Types of Immunoelectrophoresis Techniques- Principle, Uses

Immunoelectrophoresis (IEP) is the process in which proteins of a biological sample are first separated by electrophoresis and then allowed to react with specific antibodies inside a gel. It is the technique combining electrophoresis with immunodiffusion. In this method the sample like serum is placed on an agarose gel and an electric field is applied, so the proteins are separated according to their charge and size.

After separation a trough is cut parallel to the protein migration path and it is filled with specific antisera. It is the process where the antigens and antibodies slowly diffuse towards each other in the gel and at the zone of equivalence, visible precipitation lines are formed. These are called precipitin arcs which indicate the presence of particular proteins in the sample.

It is the shape, position, and thickness of these arcs that is used for detecting abnormal protein patterns, and this is referred to as the interpretation of immunoelectrophoresis.

The presence of distorted or thickened arcs is often associated with monoclonal proteins, while complete absence of a given precipitation arc may indicate immunodeficiency. Although this technique is classical and qualitative in nature, it is now mostly replaced by Immunofixation Electrophoresis (IFE) because IFE is more sensitive.

It is still used in some specialized applications. Rocket immunoelectrophoresis is the method where an antibody containing gel is used and the antigen forms rocket-shaped precipitates whose height indicates antigen concentration.

Crossed immunoelectrophoresis is another form in which two-dimensional separation is done in order to obtain high resolution of complex protein mixtures.

Types of Immunoelectrophoresis

Immunoelectrophoresis is the process where antigen is separated by electrophoresis and then it is allowed to react with the antibody in gel. It is the method used for identification and estimation of different serum proteins. The reaction is based on antigen-antibody precipitation in agar medium. Some of the main types are given below–

1. Classical Immunoelectrophoresis

It is the basic qualitative type used in studying serum proteins. In this method the antigen mixture is first separated by electrophoresis on agarose gel. A trough is made parallel to the separation line and antiserum is filled in it. It is the process where both antigen and antibody diffuse towards each other and precipitin arcs is formed at the zone of equivalence.

These arcs is useful for detection of abnormalities in proteins like IgG, IgA and IgM. It is still used when sample contains very high paraprotein concentration where other methods is not giving correct results.

2. Immunofixation Electrophoresis (IFE)

It is the process widely used in clinical laboratories for monoclonal protein detection. The proteins are separated first by high-resolution electrophoresis. After that specific antisera is directly applied over the separated protein bands. The antibody fixes the target protein immediately, and then staining is done for visualization.

It is more sensitive and has better resolution compared to classical immunoelectrophoresis. It is useful in identification of heavy and light chains in diseases like Multiple Myeloma.

3. Rocket Immunoelectrophoresis

This is referred to as Electroimmunoassay. In this process the antibody is mixed throughout the gel. Wells are cut on the gel surface and antigen is placed in them. When electrophoresis is run, the antigen migrates through antibody-containing gel and a rocket-shaped precipitin peak is formed.

The height of the rocket is directly proportional to the concentration of antigen. It is used for quick quantitative estimation of serum proteins.

4. Crossed Immunoelectrophoresis

It is also known as two-dimensional quantitative immunoelectrophoresis. The process occurs in two steps. In the first step the proteins are separated by charge. In the second step the separated strip is placed at right angle on another gel layer which contains antibody, and electrophoresis is run again.

It produces bell-shaped precipitin curves. This helps in quantifying many antigens at the same time. More than 50 proteins can be resolved in a single experiment.

5. Counter Immunoelectrophoresis

In this technique antigen and antibody are placed in separate wells opposite to each other. It is the process where both are driven rapidly towards each other by electric field. A distinct precipitin line is formed within a short time.

It is used for quick detection of microbial antigens in samples like cerebrospinal fluid.

6. Affinity Immunoelectrophoresis

This type studies the change in mobility of protein when it interacts with ligands. The ligand like lectin is added in the gel. When protein interacts, its electrophoretic movement changes.

It is used in understanding binding properties, glycan content and structural characteristics of proteins.

A. Classical Immunoelectrophoresis

• It is the qualitative laboratory technique that separates and identifies proteins based on their charge and antigenic specificity.
• It is developed by Grabar and Williams in 1953 and it was the first method used to resolve proteins in serum or urine.
• It is the process carried out in two major steps, electrophoresis and immunodiffusion.
• In the first step the patient serum is placed in a well of agarose gel and proteins is separated into albumin, alpha, beta and gamma regions.
• In the next step a trough is cut parallel to the separated proteins and specific antiserum is added into this trough.
• Antigen and antibody diffuses towards each other and precipitin arcs is formed at the zone of equivalence.
• These arcs show the pattern of different protein fractions and any distortion or thickening indicate monoclonal protein.
• It is used for diagnosing plasma cell disorders and detecting abnormal or missing immunoglobulin fractions.
• It is the method that is less sensitive than IFE but useful when sample has very high monoclonal protein where antigen excess can occur.

