Immunoelectrophoresis
Definition
A two-step technique: proteins are first separated by electrophoresis in agar gel, then antiserum diffuses from a parallel trough and forms precipitin arcs with each separated protein. It resolves complex protein mixtures that simple diffusion cannot separate.
- Technique type
- Two-step immunological and electrophoretic method
- Primary use in forensics
- Species identification of biological evidence (blood, body fluids)
- Key principle
- Antigens separated by charge, then identified by species-specific antibody reactions
Common questions
How does immunoelectrophoresis identify the species origin of blood or body fluids?+
The method works in two steps. First, antigens in the fluid are separated by electrophoresis in a gel matrix like agarose. Then a trough of antibodies (antiserum) is added to the gel, and the separated antigens diffuse toward it. Species-specific antigens react with the antibodies to form visible precipitation arcs, each arc revealing a different antigen and indicating the species of origin.
Why add antiserum after the electrophoresis step instead of mixing everything together?+
Separating the electrophoresis and immunological steps prevents antibodies from interfering with the charge-based separation of antigens. By running electrophoresis first, you resolve individual antigen bands by their electrical properties. Only after they are isolated in space do you introduce antibodies to identify which ones match the species you are testing for.
What do the precipitation arcs tell a forensic examiner?+
Each arc represents a reaction between a separated antigen and a species-specific antibody. The pattern and number of arcs reveal the species identity of the blood or body fluid sample. Different species have different antigen profiles, so comparing the arc pattern to known species references allows the examiner to confirm whether the evidence came from human, animal, or other biological sources.
Related terms
- Agarose
- Polysaccharide gel from seaweed. Large pore size, used for nucleic acids and large proteins. Standard for DNA submarine gels.
- Anti-Human Serum
- Antibodies raised in a rabbit (or goat) by repeated injection of human serum. The rabbit's immune system makes IgG against human serum...
- Antigen-Antibody Reaction
- The specific non-covalent binding between an antigen (a molecule capable of eliciting an immune response) and the antibody produced against it. In...
- Capillary Electrophoresis (CE)
- A separation technique that resolves DNA fragments by size through a polymer-filled glass capillary under an applied electric field. Single-base resolution is...
- COI Barcoding
- DNA-based species identification using a standard 658 bp segment of the mitochondrial cytochrome oxidase subunit I gene. Sequences are matched against reference...
- Cyt-B PCR
- Amplification and sequencing of the mitochondrial cytochrome-b gene. The reference marker for species identification in degraded and trace samples.
- Double Immunodiffusion (Ouchterlony)
- A gel diffusion method in which both antigen and antibody diffuse toward each other from separate wells in agar. Precipitin bands form...
- Electrophoretic Mobility (μ)
- Velocity of a charged particle per unit electric field, μ = v/E. Depends on charge, size, shape and the viscosity of the...
- Equivalence Zone
- The range of antigen-to-antibody ratios at which binding sites on both molecules are maximally cross-linked, producing the largest lattice and the heaviest...
- IEF (Isoelectric Focusing)
- Electrophoresis across an immobilised pH gradient. Each protein migrates until it reaches its pI and stops. Very high resolution; used in haemoglobin...
- Isoelectric Point (pI)
- pH at which a molecule (usually a protein) carries zero net charge and stops migrating. The basis of isoelectric focusing (IEF).
- Lattice Theory
- The structural explanation for precipitation proposed by Marrack in 1934. Multivalent antigens and bivalent antibodies can each bind more than one partner,...
Explained in these topics
- Determination of Species of Origin from Blood and Body FluidsAntigens are first separated by electrophoresis in a gel, then a trough of antiserum is added; precipitin arcs form for each species-specific antigen.
- Electrophoresis in Forensic Science: High Voltage, Low Voltage and ImmunoelectrophoresisTwo-step method: antigens are first separated by electrophoresis in agarose, then made to diffuse against an antibody trough. Precipitation arcs reveal species...
- Precipitation Reactions: Principles and ApplicationsA two-step technique: proteins are first separated by electrophoresis in agar gel, then antiserum diffuses from a parallel trough and forms precipitin arcs wit...