Development Of Monoclonal Antibodies Against HP And Its Application In Immunological Assays

Helicobacter polori(Hp)is a micro-aerobic gram-negative bacteria,which settle inthe hunman stomach and duodenum.It is one of the main risk factors of the occurren-ce and recurrence of peptic ulcer,chronic active gastritis,astric mucosal-associatedlymphoid tissue lymphoma,gastric cancer and other gastrointestinal diseases pepticulcer.The World Health Organization has listed it as class I carcinogen.More than halfof the world’s population is infected with Hp.The incidence of Hp infection is veryhigh in China,so Hp detection has important practical significance.Hp strain NCTC11637(ATCC43504) cultured in the Columbia Blood Agarmedium was sonicated and used as the immunogen. The Hsp60and UreaseB genewere amplified by PCR and pET28a-UreaseB and pET22b-Hsp60plasmid weresuccessfully constructed, Recombinant Hsp60and UreaseB were successfullyexpressed in E. coli as soluble form and used for monoclonal antibodies screening.Hp NCTC11637(ATCC43504) strain lysis was used as immunogen toimmunize Balb/c mice for monoclonal antibody preparation. After four times ofimmunizations mice were sacrificed and the spleen cells were fused with SP2/0myeloma cells.21stable hybridoma cells secreting anti-Hp monoclonal antibodies(mAbs) were established after indirect enzyme-linked immunosorbent assay（ELISA）by using HP strain lysis, recombinant Hsp60and UreaseB as antigens. MAbs werepurified from the ascites by ammonium sulfate precipitation methods.The monoclonal antibodies were labelled by horseradish peroxidase (HRP).51different coating and labeling mAb pairs that capture Hp in a sandwich ELISA wereobtained and6of them yielded a limit of detection (LOD) of0.1ng/mL.37differentcoating and labeling mAb pairs that capture Hp in a sandwich format were screenedby colloidal gold immunochromatographic assays (GICAs). Two antibody pairsconsisting of capture (mAb2-18E1) and detector (mAb2-9H5, mAb2-17E9) thatshowed a relatively higher sensitivity and better specificity to Hp were selected for later GICA tests. Our method yielded a limit of detection (LOD) of25ng/mL and theresults indicate that the developed GICA method could provide a convenient andefficient approach to detect Hp and has potential to be widely used.