Guru Nanak Institute of Pharmaceutical Sciences and Technology

Sub: Lab on Immunology Code: MSBT/MSMC/MSGN-292

Sl. No. - EXPERIMENT No.- Date:

Aim of the experiment: To study the immunodiffusion technique by Single Radial

Immunodiffusion.

Objective: 1) To detect antigen-antibody complexes.

2) Describe the circumstances under which antigen-antibody complexes precipitate
out.
3) Determine relative concentration of antigens.

PRETEST QUESTION:

1. What is radial immunodiffusion?

A. A method of measuring the size of antigens
B. A method of measuring the concentration of antibodies
C. A method of measuring the activity of T cells
D. A method of measuring the concentration of antigens

2. Which of the following is required for performing radial immunodiffusion?

A. Agarose gel
B. Antibody solution
C. Antigen solution
D. All of the above

Principle:
Single Radial immunodiffusion is used extensively for the quantitative estimation of antigen.
Here the antigen-antibody reaction is made more sensitive by the addition of antiserum into the
agarose gel and loading the antigen sample in the well. As the antigen diffuses into the agarose
radially in all directions, it’s concentration continuously falls until the equivalence point is
reached at which the antigen concentration is in equal proportion to that of the antibody present
in the agarose gel. At this point ring of precipitation is formed around the well. The diameter
of the precipitin ring is proportional to the concentration of antigen. With increasing
concentration of antigen, precipitin rings with larger diameter are formed. Thus, by having
various concentrations of a standard antigen, standard curve can be obtained from which one
can determine the amount of an antigen in an unknown sample. Thus, this is a quantitative test.
If more than one ring appears in the test, more than one antigen/antibody reaction may have
occurred. This could be due to a mixture of antigens or antibodies.

Materials required: -
Materials provided in the kit: -
 Guru Nanak Institute of Pharmaceutical Sciences and Technology
Sub: Lab on Immunology Code: MSBT/MSMC/MSGN-292

1. Agarose
2. 10X Assay buffer
3. Antiserum
4. Standard Antigen A
5. Standard Antigen B
6. Standard Antigen C
7. Standard Antigen D
8. Test Antigen 1
9. Test Antigen 2
10. Glass plate
11. Gel puncher
12. Template

Materials required but not provided in the kit: -

1. Conical flask
2. Measuring cylinder
3. Beaker
4. Distilled water
5. Alcohol
6. Incubator
7. Bunsen burner
8. Vortex mixer
9. Spatula
10. Micropipettes
11. Tips
12. Moist chamber
Procedure
1. Prepare 10ml of 1% agarose gel. Take 6 ml of this gel solution in a clean test tube.
2. Allow the solution to cool down to 55–60-degree Celsius and add 80micro litre of
antiserum to 6 ml agarose solution. Mix well for uniform distribution of the antibody.
3. Pour agarose powder containing the antiserum on a grease free glass plate placed on a
horizontal surface. Allow the gel to set for 30 minutes.
4. Place the glass plate on the template provided.
5. Punch wells with the help of gel puncher corresponding to the markings on the template.
Use gentle suction to avoid forming rugged wells.
6. Add 10 micro litre of the given standard antigen and test antigen samples to the wells.
A. Standard Antigen A (3.75 mg/ml)
B. Standard Antigen B (7.5 mg/ml)
 Guru Nanak Institute of Pharmaceutical Sciences and Technology
Sub: Lab on Immunology Code: MSBT/MSMC/MSGN-292

C. Standard Antigen C (15 mg/ml)

D. Standard Antigen D (30 mg/ml)
E. Test Antigen 1
F. Test Antigen 2
Result & discussion:

PRECAUTIONS:

QUESTION:
3. What is the principle of radial immunodiffusion?
A. Antigens and antibodies form complexes that precipitate out of solution
 Guru Nanak Institute of Pharmaceutical Sciences and Technology
Sub: Lab on Immunology Code: MSBT/MSMC/MSGN-292

B. Antigens and antibodies are separated by an electric field

C. Antigens and antibodies are separated by size exclusion chromatography
D. Antigens and antibodies are separated by isoelectric focusing
4. What is the advantage of radial immunodiffusion over other methods of quantifying
antigens?
A. It is faster than other methods
B. It is less expensive than other methods
C. It is more specific than other methods
D. It is more sensitive than other methods
5. Which of the following diseases can be diagnosed using radial immunodiffusion?
A. HIV/AIDS
B. Tuberculosis
C. Influenza
D. All of the above
6. Which of the following is an example of an antigen that can be quantified using radial
immunodiffusion?
A. Albumin
B. Glucose
C. Haemoglobin
D. Insulin
7. Which of the following is an example of an antibody that can be used in radial
immunodiffusion?
A. IgG
B. IgA
C. IgM
D. All of the above

8. What is the purpose of performing radial immunodiffusion in the laboratory?

 Guru Nanak Institute of Pharmaceutical Sciences and Technology
Sub: Lab on Immunology Code: MSBT/MSMC/MSGN-292

9. What are the advantages of using radial immunodiffusion over other methods of
quantifying antigens?

REMARKS:

Attendance Viva Performance (20) Total

(10) (10) (40)
Cleanliness Conduct Method Critical
And & accuracy thinking
Manual Involvement (4) (8)
maintenance (4)
(4)

Signature of the Teacher