Rocket Immunoelectrophoresis- Objectives, Principle, Procedure, Results, Uses
Rocket immunoelectrophoresis is a laboratory technique that allows the quantification of antigens in a sample by measuring the height of a precipitin peak formed by the antigen-antibody reaction. The technique is based on the principle of electrophoresis, which is the movement of charged particles in an electric field. In rocket immunoelectrophoresis, the antigen is negatively charged and migrates towards the positive electrode, while the antibody is immobilized in the gel matrix. As the antigen encounters the antibody, it forms a visible immune complex that resembles a rocket. The height of the rocket is proportional to the concentration of the antigen in the sample.
Rocket immunoelectrophoresis was first described by Laurell in 1966 as a method for quantifying serum proteins. Since then, it has been widely used for various applications in clinical and research settings, such as detecting and measuring immunoglobulins, enzymes, hormones, toxins, viruses, and bacteria. Rocket immunoelectrophoresis is also known as electroimmunodiffusion, immunoelectrophoretic assay, or Laurell`s method.
Rocket immunoelectrophoresis is a technique that can be used to achieve two main objectives:
- To detect antigen-antibody complexes in a sample. This can help in identifying the presence or absence of specific antigens or antibodies in a biological fluid, such as serum, plasma, urine, cerebrospinal fluid, etc. For example, rocket immunoelectrophoresis can be used to diagnose certain infections, autoimmune diseases, allergies, or immunodeficiencies by detecting the corresponding antigens or antibodies in the patient`s sample.
- To determine the concentration of antigen in an unknown sample. This can help in quantifying the amount of a specific protein or molecule in a sample, such as an enzyme, a hormone, a drug, a toxin, etc. For example, rocket immunoelectrophoresis can be used to measure the level of immunoglobulin G (IgG) in the serum of a patient with multiple myeloma, a type of blood cancer that causes overproduction of IgG.
Rocket immunoelectrophoresis is based on the principle of electrophoresis and immunodiffusion. Electrophoresis is the process of separating charged molecules by applying an electric field. Immunodiffusion is the process of forming visible precipitates when antigens and antibodies interact in a gel. By combining these two processes, rocket immunoelectrophoresis can produce conical-shaped precipitates that resemble rockets. The height and area of these rockets are proportional to the concentration of antigen in the sample. By comparing the rockets of unknown samples with those of known standards, the concentration of antigen can be calculated.
Rocket immunoelectrophoresis is a quantitative one-dimensional single electro-immunodiffusion technique. 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 principle of rocket immunoelectrophoresis can be summarized by the following equation:
h = k log C + b
where h is the height of the rocket, C is the concentration of antigen, k and b are constants that depend on the antibody-antigen system.
To perform rocket immunoelectrophoresis, you will need the following materials:
- Agarose: This is a polysaccharide derived from seaweed that forms a gel when dissolved in water and cooled. Agarose gel is used as a medium for electrophoresis because it has a uniform pore size and low electrical resistance. You will need about 15 ml of 1% agarose gel for one experiment.
- Antigen: This is the substance that you want to measure the concentration of in your sample. It can be a protein, a peptide, a hormone, an enzyme, or any other molecule that can elicit an immune response. You will need 10 µl of the antigen sample for each well.
- Antiserum: This is a solution that contains antibodies that are specific for the antigen of interest. Antibodies are proteins produced by the immune system that bind to antigens and form immune complexes. You will need 250 µl of antiserum for 13 ml of agarose solution.
- Assay Buffer: This is a solution that maintains the pH and the ionic strength of the gel and the samples. It also provides electrical conductivity for electrophoresis. You can use Tris-Borate-EDTA (TBE) buffer or any other suitable buffer for this purpose. You will need enough buffer to cover the gel in the electrophoresis tank.
- Electrophoresis Apparatus: This is a device that applies an electric field across the gel and allows the migration of charged molecules. It consists of a power supply, electrodes, wires, and a tank that holds the gel and the buffer. You will need an electrophoresis apparatus that can deliver 80-120 volts and 60-70 mA of current.
- Glass Slides: These are thin sheets of glass that are used to support the gel and allow easy handling and observation. You will need one glass slide for each gel.
- Prepare 15 ml of 1% agarose gel by dissolving agarose powder in boiling water and stirring until completely dissolved.
- Cool the solution to 55-60°C and add 250 µl of antiserum to 13 ml of agarose solution. Mix well for uniform distribution of antibodies.
- Pour the agarose solution containing the antiserum onto a grease-free glass plate placed on a horizontal surface and let the gel set for 30 minutes.
- Place the glass plate on a template and punch wells along the edge of the gel with a gel puncher.
