Immunological Techniques- Types and Examples

Immunodiffusion tests are a type of immunological technique that use the diffusion of antigens and antibodies in a gel medium to form visible precipitation bands. These bands indicate the presence and identity of specific antigens or antibodies in a sample. Immunodiffusion tests are based on the principle that when an antigen and an antibody are mixed in optimal proportions, they form an insoluble complex that precipitates out of the solution.

Immunodiffusion tests have several advantages over other immunological methods, such as:

• They are simple, inexpensive, and easy to perform.
• They do not require sophisticated equipment or reagents.
• They produce stable and permanent results that can be stored and examined later.
• They can detect multiple antigens or antibodies in a single test.
• They can differentiate between identical, partially identical, and non-identical antigens or antibodies based on the shape and pattern of the precipitation bands.

Immunodiffusion tests can be classified into different types based on the number of dimensions in which the diffusion occurs and the number of components involved in the reaction. The main types of immunodiffusion tests are:

• Single diffusion in one dimension (Oudin procedure)
• Double diffusion in one dimension (Oakley-Fulthrope procedure)
• Single diffusion in two dimensions (Radial immunodiffusion)
• Double diffusion in two dimensions (Ouchterlony procedure)

Each type of immunodiffusion test has its own applications and limitations. In the following sections, we will explain each type in detail and provide some examples of their use in clinical and research settings.

Single diffusion in one dimension is a simple immunodiffusion technique that can be used to detect different antigens in a sample. The principle of this technique is that the antigen diffuses downward through a layer of agar gel that contains the antibody, and forms a line of precipitation when it reaches the optimal concentration of antigen-antibody interaction.

The procedure for this technique is as follows:

1. Prepare a test tube with 1% agar gel and mix it with the antibody solution.
2. Pour the antigen solution over the surface of the agar gel and let it diffuse downward.
3. Observe the formation of a precipitate band after some time (usually 24 to 48 hours).

The result of this technique can be interpreted as follows:

• The number of different precipitate bands indicates the number of different antigens in the sample.
• The position of the precipitate band reflects the relative concentration of the antigen in the sample. The higher the concentration, the lower the position of the band.
• The intensity and width of the precipitate band reflect the affinity and avidity of the antibody for the antigen. The stronger the binding, the more intense and wider the band.

This technique has some advantages and disadvantages:

• It is easy to perform and requires minimal equipment and reagents.
• It can detect multiple antigens in a single test tube.
• It is not very sensitive or quantitative compared to other techniques.
• It may not distinguish between closely related antigens or show cross-reactivity between different antigens.

This technique has some applications in clinical and research settings, such as:

• Identifying different types of hemagglutinins in influenza virus strains.
• Detecting antibodies against bacterial toxins such as tetanus and diphtheria.
• Typing blood groups and Rh factors.

This is another type of immunodiffusion test that involves the movement of both antigen and antibody in one dimension. The procedure is as follows:

1. The antibody is mixed with agar in a test tube.
2. A column of plain agar is added on top of the antibody solution.
3. The antigen is poured on the plain agar column.
4. The antigen and antibody move toward each other through the intervening column of plain agar and a precipitate band will form when at the optimum concentration of the antigen and antibody.

The advantages of this technique are:

• It is more sensitive than the Oudin procedure as it allows more time for diffusion and reaction.
• It can detect small differences in antigenic composition or concentration.

The disadvantages of this technique are:

• It is slower than the Oudin procedure as it requires two steps of diffusion.
• It can be affected by factors such as temperature, pH, and ionic strength.

This technique can be used for:

• The identification of bacterial antigens such as Salmonella and Shigella.
• The detection of antibodies to viral antigens such as measles and mumps.

This is an immunological technique that uses the principle of diffusion of antigens in a gel containing antibodies. The technique is also known as Mancini method or Fahey-McKellar method. The procedure is as follows:

1. The antibody is mixed with agar gel and a layer of this mixture is formed on a glass slide.
2. Wells are cut on the surface of the gel using a template or a punch.
3. The antigen solution is added to the wells. As a result, it diffuses radially from the wells into the gel containing the antibody.
4. A ring-shaped precipitation band is formed around each well when the antigen and antibody reach an optimal concentration ratio.
5. The diameter of the band is proportional to the concentration of the antigen in the well. Therefore, by measuring the diameter of the band and comparing it with a standard curve, the concentration of the antigen can be estimated.

