Fig. 2.

Detection of H. pylori urease in the bacterial culture supernatant by sensitive ELISA using H. pylori urease-specific MAb. (A) Urease-positive H. pylori strains (isolates SS-1 and NCTC 11637) were cultured in brucella broth containing 5% horse serum for 2 days, and cell-free culture supernatant (culture sup.) was collected carefully and further sedimented by centrifugation (3,000 rpm for 5 min at 4°C) to remove debris. Compared with positive controls containing purified H. pylori urease (100 μg/ml), H. pylori urease was only slightly detected in the bacterial culture supernatant. Results are expressed as means ± SDs (n = 5). Statistical significance was determined by the Student t test, and the values are given in the graph. (B) We then tried to confirm the results by Western blotting by using whole urease-specific antibody from immune rabbit sera and two H. pylori urease-specific MAbs, L2 for the large subunit (UreB) and S2 for the small subunit (UreA), as described in Materials and Methods. We could detect both the large subunit (UreB) and the small subunit (UreA) in the culture supernatants of both SS-1 and NCTC 11637 compared with purified urease obtained from the bacteria. We have confirmed that HPP1801, a urease-negative mutant from the NCTC 11637 isolate, did not express urease in its lysates.