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Immunoelectrophoresis

Immunoelectrophoresis

Definition

Immunoelectrophoresis, also called gamma globulin electrophoresis, or immunoglobulin electrophoresis, is a method of determining the blood levels of three major immunoglobulins: immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA).

Purpose

Immunoelectrophoresis is a powerful analytical technique with high resolving power as it combines separation of antigens by electrophoresis with immunodiffusion against an antiserum. The increased resolution is of benefit in the immunological examination of serum proteins. Immunoelectrophoresis aids in the diagnosis and evaluation of the therapeutic response in many disease states affecting the immune system. It is usually requested when a different type of electrophoresis, called a serum protein electrophoresis , has indicated a rise at the immunoglobulin level. Immunoelectrophoresis is also used frequently to diagnose multiple myeloma , a disease affecting the bone marrow.

Precautions

Drugs that may cause increased immunoglobulin levels include therapeutic gamma globulin, hydralazine, isoniazid, phenytoin (Dilantin), procainamide, oral contraceptives, methadone, steroids, and tetanus toxoid and antitoxin. The laboratory should be notified if the patient has received any vaccinations or immunizations in the six months before the test. This is mainly because prior immunizations lead to the increased immunoglobulin levels resulting in false positive results.

It should be noted that, because immunoelectrophoresis is not quantitative, it is being replaced by a procedure called immunofixation, which is more sensitive and easier to interpret.

Description

Serum proteins separate in agar gels under the influence of an electric field into albumin, alpha 1, alpha 2, and beta and gamma globulins. Immunoelectrophoresis is performed by placing serum on a slide containing a gel designed specifically for the test. An electric current is then passed through the gel, and immunoglobulins, which contain an electric charge, migrate through the gel according to the difference in their individual electric charges. Antiserum is placed alongside the slide to identify the specific type of immunoglobulin present. The results are used to identify different disease entities, and to aid in monitoring the course of the disease and the therapeutic response of the patient with such conditions as immune deficiencies, autoimmune disease, chronic infections, chronic viral infections, intrauterine fetal infections, multiple myeloma, and monoclonal gammopathy of undetermined significance.

There are five classes of antibodies: IgM, IgG, IgA, IgE, and IgD.

IgM is produced upon initial exposure to an antigen. For example, when a person receives the first tetanus vaccination, antitetanus antibodies of the IgM class are produced 10 to 14 days later. IgM is abundant in the blood but is not normally present in organs or tissues. IgM is primarily responsible for ABO blood grouping and rheumatoid factor, yet is involved in the immunologic reaction to other infections, such as hepatitis. Since IgM does not cross the placenta, an elevation of this immunoglobulin in the newborn indicates intrauterine infection such as rubella, cytomegalovirus (CMV) or a sexually transmitted disease (STD).

IgG is the most prevalent type of antibody, comprising approximately 75% of the serum immunoglobulins. IgG is produced upon subsequent exposure to an antigen. As an example, after receiving a second tetanus shot, or booster, a person produces IgG antibodies in five to seven days. IgG is present in both the blood and tissues, and is the only antibody to cross the placenta from the mother to the fetus. Maternal IgG protects the newborn for the first months of life, until the infant's immune system produces its own antibodies.

IgA constitutes approximately 15% of the immunoglobulins within the body. Although it is found to some degree in the blood, it is present primarily in the secretions of the respiratory and gastrointestinal tract, in saliva, colostrum (the yellowish fluid produced by the breasts during late pregnancy and the first few days after childbirth), and in tears. IgA plays an important role in defending the body against invasion of germs through the mucous membrane-lined organs.

IgE is the antibody that causes acute allergic reactions; it is measured to detect allergic conditions. IgD, which constitutes the smallest portion of the immunoglobulins, is rarely evaluated or detected, and its function is not well understood.

Preparation

This test requires a blood sample.

Aftercare

Because this test is ordered when either very low or very high levels of immunoglobulins are suspected, the patient should be alert for any signs of infection after the test, including fever , chills, rash, or skin ulcers. Any bone pain or tenderness should also be immediately reported to the physician.

Risks

Risks for this test are minimal, but may include slight bleeding from the blood-drawing site, fainting or feeling lightheaded after venipuncture, or bruising.

