Production And Characterization Of Monoclonal Antibodies Specific To Hemagglutinin Of Avian Influenza Virus H5N1 Subtype, And Establishment Of H5N1 Hemagglutinin Antigen Capture ELISA

Influenza virus is one of the Influenza virus genus of the Orthomyxoviridae family, which can cause humans, poultries and livestocks ill. According to characteristics of nucleoprotein(N) and the matrix(M) ptoteins, influenza virus can be divided into three groups of A, B and C. Influenza B and C mainly infect humans, while the epidemic range is limited. However, Influenza virus A is the major pathogen of influenza epidemics, which can infect humans, fowls and livestocks. On the basis of the antigenicity differences of Hemagglutinin(HA) and Neuraminidase(NA) proteins, Influenza virus A can be subdivided into 16 HA subtypes and 9 NA subtypes, and among them, subtypes H1, H2, H3, N1 and N2 are the predominant pathogens accounting for human flu. Especially, influenza virus H1N1 and H3N2 caused global epidemics, such as the 1918 "Spain Flu", the epedimics in 1957 and 1968, and so on. But since the incident happened that H5N1 subtype avian influenza virus' outbreak in Hong Kong SAR caused 18 cases infected and 6 deaths, people have become to focus on the avian influenza virus and the representive H5N1 and H7N7 subtypes. After this, highly pathogenic avian influenza(HPAV) continued to prevail in East Asia, Southeast Asia and Europe several times. For this reason, hundreds of millions of poultries were slaughtered to prevent the spread of influenza in asia, which directly resulted in a loss over hundreds of billions dollars. However, the most important is that H5N1 avian influenza is under a rapid mutation, probably getting prevalent ability among human race. Similar to the other viral acute respiratory diseases in clinical symptoms, avian influenza must be diagnosed accurately through laboratory tests, instead of being diagnosed only on the basis of clinical manifestations. Therefore, the early diagnosis technologies which are convenient, rapid and easy to be commercialized for clinical use, are able to help clinicians to make early, quick and accurate diagnosises and play a critical role in prevention and control of the pest status, so that the patients can be shielded promptly and the pandemic can be controled. By now, there are several antigen detection kits based on Immunochromatography(IC) and Enzyme Linked Immunosorbent Assay(ELISA) which can be used to distinguish influenza viruses A and B, while not be able to get a classfication of subtypes. Consequently, developping subtype detection technologies to differentiate avian influenza viruses from ordinary influenza viruses, is helpful in several aspects, like prompt directive anti-virus therapy, researches on prevalence and evolution rules of avian influenza virus subtypes, monitoring newly emergent virus subtypes, large-scale screening for finding infection sources and disconnecting infection routes, and so on. Considering the facts that antigenicity is one of the standards of influenza A subtypes and the homology among virus hemagglutinins is not high, hemagglutinin is an ideal target for influenza virus subtype detection. Till now, aiming at hemagglutinin protein, laboratory influenza virus subtype detection assays comprise mainly serological test, gene test and viral antigen detection, while each has some shortages: serological test detects antibodies which is unfit for early diagnosis; gene test requires skilled operators and can't be popular in the basic units; viral antigen detection chiefly consists of Indirect Immunofluorescence Assay and Enzyme Linked Immunosorbent Assay, while the former is only carried out in big hospitals or research institutes with fluorescence microscopes, nevertheless, antigen capture ELISA based on antibody specificity aims directly at the hemagllutinin antigen and is capable of identify virus HA subtypes in the earlier period of infection whereas no corresponding commercial kits were invented. In this research, several types of hemgglutinins were used to immunize mice, then to screen potent hemagglutinin-inhibition antibodies and neutralization antibodies, and to analyze recognition epitopes, then the established kit was examined by seperated virus strains from variant hosts at different time, and double antibody sandwich ELISA was implemented, to detect hemagglutinin antigens of H5N1 avian influenza viruses.In addition, specific monoclonal antibodies to hemagglutin produced can be utilized not only to develop detection assays for influenza virus subtypes but