- Add 10 µl of the standard antigen and test antigen samples to the wells.
- Pour 1X TBE buffer into the electrophoresis tank such that it just covers the gel.
- Connect the electrodes to the power supply and run electrophoresis at 80-120 volts and 60-70 mA until the antigen travels 3-4 cm from the well.
- Incubate the glass plate in a moist chamber overnight at 37°C and interpret the results.
- Mark the tips of the precipitin peaks and measure the peak height from the upper edge of the well to the tip of the peak.
- Plot a graph of the rocket height (on Y-axis) versus the concentration of antigen (on X-axis) on a semi-log graph sheet. Determine the concentration of the unknown from the graph by finding the concentration against the rocket height.
- After the electrophoresis and incubation, the gel can be examined for the presence of rocket-shaped patterns of precipitation that spread out from the loading wells. These rockets indicate positive reactions or specific antigen-antibody interactions due to the presence of antibody specific to the antigen.
- The absence of precipitation indicates no reaction or the absence of any corresponding antibody or 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. Therefore, if a series of wells are loaded with increasing antigen concentration, then a series of rockets of increasing height should be produced.
- The height of the rocket can be measured from the upper edge of the well to the tip of the peak using a ruler or a caliper. A graph can be 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 sample can be determined from the graph by finding the concentration against the rocket height.
- Alternatively, the area under the rocket can be measured using a planimeter or a densitometer and plotted against the antigen concentration. This method may be more accurate than measuring the rocket height, especially for low concentrations of antigen.
- The results can be expressed as absolute values (µg/mL) or relative values (% of normal) depending on the purpose of the assay. For example, in estimation of immunoglobulin levels, relative values may be more useful than absolute values.
Rocket immunoelectrophoresis has various applications in the field of immunology and biochemistry. Some of the common applications are:
- Rocket immunoelectrophoresis is used mainly for quantitative estimation of antigen in the serum. It can measure concentrations of proteins as low as 1 µg/mL.
- The method has been used for quantization of human serum proteins before automated methods became available. It can detect and measure immunoglobulins, albumin, transferrin, haptoglobin, etc.
- Rocket immunoelectrophoresis can be used for determining the concentration of a specific protein in a protein mixture. For example, it can be used to measure the amount of hemoglobin in blood samples.
- Rocket immunoelectrophoresis can be used for estimation of immunoglobulin protease activity. This is useful for studying the degradation of antibodies by proteases.
- Rocket immunoelectrophoresis can be used for studies dealing with antigenic relationships between organisms. For example, it can be used to compare the antigenic similarity of different strains of bacteria or viruses.
- Rocket immunoelectrophoresis can also be used for enzyme activity electrophoresis. This is a technique where enzymes are electrophoresed in a gel containing their substrate and the product formation is visualized by staining or colorimetric reaction.
Rocket immunoelectrophoresis has several advantages over other immunological methods for quantifying antigens. Some of them are:
- It is a simple, quick, and reproducible method that does not require complex equipment or reagents.
- It allows simultaneous analysis of several unknown samples on a single plate, which saves time and resources.
- It can measure concentrations of proteins as low as 1 µg/mL, requiring as little as 20 ng of protein to be loaded in a well. This makes it suitable for samples with limited availability or high dilution.
- It can provide a visual representation of the antigen-antibody reaction, which can be useful for qualitative assessment and comparison of different samples.
- It can be easily adapted to different antigens and antibodies by changing the gel composition and the electrophoresis conditions.
- Rocket immunoelectrophoresis allows quantitative analysis of antigens, but it is not applicable to complex mixtures. This is because the antigen-antibody reaction may be influenced by the presence of other proteins or substances in the sample that may interfere with the binding or migration of the antigen.
- Rocket immunoelectrophoresis requires a specific and high-affinity antibody for each antigen to be measured. This may limit the availability and cost-effectiveness of the method for some antigens. Moreover, the antibody concentration and quality may vary from batch to batch, affecting the reproducibility and accuracy of the results.
- Rocket immunoelectrophoresis is slower, less sensitive, and more difficult to interpret than some other methods of immunoelectrophoresis, such as immunofixation electrophoresis. Rocket immunoelectrophoresis may fail to detect some small or low-concentration antigens because they may be obscured by the most abundant or rapidly migrating proteins in the sample.
- Rocket immunoelectrophoresis depends on several factors that may affect the shape and height of the rocket, such as the pH, temperature, voltage, buffer composition, gel concentration, and antigen loading volume. These factors need to be carefully controlled and standardized to ensure reliable and consistent results. Any variation or deviation from the optimal conditions may cause distortion or variation of the rocket.
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