Normal results

Reference ranges vary from laboratory to laboratory and depend upon the method used. For adults, normal values are usually found within the following ranges (1 mg = approximately.000035 oz. and 1 dL = approximately 0.33 fluid oz.):

  • IgM: 60-290 mg/dL
  • IgG: 700-1, 800 mg/dL
  • IgA: 70-440 mg/dL

Abnormal results

Increased IgM levels can indicate Waldenström's macroglobulinemia , a malignancy caused by secretion of IgM at high levels by malignant lymphoplasma cells. Increased IgM levels can also indicate chronic infections, such as hepatitis or mononucleosis and autoimmune diseases, like rheumatoid arthritis.

Decreased IgM levels can be indicative of AIDS, immunosuppression caused by certain drugs like steroids or dextran, or leukemia.

Increased levels of IgG can indicate chronic liver disease, autoimmune diseases, hyperimmunization reactions, or certain chronic infections, such as tuberculosis or sarcoidosis.

Decreased levels of IgG can indicate Wiskott-Aldrich syndrome, a genetic deficiency caused by inadequate synthesis of IgG and other immunoglobulins. Decreased IgG can also be seen with AIDS and leukemia.

Increased levels of IgA can indicate chronic liver disease, chronic infections, or inflammatory bowel disease.

Decreased levels of IgA can be found in ataxia, a condition affecting balance and gait, limb or eye movements, speech, and telangiectasia, an increase in the size and number of the small blood vessels in an area of skin, causing redness. Decreased IgA levels are also seen in conditions of low blood protein (hypoproteinemia), and drug immunosuppression.

Resources

BOOKS

Fischbach, Frances T. A Manual of Laboratory Diagnostic Tests. Philadelphia: Lippincott Williams & Wilkins, 1999.

Pagana, Kathleen D., and Timothy J. Pagana. Mosby's Manual of Diagnostic and Laboratory Tests. St. Louis, MO: Mosby, Inc., 1999.

Janis O. Flores

KEY TERMS

Antibody

A protein manufactured by the white blood cells to neutralize an antigen in the body. In some cases, excessive formation of antibodies leads to illness, allergy, or autoimmune disorders.

Antigen

A substance that can cause an immune response, resulting in production of an antibody, as part of the body's defense against infection and disease. Many antigens are foreign proteins not found naturally in the body, and include germs, toxins, and tissues from another person used in organ transplantation.

Autoimmune disorder

A condition in which antibodies are formed against the body's own tissues, for example, in some forms of arthritis.

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Immunoelectrophoresis

Immunoelectrophoresis

Definition

Immunoelectrophoresis, also called gamma globulin electrophoresis, or immunoglobulin electrophoresis, is a method of determining the blood levels of three major immunoglobulins: immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA).

Purpose

Immunoelectrophoresis is a powerful analytical technique with high resolving power as it combines separation of antigens by electrophoresis with immunodiffusion against an antiserum. The increased resolution is of benefit in the immunological examination of serum proteins. Immunoelectrophoresis aids in the diagnosis and evaluation of the therapeutic response in many disease states affecting the immune system. It is usually requested when a different type of electrophoresis, called a serum protein electrophoresis, has indicated a rise at the immunoglobulin level. Immunoelectrophoresis is also used frequently to diagnose multiple myeloma, a disease affecting the bone marrow.

Precautions

Drugs that may cause increased immunoglobulin levels include therapeutic gamma globulin, hydralazine, isoniazid, phenytoin (Dilantin), procainamide, oral contraceptives, methadone, steroids, and tetanus toxoid and antitoxin. The laboratory should be notified if the patient has received any vaccinations or immunizations in the six months before the test. This is mainly because prior immunizations lead to the increased immunoglobulin levels resulting in false positive results.

It should be noted that, because immunoelectrophoresis is not quantitative, it is being replaced by a procedure called immunofixation, which is more sensitive and easier to interpret.

Description

Serum proteins separate in agar gels under the influence of an electric field into albumin, alpha 1, alpha 2, and beta and gamma globulins. Immunoelectrophoresis is performed by placing serum on a slide containing a gel designed specifically for the test. An electric current is then passed through the gel, and immunoglobulins, which contain an electric charge, migrate through the gel according to the difference in their individual electric charges. Antiserum is placed alongside the slide to identify the specific type of immunoglobulin present. The results are used to identify different disease entities, and to aid in monitoring the course of the disease and the therapeutic response of the patient to such conditions as immune deficiencies, autoimmune disease, chronic infections, chronic viral infections, and intrauterine fetal infections.

There are five classes of antibodies: IgM, IgG, IgA, IgE, and IgD.

