Various Techniques of Immunofluorescence:
First utilized by Coon’s in the year 1942 for Pneumococcus in tissue sections.Direct method: or single layer method
In this technique antigen is treated once with antibody tagged with fluorochrome.
Illustration: Explanation of Rabies antigen in impression smear.1. Impression smear from hippocampus of infected dog is prepared and fixed.
2. This is treated with antirabies antibody tagged with Incubated and FITC.
3. It is washed to eliminate un-reacted antibody.
4. The preparation is inspected for fluorescence.
Likewise different other antigens can be explained.
1. To explain viruses and bacteria in the respiratory specimens.2. To explain Adenovirus, Chlamydia in the conjuntival smears.3. To explain GC and Mycoplasma in urethral smear.Indirect Technique or Antiglobulin Method or Double Layer Technique:
Principle:
To explain the antigen, first unlabelled particular antibody is employed. Subsequently it is treated with tagged antibody to gamma globulin. (Therefore when the first layer is rabbit antibody then the second layer is anti-rabbit gamma globulin- tagged).Procedure:
To explain an unknown antigen in fixed preparation. The test is completed in two steps:Step: 1
1. Unknown antigen is existed in a fixed preparation.
2. It is treated with particular non-fluorescent serum.
3. Incubated at 37ºC for 10 to 20 min in wet chamber.
4. Washed 3 to 4 successive baths for 5 min each in phosphate buffered saline. Antigen antibody complex is opposed to washings. The preparation is washed and then dried.Step 2:
1. To the washed dried preparation labeled anti-species anti-globulin is added and incubated at 37ºC for 15 to 30 minutes in wet chamber. It is then washed and dried as in step 1. Whenever antigen-antibody complex has been made in the first step, labeled antiglobulin will couple to particular antibody to antigen and a finely fluorescent complex will be prepared.
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