also to produce remedial antibodies. Hemagglutinin is a key factor for virulence, determines hemagglutination activity of viruses, and correlates with viral adsorption process to sialyloligosaccharide receptors on target cells' surface. Then, neutralization antibodies will bind to hemagglutinins in the earlier era, and disable virus from attaching and infecting host cells. On the whole, after obtaining specific monoclonal antibodies to hemagglutin with hemagglutination inhibition activity, antibodies' neutralization activities were then evaluated, to build foundation for research on therapic antibodies.The research is divided into three parts:Ⅰ. The production and characterization of hemagglutinin antigens of H5N1 avian influenza virusesHemagglutinins were obtained as follows: Firstly, recombinant H5 hemagglutinin protein was expressed in baculovirus/High Five insect cells, characterized by IFA anf Western Blotting, and hemagglutination titer was determined. Experimental results showed that recombinant hemagglutin protein was about 66 000 Dalton and could be recognized and combinded by animal serum against H5N1 avian influenza virus and standard anti-H5 subtype serum, and hemagglutination titer was 1:128. Secondly, plasmid pVAX1-tpA-97-H5 with the hemagglutinin gene of H5N1 avian influenza virus(A/Hong Kong/482/97) was characterized, pVAX1 vector was examined for transfection efficiency in X-gal dyeing assay, and the expression of H5 plasmid was tested at a cellular level by IFA. Experimental results exhibitted that lacZ gene expressedβ-galactosidase successfully and efficiently after the control plasmid pVAX1-lacZ was transfected into 293 cells, and 293 cells transfected could combine with animal serum against H5N1 avian influenza virus. Lastly, commercial native H5 hemgglutinin(concentrated from H5N1 avian influenza virus A/Goose/Guangdong/1/96 obtained from the veterinary surgeon institute of Harbin) was tested of its hemagglutination titer, and the results confirmed that native H5 hemagglutinin was capable of agglutinate red blood cells from guinea pig and the titer was 1:1 024.Three types of H5 hemagglutinin antigens were obtained and it was confirmed that 293 cells tranfected by H5 plasmid could be recognized and combinded by animal serum against H5N1 avian influenza virus and the standard anti-H5 subtype serum respectively as well as recombinant H5 protein, and native H5 hemagglutinin with pretty hemagglutination activity would provided good immunogens for production of antibodies specific to hemagglutinin of avian influenza virus H5N1 subtype.Ⅱ. Production, characterization and neutralization test of mAbs specific to avian influenza virus H5N1 subtype's hemagglutinin. Several types of H5 hemagglutinin antigens were utilized to immunize mice, to produce mAbs specific to hemagglutinin of avian influenza virus H5N1 subtype, and experiments were carried out to identify antibodies' immunology characteristicses, recognition sites, hemagglutination inhibition activities, and neutralization activities. Nine Balb/c mice were immunized via three methods as follows: three were immunized by native H5 hemagglutinin, three were immunized by recombinant H5 protein, and the other three received boost of native H5 hemagglutinin after injections of plasmid with the H5 gene. Then the splenocytes of the immunized mice were fused respectively with myeloma cells, to produce hybridoma cell lines secreting anti-H5 hemagglutinin antibodies via HI assay and indirect ELISA, without which reacted with normal embryo fluid, influenza virus A(H1N1 and H3N2), influenza virus B, or recombinant nucleoprotein of influenza virus A. Ultimately, after two or three cycles of cloning with limiting dilution assay, thirty two strains of hybridoma cell lines steadily secreting antibodies to hemagglutinin of H5N1 avian influenza virus were obtained, and by intraperitoneal (ip) innoculation into mice, thirty one strains of mAbs in ascitic fluid were obtained. Among all the antibodies, twelve strains were immunized by native H5 hemagglutinin in which ten strains were identified as IgG1, one as IgG2b, one as IgM; nine strains were obtained from plasmid with the H5 gene in which one strain was identified as IgG1, four as IgG2a, one as IgG2b, while the other three were excluded from the categories above; and ten strains were prepared from recombinant H5 protein in which seven strains were identified as IgG1, two as IgG2a, one as IgM. HI assay concluded that antibodies from native H5 hemagglutinin or plasmid with the H5 gene inhibit the hemagglution of red blood