IgM is produced upon initial exposure to an antigen. For example, when a person receives the first tetanus vaccination, antitetanus antibodies of the IgM class are produced 10 to 14 days later. IgM is abundant in the blood but is not normally present in organs or tissues. IgM is primarily responsible for ABO blood grouping and rheumatoid factor, yet is involved in the immunologic reaction to other infections, such as hepatitis. Since IgM does not cross the placenta, an elevation of this immunoglobulin in the newborn indicates intrauterine infection such as rubella, cytomegalovirus (CMV) or a sexually transmitted disease (STD).

IgG is the most prevalent type of antibody, comprising approximately 75% of the serum immunoglobulins. IgG is produced upon subsequent exposure to an antigen. As an example, after receiving a second tetanus shot, or booster, a person produces IgG antibodies in five to seven days. IgG is present in both the blood and tissues, and is the only antibody to cross the placenta from the mother to the fetus. Maternal IgG protects the newborn for the first months of life, until the infant's immune system produces its own antibodies.

IgA constitutes approximately 15% of the immunoglobulins within the body. Although it is found to some degree in the blood, it is present primarily in the secretions of the respiratory and gastrointestinal tract, in saliva, colostrum (the yellowish fluid produced by the breasts during late pregnancy and the first few days after childbirth ), and in tears. IgA plays an important role in defending the body against invasion of germs through the mucous membrane-lined organs.

IgE is the antibody that causes acute allergic reactions; it is measured to detect allergic conditions. IgD, which constitutes the smallest portion of the immunoglobulins, is rarely evaluated or detected, and its function is not well understood.

Preparation

This test requires a blood sample.

Aftercare

Because this test is ordered when either very low or very high levels of immunoglobulins are suspected, the patient should be alert for any signs of infection after the test, including fever, chills, rash, or skin ulcers. Any bone pain or tenderness should also be immediately reported to the physician.

Risks

Risks for this test are minimal, but may include slight bleeding from the blood-drawing site, fainting or feeling lightheaded after venipuncture, or bruising.

Normal results

Reference ranges vary from laboratory to laboratory and depend upon the method used. For adults, normal values are usually found within the following ranges (1 mg = approximately 0.000035 oz. and 1 dL = approximately 0.33 oz.):

  • IgM: 60-290 mg/dL
  • IgG: 700-1,800 mg/dL
  • IgA: 70-440 mg/dL

Abnormal results

Increased IgM levels can indicate Waldenstroöm's macroglobulinemia, a malignancy caused by secretion of IgM at high levels by malignant lymphoplasma cells. Increased IgM levels can also indicate chronic infections, such as hepatitis or mononucleosis and autoimmune diseases, like rheumatoid arthritis.

Decreased IgM levels can be indicative of AIDS, immunosuppression caused by certain drugs like steroids or dextran, or leukemia.

Increased levels of IgG can indicate chronic liver disease, autoimmune diseases, hyperimmunization reactions, or certain chronic infections, such as tuberculosis or sarcoidosis.

Decreased levels of IgG can indicate Wiskott-Aldrich syndrome, a genetic deficiency caused by inadequate synthesis of IgG and other immunoglobulins. Decreased IgG can also be seen with AIDS and leukemia.

Increased levels of IgA can indicate chronic liver disease, chronic infections, or inflammatory bowel disease.

Decreased levels of IgA can be found in ataxia, a condition affecting balance and gait, limb or eye movements, speech, and telangiectasia, an increase in the size and number of the small blood vessels in an area of skin, causing redness. Decreased IgA levels are also seen in conditions of low blood protein (hypoproteinemia), and drug immunosuppression.

Resources

BOOKS

Fischbach, Frances T. A Manual of Laboratory Diagnostic Tests. Philadelphia: Lippincott Williams & Wilkins, 1999.

Pagana, Kathleen D., and Timothy J. Pagana. Mosby's Manual of Diagnostic and Laboratory Tests. St. Louis, MO: Mosby, Inc., 1999.

KEY TERMS

Antibody A protein manufactured by the white blood cells to neutralize an antigen in the body. In some cases, excessive formation of antibodies leads to illness, allergy, or autoimmune disorders.

Antigen A substance that can cause an immune response, resulting in production of an antibody, as part of the body's defense against infection and disease. Many antigens are foreign proteins not found naturally in the body, and include germs, toxins, and tissues from another person used in organ transplantation.

Autoimmune disorder A condition in which antibodies are formed against the body's own tissues; for example, in some forms of arthritis.

