Development Of An Indirect ELISA For Detection Of Rabies Virus Antibody

Rabies is an important public health problem in the world. Globally there are over 55,000 reported human cases of rabies each year, although the actual number of cases is probably much higher. Since 1997 rabies became a re-emerging disease in China. In 2005, 2537 humans contracted rabies in our country; 3279 humans contracted rabies in 2006. Dogs are the major reservoirs and play a pivotal role in rabies transmission, particularly in countries where rabies is endemic. A method capable of measuring the presence of antibodies to rabies virus in the sera of dogs is necessary. Such a technique, if reliable and cheap, would also be useful in establishing the extent of herd immunity in control efforts involving immunization of domestic animals. It is best that ELISAs used for the detection of rabies virus-specific antibodies are rapid and relatively easy to perform, without the need for special facilities, but indirect ELISAs in present have a lower sensitivity. The objective of this experiment is to improve the sensitivity and the specificity of indirect ELISA for detection of antibody against rabies virus.Rabies virus is a RNA virus in the Rhabdoviridae family (genus Lyssavirus), and the genome is single stranded, negative sense, and nonsegmented. Virus particles are bullet shaped with an envelope covered with surface glycoprotein projections, which appear as spikes.The rabies virus glycoprotein (G-protein) was shown to be the major antigen capable of inducing virus neutralizing antibody that could confer protective immunity. against rabies virus infection. The N protein is also important in inducing protective immunity.In this study, a prokaryotic expression plasmid was constructed by enrich epitope of the rabies virus(CVS-24) glycoprotein and neucleoprotein gene and was cloned into pET-28a(+). The recombinant plasmids pET-G1,pET-G2,pET-N1,pET-N2,pET-N3, were transformed into E.coli Rosetta and expressed under the induction of 1mmol/L IPTG. Objective proteins were identified by SDS-PAGE and western-blotting. With Western-blotting analysis, there appeared special reaction bands between the specific antibody and the expressed protein, which indicated that the expressed product showed good immunoreactivity. The expressed products accounted for 42%,34%,32%,27%,36% of the total bacterium protein. Via optimization,the 96-well microtiter plates were coated with further purified Pro-N3 after the square matrix titrate determination best density is 1.17μg/ml, the blood serum best dilution ratio is 1:100. It was proved that the established ELISA has high specificity by cross reaction and block experiment.