Development of new diagnostic test of rabies infection by immunoblot

Abstract

Dot-immunoblot was developed as a new diagnostic test for rabies in dogs submitted for examination at the Queen Saovabha Memorial Institute of Thai Red Cross Society. The test specimens were applied on a piece of cut nitrocellulose membrane and stained with locally prepared rabbit antirabies immunoglobulin G (RRIG) and amplified by avidinbiotin peroxidase complex. The whole staining procedure took about 6 hours and the positive test appeared as blue dot on the nitrocellulose membrane. The results obtained with the freshly prepared parotid gland suspension were 100% sensitive and 100% specific as compared to fluorescent antibody test (FAT) and mouse inoculation test (MIT) of the brain. With fresh saliva, the sensitivity and specificity of the dot-immunoblot were 94.7% and 91.6% respectively. However, the sensitivity and specificity were diminished when stored specimens were used, i.e., 96.5% and 84% respectively for parotid gland suspension and 89.6% and 73% respectively for saliva. In order to make the test more widely applicable, the commercial primary antibody (Equine rabies immunoglobulin or ERIG from BBL) was also studied. As compared to RRIG, ERIG-BBL gave a more clearout distinction between positive and negative specimens. With ERIG-BBL, the sensitivity and specificity of stored parotid gland suspension were 81.8% and 85.7% respectively and of saliva were 72% and 85.7% respec ively. We extented our dot immunoblot study to the brain tissue. Similar to the results with stored salivary gland suspension and saliva, stored brain suspension was also less sensitive than FAT and MIT, i.e., only 75% and 61.9% sensitivity with ERIG-BBL and RRIG respectively but the specificity was 100% and 95.2% respectively. However, when freshly prepared brainstem suspensions were used, the sensitivity and specificity increased to 100% and 88.8% respectively with ERIG-BBL and 94.7% and 88.8% respectively with RRIG. The reason for this improved sensitivity and specificity may be due either to non-degraded state of the brain or to the viral predilection in the brainstem. The dot immunoblot technique was further applied for antemortem diagnosis of rabies in with quarantined animals. The results were very satisfactory with an excellent correlation with the other standard diagnostic methods and with clinical pictures of both rabid and nonrabid quarantined dogs. Our results indicated that dot-immunoblot technique, when property performed, could be of great diagnostic value in both antemortem and postmortem diagnosis of rabies. After a larger scale of study, it may replace the time-consuming MIT and be used as the confirmatory test for FAT or even replace FAT in small laboratories where fluorescent microscope is not available.