Immunoperoxidase inhibition assay for rabies antibody detection.

An immunoperoxidase inhibition assay (IIA) for detection of rabies antibodies in human sera is described. Diluted test sera are added to microplates with paraformaldehyde-fixed, CER cells infected with rabies virus. Antibodies in test sera compete with a rabies polyclonal rabbit antiserum which was added subsequently. Next, an anti-rabbit IgG-peroxidase conjugate is added and the reaction developed by the addition of the substrate 3-amino-9-ethylcarbazole (AEC). The performance of the assay was compared to that of the "simplified fluorescence inhibition microtest" (SFIMT), an established virus neutralization assay, by testing 422 human sera. The IIA displayed 97.6% sensitivity, 98% specificity and 97.6% accuracy (Kappa correlation coefficient=0.9). The IIA results can be read by standard light microscopy, where the clearly identifiable specific staining is visible in antibody-negative sera, in contrast to the absence of staining in antibody-positive samples. The assay does not require monoclonal antibodies or production of large amounts of virus; furthermore, protein purification steps or specialized equipment are not necessary for its performance. The IIA was shown to be suitable for detection of rabies antibodies in human sera, with sensitivity, specificity and accuracy comparable to that of a neutralization-based assay. This assay may be advantageous over other similar methods designed to detect rabies-specific binding antibodies, in that it can be easily introduced into laboratories, provided basic cell culture facilities are available.

Neutralizing antibodies to bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) and its subtypes.

This study was carried out to determine whether the sensitivity of serum neutralization (SN) tests would be affected by the use of distinct subtypes of bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) as test challenge viruses. Bovine sera collected from a randomized sample (n=287) were tested in a 24h incubation SN against three type 1 viruses (BoHV-1.1 strains "Los Angeles" (LA) and "EVI 123"; BoHV-1.2a strain "SV 265") and three type 5 viruses (BoHV-5a strain "EVI 88"; BoHV-5b strain "A 663" and BoHV-5c "ISO 97"). SN sensitivity varied greatly depending on the test challenge virus used in the test, particularly when results against each virus were considered individually, where it ranged from 77% (detecting 80 out of 104 antibody-positive sera) with ISO 97 to 91% (95/104) with BoHV-1.1 strain LA. All tests to single viruses revealed a significantly low sensitivity (McNemar's; p<0.05). Maximum sensitivity (104/104) was achieved when positive results to a particular combination of four of the challenge viruses (LA+EVI 123+SV 265+A 663) or some combinations of five viruses (or all six viruses) were added cumulatively. These results provide evidence for no association between any particular virus type/subtype and higher SN sensitivity. In addition, it was clearly shown that when SN is performed with single test challenge viruses, sensitivity can vary so significantly that might compromise control or eradication efforts. Performing SN against a number of different viruses demonstrated to improve significantly the test's sensitivity.

A sandwich enzyme linked immunosorbent assay to detect rabies antibodiesin sera of different species

A sandwich ELISA (S-ELISA) was developed to detect antibodies to rabies virus in seraof different species. The test was performed as follows: ELISA plates were coated withpolyclonal mouse/anti-rabies antibodies for 2 hours at 37ºC. After adsorption, plateswere washed and non-specific binding blocked with 2% powdered milk. In a separateplate, serial threefold dilutions of test sera were incubated with inactivated rabies virusantigen. The mixtures were then placed on the rabies antibody-coated plates andincubated. These were then washed and incubated with polyclonal rabbit/anti-rabiesantibodies. Subsequently, a rabbit/IgG-peroxidase conjugate was added and platesincubated. After washing, the chromogen (ABTS with 0.15% H2O2) was added to platesand after incubation for 30 min were read in a spectrophotometer (OD405). To validatethe S-ELISA, 128 serum samples including humans, cattle, hematophagous and nonhaematophagous bats, mice, marmosets, ocelots - Leopardus pardalis, raccoons -Procyon lotor, jaguarondi - Herpailurus yaguarondi, fox - Cerdocyon thous and coati -Nasua nasua, were tested and compared to a standard fluorescent antibody virusneutralization test (FAVN). In comparison to FAVN, the S-ELISA showed highsensitivity (82.98%) and specificity (100%), with an accuracy of 87.5%. Subsequently,738 serum samples from different species were tested in the S-ELISA. Antibodies torabies were detected by S-ELISA in all species tested, with the exception of the threeserum samples from raccoons. The S-ELISA was shown to be a serological test of lowcost that can be easily implemented in diagnostic laboratories. In addition, no liveanimals, infectious virus, cell culture or fluorescence microscopy are required forperformance of the test. This is an additional advantage of the S-ELISA over othermethods of rabies antibody detection.