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Immunoelectrophoresis

Immunoelectrophoresis

Immunoelectrophoresis is a technique that separates proteins on the basis of both their net charge (and so their movement in an electric field) and on the response of the immune system to the proteins. The technique is widely used in both clinical and research laboratories as a diagnostic tool to probe the protein composition of serum.

Petr Nikolaevich Grabar, a French immunologist, devised the technique in the 1950s. In essence, immunoelectrophoresis separates the various proteins in a sample in an electric field and then probes the separated proteins using the desired antiserum .

The most widely used version of the technique employs an apparatus, which consists basically of a microscope slide-sized plate. The plate is the support for a gel that is poured over top and allowed to congeal. The construction of the gel can vary, depending on the separation to be performed. Agar , such as that used in microbiological growth media, and another material called agarose can be used. Another popular choice is a linked network of a chemical known as acrylamide. The linked up acrylamide chains form what is designated as polyacrylamide.

The different types of gel networks can be most productively envisioned as a three-dimensional overlay of the crossed linked chains. The effect is to produce snaking tunnels through the matrix of various diameters. These diameters, which are also referred to as pore sizes, can be changed to a certain extent by varying the concentrations of some of the ingredients of the gel suspension. Depending on the size and the shape of the protein, movement through this matrix will be relatively slow or fast. As well, depending on the net charge a protein molecule has, the protein will migrate towards the positively charged electrode or the negatively charged electrode when the electric current is passed through the gel matrix. Thus, the various species of protein will separate from each other along the length of the gel.

In some configurations of the immunoelectrophoretic set-up, the samples that contain the proteins to be analyzed are added to holes on either side of the gel plate. For example, one sample could contain serum from a health individual and another sample could contain serum from someone with an infection. The middle portion of the plate contains a trough, into which a single purified species of antibody or known mixture of antibodies is added. The antibody molecules diffuse outward from the trough solution into the gel. Where an antibody encounters a corresponding antigen , a reaction causes the formation of a visual precipitate. Typically, the precipitation occurs in arc around the antigen-containing sample. In the example, the pattern of precipitation can reveal antigenic differences between the normal serum and the serum from a infected person.

This type of immunoelectrophoresis provides a qualitative ("yes or no") answer with respect to the presence or absence of proteins, and can be semi-quantitative. The shape of the arc of precipitation is also important. An irregularly shaped arc can be indicative of an abnormal protein or the presence of more than one antigenically similar protein.

Immunoelectrophoresis can also be used to detect a particular antigenic site following the transfer of the proteins from a gel to a special support, such as nitrocellulose. Addition of the antibody followed by a chemical to which bound antibody reacts produces a darkening on the support wherever antibody has bound to antigen. One version of this technique is termed Western Blotting. An advantage of this technique is that, by running two gels and using just one gel for the transfer of proteins to the nitrocellulose, the immune detection of a protein can be performed without affecting the protein residing in the other gel.

Another application of immunoelectrophoresis is known as capillary immunoelectrophoresis. In this application, a sample can be simultaneously drawn up into many capillary tubes. The very small diameter of the tubes means that little sample is required to fill a tube. Thus, a sample can be subdivided into very many sub volumes. Each volume can be tested against a different antibody preparation. Often, the reaction between antigen and antibody can be followed by the use of compounds that fluoresces when exposed to laser light of a specific wavelength. Capillary immunoelectrophoresis is proving to be useful in the study of Bovine Spongiform Encephalopathy in cattle, where sample sizes can be very small.

In the clinical laboratory setting, immunoelectophoresis is used to examine alterations in the content of serum, especially changes concerned with immunoglobulins . Change in the immunoglobulin profile can be the result of immunodeficiencies, chronic bacterial or viral infections, and infections of a fetus. The immunoglobulin most commonly assayed for are IgM, IgG, and IgA. Some of the fluids that can be examined using immunoelectrophoresis include urine, cerebrospinal fluid and serum. When concerned with immunoglobulins, the technique can also be called gamma globulin electrophoresis or immunoglobulin electrophoresis.

See also Antibody-antigen, biochemical and molecular reactions; Immunological analysis techniques

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immunoelectrophoresis

immunoelectrophoresis An analytical technique used to identify antigens that have been separated by electrophoresis. The gel medium used for the electrophoresis is irrigated with specific antibodies for the required antigens: when an antigen meets its corresponding antibody a precipitate forms, which can be seen and isolated.

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