Types of Immunoelectrophoresis Techniques

Immunoelectrophoresis is a biochemical technique which is used for the separation and identification of proteins, mainly immunoglobulins in biological samples. It is a combined method of electrophoresis and immunodiffusion. In this technique, proteins are first separated in agarose gel by applying electric current.

After separation, specific antibody is added into the gel. The separated proteins act as antigens and they diffuse toward the antibody. When the antigen and antibody meet in proper proportion, they form visible precipitation arcs or lines.

These precipitation arcs help to identify the presence and nature of particular proteins in the sample. The pattern of arcs is used for studying normal and abnormal serum proteins. So, immunoelectrophoresis is useful in clinical diagnosis and in analysis of antigen-antibody reactions.

Principle of Immunoelectrophoresis Technique

Principle of Immunoelectrophoresis Technique is based on the separation of proteins by electrophoresis and then their reaction with specific antibody by diffusion in gel. The biological sample is placed in agarose gel and electric current is passed through it. At alkaline pH, generally around 8.6, most proteins become negatively charged and move toward the positive electrode or anode.

During this movement, different proteins migrate at different rate according to their size, charge and molecular mass. In the same condition, immunoglobulins or antibodies remain almost uncharged and may move slowly toward the opposite side due to electroendosmosis. Thus the antigenic proteins become separated in the gel.

After electrophoresis, specific antiserum is added in a trough cut in the gel. The separated proteins act as antigens and they diffuse toward the antibody. The antibody also diffuses toward the separated antigen.

When antigen and antibody meet in proper concentration, they combine with each other. This region is called the zone of equivalence. In this zone, large immune complexes are formed and they become insoluble.

These insoluble complexes are trapped in the pores of gel and form visible precipitin arcs or lines. The arcs indicate the presence and nature of specific protein in the sample. Thus immunoelectrophoresis is used for identification of antigenic proteins by their electrophoretic movement and antibody reaction.

Types of Immunoelectrophoresis Techniques

1. Classical Immunoelectrophoresis (IEP)
   It is also known as one dimensional immunoelectrophoresis. In this method, proteins are first separated in agarose gel by electrophoresis. Then antiserum is added in a parallel trough and antigen-antibody reaction takes place by diffusion. It forms visible precipitin arcs.
2. Countercurrent Immunoelectrophoresis (CIEP)
   It is a rapid type of immunoelectrophoresis. In this method, antigen and antibody are placed in opposite wells. Under electric field, they move toward each other and form a sharp precipitin band. It is used for quick detection of antigen or antibody.
3. Rocket Immunoelectrophoresis
   It is also called Laurell’s method. In this method, the gel contains specific antibody uniformly. The antigen moves through the gel during electrophoresis and forms cone shaped or rocket shaped precipitin peak. The height of rocket is proportional to the concentration of antigen.
4. Crossed Immunoelectrophoresis
   It is a two dimensional immunoelectrophoresis technique. In the first step, proteins are separated by ordinary electrophoresis. In the second step, they are electrophoresed at right angle into a gel containing antibody. It forms different bell shaped precipitin peaks.
5. Fused Rocket Immunoelectrophoresis
   It is a modified form of rocket immunoelectrophoresis. In this method, samples are placed in closely spaced wells and allowed to migrate into antibody containing gel. It is mainly used for comparing protein concentration in different fractions.
6. Affinity Immunoelectrophoresis
   This technique is based on the specific interaction of proteins with other molecules or ligands present in the gel. Due to this interaction, the electrophoretic movement of protein is changed. It is used to study binding nature and complex formation of proteins.
7. Immunofixation Electrophoresis (IFE)
   It is a sensitive clinical technique used for detection of specific immunoglobulins. Proteins are first separated in parallel lanes by electrophoresis. Then specific antisera is added directly over the lanes. The antigen-antibody reaction fixes the specific protein band.
8. Two Dimensional Immunoelectrophoresis
   It is an advanced type of immunoelectrophoresis. In this method, proteins are separated in two directions based on different properties. The first separation may depend on size or native molecular form and the second separation may depend on charge or isoelectric point. It gives more detailed protein pattern.

