The document discusses immunodiffusion techniques, focusing on single (Mancini test) and double (Ouchterlony test) immunodiffusion, along with immunoadsorption therapy. Single immunodiffusion quantifies antigens using a precipitation ring formed in an agarose gel, while double immunodiffusion detects specific antigen-antibody interactions through visible precipitin lines. Immunoadsorption is utilized for selectively removing harmful antibodies from blood in autoimmune diseases and has seen advancements in technology and methodologies over the years.

1. TOPICS
1) Single immunodiffusion(Mancini Test)
2) Double Immunodiffusion technique(Ouchterlony Test)
3) Immunoadsorption

2. Single immunodiffusion( Mancini Test)
• Single Immunodiffusion, also known as the Mancini Test or Radial Immunodiffusion (RID), is an immunological
technique used to quantify the concentration of antigens in a sample.
Principle
• The principle is based on the diffusion of antigens and antibodies in a gel medium, leading to the formation of a
precipitation ring. In this test, an antibody is uniformly distributed within an agarose gel, and the antigen diffuses
radially from a well cut into the gel. The antigen-antibody interaction forms a precipitate, visible as a ring around
the well. The diameter of this ring is proportional to the concentration of the antigen.

3. History:
• In the early 1960s, Dr. Giovanni Mancini, along with his colleagues at the University of Genoa in Italy, developed single
radial immunodiffusion (RID), often referred to as the Mancini method. Their goal was to create a simple, yet effective,
method to measure the concentration of antigen. This method provided a more quantitative approach compared to earlier
qualitative immunodiffusion assays developed by Ouchterlony and others.
• In 1970s-1980s,the method quickly gained popularity due to its simplicity and effectiveness. It was widely adopted in
clinical laboratories for quantifying serum proteins and other antigens. Improvements were made to the gel formulations
and the preparation of antigen and antibody solutions, enhancing the accuracy and reproducibility of the technique.
• Emergence of competing technologies in 1990s such enzyme linked immunosorbent assay provided a more sensitive and
versatile alternative to Mancini test.
• While advanced immunoassays like ELISA, Western blotting, and multiplex assays have largely supplanted Mancini test
in many areas, the method is still employed in certain diagnostic and research settings due to its simplicity and reliability

4. Method
1. Agarose is dissolved in a buffer solution and poured into a Petri dish or
slide to form a uniform gel layer and allowed to solidify.
2. The specific antibody is mixed with the agarose gel before it solidifies or
is layered on the surface of the solidified gel.
3. Wells are punched into the agarose gel, and antigen samples of known
and unknown concentrations are placed into these wells.
4. The plate is incubated to allow antigen diffusion radially from the wells.
The antigen interacts with the antibody in the gel to form a precipitate.

5. CONT…..
6.After an appropriate incubation period, the diameter of the precipitin ring is measured.
7.A standard curve is prepared using the ring diameters of the standards versus their
concentrations. This curve is then used to determine the concentration of the control and
unknown samples.
8. The presence of a precipitin ring around the antigen wells indicate specific antigen-
antibody interaction.
9. Absence of precipitin ring suggest absence of reaction.
10.The greater the amount of antigen in the well, the further the ring will form from the
well.

6. Results interpretation

7. Single immunodiffusion standard
curve

8. Applications
1) For allergen quantification.
2) To determine quantification of serum proteins
3) Vaccine potency testing -to determine the concentration of
antigens in vaccines.
4) Evaluation of immune response
5) For clinical diagnosis of immune disorders

9. Latest Developments/Future
1) Automation and Digital Imaging: Advances in imaging technology and software have allowed for automated
measurement and analysis of precipitin rings, increasing accuracy and efficiency.
2) High-Throughput Systems: Development of multi-well formats and microfluidic systems for parallel
processing of multiple samples.
3) Enhanced Sensitivity: Improvements in gel formulations and detection methods to increase the sensitivity of
the assay.
4) Combination with Other Techniques: Integration with other immunoassays (e.g., ELISA, Western Blot) for
comprehensive protein analysis.
5) Biomarker Discovery: Using single immunodiffusion in the discovery and validation of new biomarkers for
various diseases.

10. 2. Double Immunodiffusion (Ouchterlony Test)
Ouchterlony double immunodiffusion (also known as passive double immunodiffusion) is an immunological technique used in the
detection, identification and quantification of antibodies and antigens, such as immunoglobulins and extractable nuclear antigens.
Principle
The principles is based on the diffusion of antigens and antibodies through a semi-solid agar gel. When these molecules diffuse
towards each other and meet, they form visible precipitin lines if they are specific to each other. This occurs because antigen-
antibody complexes precipitate out of solution. The formation and pattern of these precipitin lines indicate the presence and
identity of specific antigen-antibody reactions, allowing for the detection of and analysis of antigen-antibody interaction.

11. HISTORY
 The technique is named after Örjan Ouchterlony, the Swedish physician who developed the test in 1940’s and 1950’s to
evaluate the production of diphtheria toxins from isolated bacteria. Ouchterlony's work focused on improving methods for
studying antigen-antibody interactions, which were crucial for advancing immunology and diagnosing infectious diseases.
His test provided a simple yet effective way to visualize these interactions.
 1960s-1970s: The method gained widespread acceptance in laboratories for the analysis of complex antigen mixtures and
the characterization of antibodies. The Ouchterlony test enabled the identification and characterization of multiple
antigens and antibodies in a single assay, based on the patterns and number of precipitin lines formed.
 The techniques, remains in use today and its utilized in educational settings to teach basic immunology principles and in
some clinical and research laboratories for specific antigen-antibody interaction studies.