This technique is simple, sensitive and specific. It can be used for the quantitation of various antigens such as immunoglobulins, complement components, hormones, enzymes and bacterial toxins. It can also be used for the detection of antibodies by adding known antigens to the wells. Some examples of applications of this technique are:

• Estimation of IgG, IgM and IgA in sera
• Screening of antibodies of influenza virus
• Diagnosis of rheumatoid arthritis by measuring rheumatoid factor
• Diagnosis of toxoplasmosis by measuring anti-toxoplasma antibodies

This technique is also known as Ouchterlony double immunodiffusion or passive double immunodiffusion.

It involves the diffusion of both antigen and antibody solutions in two dimensions (horizontally and vertically) through an agar gel layer.

A layer of agar gel is formed on a Petri plate. Then wells are formed by using a template.

Antibody solution is added to the central well and different antigens are added to the surrounding wells.

The antigen and antibody solutions diffuse from the wells toward each other and form precipitin lines where they meet at equivalent proportions.

The pattern of the precipitin lines can reveal the relationship between the antigens:

• If two adjacent antigens are identical, the precipitation lines will fuse.
• If two adjacent antigens are unrelated, the precipitation lines will cross.
• If two adjacent antigens are partially related, spur formation will be observed.

This technique is used for the toxicity test of C. diphtheria (Elck’s Test), detection of antibodies against protein or complex carbohydrate antigens, and comparison of antigenic similarity.

Immunoelectrophoresis is a technique that combines electrophoresis and immunodiffusion to separate and identify proteins based on their antigenic properties. Electrophoresis is a process that uses an electric field to move charged molecules through a gel matrix. Immunodiffusion is a process that uses antibodies to form visible precipitates with antigens in the gel.

The procedure of immunoelectrophoresis is as follows:

1. A glass slide is coated with a thin layer of agarose gel.
2. A well is cut on one end of the gel and the sample containing the antigen mixture is added to the well.
3. Electrophoresis is performed for about an hour, which separates the antigens according to their size and charge in the gel.
4. A rectangular trough is cut parallel to the direction of electrophoresis and filled with a specific antibody solution.
5. The antibody diffuses into the gel and reacts with the corresponding antigens, forming lines of precipitation.
6. The pattern of precipitation lines indicates the number and identity of the antigens in the sample.

Immunoelectrophoresis can be used for various purposes, such as:

• Detection of different antigens in human serum, such as immunoglobulins, complement components, and transferrin.
• Diagnosis of abnormal serum proteins, such as monoclonal gammopathies (e.g., multiple myeloma) and dysgammaglobulinemias (e.g., selective IgA deficiency).
• Identification of bacterial, viral, or fungal antigens in clinical specimens, such as cerebrospinal fluid, urine, or sputum.

Counter immunoelectrophoresis (CIE) is a one-dimensional double electro-immunodiffusion test. The test is based on the movement of antigen and antibody in the opposite direction under an electric field. This test is faster and more sensitive than simple immunodiffusion tests.

The test is performed on a glass slide which is layered with agar gel. Two different wells are formed on the surface of the gel. In one well, antigen solution is added and another well contains antibody solution. The wells are placed at a certain distance from each other and connected by electrodes. The electricity is passed through the gel which accelerates the movement of antigen and antibody towards each other. A precipitation line will be formed at a specific point between the two wells when the antigen and antibody reach their optimal concentration and ratio.

The position and intensity of the precipitation line can indicate the presence and quantity of the antigen or antibody in the test sample. The closer the line is to the antigen well, the higher the concentration of the antigen. The thicker the line is, the more antigen or antibody is present in the sample.

CIE is a standard technique that requires around 30 minutes to perform. It can be used for clinical detection of various antigens and antibodies, such as hepatitis B antigens and antibodies, antigens of Cryptococcus in cerebrospinal fluid, bacterial toxins, etc.