Classical Immunoelectrophoresis (IEP)

• Classical Immunoelectrophoresis (IEP) is a traditional immunoelectrophoresis technique used for the analysis of proteins in biological fluids. It is also known as one dimensional immunoelectrophoresis or ad modum Grabar method. It was developed by Pierre Grabar and Curtis A. Williams in 1953.
• This method is mainly a qualitative and semi-quantitative method. It is used to study complex protein mixture like serum proteins and immunoglobulins. It combines electrophoretic separation with immunodiffusion reaction in gel.
• In the first step, the antigen sample is placed in a well made on agarose gel. Then electric current is passed through the gel. During this process, the proteins are separated according to their charge, size and mass.
• After electrophoresis, the electric current is stopped. A trough is cut parallel to the path of protein migration. This trough is then filled with specific antiserum.
• The gel is then kept in a moist chamber for about 18 to 24 hours at room temperature. During this time, the separated proteins diffuse outward from their position. The antibodies also diffuse laterally from the trough.
• When the diffusing antigen and antibody meet in proper proportion, they combine and form insoluble immune complex. This region is called zone of equivalence. The immune complex is seen as curved or elliptical precipitin arcs in the gel.
• The number, shape and position of precipitin arcs are used for identification of different proteins. These arcs may be overlapping also because many proteins are present in serum. So interpretation needs careful observation.
• Classical IEP is useful for detection of abnormal immunoglobulins, complement deficiencies and paraproteinemia. It was also important in early protein chemistry because it showed many different proteins present in human serum.
• The method is slow and needs long diffusion time. Its detection limit is about 20 to 50 μg/mL. It is also difficult to interpret than immunofixation electrophoresis (IFE), so now it is replaced in many clinical laboratories.

Countercurrent Immunoelectrophoresis (CIEP)

• Countercurrent Immunoelectrophoresis (CIEP) is a rapid immunoelectrophoresis technique used for detection of antigen or antibody. It is also known as crossed-over immunoelectrophoresis or voltage facilitated double immunodiffusion. It is a modified form of Ouchterlony double diffusion method.
• This test is done on agarose gel slide. The gel used has high electro-endosmotic flow (EEO). In this method, the antigen and antibody are placed in two separate wells opposite to each other.
• The antigen is usually placed in the well near the cathode side. The specific antibody is placed in the well near the anode side. Then direct electric current is passed through the gel.
• When electric current is applied, negatively charged antigen moves toward the anode. At the same time, antibody moves toward the cathode due to electro-endosmotic flow. So both antigen and antibody migrate toward each other.
• When antigen and antibody meet between the two wells, they combine with each other. The reaction occurs in the zone of equivalence. A sharp visible precipitin band or arc is formed between the wells.
• The result is obtained very fast, usually within 15 to 60 minutes. In many cases, the reaction can be seen within 30 to 45 minutes. So it is more rapid than ordinary immunodiffusion method.
• CIEP is used in rapid diagnostic microbiology. It is used for detection of bacterial, viral and fungal antigens in body fluids like CSF, serum and sputum. It is also used for detection of Hepatitis B surface antigen (HBsAg).
• It is used for identification of capsular antigens of Cryptococcus, Meningococcus, Haemophilus influenzae and Pneumococcus. These antigens can be detected quickly by this method.
• CIEP is also used in forensic science. It helps to detect the species and origin of biological stains like blood, semen and saliva. So it is useful in medico-legal sample analysis.
• The sensitivity and visibility of this method can be increased by using fluorescent polymer. In this modification, the precipitin line is seen under UV light. This helps to make the band more clear.
• The main advantage of CIEP is that it is fast and more sensitive than simple double diffusion technique. But it is less simple and sometimes less sensitive than modern latex agglutination test. It also needs electric current and proper gel condition.

Rocket Immunoelectrophoresis (Laurell’s Method)

• Rocket Immunoelectrophoresis is a quantitative immunoelectrophoresis technique. It is also called Laurell’s method, electroimmunoassay or electroimmunodiffusion. It is used for estimation of specific antigen in a sample.
• In this method, specific monospecific antibody is mixed uniformly with agarose gel. The pH of gel is generally maintained around 8.6. At this pH, antibody remains almost fixed in the gel and does not migrate much.
• The antigen samples are placed in small circular wells made at the cathode side of the gel. Known standard antigen samples are also placed in separate wells. These standards are needed for comparison with unknown sample.
• When electric current is passed, the negatively charged antigen moves toward the anode. The antigen enters into the gel which already contains antibody. In the beginning, antigen is in excess and soluble antigen-antibody complexes are formed.
• As the antigen moves forward, it meets more and more fresh antibody in the gel. At one point, antigen and antibody reach proper proportion. This point is called zone of equivalence.
• In the zone of equivalence, insoluble immune complex is formed. It gives a cone shaped precipitin line. This cone shaped precipitate looks like a rocket, so the method is called rocket immunoelectrophoresis.
• The height of the rocket is measured from the upper margin of the well to the tip of the rocket. The rocket height is directly proportional to the concentration of antigen present in the sample. Higher antigen concentration gives taller rocket.
• The concentration of unknown antigen is calculated by comparing with standard antigen rockets. A standard curve is prepared by plotting rocket height of known antigen concentration. Then the unknown concentration is found from this curve.
• This method is very sensitive and can detect small amount of protein. It may measure protein concentration up to about 1 μg/mL. Very small amount of antigen may be needed in the well.
• Rocket immunoelectrophoresis is simple, rapid and reproducible method. Many samples can be tested at the same time on one gel plate. So it is useful for routine quantitative estimation.
• It is used for estimation of serum proteins, immunoglobulins, enzymes and other specific antigens. It is also used to study antigenic relationship between different organisms.
• This method is not suitable for complex protein mixture. It is mainly useful for single antigen estimation. The gel should be handled carefully, because uneven gel or overflow of sample may give distorted or blurred rocket.
• The antiserum should not be added in very hot agarose. If agarose is not cooled to about 55-60°C, the antibody may denature. Then proper rocket shaped precipitin peaks will not form.

