| Antibody, an immunoglobulin family protein that binds to specific epitopes, is synthesized by lymphocytes or generated by aggressive(or non self) organisms.The development and maturation of monoclonal antibody(mAb) technology has laid the foundation for the application of antibody. MAb has a very obvious advantage in the diagnosis and treatment of disease, for the characteristics of single epitope recognition, infinite passage and stable expression. Besides, the antibody can be used as a basic research tool, because of its specific binding with antigen. Therefore, many researches have focus on how to obtain an antibody with good activity and high titer. In this study, different immunogen including natural protein and recombinant protein were immunized. Then, according to the interested antibody applications and characteristics, ELISA, hemagglutination inhibition and neutralization test were combined to compare more efficient screening method that satisfy the antibody demand, which will offer a useful reference for future antibody preparation strategy.In the present study, twenty-one mAbs were obtained against different protein from 3 kinds of animal disease virus, avian influenza virus(AIV), Newcastle disease virus(NDV) and canine distemper virus(CDV), respectively. We identified the characteristics, epitopes of the monoclonal antibodies and investigated their application starting from the immunogen, screening method and preparation process.Seven mAbs were obtained against AIV. BALB/c mice were immunized with a purified recombinant NS1 expressed in E.coli. Besides, AIV viruses was purified by sucrose density gradient centrifugation. After been observed under electron microscope, the virus particles were used to immunized BALB/c mice. The spleen cells from the immunized mice were fused with myeloma cells SP2/0. Through screening via ELISA coated with recombinant NS1, two mAbs against NS1, named D7 and D9, was identified. The five mAbs of anti AIV-HN were obtained by hemagglutination inhibition test and ELISA coated with AIV, respectively. The five mAbs were as follows: D1C12, D3B2, D3B3, D3A11 and D12B3, but none of them has neutralizing activity. Epitope identification of D7 and D9 against AIV-NS1 protein were firstly used phage display peptide technology with 3 rounds of biopanning, then combined with a series of truncated proteins and polypeptide chip, respectively, to map the minimal unit B cell epitopes. D7 recognized the 29DAPF32 epitope, while D9 recognized the 182WNDNT186 epitope.NDV were propogated by SPF chicken embryos, concentrated for the BALB/c mice immunization. Three methods were applied for screening NDV antibodies. Using NDV-HN prokaryotic expression protein ELISA for antibody screening, 3 strains named D5D2, D5G2 and D6A2 were obtained. None of them had hemagglutination inhibition activity and neutralizing activity. 5 strains of antibodies screened by NDV virus ELISA method named D1B4 and D1D9. D3F1, D4F8 and D5E11, with 2 strains had hemagglutination inhibition activity and all 5 had no neutralizing activity. By hemagglutination inhibition test for the antibody screening, 3 strains named D1H12, D5C5 and D3C2 were obtained. D5C5 in embryo neutralization test indicated that the neutralization of virulent F48E9 strains, moderate toxicity Mukteswar strains and attenuated LaSota virus neutralization titers were greater than 1:2048. Truncated expression was utilized to identify the epitope that D5C5 recognized, and 353DEQDYQIR360 was a neutralized epitope of NDV.An antigen-capture enzyme-linked immunosorbent assay(AC-ELISA) and a colloidal gold test were developed for the detection of NDV based on two monoclonal antibodies against HN protein and a polyclonal anti-serum. The specificity of the optimized testing methods was evaluated using NDV-F48E9, Mukterswar LaSota strains, and avian pox virus, infectious bronchitis virus, infectious laryngotracheitis virus, goose plague virus, mycoplasma gallisepticum, infectious bursal disease virus, avian influenza virus. The specimens that showed strong signal were NDV-F48E9, Mukterswar LaSota strains, indicating that this assay is suitable for the detection of NDV. The limit of detection of AC-ELISA was 10-3 dilution as same as RT-PCR, and colloidal gold test could detect 10-2 diluted allantoic fluid in five minutes.MAbs also play an important role in the study of the interaction between viral proteins and host proteins. The DF1 cells infected by highly virulent NDV strains F48E9 were lysed and immunoprecipitated with anti- NDV- HN mAb with neutralizing activity. The host cellular proteins that interact with the HN of NDV were identified by LTQ mass spectrometry analysis. Two proteinscaveolin and apolipoprotein B were identified. Caveolin is a protein component of caveolae membrane coats, which involves in caveolae formation and location. It also involves in endocytosis, endosome vesicle transportation, signal transduction, cholesterol homeostasis and transportation. Apolipoprotein B plays vital role in the transport and metabolism of lipids and cholesterol.To generate CDV antibodies, CDV-F protein was expressed by prokaryotic expression and CDV was amplified in Vero cells. Virus was purified by sucrose density gradient centrifugation. CDV-F protein and purified virus were used to immunized BALB/c mice separately. 2 strains of antibodies named D1G8 and D3B1, were obtained by CDV-F ELISA, but the reaction of antibodies and virus was week. Both of the antibodies showed no neutralizing activity. CDV-ELISA combined with virus neutralization test methods were utilized for screening neutralizing antibody. One strain named DC2 was obtained with a 1:3200 neutralizing titer. The result showed the aim of the using of antibodies should be clear at the very beginning. Based on the character of proteins or the use of antibodies, successful rate would be significantly increased.NDV and CDV high neutralizing activity antibodies D5C5 and DC2 were tested the therapeutic effect in vivo. NDV moderate virulent vaccine Mukteswar strain were selected for chicks’ infection to establish NDV model, determined to attack toxic dose for 106EID50/chick, after neutralizing antibody in muscle injection and chick survival rate increased from 15% to 60%, it showed a clear therapeutic effects in vivo. CDV nucleic acid positive infection fox was injected by mAb DC2. After the injection, status of fox was improved significantly and no CDV nucleic acid detection in oral, nasal swabs and blood. Two strains with neutralizing activity were aimed to sequence variable region, based on 5’RACE, high degenerate primers and nested mixed primer PCR methods, and ultimately determined that the mixed nested primer PCR has high sensitivity and specificity of mAb sequencing can be used in this method. The sequence of variable regions of heavy chain and light chain of D5C5 and DC2 were obtained. Variable regions were amplified by PCR and connected with the IgFc segment of mouse, and whole Ig DNA segments were obtained.Recently, flow cytometry screening individual B lymphocytes secreted antibodies has been a new research hotpot. In present study, anti-CD19-FITC was used as a probe to identify and sort fox B lymphocytes, which were isolated from peripheral blood lymphocytes of economic animal fox. The specific binding between anti-CD19-FITC with the fox B lymphocytes provides technical support for future fox related disease antibody screening.In this study, the methods of antibody preparation and screening were used to analyze and explain the strategy of obtaining the antibody. From the point of view of mAb application, the mAbs obtained in this study were investigated as a testing tool, the establishment of diagnostic methods as well as in vivo treatment and other aspects of the application. In addition, the commonly used methods of epitope identification were compared and analyzed, and three epitopes were identified. The variable region of two mAbs with neutralizing activity was sequenced, and connected with the mice IgFc segment to obtain a complete DNA. Also, the mAb that can be used for flow sorting of fox B lymphocytes was determined. These mAbs and their applications contribute to the preparation, identification and application of animal antibodies in future studies. |
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