Anti-bile Helicobacter Monoclonal Antibody Development And Initial Applications

Infection by Rodent Helicobacter on lab rodent animals has aroused remarkable attention by researchers mainly including Helicobacter bilis, and this kind of pathogen was gradually classified as one which has to be eliminated on rodent animals by Government Standard on microbiology mass detection in most countries. However, it hasn't been listed into our country's Government Standard owing to absence of one effective detecting method. So far the serological assays have been diffusely applied into lab detection of pathogenic germ infection in possession of sorts of advantages, such as high sensitivity, convenience and low expense, and its accuracy lied on the quality of the diagnostic antigens or antibodies. In this study we used hybridoma technique to prepare the monoclonal antibodies (McAbs) specific for H. bilis, and developed the double antibody sandwich ELISA (BASE) and indirect immunofluoescence assay (IFA) in use of these McAbs for the purpose to detect H. bilis on lab animals effectively and rapidly. With this reason, some aspects were researched as the following.First of all, McAbs specific for H. bilis were prepared and identified. Through immunizing BALB/c mice hypodermically with B2m H. bilis isolated from lab mice by ourselves and routine cell fusing process, we obtained 11 positive hybridoma subcell lines screened by ELISA. McAbs secreted by upper hybridoma cells didn't cross-react with 15 kinds of common pathogenic bacterial strains on lab animals and the highest value was greater than 1 : 4×10⁵; The IgG belonged to IgG_2a and IgG_2b subclasses ; Western blotting results indicated that immunodominant antigens in H.bilis binding 6 McAbs (A～F) were located at about molecular weight (17,20,21,30,52,66)×10³ respectively via WB while 5 McAbs (G～K) all at about (52,82)×10³, suggesting that McAbs A～F aimed at specific antigen H.bilis and McAbs G～K probably possessed of genus specificity.Further more, BASE and IFA based on McAbs were established, and the feasibility of their application in subclinical infection by H.bilis on lab animals was elementarily exploded.On BASE aspects, after fine pairing experiment the best two matches were screened out, i.e., using the McAbs D or E as the coating antibody and the McAbs C labeled by horse radish peroxide enzyme (C-HRP) as the diagnostic antibody. Their sensitivity in H. bilis antigen detection could achieve the nanogram level (ng), and the positive rates of 5/10 and 4/10 could be got when applied in mice cecum contents' detection in mice cluster infected with H. bilis.On IFA aspects, McAbs C was selected as the best diagnostic antibody after comparison between the six McAbs A～F. This McAbs could react with mice cecum contents in mice cluster infected with H. bilis, and as a result the spiral-shaped bacteria encircled with periplasm cilia could be clearly observed by fluorescence microscope, which is the typical characteristic of H. bilis. When compared with immune sera it could more easily eliminate false positive results because of possessing clearer background. This method could display positive rate of 6/10 when applied into in mice cecum contents' detection in mice cluster infected with H. bilis.In final term, polymerase chain reaction (PCR) was used to detect the upper positive mice cluster and finally positive rate of 8/10 was got. We could deduce that there is 50～60% rate coincidence when PCR was compared with BASE and 75% with IFA. Therefore, the conclusion could achieve that the two serological methods referred in this thesis could basically satisfy the requirements with subclinical infection detecting on lab rodent animals by H. bilis.In sum, 11 McAbs against H. bilis were prepared with high specificity and sensitivity and two detecting methods BASE and IFA based on them were established in order to detect H. bilis, which could basically satisfy the requirements with fine mass detection on lab animals when applied in infection detecting on lab mice by H. bilis. McAbs against several Helicobacter species prepared in this study would be used in further researches on detecting methods with significance. The upper results would make for establishment of serological assays of detecting H. bilis and other rodent Helicobacter species.