It is also known as electroimmunoassay or electroimmunodiffusion. The migration of the antigen toward the anode gives rise to rocket-shaped patterns of precipitation. The area under the rocket is proportional to antigen concentration. In this method antibody is incorporated in the gel at a pH value at which the antibodies remain essentially immobile. Antigen is placed in wells cut in the gel. Electric current is then passed through the gel, which facilitates the migration of negatively charged antigens into the agar.
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It is also known as electroimmunoassay or electroimmunodiffusion. The migration of the antigen toward the anode gives rise to rocket-shaped patterns of precipitation. The area under the rocket is proportional to antigen concentration. In this method antibody is incorporated in the gel at a pH value at which the antibodies remain essentially immobile. Antigen is placed in wells cut in the gel.
Electric current is then passed through the gel, which facilitates the migration of negatively charged antigens into the agar. As the antigen moves out of the well and enters the agarose gel, it combines with the antibody to form immune complex which becomes visible. During the initial phase there is considerable antigen excess over antibody and no visible precipitation occurs. However, as the antigen sample migrates further through the agarose gel, more antibody molecules are encountered that interact with the antigen to form immune complex.
This results in formation of a precipitin line that is conical in shape, resembling a rocket. The greater the amount of antigen loaded in a well, the further the antigen will have to travel through the gel before it can interact with sufficient antibody to form a precipitate. Thus, the height of the rocket, measured from the well to the apex and area are directly proportional to the amount of antigen in the sample.
The solution is cooled to oC and ml of antiserum added to 13 ml of agarose solution. It is well mixed for uniform distribution of antibody. Agarose solution containing the antiserum is poured on to grease free glass plate placed on a horizontal surface and the gel is allowed to set for 30 minutes.
The glass plate is on the template and wells punched with the help of gel puncher. Electrophoresis is carried out at volts and mA, until the antigen travels cms from the well. The glass plate is incubated in a moist chamber overnight at 37o C and results interpreted.
In case positive for reaction, the tips of the precipitin peaks are marked and the peak height measured from the upper edge of the well to the tip of the peak. A graph is plotted of the rocket height on Y-axis versus the concentration of antigen on X-axis on a semi-log graph sheet.
The concentration of the unknown is determined from the graph by finding the concentration against the rocket height. The absence of the precipitation indicates no reaction or the absence of any corresponding antibody — antigen. The height of the rocket, and its area are directly proportional to the amount of antigen in the sample, that is, the height of the precipitin peak depends on the concentration of antigens loaded in the corresponding wells.
Applications of Rocket Immunoelectrophoresis Rocket electrophoresis is used mainly for quantitative estimation of antigen in the serum. The method has been used for quantization of human serum proteins before automated methods became available.
Determining the concentration of a specific protein in a protein mixture. In estimation of immunoglobulin protease activity. In enzyme activity electrophoresis. Simple, quick, and reproducible method. Several unknown samples can be analyzed on a single plate. Limitations of Rocket Immunoelectrophoresis These techniques allow quantitative analysis of antigens, but are not applicable to complex mixtures. Two-dimensional immunoelectrophoresis is a variant of rocket electrophoresis. The test is a two-stage procedure.
In the first stage, antigens in solution are separated by electrophoresis. In the second stage, electrophoresis is carried out again, but perpendicular to that of first stage to obtain rocket-like precipitation. References Walker JM Rocket immunoelectrophoresis. Methods Mol Biol. Immunology 2 ed. Parija S. India: Elsevier India. Sastry A. Essentials of Medical Microbiology.
Jump to navigation Jump to search Biochemical methods of separation and characterization of proteins Crossed immunoelectrophoresis of 2 microLitre of normal human serum. The electrophoresis was performed in thin layers of agarose, the pictured gel is about 7x7 cm. The lower part is the first dimension gel without antibodies, where the serum was applied into the slot at the lower left. The upper part is the second dimension gel with Dako antibodies against human serum proteins. More than 50 major serum proteins can be named. Fused rocket immunoelectrophoresis of an affinity chromatographic separation of human serum proteins on con A. A 15 microlitre sample from each fraction is applied starting from left and let to diffuse about one hour, then electrophorsis was performed over night.
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