Here is a diagram of CIE:

+---------------------+
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|                     |    +   -   +
|    Ag    Ab         |    |   |   |
|    +     +          |    |   |   |
+----+-----+----------+----+---+---+
     |     |               |   |
     |     |               |   |
     V     ^               V   ^
 Antigen  Antibody      Cathode Anode

Rocket electrophoresis is a one-dimensional single electroimmunodiffusion test. It is mostly used for the quantitation of antigens. In this case, the antibody is mixed with the agarose gel and this mixture will be used to form a layer on the glass slide. Wells will be formed on the surface of the gel and antigens are added to those wells in increasing concentration. Electrophoresis will be performed. As a result, cone-like precipitation bands (rocket-like structures) will be observed. The length of the rocket-like structure is directly connected with the concentration of antigens.

To illustrate this technique, let`s take an example of measuring serum albumin levels. Albumin is a protein that is produced by the liver and helps to maintain fluid balance in the body. Low levels of albumin can indicate liver disease, kidney disease, malnutrition, or inflammation.

To perform rocket electrophoresis for albumin measurement, we need:

• A glass slide coated with agarose gel containing anti-albumin antibodies
• A standard solution of albumin with known concentration
• A test solution of serum with unknown concentration of albumin
• A power supply to apply an electric field

The steps are as follows:

1. Cut wells on one end of the gel and load them with different volumes of the standard and test solutions. The more volume, the more antigen present in the well.
2. Connect the power supply and run electrophoresis for a fixed time. The albumin molecules will migrate towards the anode (positive electrode) and form complexes with the antibodies in the gel.
3. Observe the formation of rocket-like bands in the gel. The length of each band is proportional to the amount of albumin in each well.
4. Measure the length of each band and plot them against the corresponding concentration or volume of albumin. This will give a standard curve that can be used to calculate the unknown concentration of albumin in the test solution.

The following image shows an example of rocket electrophoresis for albumin measurement:

Rocket electrophoresis is a simple, fast, and sensitive technique that can measure antigens in nanogram quantities. However, it also has some limitations, such as:

• It requires specific antibodies for each antigen.
• It can be affected by variations in temperature, pH, voltage, and buffer composition.
• It can only measure one antigen at a time.

Radioimmunoassay (RIA) is a technique that uses radioisotopes to measure the concentration of antigens in a sample. It was first developed by Berson and Yalow in 1959 to measure insulin levels in blood.

The principle of RIA is based on the competition between a known amount of radiolabelled antigen and an unknown amount of test antigen for a limited number of specific antibodies. The radiolabelled antigen can be detected by a device that measures radioactivity, such as a gamma counter or a scintillation counter.

The steps involved in RIA are as follows:

1. A fixed amount of antibody is added to a series of tubes containing different concentrations of test antigen and radiolabelled antigen. The tubes are incubated for a sufficient time to allow the antigen-antibody reaction to reach equilibrium.
2. The tubes are then separated into two fractions: bound and free. The bound fraction contains the antibody-bound antigen, while the free fraction contains the unbound antigen. This can be done by various methods, such as precipitation, adsorption, or immunomagnetic separation.
3. The radioactivity of both fractions is measured and recorded. The bound fraction represents the amount of antigen that has been captured by the antibody, while the free fraction represents the amount of antigen that has remained in solution.
4. A standard curve is plotted by plotting the percentage of bound antigen versus the concentration of test antigen. The concentration of test antigen in an unknown sample can be determined by interpolating its percentage of bound antigen on the standard curve.

RIA has several advantages, such as high sensitivity, specificity, accuracy, and versatility. It can be used to measure various types of antigens, such as hormones, drugs, hepatitis B surface antigen, IgE, and viral antigens. However, RIA also has some limitations, such as the use of radioactive materials, which poses safety and environmental hazards, the short half-life of some radioisotopes, which requires frequent calibration and standardization, and the possibility of interference from other substances in the sample.

References:

Berson SA, Yalow RS (1959). Assay of plasma insulin in human subjects by immunological methods. Nature 184: 1648–1649.

Chard T (1995). An introduction to radioimmunoassay and related techniques. Elsevier.

Voller A (1978). The enzyme linked immunosorbent assay (ELISA). Diagnostic Horizons 2: 1–7.

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