12. METHOD
1) Prepare an agar or agarose gel plate
2) Punch a series of wells into the gel in a specific pattern(commonly a central well surrounded by
peripheral wells)
3) A gel plate (usually agar or agarose) is cut to create a series of wells (holes).
4) Add antigen solution in one set of wells and the antibody solution to another set of wells.
5) Incubate the plates at an appropriate temperature to enable diffusion of antigens and antibodies
towards each other
6) If antibodies recognize the antigens, they interact to form immune complexes, resulting in a thin
white precipitin line in the gel.
7) The patterns of the precipitin lines is analyzed to determine the relationship between antigens and
antibodies

13. Results interpretation
i. Identity line –a continuous arc of precipitation forms between the
antigen and antibody wells. This indicates that the antibodies and
antigens are identical or share very similar epitopes
ii. Partial identity line –a spur of precipitation forms between the
antigen and antibody wells. This suggests that the antigen and
antibody share some but not all epitopes.
iii. Non identity line- no precipitation lines forms between the antigen
and antibody. This indicates that the antibodies and antigens are
different and do not share epitopes

14. Results interpretation

15. Applications
1. Diagnosis of Infectious Diseases: Detecting specific pathogens by identifying the
presence of their antigens or antibodies.
2. Allergy Testing: Determining the presence of specific antibodies against allergens.
3. Vaccine Development: Evaluating the immune response to vaccine candidates.
4. Protein Purity Testing: Verifying the presence and purity of specific proteins in a
sample.
5. Research: Studying antigen-antibody interactions and their kinetics.

16. Latest Developments / Future
1. Improved Sensitivity: Advances in gel composition and preparation have increased the sensitivity of double
immunodiffusion, allowing for the detection of lower concentrations of antigens and antibodies.
2. Automation and Digitalization: The integration of automated systems and digital imaging has streamlined the
process, enabling high-throughput analysis and more accurate interpretation of results.
3. Integration with Molecular Techniques: Combining double immunodiffusion with molecular methods such as
PCR and sequencing could provide more comprehensive insights into antigen-antibody interactions.
4. Educational and Research Tools: Continued use and development of double immunodiffusion in educational
settings and basic research will help train new scientists and contribute to a deeper understanding of
immunological principles.

17. 3.IMMUNOADSORPTION
Immunoadsorption, also known as immunoapheresis, is a specialized form of apheresis therapy designed
to remove harmful antibodies or immune complexes from the blood. It plays a crucial role in managing
autoimmune diseases and other conditions involving abnormal immune responses.
Principle
The principle of immunoadsorption is based on the selective removal of specific antibodies or immune
complexes from the blood using an adsorbent material with a high affinity for the target molecules.
Blood is circulated through a column containing this adsorbent, which binds the antibodies or immune
complexes while allowing other blood components to pass through. Once the harmful substances are
adsorbed onto the column, the treated blood is returned to the patient's circulation.

18. Method
1. Blood is drawn from the patient and directed into the
apheresis machine.
2. The apheresis machine separates plasma from the blood
cells. The plasma contains the targeted antibodies or
immune components
3. The plasma passes through the immunoadsorption column
where the targeted antibodies are selectively bound to the
adsorbent.
4. The purified plasma, now free of the targeted antibodies, is
recombined with the blood cells
5. The reconstituted blood is returned to the patient.

19. Immunoadsorption

20. HISTORY
 (1960s-1970s):The concept of immunoadsorption emerged from the need to selectively remove specific antibodies from
the blood. While effective, it was non-selective, removing both harmful and beneficial plasma components.
 1970s: Researchers began exploring more targeted approaches to plasma exchange. Protein A, derived from the cell wall
of Staphylococcus aureus, was identified for its ability to bind IgG antibodies. This discovery led to the first attempts at
using protein A in immunoadsorption columns to selectively remove IgG antibodies from the blood.
 In (1980s-1990s):The technique gained traction in clinical settings for treating autoimmune diseases and preparing
patients for organ transplantation. The introduction of synthetic adsorbents and other protein-based adsorptive materials,
such as protein G and protein L, expanded the range of antibodies and immune complexes that could be selectively
removed.
 2000s-Present: Technological advancements have continued to enhance the effectiveness and safety of
immunoadsorption. Innovations include the development of columns with higher specificity and affinity for various
pathogenic antibodies, automated systems for easier operation, and improved patient monitoring techniques.

21. APPLICATIONS
1) Clinical diagnosis and treatment of autoimmune diseases,such as systemic lupus erythematosus,rheumatoid arthritis.
2) Research: It is also used in research settings to purify antibodies or antigens for further study
3) .Treatment of hematological disorders such as immunethrombocytopenia,by removing antibodies that cause abnormal platelets
destruction
4) Management of neurological conditions such as chronic inflammatory demyelinating polyneuropathy.
5) Prevetion and treatment of antibody-mediated rejection in organ transplation by removing donor specific antibodies that target
the transplated organ.

22. Latest Developments / Future
1. Advancements in Adsorbent Materials: Researchers have been developing more efficient and selective
adsorbent materials to improve the efficacy of immunoadsorption.
2. Combination Therapies: There is an increasing focus on combining immunoadsorption with other therapies,
such as immunosuppressive drugs or biologics, to enhance treatment outcomes.s
3. Personalized Medicine: Advances in personalized medicine are being integrated into immunoadsorption
therapies. By tailoring the treatment to the specific antibody profile of the patient, clinicians can achieve better
results and minimize adverse effects.
4. Enhanced Selectivity: Future research is likely to focus on enhancing the selectivity of immunoadsorption
techniques.
5. Integration with Diagnostic Tools: The integration of advanced diagnostic tools with immunoadsorption
procedures could allow for real-time monitoring and adjustment of the treatment.