Preparation And Characterization Of Monoclonal Antibodies Against The Outer-membrane-protein Of Bordetella Avium

Bordetalla avium disease was a high-contacted infectious disease.In our country,it was discovered by Rui-liang Zhu in 1990.It could arouse that chicken-embryo death;low incubation;chicking acuting death and chicken opHthalmia so on.The infection rate of this disease was 10%~50%,it could bring up illness-chicken grew up lowly, the low redound upon of feedingstuff,and great economy loss.In China, the pathogen can induce the death of embryo in hatching and the acute death of chicken after birth. Chicken,less than one-week,is most susceptive.The diseased chicken charactered by rhinotrachititis,depressing,food uptake reduce,and the most died of exhaust or infection by other pathogens.The chickens more than one-month have some resistance to this disease,and the mature chicks do not present symptom after infection,but the eggs can be contaminated,and it causes vertical spread of this disease.Recent years, the immunity of outer-membrane-protein of bacteria in cell-wall was paid attention to by science workers. The test testified that OMP with well immunity could stimulate not only humoral immunity and cell immunity.Because the traditional methods of microbe seperation and identification need much time and effort,it can not aquire exact result in 2~3 days.But monoclonal antibody(McAb) is specially,sensitivivity and can identify single antigen dots .In this study ,Balb/c mice were immunized with the puirfied Bordetella avium OMP , the spleen cell was taken and fused with SP2/0 bone myeloma cell by 50% PEG 4000, we have successfully prepared Bordetella avium OMP specific McAb. This study will found an important base for further developing fast diagnostic method to detect Bordetella avium,analyse antigen dots and the system of bacterial typing . The research divides three parts:Part One: Development of Indirect ELISA for Detection of Antibodies to Bordetella avium OMPIn this study, out membrane proteins of B.avium strains P5 was prepared by improved Wooldridge method. We could determine contents of Bordetella avium OMP by Bradford, do SDS-PAGE electropHoresis of Bordetella avium OMP .The result showed that: the contents of Bordetella avium OMP was 640 ug/mL. The protein molecular weight of P5 strain is respectively 58,47,41,36,24KD. Then we purified Bordetella avium OMP . Balb/c mice were immunized with the puirfied Bordetella avium OMP , for preparation of high titer immuno serum. And then detections of indirect ELISA was established by optimization and screening of conditions. HRP-labelled goat anti mouse IgG diluted by PBST(pH 7.4,0.1 mol/L)in l:10000 was choosed as secondary antibody,coated antigen 10μg/mL Bordetella avium OMP dissolved in carbonate buffer solution(CBS,pH 9.6,0.05 mol/L),1.0%BSA as blocking agent,and 1.0%TMB as a substrate.Part two: Preparationstrains hybridoma cell lines and McAb of Bordetella avium OMPIn this study , balb/c mice were immunized with the puirfied Bordetella avium OMP . Then,we make use of the method Cell-fusion . Selected the high blood serum antibody titer Balb/C mice,the spleen cell was taken and fused with SP2/0 bone myeloma cell by 50% PEG 4000. The fusion rate was about 65%, positive clones were got by indirect ELISA screening, positive rate was about 12%. Subcloned to get monoclone cell lines by limit dilution. After fourth subcloning we got three strains hybridoma cell lines steadily secreting Bordetella avium OMP specific mono-clonal antibodies ,they were named 3G10,4A3and 4E8, monoclones were enlarged and frozen timely.by the strains hybridoma cell lines ,we prepared and purified Bordetella avium OMP specific McAb. Antigen dots and specific tests of the two McAbs were analysed.Part three: Characterization of monoclonal antibodies against the outer-membrane-protein of Bordetella aviumIn this study we identified the McAb of Bordetella avium OMP. The indirect ELISA titer for Bordetella avium OMP in culture supernatant were 1:320,1:640 and 1:640, the ascites obtained by mice were1:6400,1:12800 and 1:12800, Test for the subgroup of the monoclonal antibodies revealed that they were subgroup IgG1(2) andIgG3(1) . Western -blot test revealed, they were Bordetella avium OMP specific monoclonal antibodies. These strains hybrid- oma cell lines can steadily secrete Bordetella avium OMP specific monoclonal antibodies after three times frozen and resuscitated. It indicates that, we have successfully prepared Bordetella avium OMP specific McAb. This study will found an important base for further developing fast diagnostic method to detect Bordetella avium,analyse antigen dots and the system of bacterial typing .