Preparation Of Monoclonal Antibodies Against Helicobacter Pylori UreB And Development Of A Sandwich ELISA

Helicobacter pyori is a spiral shaped, microaerophilic, Gram-negative bacterium. It has been classified asa group I carcinogen by the World Health Organization. Infection with Hp highly associated with chronicactive gastritis, gastric ulceration, and doudenal ulceration, lymphoma of mucosa-associated lymphoid tissueand gastric. Currently, More than50%population infects in the world. Hp produces a large amount of urease,which is essential for the survival and pathogenesis of the bacterium. Urease subunit B (UreB) in particular hasproven to be more protective. This research includes the following three parts:The UreB gene of Hp was amplified by PCR, digested with restriction enzyme and inserted into plasmidpET-28a. The results indicated that the homology of nucleotide sequence of the cloned UreB gene was to96.73%in comparison with the reported26695standard strain sequences in GenBank, while the homology ofits putative amino acid sequence was99.30%. The recombinant expression vector pET28a-UreB wastransformed into the E.coli BL21(DE3) strains, recombinant protein expressed by IPTG induction.SDS-PAGE analysis showed that the molecular weight of recombinant protein was about66kDa and existedin the medium supernatant. BALB/c mice were immunized with purified recombinant UreB protein, antibodytiter was measured by ELISA assay.The immunoreactivity of recombinant protein was identified byWestern-blot. The purified recombinant protein could react with serum from patients infected by Hp inWestern blotting; specific humoral immune response could be induced by purified protein, which showed thatthe recombinant protein had satisfied immunogenicity. The results showed that recombinant protein wasexpressed successfully and showed strong immunogeicity.Female BALB/c mice were immunized with purified recombinant UreB from Hp and McAb against UreBwas prepared with hybridoma cell fusion technique, positive clones of hybridoma cell strains werescreened.The secreted McAb was identified for specificity by indirect ELISA, determined for titer in cellculture supernatant and ascites McAb by indirect ELISA, then identified for subtype, and the stability ofhybridoma cell strain was analyzed.9hybridoma cell strains stably secreting McAbs were obtained by cellfusion and screening of clones, and the secreted McAbs showed no cross reactions with other intestinalbacteria but specific reaction with whole cell, the subtype of identified McAbs were IgG1、IgG2a、IgG2b,SDS-PAGE showed molecular weight of heavy chains of the9McAbs is50kDa roughly and the light chain is25kDa approximately. The titers of McAbs in culture supernatants of the nine hybridoma cell strains were1:1280~1:5120, while those in ascites were1:2.6×103~1:5.1×106. High titer McAbs were secreted from thehybridoma cells after repeat storage in frozen.A double-antibody sandwich-ELISA was developed to detect Hp, using6H4as the capture-antibody and6F11-HRP as the detection antibody. Stool specimens were collected from49patients who had gastro copy forgastrointestinal symptoms. Double-antibody sandwich ELASA was used to determinant Hp antigen in the stool,Hp status was evaluated by the14C-UBT tests. With14C-UBT,31cases had positive results and the other18had negative result. With A double-antibody sandwich-ELISA,29cases out of the31positive cases werediagnosed with the same positive results and the other2were negative.18cases with negative results, Adouble-antibody sandwich-ELISA found18negative. Compared with the14C-UBT method, double-antibodysandwich-ELISA is a simple, reliable, non-invasive method and has highly sensitivity and specificity and maybe used as a routine test of Hp infection in clinical laboratory.