresis QUALITATIVE IMMUNO ELECTROPHORESIS: i.

Qualitative I E combines the specificity of immunopreciptin reaction with the separation of molecules by electrophoresis in a molecular sieving medium.

ii. The analysis is carried out in an agarose gel containing barbitone buffer on a microscopic slide. iii. A suitable pattern as shown in the figure is cut with gel punch and 1-5 containing 1 - 100 of antigen are added to the wells.

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iv. The slides are connected by thick wet filter paper wicks to the electrode wells, and a direct electric current of about 8 mA per slide is passed for 1 - 2 hours giving a voltage drop of 1 - 8 rcm-1 STAGES IN MICRO IMMUNO ELECTROPHORESIS: (QUALITATIVE IE)

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB.

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Charged molecules will have been separated electrophoretically, but will not usually be visible. Immediately after the voltage supply has been dis connected, the troughs are filled with appropriate antisera and incubated ovar right at room temperature a humid chamber. The antigens diffuse radially and the antibodies diffuse laterally as shown the figure, resulting in the antigen - antibody precipitation areas. Despite the use of agarose which acquires a smaller charge than agar, the electrosinotic flow of water during electrophoresis moves all the antigens towards the cathode. This results in the apparent cathodic migration. K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB.

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Application: Qualitative IE may be used to investigate the purity and to detect the particular antigens in sera, culture filtrates, tissue or cell extracts or fraction from any preparative procedure. CROSS OVER ELECTROPHORESIS: Most proteins show anodic migration at pH 8.0 but globulins is exceptional apparently migrating towards cathode, due to electroosmosis. Cross - over IE as shown in the figure takes advantage of this by removing. IgG antibodies (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. Upto 12 samples may be tested on one agar - covered microscopic slide, saving time valuable reagents band samples.

Principles of cross – over immune electophoresis K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB.

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APPLICATION: Cross - over IE is particularly useful in forensic science for establishing the species of origin of body fluids such as blood, semen and saliva. QUANTITATIVE (OR) ROCKET IMMUNO ELECTROPHORESIS: Laurells 'rocket' electrophoresis is related single radical immuno diffusion.

The antigen sample is placed in wells cut in agar containing. When a direct electric current in applied most antigens migrate towards the antigen and the antibodies migrates towards the cathodes initially antigen samples are formed in the presence of excess antigen. When an antigen has into the gel is removed and antigen antibody complexes precipitate. The area under the rocket shape is directly proportional to the antigen concentration. When the precipitation area have become stationary (1-10 hours) plot of rocket height against concentration will be linear. Thus by the use of and the preparation of a calibration curve the concentration of antigen solutions may be determine

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB.

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TWO DIMENTIONAL IMMUNOELECTROPHORESIS: i.

Two dimensions IE combines the electrophoretic separation to a molecular sieving medium with specificity and speed of rocket electrophoresis.

ii. Initially antigens are separated by agar electrophoresis one dimension (as in qualitative IE) 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. Filter rocket electrophoresis in the second dimensions, precipitins area 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.

Gel has been stained with coomassive brilliant blue to enhance the immuno precipitator (The large side in human serum albumin, the major serum protein).

K. VINOTH KALAISELVEN., Academia.edu/ Asst. Prof., Islamiah College, VNB.

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