Preparation And Application Rearch On Monoclonal Antibodies Against Rabies Virus

Rabies is a serious zoonosis caused by Rabies Virus which damage brain and central nervous system. People had recognized this disease before 2300, BC. Once symptoms of the disease develop, rabies is 100% fatal to both animals and human. Rabies has been eliminated as a significant public health risk in most parts of the developed world. However, annually 55,000 deaths are reported, most of them in the developing world, especially in Asia and Aferica(55,000 died of Rabies every year). There are many Rabies cases in China, morbility and mortality are second only to India of the world. China has take great efforts to prevent Rabies, such as limit people to breed dogs, vaccinate dogs, and applies prophylactic treatment to susceptive and bited people, howerer, morbility continue to rise recent years. Date about intensive communicable diseases from China Department of Health showed that people died of rabies is the Number 1 among 37 legal communicable diseases. Every year many thounsands of people died of Rabies, and mortality is 100%. Especially from 2006, media reportesd that Rabies is spreading at a high velocity. Number of cities where newly occurred rabies is increased at a large scale, peole's panic emotion to the Rabies is greatly rised and regards Rabies as the pronoun of death. So, Rabies is a serious infectious desease that do great harm to public health.Rabies virus belongs to the order Mononegavirales viruses with a nonsegmented, negative-stranded RNA genomes.There are 5 genes which encode 5 structural protein. Rhabdoviruses are approximately 180 nm long and 75 nm wide. The rabies genome encodes five proteins: nucleoprotein(N), phosphoprotein(P), matrix protein (M), glycoprotein(G)and polymerase(L). All rhabdoviruses have two major structural components: a helical ribonucleoprotein core(RNP) and a surrounding envelope. In the RNP, genomic RNA is tightly encased by the nucleoprotein. Two other viral proteins, the phospoprotein and the large protein (L-protein or polymerase)are associated with the RNP. The glycoprotein spikes are tightly arranged on the surface of the virus. Typical RV particles is like a bullet, with length 130~200 nm , width 75 nm, and 11,928~11,932 spikes anchoraged at suffice.People have studied deeply and carefully on the Rabies and RV for many years, however, many hard work and research are necessary to thoroughly recognize them. Being high specific, homogeneous, high efficient and infinite accommodate, monoclonal antibody plays a more and more important role in detection, diagnosis and treating the Rabies, and analyse the antigen variation as well. The aim of this research is to obtain a set of stabile, special, and efficient monoclonal antibody(McAb); further more, use these monoclonal antibodies to analyze some popular RV strains of China; also, these McAb can be used in research and work in RV and Rabies field.At the beginning of the research, we prepared the antigen(Rabies Virus) elaborately. For rejuvenate the high attenuated RV-SRV9, innoculate cell-cultured SRV9 into brain of suckling mices, and retrieve RV when suckling mices die, and repeat this process twice. Then, inoculate the retrieved RV to BHK-21cell, and cultivate with 1% serum at 33℃, to avoid RV degenerate which result in many"T"particles with low antigenicity. Then, passage the infected cell for 3 times to gain more viruses. At the same time, inspect virus quantity and its morphogenesis by electronmicroscope(EM)to determinate the best time to harvest virus. After carefully observation and compare, we draw the conclusion that the time after passage the infected cell for 3times and cultivate for 96 h is the opportunity to harvest virus, because virus quantity is large and many virus budding from the cellmembrane.Then concentrated and purified the harvested virus. Because cell composition can intervene the concentrate procedure, as well as loss purity, we gathered supernatant and discarded the cell. Add zinc acetate to precipitate the supernatant, then centrifugate in a sucrose density gradient and retrieve the virus banding. Finally obtained Rabies Virus with high concentration and purity, as well as fine and typical shape. Typical particles possess good antigenicity, while atypical"T"particles with low antigenicity. In the EM scope, we noticed more than 90% of particles are typical, that is, with a bullet shape, glycoprotein spikes surround the particles, 130~200 nm length and 75 nm width. However, atypical"T"particles is less than 10%, with 90 nm length and 90 nm width, and a triangle shape. Concentration of virus protein is about 9.7 mg.mL-1, suffice to immune procedure.Immunize the Balb/c mices with purified virus with the dosage of 50μg per mice. Immune effect is detected by indirect ELISA. After 3 times immune, serum antibody titer is higher than 200,000, suffice to cell fusion produce.Then the study carries out cell fusion produce, and screen positive cell by indirect ELISA and IFA assay. After sevrral times of cell fusing, cultivating, screening and subclone, 10 monoclonal antibodies against Rabies Virus were obtained with highly stabile, special, and efficient, this greatly supplied the Rabies and Rabies Virus research and work.And we successfully labeled some monoclonal antibodies with FITC(FITC-McAb), and got satisfied result when they were used in practical work. Use both McAb and FITC-McAb, we analysed the reaction pattern of some popular RV strains of China with different McAbs. The study finds that some strains from the same area showed different pattern, and we postponed that one of their epitope was mutanted. Deep research on the epitope has been carried out. This research looked deeply into the morph and morphogenesis of SRV9 strain by EM and result was satisfied. This result can be refered to virus concentrate and purify, and offer some guide to the vaccine manufacture produce. Immune Balb/c mice with the antinge-concentrated and purified virus, via cell fusion, screen, subclone, we obtained several monoclonal antibody strains, and some were labeled with FITC, which can be effectively used in RV diagnose and diction; based on the reaction pattern between McAbs and different RV strains, we find one strain is different from other strains in the same area, which epitope may have mutanted.