Immunodiffusion techniques use the reaction between antigens and antibodies to form visible precipitates. They can be used to determine relative concentrations, compare antigens, and assess purity. Key techniques include radial immunodiffusion, Ouchterlony double diffusion, and immunoelectrophoresis. Factors like size, temperature, gel viscosity affect the rate of diffusion and precipitation formation between antigens and antibodies.
Immunodiffusion techniques use the reaction between antigens and antibodies to form visible precipitates. They can be used to determine relative concentrations, compare antigens, and assess purity. Key techniques include radial immunodiffusion, Ouchterlony double diffusion, and immunoelectrophoresis. Factors like size, temperature, gel viscosity affect the rate of diffusion and precipitation formation between antigens and antibodies.
Immunodiffusion techniques use the reaction between antigens and antibodies to form visible precipitates. They can be used to determine relative concentrations, compare antigens, and assess purity. Key techniques include radial immunodiffusion, Ouchterlony double diffusion, and immunoelectrophoresis. Factors like size, temperature, gel viscosity affect the rate of diffusion and precipitation formation between antigens and antibodies.
Immunodiffusion techniques use the reaction between antigens and antibodies to form visible precipitates. They can be used to determine relative concentrations, compare antigens, and assess purity. Key techniques include radial immunodiffusion, Ouchterlony double diffusion, and immunoelectrophoresis. Factors like size, temperature, gel viscosity affect the rate of diffusion and precipitation formation between antigens and antibodies.
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Immunodiffusion techniques
• APPLICATIONS • To determine relative concentrations of Antibodies / Antigens. • To compare Antigens or • To determine the relative purity of an Antigen preparation. • For disease diagnosis. • Serological surveys. The combination of antibody (Ab) with antigen(Ag) is the fundamental reaction of immunology.
Primary binding tests
Secondary binding tests Tertiary binding tests Principle • Soluble Ab & soluble Ag interacting in aqueous solution form lattice that develops into insoluble visible precipitate. • Soluble Ag: Toxins, toxoids, proteins, carbohydrates, glycoproteins, Liproproteins • Both qualitatively & quantitatively in solutions & gels.
• Formation of Ag-Ab lattice depends on the valency of both Ag & Ab:
Ab must be bivalent Fab fragments - Ag must be bivalent / polyvalent. Precipitation
•Insoluble complexes •Visible to the eyes Precipitation Curve
• Zone of Equivalence optimum precipitation • Prozone excess antibody is present • Postzone excess antigen is present
Prozone and Postzone
phenomena are negative reactions. Immunodiffusion
Precipitation Reactions Immunodiffusion o Radial Immunodiffusion (Mancini method). o Ouchterlony Double Diffusion
Electrophoresis o Rocket Immunoelectrophoresis o Immunoelectrophoresis
• Precipitation is best demonstrated
• Random movement of Ag or Ab to form Ag-Ab complexes in medium, such as gel.
MEDIUM Agar - high molecular weight complex polysaccharide - from seaweed Agarose- purified agar - Used to help stabilize the diffusion process and allow visualization of precipitin bands.
0.3 – 1.5 % Agar concentration: diffusion of most reactants
Agarose- more preferred than agar
Agar has strong negative charge; Agarose has almost none (no charge) - interactions between gel and reactants are minimized. Passive Immunodiffusion • Passive diffusion method in which a concentration gradient is established for an antigen and/or antibody - diffusion of reactants to form Ag - Ab reactions without electric current to speed up reaction.
Rate of Diffusion 1. Size of particles 2. Temperature 3. Gel viscosity 4. Amount of hydration 5. Interactions between matrix and reactants. Immunodiffusion Techniques • Radial Immunodiffusion - A single diffusion technique where Ab is put into gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel.
• Ouchterlony Double Diffusion
- Both Ab and Ag diffuse from wells into a gel medium. Radial Immunodiffusion
• Ag is added to an antibody rich media. • The two continue to react until the zone of equivalence is reached. • The area of ring is a measure of the Ag concentration. • Method – Ab in gel – Ag in a well
• Interpretation – Diameter of ring is proportional to the concentration
diffuse independently through a semi solid medium (agar) Wells are cut in the gel
Reactants are added in the well
Incubate (12-48 hrs) in a moist chamber
Precipitin lines will form
(where the moving front of the antigen meets antibody) The density of the line reflects the amount of immune complexes formed Ouchterlony
• Both antigen and
antibody can diffuse independently. • Antibody that is multispecific is placed in the central well
• Different antigens are placed
in surrounding wells
0.5% Amido-black 0.5% Coomassie Brilliant Blue
**Position of the precipitin bands between wells allows for the
antigens to be compared with one another. Diffusion patterns
Fusion of lines at their
junction to form an arc - Serologic identity /
presence of common epitope
Crossed lines
- Demonstrates 2 separate reactions - Compared antigens shared no common epitopes Fusion of 2 lines with spur
- Partial identity IMMUNOELECTROPHORESIS
• Double-diffusion technique that utilizes electric current to enhance
results. • SPEED, Specificity. • Introduced by Grabar and Williams in 1953.
• Combine immunodiffusion with electrophoresis
• Can be used for semiquantitaion of wide range of antigens • Qualitative • Antigen source: serum. Electrophoresis Techniques
• Electrophoresis separates molecules according to differences in
their electrical charge. – Rocket Immunoelectrophoresis – Countercurrent Immunoelectrophoresis Principle 2 step process
Trough is cut in the gel parallel Antigen from electrophoresed to the line of serum separation
•double diffusion Antiserum is
occurs at right Incubate: 18- 24 hours placed in the angles to the trough electrophoresis separation •Precipitin lines develop where Ag- Lines or arcs shape, intensity and Ab combination location: compared with normal serum takes place. control Immunoeletrophoresis • Method – Ags are separated by electrophoresis – Ab is placed in trough cut in the agar
• Interpretation – Precipitin arc represent individual antigens Uses • Immunodeficiencies can be detected by this procedure, if no precipitin band is formed for a particular Ig. • Overproduction of serum proteins. • Deficiencies in complement can also be detected. • Identification of monoclonal protein – Free kappa and lambda light chains • May be used to identify urine proteins. • Testing normal & abnormal proteins in serum/urine. • Purity of Ag. Countercurrent electrophoresis Method – Ag and Ab migrate toward each other by electrophoresis – Used only when Ag and Ab have opposite charges
- + Ag Ab
• Qualitative- Rapid. • Detection of Antinuclear- Ribonuclear protein. Rocket Immunoelectrophoresis ( LAURELL TECHNIQUE ) • One dimension electroimmunodiffusion - Adaptation of RID
• Electrophoresis is used to facilitate migration of antigen in agar
- Ag diffuses out of the well: precipitation begins - Change in Ag concentration: dissolution and reformation of precipitate.
End result: precipitin line = conical-shaped, resembles a racket
• . The height of the racket is measured - Height of the racket is directly proportional to the amount of antigen present • Racket electrophoresis is more rapid than RID. Rocket electrophoresis Uses of Racket Immunoelectrophoresis
• Quantitate Igs - (using a buffer = pH 8.6)
• Assay of proteins - When concentration is too low to be detected by nephelometry and too high for RID - Ex: alpha-fetoprotein, Igs in urine and spinal fluid, Complement components in body fluid. Applications
• Screening tool for differentiation of more than 30 serum
proteins. - Major classes of immunoglobulins • Used for the detection of: - Myelomas - Malignant lypmhomas - Other lymphoproliferative disorder.
