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This article was published in 1966
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INSTITUTE OF INSPECTORS OF STOCK OF N.S.W. YEAR BOOK.
The Fluorescent Antibody Technique (F.A.T.)
D. HELWIG, B.V.Sc., Special Veterinary Research Officer, Glenfield
This technique was pioneered by Albert Coons and his co-workers in 1942. Over the intervening years the method has achieved great popularity as both a diagnostic and research tool in the fields of bacteriology, virology, and immunology.
Basically the method consists of labelling antibodies with a fluorescent dye, allowing the labelled antibodies to react with their specific antigen, and observing the reaction produced under a fluorescent microscope. The latter consists of a standard microscope with, usually, a dark field condenser and a high intensity light source containing a useful amount of ultra-violet light. Suitable filters are used to permit ultraviolet illumination of the specimen and to protect the eyes of the observer against harmful U-V radiation.
Two staining procedures can be employed:
1. Direct Immunofluorescent Staining where the labelled antisera is applied directly to the antigen preparation. This is commonly used for the identification of antigen.
2. Indirect Immunofluorescent Staining, also called the "Sandwich technique". In this method a known antigen is allowed to react with unconjugated (unlabelled) antisera, the antibody status of which may be unknown. Any reaction which may occur is "visualised" by treating the complex with conjugated antisera prepared against the animal species from which the unlabelled antisera was obtained. This method lends itself to the detection of specific antibody.
The two fluorescent dyes most commonly used for labelling antisera are fluorescein (green fluorescence) and rhodamine (orange-red fluorescence). The isothiocyanate or sulphonyl chloride form of the dye is required to ensure firm bonding with serum proteins.
A considerable range of conjugated antisera is available commercially, particularly in the field of human medicine, but such preparations are expensive. Any laboratory which can produce its own conjugates will find the results rewarding as the cost is greatly decreased and, in some cases, the quality of the product is superior to the commercial preparation.
APPLICATIONS OF THE TECHNIQUE AT GLENFIELD
The F.A.T. is now being used routinely to facilitate rapid diagnosis of some clostridial infections. This station is now preparing conjugated antisera against Cl. septicum, Cl. chauvoei and Cl. oedematiens Type B. The results so far have been very encouraging, when the technique has been used on muscle, liver or inflammatory exudate smears prepared from lesions in affected animals.
It is recommended that at least three smears should be prepared from each site and air dried. This will permit one smear to be stained by Gram in order to ascertain the numbers of clostridial organisms present per field, after which one smear is stained with fluorescein conjugated Cl. septicum and rhodamine conjugated Cl. chauvoei antisera, while the third smear is stained with fluorescein conjugated Cl. oedemariens antiserum. Impression smears should be made from muscle tissue. When preparing smears from Black Disease-like liver lesions it is advisable to collect tissue from the periphery of the lesion, just inside the inflammatory zone, with the corner of a glass slide, and then prepare three smears from the material so obtained.
A Swine Fever conjugate has also been prepared at the V.R.S. and while initial results were encouraging, when impression smears from lymphoid and other tissues of experimentally infected pigs were examined, further work will have to be delayed until a more economical source of fresh infective material is available; such as tissue culture preparations.
Bibliography:
- "Fluorescent Antibody Methods". Coons, A. H. (1958) in General Cytochemical Methods. Vol. I, pp. 399-422. Edited by J. F. Danielli, Academic Press Inc., New York
- "Immunofluorescent Staining: The Fluorescent Antibody Method". Beutner, E. H. (1961) Bacteriological Reviews. 25:49
- "The Beginnings of Immunofluorescence". Coons, A. H. (1961) J. Immunology. 87:499
- "Immunofluorescence as Applied to Pathology". Smoth, C. W., Metzger, J. F., and Hoggan, M. D. (1962) The American Journal of Clinical Parhology. 38:26