Principle of Classical Immunoelectrophoresis

• It is the process where two techniques is combined, the zone electrophoresis and the double immunodiffusion.
• The antigen mixture is first separated in the gel according to their mobility under an electric field.
• It is carried out mostly in agarose gel where proteins move based on their electrical charge and size.
• After electrophoresis, a trough is cut parallel to the direction of migration and it is filled with specific antiserum.
• The antigen and antibody now diffuse toward each other in the gel medium.
• This process occurs when both reach an optimum concentration ratio called the zone of equivalence.
• At this zone, a visible precipitin line is formed which is referred to as the precipitin arc.
• These arcs indicate the presence of specific proteins and the pattern helps in identifying abnormalities in serum proteins.
• The reaction is dependent on diffusion rate, charge of proteins, and the specificity of antigen–antibody interaction.

Uses of Classical Immunoelectrophoresis

• It is used for detecting and characterizing monoclonal gammopathies such as multiple myeloma and Waldenström’s macroglobulinemia.
• It is the method that identifies M-proteins by observing thickened or distorted precipitin arcs.
• It is used for analysing urine samples to detect Bence Jones proteins (kappa or lambda light chains).
• It helps in diagnosing light-chain diseases and renal complications in plasma cell disorders.
• It is used to detect immunodeficiencies by observing reduced or absent arcs of IgG, IgA or IgM.
• It is helpful when antigen concentration is very high because diffusion helps to avoid prozone effect.
• It is useful for typing large monoclonal gammopathies where IFE may give false negative results.
• It helps in differentiating monoclonal and polyclonal gammopathies by the pattern of precipitin arcs.
• It is used in research for checking protein purity and studying antigen identity or non-identity.
• It can resolve serum proteins into many bands and provide a general profile of protein heterogeneity.

B. Immunofixation Electrophoresis (IFE)

• It is the technique used to detect and characterize monoclonal proteins in serum, urine or CSF samples.
• It is a two-stage procedure where proteins is first separated on agarose gel by electrophoresis.
• It is the process where specific antisera (IgG, IgA, IgM, Kappa, Lambda) is applied directly on the separated lanes.
• The antigen–antibody reaction is formed on the gel surface and an insoluble immune complex is fixed in the gel.
• These immune complexes remain after washing the gel, while unreacted proteins is removed.
• After staining, discrete narrow bands appear which indicate the presence of monoclonal proteins.
• It is the method used for identifying the heavy chain and light chain type of paraprotein such as IgG Kappa.
• In this method antigen excess can occur when the protein concentration is too high, so the sample is diluted and retested.

Principle of Immunofixation Electrophoresis (IFE)

• It is the process where zone electrophoresis is combined with specific immunoprecipitation for identifying monoclonal immunoglobulins.
• In the first stage the patient sample is separated in agarose gel according to electrical charge and molecular size.
• One lane gives the reference protein pattern while other lanes are used for applying specific antisera.
• It is the process where monospecific antibodies diffuse into the separated zones and react with their complementary antigen.
• When antigen and antibody meet in the zone of equivalence, an insoluble precipitate is formed which fixes the protein in the gel.
• The gel is washed so that only the fixed antigen–antibody complexes remain while other proteins is removed.
• Staining is carried out to visualize the fixed bands in each lane.
• The position of these bands helps in determining the heavy and light chain type of the monoclonal protein.
• If antigen is present in excess, the precipitate may not form properly and the sample needs dilution.

Uses of Immunofixation Electrophoresis (IFE)

• It is used for diagnosis of monoclonal gammopathies such as multiple myeloma and Waldenström’s macroglobulinemia.
• It helps in typing the exact heavy chain and light chain of the monoclonal protein.
• It is the process applied for detecting small abnormal bands which cannot be resolved by routine electrophoresis.
• It is used to detect Bence Jones proteins in urine samples, often after concentrating the sample.
• It is important for diagnosing light chain myelomas and studying renal complications in such disorders.
• It is used in analysing cerebrospinal fluid to detect oligoclonal bands in neurological diseases.
• It helps in differentiating monoclonal and polyclonal gammopathies.
• It is also used for monitoring disease progression and treatment response in myeloma patients.
• It can detect genetic polymorphisms of certain proteins like ceruloplasmin and complement components.
• It is used in identifying variants of alpha-1 antitrypsin in research and clinical investigations.