Crossed Immunoelectrophoresis (Two-Dimensional CIE)

• Crossed Immunoelectrophoresis is a two dimensional immunoelectrophoresis technique. It is also known as two-dimensional quantitative immunoelectrophoresis. It is a modified and high resolution form of rocket immunoelectrophoresis.
• This method is used for analysis of complex protein mixture. It is useful when many antigens are present in same sample. It gives better separation and better identification than classical immunoelectrophoresis.
• In the first step, the protein sample is placed in a narrow lane of agarose gel. Then ordinary electrophoresis is done. During this process, different proteins are separated according to their charge and mobility.
• After first electrophoresis, the gel strip containing separated proteins is cut out. This strip is then placed along the side of another fresh agarose gel. This second gel contains a fixed amount of polyspecific antiserum.
• In the second step, electrophoresis is done again at right angle to the first direction. This means the proteins move perpendicular or at 90° angle into the antibody containing gel.
• The separated proteins are not allowed to diffuse passively only. They are pushed by electric current into the gel containing antibodies. So the reaction becomes faster and more clear than simple diffusion.
• When each migrating antigen meets its specific antibody, antigen-antibody reaction takes place. Insoluble immune complex is formed. These complexes appear as separate bell shaped precipitin peaks.
• Each bell shaped peak represents one antigenic protein. The number and position of the peaks show the different proteins present in the sample. The peaks may be small or large according to antigen amount.
• The area under each peak is proportional to the concentration of that particular antigen. So this method can be used for quantitative study also. Many proteins can be measured in one run.
• Crossed immunoelectrophoresis has high sensitivity and high resolving power. It is more useful than classical immunoelectrophoresis for complex biological fluids like human serum and tissue extracts.
• This method is also used in microbiology for studying antigenic relationship between organisms. It can be used for bacterial antigen analysis, such as in Brucella melitensis and other complex microbial extracts.

Fused Rocket Immunoelectrophoresis

• Fused Rocket Immunoelectrophoresis is a modified form of rocket immunoelectrophoresis. It was originally designed by Svendsen and Harboe. It is a one dimensional quantitative electroimmunoassay method.
• This technique is not mainly used for single separate patient sample. It is used for studying protein fractions collected during separation methods like ion-exchange chromatography and gel filtration.
• In this method, an agarose gel plate is used. A continuous row of closely placed parallel wells are made along the cathode side of the gel. The wells are arranged in order for loading different fractions.
• The samples collected from consecutive chromatography fractions are added into these wells one by one. The fractions are loaded in the same order as they are collected. After loading, the antigen is allowed to diffuse for short time.
• Then electric current is passed through the gel. The diffused antigens move forward from the wells into the nearby gel region. This region contains polyspecific antibody.
• When the moving antigens meet the antibodies, antigen-antibody reaction occurs. The precipitin patterns of different wells join with each other. So separate rockets are not seen clearly like normal rocket method.
• A continuous fused line is formed in the gel. This line looks like wave or undulating line. The line is produced due to different amount of protein present in different fractions.
• The high part of the fused rocket line indicates higher concentration of the protein. The low part indicates lower concentration. So the peaks and valleys show the elution pattern of proteins.
• Fused rocket immunoelectrophoresis is useful for mapping protein concentration across chromatography fractions. It helps to study purification efficiency and distribution of antigenic proteins in collected fractions.

