Identification Of The Neutralizing Monoclonal Antibody To Newcastle Disease Virus And Analysis Of Its Directed Epitope

The hemagglutinin-neuraminidase (HN) protein and the fusion (F) protein of Newcastle disease virus (NDV) are the principal targets of neutralizing and protective antiboedies against Newcastle disease. Newcastle disease (ND) is regarded worldwide as one of the most devastating diseases for poultry. Newcastle disease virus (NDV) is classified as one member of the family of Paramyxoviridae. Because of the severe nature of the disease and the associated consequences, ND was included as A class disease on the office International of Epizooties (OIE) list. ND in the chicken flocks has been more and more frequently reported since the late1990s, even some chicken flocks with high antibody level can also suffer ND. Some scholars think that the emergence of new genotypes or subgenotypes could be responsible for NDV outbreak in vaccinated flocks. So the vaccine of NDV is key point. This study focus to screening of the neutralization monoclonal antibodies producted in our laboratory and to further analysis yheir epitopes to provid theoretical basis for the research of vaccine of NDV.First the titers of three Mab samples, F306, L27and H55, were detected with the indirect ELISA respectively. The antigen of NDV were diluted from1.5μg/mL to0.04μg/mL, the polyclonal antibodies of NDV were diluted form1:500to1:16000. A matrix titration results indicated that the optimal concentration of NDV for coating was0.37ug/ml and in4℃overnight. The ascites of F306, L27and H55were diluted gradiently from1:1000, and then reacted with coated NDV antigen with the indirect ELISA respectively. The rusults showed F306didn’t react to NDV, the titers of L27and H55were1:16000and1:32000respectively. Acroding to the calss appraisal kit, F306and L27belong to IgGl, H55was belongs to IgG2b. Then these Mabs were purified by a saturated ammonium sulfate method for the next experiments.Secondly, the ability of neutralize NDV of L27and H55was evaluated in a micro cell culture for neutralization test. BHK-21was inoculated with NDV in micro cell culture plate for evaluating its titre of TCID50. Then the liquid containing NDV was diluted as a work concentration, and after a reaction of DNA with the diluted ascites of L27and H55, the mixture were added into micro wells respectively, in which a cell monolayer of BHK-21was cultured, and Cytopathic effect (CPC) was farther observed. Acoording to CPE and Reed-Muench the dose of antiserum protects50%of cell challenged (PD50) of Mabs was evaluated respectively. The results showed that only H55was the protection antibody, its dose of the ascites counted for10-3.5, comparison of the control without antibody the cell cytopathic delayed for13hours. The other Mabs had no protection.Finally, analyse the epitopes of F306and H55. The result in Western blot showed that the two Mabs could not react with NDV antigen due to no band in the test. This suggests the Mabs could only recognized the conformation epitopes. In the12-mer peptide library of phage display the some phage clones were screened with H55and F306after three rounds of biopanning respectively. According to sequencing and homologous analysis with Blast the amino acid sequence of IQPRMINS(M)PRPfor H55was obtained.In summary, in this study a neutralization Mab (H55) to NDV from three Mabs in our laboratory producted was screened, and two amino acid sequences of epitopes for NDV were identified with a phage display random12-mer peptide library respectively. These results will be basis for further studies of disease mechanism of ND and vaccine research.