Immunodiffusion (Gel Diffusion)

The gel diffusion test involves a precipitation reaction in a semisolid medium. The object of the gel diffusion test is to bring together, through diffusion, optimal concentrations of antigen and antibody to form visible bands of precipitation. The number of bands indicates the minimal number of antigen-antibody systems present. Refer to textbook and class notes for more background information.

Materials required per student:

2-3 glass slides

1 large tube of agarose gel

1 10 ml pipette

2 (1 ml) pipettes

pasteur pipettes

1(25 ul)micropipet

assorted antisera

assorted corresponding antigen

1 test tube rack

1 marking pen

methylene blue

2-3 petri dishes

2-3 pieces of filter paper

wooden applicator sticks

6 ml saline

10 small test tubes


1. Melt the agar in a boiling water bath.

2. Wash two glass slides with soap and water, let dry on a paper towel.

3. Place dried clean slides on a paper towel and use a 10 ml pipette to transfer 3 to 4 ml of melted agar onto each slide. Do not let the agar spill over the edge of the slide but cover the slide completely.

4. Let the gel on the slides solidify for 30 minutes. While waiting for slides to harden, work on design.

5. After the agar gel on the slides has solidified, punch a 7 hole template using the Gelman immunodiffusion punch.

6. Aspirate with a pasteur pipette the gel from the holes in the agar as demonstrated by the instructor.


1.Decide on the patterns you wish to use for your experiment. One possible protocol is as follows:

Make serial two fold dilutions of the antigen preparation.

    1. Select your best slides and with the use of a micropipette fill the holes in your slide starting with the 1:32 dilution (place in well #8) and working towards the undiluted sample (well #1). If you go from the most diluted to the most concentrated tubes, you need only one pipette. Fill the holes to the top of the gel but do not let them run over start with 10ul to test capacity of well.

2. Fill the center hole with the antisera undiluted.

2.You should put antisera in the middle well and antigen in the outside wells. You can mix different antigen and antisera together. Please note the limited volumes of materials available to you as you design your experiment

3.When you have finished filling the wells they are ready for incubation.

4.Place filter paper in the petri dishes, moisten with water, and place two applicator sticks on filter paper to support the slides.

5.Put the completed slide in the petri dish on top of the wooden sticks. Place the cover (top) on your petri dish and label it with a marking pen. This is a humidor and will prevent your slide from drying out.

6.Incubate at room temperature for 18 to 24 hours.

7.The next day, view the slide and record the results by drawing all the bands, etc. Some may be difficult to see, so check with the instructor if you can't see anything! When you have finished recording the results, place the slides into the petri dishes and store in the refrigerator until you are ready to begin the washing and staining process.

Staining Immunodiffusion Slides

1. Check that the precipitin bands are still present on the slides. (Mon.)

2. Place each slide into a petri dish using 2 additional blank slides on either end to prevent gel from sliding off. Fill petri dish carefully with saline. Allow this to set for one day.

3. The next day, aspirate the saline and replace with distilled water. Allow this to set for one day. (Tues.)

4. The next day cut a piece of filter paper using a glass slide as a template, wet it and place it on your slide. (Wed.)

5. Place the filter paper covered slide in the incubator.

6. That evening or when the agar has dried to a thin film, moisten the filter paper and gently remove it from slide by washing the surface of the dried agar with your finger using tap water. (Thurs)

7. Stain slide by immersing it in the stain with the following composition for 6-10 minutes.

Amido black 10B 1 gr

Glacial acetic acid 100 ml

Methanol 700 ml

Distilled water 200 ml

8. Rinse off the excess stain by placing the slide into a bath of the following composition:

Glacial acetic acid 100 ml

Methanol 700 ml

Distilled water 200 ml


9. After approximately 5 minutes in the wash solution, take slide out and allow to air dry on a piece of paper towel.

10. Record the results again by drawing from these stained slides. Compare to the earlier results from the unstained slides.

11. Interpret your results and write a lab report.