Immunology Lab Report

Radial Immunodiffussion (RID)

Christian Crespo
18 October 2013
Immunology
Lab Report

Purpose of the Experiment: The objective of this experiment is to quantitatively observe the foundational reaction in our Immune system; the Antigen-Antibody interactions. The Ouchterlony procedure is what will be used in this lab to detect nature of the antibody interaction. The orientations of the band will provide more information about the interaction of antibody and antigen.
Hypothesis:
For this experiment, antibodies will be placed in wells on an agar plate, surrounded by wells containing serum (proteins, antigens etc.). If the antibody interacts with the antigen of surrounding wells, then the presence
of a precipitate band will form and will further illustrate the specificity between the antibody and antigen.
Materials:
Pipette
Small vials containing antibodies or various antigens
3 Agar plates 1 sample agar plate
Straw for Making wells
Template for well making

Procedure:
To begin the Experiment, the agar plates needed to be prepared with wells. A template for the wells was provided on the lab handout and was used along with a straw to prepare the wells on the agar plate. Next, on the sample agar plates, practice runs were made by prepping sample solution in the wells to become familiar with using the transfer pipettes. After the practice run, 3 plates were prepared as follows:

The follow table demonstrates what was observed in wells AD:
Vials (AD)
Contents?
Amount?
A
Antiserum Containing Antibodies
2 Drops w/ Transfer pipette
B
Whole Serum (Protein, Antigen etc.)
2 Drops w/ Transfer pipette
C
Albumin (protein)
2 Drops w/ Transfer pipette
D
IgG
2 Drops w/ Transfer pipette

After the wells were prepared on the agar plate, they were allowed to incubate for 24-48 hours. It is important that the plates are oriented with the agar end of plate against the surface. The results will not be correct if not correctly placed.
Observations, Results, and Conclusion:
For this experiment, it is important to understand antigen-antibody interactions.
In this interaction, the antibodies will bind specifically to the epitopes of an antigen that it is specific for. The epitopes that are found on the antigen are also known as antigenic determinants. There are two types of antigens: 1) Particulate antigens & 2) Soluble antigens. In this experiment, the antigen reacting with the antibody was observed to be a soluble antigen. When the soluble antigen binds to antibody it will form antigen-antibody complex. As a result to this binding and forming of the complex, the complex will precipitate in the gel which will be visible to the naked eye (“Antigent-Antibody Interaction,” 4). This formation of a precipitate will allow one to characterize these interactions and make conclusions. However, it is important to consider some limitations in receiving a precipitate in the forming of this complex. The “visible precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of antibody or antigen excess ("Ouchterlony test", p. 1). Therefore, when you observe a faint precipitate or no precipitate, this is because you do not have enough antigens for antibody to bind to or the latter. When the antibodies are placed in well A, they will diffuse in the agar gel toward the antigen and the antigen vice versa. There are three main results that will be observed in this experiment: pattern of identity, pattern of partial identity,
and pattern of non-identity (“Antigent-Antibody Interaction,” 5). In the pattern of identity, the antibodies in the antiserum of well A react with both antigens which results in a smooth line of precipitate. The antibodies cannot distinguish between the two antigens; therefore this means that the two antigens are identical ("Ouchterlony Double Diffusion for Antigen-Antibody Patterns - Amrita University"). This is why I observed a curved band in Plate #1 (Vials B, Whole serum). The antibodies in the antiserum of well A recognized the antigenic determinants (epitopes) of the antigens of wells B and since there was a nice curved precipitin band, I can confirm I received my expected result of antibody reacting specifically with identical antigens. In the pattern of partial identity, the antibodies in the antiserum react with more of one antigen than the other ("Ouchterlony Double Diffusion for Antigen-Antibody Patterns - Amrita University"). This result will represent a long band with a “spur” because the antibody is able to react with the epitopes of one antigen (B, Whole Serum) but is unable to interact with all the epitopes of the other antigens of the other well (C, Albumin). Lastly, in the pattern of non-identity, none of the antibodies in the antiserum react with the antigenic determinants present in both antigens. This will result in a crossing over of the bands because the antibodies are not interacting with any identical antigens (C antigen is not indentical with D antigen) (“Antigent-Antibody Interaction,” 12). In conclusion, after performing ouchterlony procedure, I can conclude that I did receive my results as expected and the antibodies in the antiserum reacted with different antigens based on their specificity for their antigenic determinants. Below demonstrates precisely what I observed as my results for the ouchterlony procedure:
Plate # 1 (Identity)

Plate #2 (Partial Identity)

Plate #3 (Non-identity)

References
"Antibody-Antigen Interaction: The Ouchterlony Procedure". (n.d.). Retrieved 4, 2013, from http://www.austincc.edu/mlt/ser/OuchterlonyGelDiffuseionFall2009Edvotek.pdf
Ouchterlony Double Diffusion for Antigen-Antibody Patterns - Amrita University [Video file]. (n.d.). Retrieved from http://www.youtube.com/watch?v=hmK7yYr2T54
Ouchterlony test. (n.d.). Retrieved from http://immunopaedia.org.za/index.php?id=526