Affinity Immunoelectrophoresis (AE)

• Affinity Immunoelectrophoresis (AE) is a type of immunoelectrophoresis technique used to study the interaction of proteins with specific ligand or other macromolecules. It is based on change in electrophoretic movement of protein after binding with ligand.
• In this method, a specific ligand is mixed or fixed inside the porous gel before electrophoresis. The ligand remains immobilized in the gel matrix. Then the protein sample is placed in the gel and electric current is passed.
• During electrophoresis, proteins move through the gel under electric field. The proteins which have high affinity for the immobilized ligand bind with it. This binding is reversible and it slows down the movement of that protein.
• Proteins with less affinity move faster through the gel. Proteins with more affinity migrate slowly or show retarded movement. So the change in mobility is used for studying protein-ligand binding.
• Sometimes free competing ligand is added in the buffer. This free ligand competes with the immobilized ligand for binding sites of protein. Due to this, the protein cannot bind properly with gel ligand and its normal electrophoretic mobility is restored.
• Affinity immunoelectrophoresis is used as quantitative method also. It can be used to calculate binding strength of protein-ligand complex. The apparent dissociation constant or Kᴅ can be calculated by mathematical methods like Takeo-Nakamura equation.
• This method is commonly used for study of lectin-glycoprotein interaction. It is also used for characterization of proteins on the basis of glycan content and ligand binding property.
• AE can separate different glycosylated protein isoforms. These isoforms differ in their binding affinity and so they show different mobility in the gel.
• The procedure usually takes about 2 to 8 hours. It is useful for studying specific binding nature of proteins, but it needs proper ligand and controlled gel condition.

Immunofixation Electrophoresis (IFE)

• Immunofixation Electrophoresis (IFE) is a sensitive clinical diagnostic technique. It combines protein electrophoresis with antigen-antibody reaction using monospecific antisera. It is mainly a qualitative method.
• This technique is commonly used for detection and identification of monoclonal immunoglobulins. It is considered as important method for diagnosis of monoclonal gammopathies like Multiple Myeloma, Waldenström’s macroglobulinemia and Bence-Jones gammopathy.
• The sample used for IFE is mostly human serum or concentrated urine. Serum is used for detecting abnormal immunoglobulins in blood. Urine is used especially for detection of light chains or Bence-Jones proteins.
• In the first step, the patient sample is diluted. This is done to prevent prozone effect, which occurs due to excess antigen. Proper dilution helps in better antigen-antibody reaction.
• In the second step, the sample is loaded in parallel lanes on alkaline agarose gel. The pH is usually around 9.1. Then electric current is applied and proteins are separated into different regions like albumin, α₁, α₂, β and γ globulin.
• After electrophoresis, specific antisera are added directly over separate lanes. These antisera may be against heavy chains like IgG, IgA, IgM and light chains like κ and λ. One reference lane is fixed by acid fixative to show all protein bands.
• The gel is then incubated for antigen-antibody reaction. The specific protein combines with its antibody and forms insoluble immune complex. This step fixes the particular protein band in its place.
• After incubation, the gel is washed to remove soluble and unprecipitated proteins. Then staining is done by dyes like Acid Violet or Coomassie Blue. The fixed bands become visible after staining.
• In normal condition, polyclonal immunoglobulins appear as broad and diffuse smear in the γ-globulin region. It is not a sharp band. This indicates normal mixed immunoglobulin population.
• In monoclonal gammopathy, one clone of plasma cell produces one type of immunoglobulin. This M-protein appears as narrow, dense and sharp band. The band in heavy chain lane matches horizontally with the same band in light chain lane.
• IFE is more sensitive than ordinary serum protein electrophoresis (SPE). Its detection limit is about 3 to 14 mg/dL. So it can detect small amount of monoclonal protein.
• This method is useful for diagnosis, typing and monitoring of monoclonal gammopathy. It may detect relapse of multiple myeloma earlier than SPE. The test usually takes about 1 to 2 hours.

Two-Dimensional (Non-Laurell) Immunoelectrophoresis

• Two-Dimensional (Non-Laurell) Immunoelectrophoresis is a special type of two dimensional immunoelectrophoresis technique. It is different from crossed immunoelectrophoresis or Laurell’s two dimensional method. It is used for more detailed study of complex protein mixture.
• In the first dimension, proteins are separated horizontally through agarose gel. The separation is mainly based on native molecular mass or apparent conformation of proteins. Smaller proteins move more distance through the gel.
• In the second dimension, the proteins are separated again by another property. This separation may be based on isoelectric point (pI) or relative protein abundance. So the same protein mixture is separated in two different directions.
• After both separations, many protein spots are formed on the gel. Each spot represents a particular protein with its own molecular mass, charge or abundance. So it gives a more detailed protein pattern.
• This technique is useful when many proteins are present together in one sample. The two dimensional separation helps to reduce overlapping of proteins. It makes individual protein identification more clear.
• Two-dimensional non-Laurell immunoelectrophoresis is used in advanced immunological research. It is used to study complex mechanisms like protein opsonization and activation of intracellular signal transduction pathways.
• It is also used in nanomedicine study. It helps to examine how therapeutic nanoparticles interact with C3 complement protein before use in the body.
• This interaction with C3 protein is important because it affects the life span of nanoparticle in blood. It also affects biological distribution and final destination of nanoparticle inside the body.

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