USER MANUAL

Double Radial Immunodiffusion

(Ab Pattern) Teaching Kit

Catalog No: IMI-KIT-1012

Consumable for 10 experiments
Experiment duration: 2 h

Double Radial Immunodiffusion Teaching Kit contains:

Standard reagents
and
Protocol

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Double Radial Immunodiffusion Teaching Manual

Double Radial Immunodiffusion Teaching Kit

I. PRINCIPLE

Interaction between Antibody and Antigen is one of the fundamental reactions in immunology, which
forms the basis of all immunological techniques. Antibodies are reasonably specific about the antigen with
which they bind or react to, so they can be used to distinguish Antigens (protein). The Ouchterlony
procedure is one of the several ways in which Antibodies are used to characterize Antigens. Double radial
immunodiffusion is a test determining not only the minimum number of antigen-antibody systems reacting
in a given mixture, but also indicates the relationship among various antigens. To determine the
complexity of a mixture of the antigens, it is important to utilize an antiserum containing antibodies to all
the antigens involved. In the process of double radial immunodiffusion both the antigen and the antibody
migrate towards each other through a semisolid medium such as an agarose gel to a point in the medium
where optimum concentration of each is reached. Thus both the reagents come in contact with each other
and react to form a precipitation arc/line in the gel. Depending upon the different types of antigens used
and the degree of similarity to each other precipitin arcs of different patterns are formed.

The technique involves cutting wells into an agarose solidified in a glass plate and filling the wells with
respective antibody and antigen and allowing it to incubate. When homologous antigen and antibody
diffuse toward each other from the individual wells, a precipitin line occurs somewhere between the two
wells. This precipitation occurs due to the multivalent nature of antigen (i, e., has several antigenic
determinants per molecule) to which antibodies can bind. Antibodies have at least two antigen binding
sites, thus large aggregates or lattices of antigen and antibody are formed. Precipitation/Cross-linking
does not occur in the presence of excess antigen or antibody but only at optimal antigen and antibody
concentrations. In the process of Double radial immunodiffusion antibodies developed against a single
antigen species are allowed to diffuse along with different antigen preparations in separate wells in the
agarose medium. Precipitin lines of different patterns are formed after incubation depicting the similarity
between the antigens. The sensitivity of the double radial immunodiffusion test depends to a large extent
on the distance between wells and the relative concentration of reactants. Sensitivity also depends on the
concentration, thickness and the viscosity of the gel employed. The gel diffusion test is suited for the
enumeration of antigens in a given mixture and for the determination of antigenic relationships among
various antigens and has been used extensively for these purposes in systematic biology. This assay is
commonly used to compare different preparations of antigen.

II. OVERVIEW

Double radial immunodiffusion test is a simple method used to check antisera for the presence of
antibodies against a particular antigen. This method has been widely used for detection and qualitative
diagnostic procedures. The method is called "double" referring to the fact that in this procedure, antigen
and antibody are allowed to migrate towards each other in a gel and a line of precipitation is formed
where the two reactants meet. This precipitation reaction is highly specific and precipitin lines of different
shapes are formed depending on the similarity between the antigens used. The method is even today
widespread and used by people working with diagnoses or protein detection or comparing antigens or
antisera. The method is not very sensitive but very useful when you have enough of antigen or antibody.

III. ADVANTAGES

• This Kit helps the student to understand the principle and basic function of the technique. It can
be used to determine the antigen quantitatively.
• The technique is relatively simple, rapid to perform and of low cost because it requires no special
equipment
• Contains Standard reagents and protocol for better results.

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Double Radial Immunodiffusion Teaching Manual

IV. KIT COMPONENTS AND STORAGE

All the reagents should be stored at 2-8°C when not in use.

Quantity
Sl No Components 5 Exp. 10Exp. 20 Exp.
1 Agarose 1.0 g 2.0 g 4.0 g

2 5X Assay buffer 30 ml 60 ml 120 ml

3 Antigen (A1, A2, B1, B2) 200 μl 400 μl 800 μl

4 Antiserum 500 μl 1 ml 2 ml

5 Glass slides 2 2 2

6 Manual 1 1 1

Materials Required:
Glassware: Conical flask, measuring cylinder, test tubes, glass slides.
Reagent: Distilled water.
Other Requirements: Micropipette, tips, moist chamber (box with wet cotton).

V. PREPARATION OF REAGENTS
NOTE: The included buffers and reagents are optimized for use with this kit. Substitution with other
reagents may not give optimal results.

Assay buffer: Prepare 1X of Assay buffer by diluting it with distilled water (add 20 ml distilled water to 5
ml of 5X Assay buffer). The diluted Assay buffer may be stored at 4°C. However, we recommend
preparing fresh 1X Assay buffer as per experimental requirement.

VI. PROTOCOL
Preparation of Agarose Gel
• Prepare 5 ml of 1.0% agarose (0.01 g/ml) in 1X Assay buffer by heating slowly till agarose
dissolves completely. Take care not to froth the solution.
• Pour 5 ml of 1.0% agarose solution in to the alcohol wiped clean grease free glass slide and allow
it to solidify for 15-20 minutes.
• If the molten agarose contains bubbles, gently swirl to remove the bubbles.
• After solidification, the gel will appear slightly opaque.
• Now punch the wells using gel puncher or with a 200μl bore tip corresponding to the template
given in figure 1.

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Double Radial Immunodiffusion Teaching Manual

ANTISERUM

A1 A2 B1 B2
ANTIGEN
Figure 1: Template for Double Radial Immunodiffusion
Loading of sample
• Place the template under the glass slide so that the pattern is aligned properly with the slide. The
distances between the wells is important and should be between 3-5 mm. Try to follow the
template as accurately as possible.
• With the help of a micropipette, fill the wells marked as antibody with 40 µl of antiserum solution
provided and the lower wells with 30 µl of the antigen samples corresponding to the given
template. Wells should appear full, but be careful not to overfill the wells and cause spillage on
the agarose surface. This may affect your results.

Incubation
• Cover the plate and place carefully (do not invert) inside the moist chamber (box containing wet
cotton) and incubate it at 37°C for overnight or at room temperature for 24 to 48 hours.

VII. READING THE RESULTS

• The precipitin lines will be visible in 24-48 hours. Carefully hold a plate up so that the overhead
room lights shine through it. You should be able to see opaque white arcs in each side of the
plate where the antibody and antigen precipitated. Note down the pattern of precipitin lines
observed between the different antigen and antibody wells and interpret the result according to
the template provided.

Note: If the precipitin line is not visible then wash the gel with 1X Assay buffer for 30 minutes.

VIII. INTERPRETATION OF RESULT

Figure 2: Different patterns of line obtained in Double Radial Immunodiffuision.

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Double Radial Immunodiffusion Teaching Manual

1. Result “A” indicates that both antigen A1 and A2 are same and the two immunogens are
immunologically identical.
2. Result “B” indicates that both antigen B1 and B2 are different and the two immunogens are
immunologically unrelated.

IX. REFERENCES
1. Ouchterlony Test: World of Forensic Science.
2. Ouchterlony, O., L.-A. Nilsson, 1978. Immunodiffusion and immunoelectrophoresis. In: Handbook of
experimental immunology, D. M. Weir (Ed), 3rd ed., Oxford: Blackwell Scientific Publication, 19.16.-19.28
3. Mehta, P.D. Immunological techniques. Indian J Ophthalmol 20:49-54 (1972)

VIII. TROUBLESHOOTING

Problems Probable Cause Suggestion

No precipitin line Less incubation Increase the incubation time.

Wash the gel with 1X assay
No precipitin line Improper washing buffer.

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