Purification Of Helicobacter Pylori Urease And Preparation Of McAb Against Urease From Helicobacter Pylori

Suuunary H. pylory NCTC 11637 were inoculated in HP broth supplemented with 5 % (v/v) horse blood in a flask on a gyratory water bath shaker at 150 rpm and incubated under microaerobic conditions for more than 72 h at 37G. The crude were precipitated from the fitrates by using a 50% (v/v) saturated solution of amnionium sulphate. The purified was extracted on a Sourse Q15 column by ion- exchange chromatography.The eluate from the colunm were analyzed by SDS- PAGE. It is indicated that the urease of H. pyloiy was consisted of two subunits, UreaB(65 Ku) and UreaA(29.5 Ku). The Balb/c mice were immunized intraperitoneally with the crude urease plus 2 CFA/IFA, B lymphocytes hybrized with myeloma cells SP/0 by PEG(MW4000). The antibody to urease from H.pylori were screened out by indirected ELISA. Using limited dilution four times cloning and subcloning, one strain of hybridoma cells(HPUD53) which secrete antibody(McAb D53)against Hpy?ori urease was obtained. HPUD53 was characterized by better stability and more chromosome numbers(100 + 5). There were four characteristics of McAb 1)53: (II) McAbD53 belonged class IgG, subclass IgG2a; ?McAb 1)53 possessed of higher titer, titers of McAb D53 from both supernant and ascite was 1: 10(s~ ?McAb D53 had higher specificity, no cross-reactions occurred to E. coil. Campvbacter jejunz, Salmonella, Shigolla and so on; ?McAb 1)53 is antibody against ureafl from H.pviori. In indirected Enzyme-Linked Immunosorbent Assay(ELIS A), an unknown test antigen was coated to surface of the wells of a niicrotiter plate, the McAb D53subunit ureai3 specific McAb was added .After washing, the presence of the test antiQen was detected by addition of the second-color development antibody sheep against mouse IgG,A.M) labelled with an enzyme AKP and enzyme substrate, following incubation a color reaction occurred. The ELISA for detecting urease from H.pviori was developed. This method is very acurate. higher sensitivity and higher specificity. therefore, it would be useful for screening H.p yiorz infection. With the assumption that antigen minimum of ihis method results was 0.2 p.giml for urease, and 4 X 106 cells/mI for H. pylon, respectivel...