Microbiology and Immunology

Immunoelectrophoresis WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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IImmunoelectrophoresisMMUNOELECTROPHORESIS

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 systemto

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, immunoelec-

trophoresis 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 microscopeslide-

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 pro-

ductively 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 pos-

itively 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 antibodyor known mix-

ture of antibodies is added. The antibody molecules diffuse

outward from the trough solution into the gel. Where an anti-

body encounters a corresponding antigen, a reaction causes

the formation of a visual precipitate. Typically, the precipita-

tion occurs in arc around the antigen-containing sample. In the

example, the pattern of precipitation can reveal antigenic dif-

ferences between the normal serum and the serum from a

infected person.

This type of immunoelectrophoresis provides a qualita-

tive (“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 par-

ticular 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 anti-

body 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 trans-

fer of proteins to the nitrocellulose, the immune detection of a

protein can be performed without affecting the protein resid-

ing in the other gel.

Another application of immunoelectrophoresis is

known as capillary immunoelectrophoresis. In this applica-

tion, a sample can be simultaneously drawn up into many cap-

illary 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 reac-

tion 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, espe-

cially changes concerned with immunoglobulins. Change in

the immunoglobulin profile can be the result of immunodefi-

ciencies, 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 alsoAntibody-antigen, biochemical and molecular reac-

tions; Immunological analysis techniques

IImmunofluorescenceMMUNOFLUORESCENCE

Immunofluorescence refers to the combination of an antibody

and a compound that will fluoresce when illuminated by light

of a specific wavelength. The duo is also referred to as a fluo-

rescently labeled antibody. Such an antibody can be used to

visually determine the location of a target antigenin biologi-

cal samples, typically by microscopic observation.

The fluorescent compound that is attached to an anti-

body is able to absorb light of a certain wavelength, the par-

ticular wavelength being dependent on the molecular

construction of the compound. The absorption of the light con-

fers additional energy to the compound. The energy must be

relieved. This is accomplished by the emission of light, at a

higher wavelength (and so a different color) than the absorbed

radiation. It is this release of radiant energy that is the under-

pinning for immunofluorescence.

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