Preparation Of Monoclonal Antibodies Against Canine Distemper Virus H Protein And Development Of Competitive ELISA

Canine distemper(CD)is an acute,fever,highly contagious disease caused by canine distemper virus(CDV),which can invade respiratory,digestive,nervous systems,and leading to immunosuppression.In recent years,CD exhibited new epidemic characteristics in China,including rapid mutation of the strains,continuous expansion of the infection spectrum,and widespread mixed infections.The immunosuppression caused by CDV lead to the failure of immunization.The direct and indirect economic losses to the canine industry and fur animal breeding are huge.Preventing and controlling CD has become a major scientific and practical problem.Thus,this study will understand the epidemic situation of CD in Jiangsu,analyze the genetic variation of CDV H protein,and enrich the CDV epidemic strain resource library.At the same time,the CDV monoclonal antibody hybridoma cell library will be established and expanded,and monoclonal antibodies with broad-spectrum neutralizing activity will be screened out.Based on recombinant H protein and monoclonal antibody,CDV antigen and antibody competitive ELISA method was established for pathogenic and serological diagnosis,providing technical support for CDV infection monitoring.The main research contents include:Sixty samples suspected canine distemper in Jiangsu province were collected from 2017 to 2019.Using RT-PCR method,twenty-two samples were identified as positive for the diagnosis of CDV nucleic acid.Through amplifying complete H gene,nucleic acid sequencing,BLAST,homology comparison,genetic evolution analysis and statistical analysis of N-glycosylation sites showed that eighteen America-1 strains were highly homologous to the vaccine Ondetstepoort strain.Four strains belonged to Asia-1 type strains(named YZ-4,NJ-13,NJ-21,YZ-27),and the amino acid homology with the vaccine strain was between90.1%-91.2%,which was high homology to the Asia-1 type strains(99.7%-100%).The H protein of four Asia-1 strains had potential N-glycosylation at positions 584-586,which had the molecular characteristics of the virulent Asia-1 strains.In addition,the SLAM receptor binding region of H protein from the YZ-4 strain,NJ-21 strain and YZ-27 strain has not changed(519R/530G/549Y),while that of the NJ-13 strain is mutated to 519R/530G/549H,suggesting that the binding ability to the host receptor may be changed.In this study,the hybridoma cell line library of CDV monoclonal antibody was generated and expanded,and the seven hybridoma cell lines(named as 3B1,3B5,3C5,3D5,5A6,4G12 and 7B2)with stable secretion of anti-H protein monoclonal antibody were screened by indirect ELISA method of recombinant H protein.The subclasses of antibodies produced by hybridoma cell lines were identified as Ig G1?type.Indirect immunofluorescence test showed that the monoclonal antibodies could react with CDV,rather than reacting with canine parvovirus,canine coronavirus and canine influenza virus,indicating that these monoclonal antibodies had good specificity.The virus neutralization titers of the monoclonal antibodies were 2⁴?2⁸ and 10²?10⁶in the cultural supernatant and the ascetic fluid,respectively.Among them,the superposition coefficient between 3B5,4G12,5A6 and 7B2 was above 50%,indicating that these four monoclonal antibodies recognize different epitopes.The continuous passage test showed that the hybridoma cells still had the ability to secrete antibody efficiently in the20th generation.In addition,these four monoclonal antibodies could also neutralize the Asia-1 type strains with good broad-spectrum neutralization activity.Among them,the neutralization titer of 3B5 anti-Ondetstepoort strain and anti-YZ-4 strain were 2⁸ and 2⁷,respectively.Based on the recombinant H protein and 3B5 monoclonal antibody,the competitive ELISA for CDV antibody was established.The optimal coating antigen(recombinant H protein)concentration of CDV antibody competition ELISA detection method is 4ug/m L;the optimal dilution concentration of HRP enzyme labeled 3B5 antibody is 1:3 200;the optimal dilution concentration of serum to be tested is 1:8;the serum sample inhibition rate(PI)?45%is considered positive.Compared with foreign commercial canine distemper antibody kits,this method has a coincidence rate of 97%,which has good specificity and sensitivity.In this study,based on the recombinant H protein and 3B5monoclonal antibody,the competitive ELISA for CDV antigen and antibody was established.Using square matrix titration to determine the optimal coating antibody(3B5)concentration is 2?g/m L and the optimal dilution concentration of HRP enzyme-labeled recombinant H protein is1:1 600.After optimization of reaction conditions,the minimum virus detection amount of this method was 10³TCID₅₀/m L,with good specificity and repeatability.The coincidence rate of this method and RT-PCR for detecting samples at the same time is 100%.In summary,this study carried out a molecular epidemiological survey of CDV in Jiangsu Province,analyzed the genetic variation of epidemic strains and summarized its epidemiological situation and molecular variation in a timely manner.Meanwhile,this experiment expanded the CDV strain resource database and CDV monoclonal antibody hybridoma cell line library,screened anti-H protein monoclonal antibodies with broad-spectrum neutralizing activity,which the epitope recognized by 3B5 monoclonal antibody was reported for the first time;Based on these,the competitive ELISA for CDV antigen and antibody was established to improve the detection methods of CD etiology and serology and to evaluate the level of CDV infection,so as to provide technical support for scientific prevention and control of CD.