The fluorescent antibody test (FAT) is the major method for the diagnosis of rabies . It is based on the tests of chapter 3.1.18 of "Manual of Diagnostic Tests and Vaccines for Terrestrial Animals” which was published by World Organization for Animal Health (WOAH) (https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.01.18_RABIES).

FAT is the major method for rabies diagnosis worldwide, which is recommended by both WHO and WOAH. FAT provides a reliable diagnosis in 98–100% of cases for all rabies virus strains if a potent conjugate is used.

1. Collection of brain samples

Usually the brain is collected following the opening of the skull in a necropsy room, and the appropriate samples are collected preferably Ammon’s horn, thalamus, cerebral cortex and medulla oblongata.

2. The procedule of fluorescent antibody test (FAT)

(1) Preparation of brain smear

The brain smears prepared from a composite sample of brain tissue, that includes the brain stem, are fixed in 100% high-grade cold acetone (put in a –20 °C freezer) for at least 30 minutes. After air-dried, the brain smear is stored in –20 °C freezer until use.

(2) Immunofluorecent stain

Brain smear is removed from the –20 °C freezer and puts in biosafety cabinet to return to room temperature. Then stain with 1-2 drops of  diluted fluorescent conjugate for 30 minutes at 37°C incubator. Discard fluorescent conjugate and wash slides with phosphate saline buffer (PBS) for 10 times. FAT slides should then be examined for specific fluorescence using a fluorescent microscope and filter appropriate for the wavelength of 490 nm.

(3) Interpretation the result of FAT

Aggregates of nucleocapsid protein are identified by specific fluorescence of bound conjugate. The determination of final result is based on two factors, including staining intensity (D1-4) and antigen distribution (+1-4). Only the staining intensity reaches D3 or D4 and antigen distribution is greater than +2, then the sample is determined as rabies positive.