cells by H5 hemagglutinin with titers from 1:100 to 1:51 200, and couldn't cross-react with hemagglutinins of H1, H3, H7 and H9 subtypes of influenza virus A or influenza B, while antibodies obtained from recombinant H5 protein didn't gain the abilities to inhibit hemagglutination. Then, eighteen strains of antibodies in ascitic fluid with HI titers above 1:100 were chosen to examine neutralization avtivities, and the results revealed that, by index of H5N1 avian influenza virus(A/Vietnam/3028/04), there were no concordances between neutralization and HI titers, and neutralization titers of fourteen strains of the total were no more than 1:40, while the other four obtained from H5 hemagglutinin presented titers over 1:160 each.By methods of caprylic acid-ammonium suIphate precipitation(CAASP), twenty eight mAbs were purified totally, and SDS-PAGE revealed that purities were all over ninety percent. Using improved method of NaIO4, twenty six strains of purified mAbs subtypes known(two strains of mAbs subtypes unknown excluded) were marked with horse radish peroxidase(HRP), and the optimal working concentration were definited to between 1:100 to 1:100 000 in indirect ELISA. And after divided into two groups, positive in HI assay or not, competitive inhibition assays were applied to judge sites the antibodies recognize, and the resluts showed that mAbs which were positive in HI assay recognized two different antigenic sites, and antibodies recognizing distinct sites were different so much in HI titers, 1:3 200 to 1:51 200 and 1:100 to 1:400 respectively, and mAbs which were negative in HI assay recognized at least four sites, and all the conclusions were substantial bases for establishments of the consequent double antibody sandwich capture ELISA.Ⅲ. Establishment of double antibody sandwich capture ELISA specific to H5 hemagglutininBeing arrayed and paired, the best sensitive Abs couple to detect H5 hemagglutinin was screened, to set up double antibody sandwich capture ELISA specific to H5 hemagglutinin. First of all, ten strains of Abs negative in HI assay were cross-matched to detect gradient-diluted native H5 hemagglutinins, recombinant H5 protein and two strains of H5N1 avian influenza viruses, and the final Abs couple of H5M21 and H5M15-HRP selected were used to organize double antibody sandwich capture ELISA, while the sensitivity detecting H5 hemagglutinin and H5N1 avian influenza viruses was under sixteen hemagglutinating units(HAU). At the same time, to ensure the sensitivity, sixteen strains of Abs positive in HI assay were matched to detect gradient-diluted native H5 hemagglutinins and six strains of H5N1 avian influenza viruses from various hosts at different time, and the final Abs couple of H5M9 and H5M11-HRP were utilized to set up a specific double Abs sandwich capture ELISA, to get a sensitivity of 1/32 HAU in detecting H5 hemagglutinin and H5N1 avian influenza viruses and no cross-reactivity with the other subtypes of influenza virus A and influenza virus B. In this part, double antibody capture sandwich ELISA-based virus detecion method was sensitive and specific extremely, applying a shorcut and specific laboratory diagnosis approach for earlier diagnosis of avian influenza virus infection.In a word, the conclusion of this research are as follows:Ⅰ. Ten strains of anti-H5 mAbs obtained from recombinant H5 protein had no HI activity, while sixteen strains obtained from plasmid with the H5 gene and native H5 hemagglutinin were positive in HI assay, which showed us that the native conformation of hemagglutinin was important to maintain Abs' HI activities.Ⅱ. The neutralizatination titers disagreed with the HI titers, because of the different strains used, in other words, hemagglutinin used in HI assay was concentrated from avian influenza virus (A/Goose/Guangdong/1/96) isolated from poultries, while avian influenza virus used in neutralization assay was isolated from humans, and the differences betweeen this two strains induced the imcompatibility of neutralizating titers and HI titers. On the other hand, it was possibly resulted in by the disagreement between the two kinds of recognized antigenic site, however the genuine reason was to be investigated.Ⅲ. Doule antibody sandwich capture ELISA was established by Abs positive in HI assay, whose sensitivity to detect H5 hemagglutinin and H5N1 avian influenza viruses was superior to that of the method established by Abs negative in HI assay, demonstrating that only Abs positive in HI assay could recognize native conformational epitopes of hemagglutinin effectively.