Diagnostic procedures for antigen detection
Fluorescent antibody (FA) test
A quick and easy procedure for the diagnosis of rabies is the use of a suitable dye for the detection of Negri bodies. Histopathological techniques have, however, been replaced in most laboratories by the fluorescent antibody (FA) test, which was first developed in 1958 by Goldwasser & Kissling. The FA test is now the most widely used method for diagnosing rabies infection in animals and humans. It is based on microscopic examination, under ultraviolet light, of impressions, smears or frozen sections of brain or nervous tisue after treatment with antirabies serum or globulin conjugated with fluorescein isothiocyanate. The test is accurate and results can often be obtained within 30 minutes of receipt of the specimen, although for routine purposes a period of 2-4 hours is desirable for the fixation in cold acetone.
Apart from an appropriate microscope, the two main requirements for success in using this technique are well trained personnel and conjugated antiserum or globulin of good quality. After one year’s experience, most laboratories find over 99% agreement between the FA test and the mouse inoculation (MI) test. In the first year, however, some laboratories may miss up to 10% or even 20% of the positives with the FA test and for this reason, both tests (MI and FA) should be run in parallel during this period. Stringent control of the labelled antirabies antibodies should be carried out to determine the specificity of the fluorescence and to minimize the number of false-positives. Appropriate tissue sampling is also important. Examination of impressions or smears of tissue samples from Ammon’s horn and brain stem are recommended. Labelled rabies antibodies can be prepared against the whole rabies virus. More highly potent antisera can be prepared using purified and concentrated rabies virus or virion components such as ribonucleoprotein. Conjugated monoclonal rabies antibodies are being increasingly used in routine diagnosis. The specificity of these latter conjugates is greater than those prepared against the whole virion or virion components. A conjugate composed of two labelled monoclonal antibodies is now widely used and is available commercially. Panels of monoclonal antibodies are also used in studying the epidemiology of rabies.
The FA technique is a highly sensitive method for detecting rabies antigen in fresh specimens. However, it may also be performed on fixed specimens. The specimen should be treated with one or more proteolytic enzymes such as trypsin or pepsin before staining to unmask the antigenic sites. The sensitivity of the test using fixed specimens has been reported to be 90-100% of that obtained using fresh specimens. However, it is recommended that fresh tissue be examined where possible.
When specimens are received in 50% glycerol-saline, it is imperative that the tissue be washed several times in saline before staining.