C. Rocket Immunoelectrophoresis

• It is the quantitative technique used to measure the concentration of specific proteins in biological samples.
• It is also called electroimmunoassay and it uses an electric current instead of passive diffusion.
• In this method the antibody is mixed uniformly with molten agarose gel and the gel is poured on a plate.
• Small wells is cut on one side of the gel and antigen samples is loaded into these wells.
• When current is applied the antigen migrates into the antibody-containing gel and forms immune complexes.
• It is the process where precipitation occurs at the zone of equivalence and a cone-shaped band called rocket is produced.
• The height of the rocket is directly proportional to the concentration of antigen present in the sample.
• Standard proteins is used to prepare a calibration curve and the unknown sample concentration is then estimated.
• It is mainly used for quantifying serum proteins such as albumin and transferrin and also used to estimate immunoglobulin protease activity.

Principle of Rocket Immunoelectrophoresis

• It is the process where electrophoresis is combined with immunodiffusion for quantifying specific proteins.
• The antibody is mixed uniformly within the agarose gel and the pH is kept so that the antibody is almost stationary in the gel.
• Antigen samples are loaded in wells cut on one side of the gel surface.
• When electric current is applied, the antigen is forced to migrate into the antibody-containing gel.
• It is the process where the antigen first moves in excess condition and forms soluble complexes that do not precipitate.
• As the antigen travels further, it meets more antibody until both reach the zone of equivalence.
• At this point stable precipitin complexes are formed and a conical precipitate appears which is referred to as the rocket shape.
• The height of the rocket is directly proportional to the concentration of antigen present in the sample.
• Standard antigen solutions are run along with the unknown samples to prepare a calibration curve for estimating the protein concentration.

Uses of Rocket Immunoelectrophoresis

• It is used for quantification of serum proteins such as albumin and transferrin.
• It allows measurement of protein concentration in different biological fluids like urine, CSF, and tissue extracts.
• It is the process used in estimating certain enzyme or protease activities including immunoglobulin protease.
• It helps in studying antigenic relationships among different microbial species.
• It is applied for screening of allergens and for detecting complement components like C3d.
• It is used when multiple unknown samples is required to be analysed together with standard controls.
• It is useful in research laboratories because it provides quick and reproducible protein estimation.

D. Crossed Immunoelectrophoresis

• It is the two-dimensional technique used to separate, identify and quantify specific proteins from complex antigen mixtures.
• It is also called quantitative immunoelectrophoresis because electrophoresis is used in both steps.
• In the first step the antigen sample is separated on agarose gel according to electrophoretic mobility.
• The separated strip is then placed on a fresh gel that contains uniformly mixed antibodies.
• A current is applied at right angle to the first run so the proteins migrate into the antibody-containing gel.
• It is the process where continuous antigen–antibody reaction forms bell-shaped peaks instead of simple arcs.
• The area of each peak is proportional to the concentration of that antigen present in the sample.
• It has high resolution and can separate many serum proteins in a single experiment.
• It is mainly used in research laboratories for studying complex biological fluids and antigenic variations.

Principle of Crossed Immunoelectrophoresis

• It is the process where two electrophoretic steps is carried out at right angles to analyse complex protein mixtures.
• The first electrophoresis separates the antigen mixture in agarose gel according to their electrophoretic mobility.
• The separated proteins form distinct fractions based on their charge and size.
• The gel strip is then placed on another agarose layer which already contains specific antibody mixed uniformly.
• Electric current is applied in a direction perpendicular to the first run so that the antigens migrate into the antibody-containing gel.
• It is the process where the antigens interact with antibody forming insoluble precipitates during the second electrophoresis.
• These precipitates form characteristic bell-shaped peaks instead of simple arcs.
• The position and height of each peak depends on the mobility of antigen and the antigen–antibody ratio.
• The area under each peak is directly proportional to the concentration of that antigen in the sample.

