used immunized spleen cells as the primary source for their anti-monoclonal antibodies. gastro-duodenal diseases, such as gastric and duodenal ulceration, gastric atrophy, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT)-type lymphoma. During the past few years it became evident that it is not only certain bacterial virulence factors, such as the vacuolating cytotoxin VacA and some of the products of the Cag pathogenicity island (Cag PAI), that determine the pathogenesis of the infection. It has been shown that differences in the hosts’ immune responses are responsible for the various inflammatory patterns in the gastric mucosa and for the development of certain clinical complications of the infection. There is increasing evidence that Rabbit Polyclonal to ARFGAP3 a predominant T-helper-1 response seems to lead to a more aggressive course of infection, while a predominant T-helper-2 response might be protective for the gastric mucosa (12, 26). Furthermore, about 30% of infection might give new insight into the pathogenesis of gastritis. So far, investigation of the humoral immune response has focused on the analysis of the polyclonal repertoire in human sera and/or mucosa. A more detailed insight into humoral immunity in infection could be achieved if monoclonal antibodies against antigens could be generated and characterized. However, to the best of our knowledge human monoclonal antibodies against antigens have been established only by one research group (16, 30). This might be due to the labor-intensive and time-consuming hybridoma technique. However, during the last few years a new method for the generation of human single-chain Fv (scFv) antibody fragments in bacteria has been developed and optimized (3, 20, 21). Instead of immortalizing human B cells for the production Lestaurtinib of monoclonal antibodies, the genes coding for the variable regions of the heavy and light chains of human immunoglobulins (V genes) are amplified by reverse transcription-PCR and cloned into (XL1-blue). For the generation of antibody fragments of a desired specificity, the amplified V-genes are expressed on the surface of filamentous phage M13, and antigen-binding scFvs are isolated by affinity selection. Human scFvs against several relevant antigens have already been generated and have brought new insight into the pathogenesis of various disorders, such as neoplastic (14, 25), infectious (2), and autoimmune diseases (7, 13). So far, only one human scFv against has been described (17). However, the V-gene repertoire used in that study was derived from uninfected donors (29). Therefore, the aim of our study was to construct an immune V-gene library using peripheral blood lymphocytes (PBLs) of an antigens. Two different antigens were used: first, a lysate of the Sydney strain (19), and second, the recombinant urease, which is known to be a Lestaurtinib major immunogen and was used in several vaccination trials (22). MATERIALS AND METHODS Assay of donor serum for presence of anti-antibodies. Sera of 21 patients with upper abdominal complaints were screened for antibodies against using a standard enzyme-linked immunosorbent assay (ELISA) test (Pyloriset; Orion, Espoo, Finland) according to the manufacturer’s instructions. Additionally, infection was tested by routine histological analysis of gastric biopsy specimens using hematoxylin-eosin (H&E) and Warthin-Starry stains. cDNA synthesis and PCR amplification of human V genes. Peripheral blood mononuclear cells were purified from 10 ml of peripheral blood that was taken from the antibody titer. This 86-year-old female patient had chronic active and antrum-predominant gastritis. Poly(A)+ RNA was isolated from the isolated cells using the Oligotex Direct mRNA Kit (Qiagen, Hilden, Germany). Lestaurtinib Eluted RNA (10-l samples) was used for oligo(dT)12-18-primed first-strand cDNA synthesis with the reverse transcriptase Superscript II (Life Technologies, Karlsruhe, Germany) according to the supplier’s protocol. The variable parts of the heavy chains (VH) and light chains (VL) of human immunoglobulin genes (V genes) were then amplified by PCR using a set of 15 primers that was previously described (31). Reaction mixtures (50 l) were prepared containing 1 l of cDNA, 20 pmol each of forward and back primers, 200 M concentrations of deoxynucleoside triphosphates, 5 l of reaction buffer (10), 1 mM MgCl2 and 0.2 l (1 U) of DNA polymerase (HotGoldstar; Eurogentec, Cologne, Germany). The reactions were overlaid with mineral oil and subjected to 30 cycles of.
used immunized spleen cells as the primary source for their anti-monoclonal antibodies