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  Section: Plant Lab Protocols
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Methodology for Amino Acids and Proteins

Immunodiffusion in Agarose Gels
The antigen-antibody complex forms an insoluble precipitate when the reaction is carried out both in solution and gels. Performing such reactions in agarose gel is advantageous because of higher sensitivity and resolution.
Immunodiffusion in gels is often classified as single or double diffusion. In single diffusion technique, the antigen (Ag) is usually allowed to diffuse into a gel containing the gel antiserum/antibody (Ab). On the other hand, in the double diffusion technique, both the antigen and antibody are allowed to diffuse into the gel and meet each other. A lot of qualitative and quantitative information on the antigen can be derived from the diffusion techniques. This is the simple technique to test for the antibody titre during immunization and to study the antigen-antibody reactions in gels.
The antigen and antibody react to produce the antigen-antibody complex. At equilibrium, the complex produced is immobile and forms a thin band of (protein) precipitin. The precipitin is visualized either directly or after protein staining for interpretation.

0.05M Borate Buffer (pH 8.0): (a) Dissolve 1.90g borax (Na2B4O7.10H2O) in 100mL water; (b) Dissolve 1.24g boric acid in 100mL water. Mix 30mL of 9a) and 70mL of (b) dilute to 400mL by adding 300mL water.
Solutions of Antigen and Antiserum.
Glass Petri Dishes or Rectangular Plates or Microscope Slides.
Gel Punch and Template – various sizes.
Humidity chamber.

normal;"> Physiological Saline Solution: Dissolve 0.9g sodium chloride in 100mL water.
Staining Solution: 0.1% Coomassie brilliant blue R250 in methanol:acetic acid:water (4:1:5).
Destaining Solution: Methanol:acetic acid:water (4:1:5).
A. Ouchterlony – Double Immunodiffusion
The double diffusion of the antigen and antibody was first described by Ouchterlony. This technique is most widely used in the qualitative analysis of antigens and antisera.
Prepare 100mL of 0.05M borate buffer (pH 8.0) containing 0.9% NaCl and 3% polyethylene glycol.
Dissolve 0.9g of agarose in the above buffer solution by heating to 90°C on a water bath with constant stirring or by autoclaving.
Pour the agarose solution to a depth of 2-3mm into the sterile Petri dishes or onto the rectangular plates or on a number of slides placed on a horizontal level surface. Allow the gel to set for 10-15min and store in a humid chamber.
Place a template of a suitable pattern depending upon the number of samples to be analyzed on top of the gel. The gel punch that is connected to a water vacuum pump is carefully inserted into the gel through each hole of the template to suck out the agarose plugs thus to form wells.
Fill the centre or inner well with the antiserum and the outer or radial wells with the antigen(s) solutions. Use serially diluted solutions of the same antigen or the same volume of different antigens. The concentrations of antigen solutions and the dilutions and the dilution of the antiserum to be used have to be established largely by trail and error by running pilot experiments.
Keep the gel plate in a humidity chamber at a constant temperature (between 16 and 20°C) for 1-3 days and examine for the precipitin line formation.
The precipitin line is visualized directly. The presence of polyethylene glycol in the gel enhances the visibility of the precipitin. Alternatively, the gel can be stained for proteins, especially if the precipitin line is vey faint.
Prior to staining remove excess moisture from the gel plate by placing some weight to a wad of filter paper placed on top of the gel for 15-30min. wash out the unreacted antigen and antiserum with several changes in the physiological saline for 2-3h. dry the gel after further pressing, in a stream of cold air from a hair-dryer.
Stain the dried gel for 10-15min with coomassie brilliant blue dye. Destain the plate for appropriate period to visualize the precipitin lines clearly. The gel may be photographed and dried subsequently for a permanent record.
B. Single Radial Immunodiffusion
This method is widely used for the quantitative analysis of antigens.
The method followed is essentially as described for the Ouchterlony double diffusion with the following modifications:
Twice the amount of agarose (1.8g/100mL) is dissolved.
The agarose solution is allowed to cool to 50-55°C, and is mixed with an equal volume of a suitable dilution of the monospecific antiserum also at 55°C; the gel is then poured on the gel plates. The plates are used within a day; may be stored for a week or so at 4°C.
The wells of 2mm diameter are cut in a row on the plate using a gel punch and a template. Central and radial well template is not used.
A few wells in the row are filled with standard solutions of antigen of known concentration. The other wells are filled with the solution containing the antigen at unknown concentration. The volume of antigens filled should be precisely known and equal in all wells.
A disc of precipitin is formed around the antigen well. The disc size enlarges with the progress of period of diffusion.
The area of each disc is measured in terms of its diameter at every 12h until no more increase is noticed. A magnifying lens on suitable oblique illumination such as colony counter may be used for measurement.
At the end of diffusion process the gel may be stained and preserved as described in the previous section.
A standard graph is constructed by plotting the diameters of the disc against the logarithm of the antigen solutions of known concentration. The concentration of the antigen in the test  samples can then be determined using the standard graph.
Double Immunodiffusion
It is suggested to precoat with 0.4% agarose gel prior to formation of the main gel to prevent the latter from being detached during the staining procedures when Petri dishes or glass plates are used.
Microscope slides are ideal for rapid and microanalysis.
Phosphate and veronal buffers are also used, instead.
Immunodiffusion can be run at high temperatures (up to 37°C) but factors such as drying of gels, denaturation of proteins pose problems.
When the antigen and antiserum wells are closer the sensitivity is greater, and the time required for diffusion is shorter.
It is important to run a few pilot experiments to standardize the antigen concentration and the dilution of antiserum for satisfactory results.
If the antigen used is not highly purified, the antiserum will contain antibodies to the protein impurities as well. Consequently, there will be many precipitin lines in the diffusion pattern.
The patterns of precipitin line are influenced by the similarities of antigens.
1. Ouchterlony, O (1968) Hand-book of Immunodiffusion and Immunoelectrophoresis Ann Arbor Science Publications Michigan.
2. Oudin, J (1980) Methods in Enzymology 70 (Eds van Vunakis, H and Langone, J J) p 166 Academic Press New York.


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