Uses of Crossed Immunoelectrophoresis

• It is used for simultaneous identification and quantification of many proteins in a single sample.
• The area under each precipitin peak helps in estimating the concentration of different antigens.
• The position and pattern of peaks is used to identify proteins based on their electrophoretic mobility.
• It is the process applied for analysing complex biological mixtures like serum, urine, cytosol extracts and microbial homogenates.
• It provides high resolution, and many distinct proteins can be separated in one experiment.
• It is used for studying antigenic heterogeneity, protein polymorphism and micro-heterogeneity.
• Researchers use it to observe protein fragmentation and association or dissociation behaviour.
• It helps in standardizing antigen–antibody systems, especially in microbial studies.
• It is used to detect and enumerate antigens in crude microbial extracts without purification.
• It is mainly preferred in biochemical and immunological research rather than routine clinical testing.

E. Counter Immunoelectrophoresis

• It is the rapid technique used to detect specific antigens or antibodies by applying an electric current.
• It is the modification of Ouchterlony double immunodiffusion to overcome slow passive diffusion.
• Two wells is cut on an agarose gel, one for antigen near cathode and one for antibody near anode.
• When current is applied the antigen migrates toward the anode while the antibody moves toward the cathode due to electroendosmosis.
• It is the process where both reactants meet in between the wells and form a visible precipitin line.
• The line appears only when antigen–antibody reaction occurs at the zone of equivalence.
• The method is fast and results is produced within 30 to 60 minutes.
• It is specific and more sensitive than ordinary double diffusion.
• It is mainly used for detecting microbial antigens in CSF, sputum or urine and also for screening HBsAg in serum.

Principle of Counter Immunoelectrophoresis

• It is the process where an electric field is applied to move antigen and antibody toward each other in a gel medium.
• The antigen is usually negatively charged and moves quickly toward the anode under electric current.
• The antibody migrates toward the cathode mainly due to electroendosmosis within the gel.
• The wells are arranged so that both reactants move directly in opposite directions and meet between the wells.
• It is the process where precipitation occurs when antigen and antibody reach the zone of equivalence.
• A visible precipitin line or arc is formed at this meeting point.
• The reaction is completed within a short time because migration is active instead of passive diffusion.
• It is more sensitive and specific compared to ordinary double immunodiffusion.

Uses of Counter Immunoelectrophoresis

• It is used for rapid detection of microbial antigens in clinical samples like serum, urine, sputum and CSF.
• It is the process applied in screening meningitis by identifying capsular antigens of organisms such as Meningococcus, Cryptococcus and Pneumococcus.
• It helps in detecting Hepatitis B surface antigen (HBsAg) in serum.
• It is used for identifying hydatid, amoebic antigens and alpha-fetoprotein in patient samples.
• It is also used in veterinary laboratories for diagnosing infectious diseases in animals.
• It helps in checking the quality and specificity of antisera in research and production laboratories.

F. Affinity Immunoelectrophoresis

• It is the specialized type of immunoelectrophoresis used to study proteins based on their biological interactions.
• It is the process where a ligand such as lectin, drug or macromolecule is mixed with gel to observe change in protein mobility.
• When a protein binds with a ligand its charge or molecular size changes and its electrophoretic movement is altered.
• The migration pattern in a normal gel is compared with the pattern in a ligand-containing gel.
• Any shift in position indicates the protein that is interacting with that ligand.
• It is the method used to predict protein behaviour during affinity chromatography.
• It is also used for characterizing glycoproteins by observing their binding with lectins.
• It is the process helpful for calculating binding constants of protein–ligand interaction.
• It is used in detecting protein variants that appear similar but show different mobility when ligand is present.

Principle of Affinity Immunoelectrophoresis

• It is the process where electrophoresis is combined with a specific biological ligand to study proteins based on their functional properties.
• The ligand is mixed in the gel so that proteins can interact with it during migration.
• It is the biospecific interaction where a protein binds to the ligand if it has affinity for it.
• The binding forms a complex which changes the mobility of that protein in the gel.
• The complex usually migrates slower compared to the free protein.
• After electrophoresis, the shifted proteins are visualized using antibody in a second dimension.
• The position of precipitin peaks is compared with a control gel to confirm binding.
• It helps in characterizing proteins such as detecting glycoproteins using lectins.
• It is also used to estimate binding strength between a protein and ligand.

Uses of Affinity Immunoelectrophoresis

• It is used for characterizing glycoproteins by studying their binding with lectins in the gel.
• It helps in determining the sugar content and the type of glycan present in a protein.
• It is the process applied to predict the behaviour of a protein before performing affinity chromatography.
• It allows estimation of binding constants between a protein and a specific ligand.
• It is used for identifying components which have specific molecular structures.
• It can also detect allergens by observing their interaction with antibody like IgE.

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