Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library.

Lpp20, an outer membrane protein of Helicobacter pylori (H. pylori), has been identified as an immunodominant antigen. To obtain mouse monoclonal antibodies (mAbs) against it and to map its antigenic epitope is potentially to develop a vaccine for prevention and treatment of H. pylori infection. In our study, the Lpp20 gene was obtained from H. pylori genomic DNA by PCR (GenBank accession no. DQ106902), cloned into pGEX-4T-1 vector and expressed in Escherichia coli (E. coli) as a recombinant fusion protein with glutathione-S-transferase (GST), which was purified by GST-affinity chromatography. mAbs were produced by the hybridoma technique using Lpp20-GST as the immunogen. Using mAb as the target molecule and immunoscreening phage-displayed random dodecapeptide library (Ph.D.-12), the positive phage clones were sequenced and analyzed. Phage clones were chosen to immunize mice to evaluate the potential of phagotopes as effective vaccines. One mimotope (SWPLYSDASGLG) showed a good match with the Lpp20 proteins at 114-117aa (DASG) and the serum of mice induced by the phage clone clearly recognized Lpp20 protein. Our work suggests that the antigenic epitope could be mapped through screening the phage-displayed peptide libraries with mAb and a mimotope of Lpp20 providing an alternative approach for the diagnosis and development of a vaccine for H. pylori.

[Preparation of monoclonal antibodies against multiple antigens].

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against multiple antigens by single cell fusion. METHODS: BALB/c mice were immunized with the multiple antigens, namely alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), HBsAgiHBcAg and HBeAg, and hybridomas were employed using PEG as the fusing agent. The hybridoma cells were respectively screened with AFP, CEA, HBsAg, HBcAg and HBeAg by enzyme-linked immunosorbent assay and limited dilution. The mAbs were purified by protein G affinity chromatography, its subtype was identified, the affinity constants (K(a)) were determined and the specificity was analyzed by Western blotting. RESULTS: Twenty hybridoma cell lines were obtained by single cell fusion, including 5 cell lines against AFP, 6 against CEA, 3 against HBsAg, 4 against HBcAg, and 2 against HBeAg. The subtypes of some hybridoma cell lines positive for the mAbs were identified as the immunoglobulin G1 (IgG1), with K(a) ranging from 1x10(9) M(-1) to x10(11) M(-1). Western blot analysis showed that all the mAbs strongly and specifically bound to their respective antigens. CONCLUSION: The mAbs against multiple antigens have been obtained by single cell-fusion, which increases the production of mAbs and reduces the time of preparation.

[Preparation and characterization of monoclonal antibodies against erythrocyte-binding antigen 175 of Plasmodium falciparum].

OBJECTIVE: To prepare and characterize the monoclonal antibodies (mAbs) against erythrocyte-binding antigen 175 of Plasmodium falciparum (EBA-175). METHODS: BALB/ c mice were immunized with purified recombinant EBA-175 and mAbs against EBA-175 were prepared by means of hybridoma technique. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were employed for characterization of the mAbs. RESULTS: Six McAbs against EBA-175 antigen were obtained, 5 of which were identified as IgG1 and one as IgG2a. The titer of these aAbs was 1:12,800 to 1:25,600 in the ascites and 1:256 to 1:512 in supernatant, and ELISA demonstrated specific binding of the 4 mAbs (1F3, 2H5, 4A1 and 4H9)with Plasmodium falciparum. Three of these mAbs recognized the protein of EBA-175 as shown by Western blotting. CONCLUSION: Six hybridoma cell lines secreting the mAbs against EBA175 with high specificity are successfully established.

[Preparation and identification of monoclonal antibodies against Helicobacter pylori].

OBJECTIVE: To prepare and identify monoclonal antibodies (mAbs) against Helicobacter pylori (Hp). METHODS: BALB/c mice were immunized with the supernatant and precipitation of cultured Hp after ultrasonication and mAbs were obtained by means of hybridoma technique. The resultant mAbs was evaluated for subtype, titer, affinity, and further identified with Lpp20, HspA, urease A, CagA, urease B, and catalase prepared by recombinant expression. RESULTS: Totally 34 hybridoma cell lines were established which secreted specific mAbs, including 31 against the supernatant and 3 against the precipitation of Hp, and the prepared mAbs showed specific reaction against Lpp20 (3 strains), HspA (2 strains), urease A (4 strains), CagA (1 strain), urease B (5 strains), and catalase (2 strains) antigens, respectively. The mAbs was all identified as immunoglobulin G1 (IgG1) and theirs titer in the culture supernatant and ascites was 1:16 to 1:32 and 1:32000 to 1:64000 respectively with affinity constants (K(aff)) ranging from 1 x 10(-10) to 5.2 x 10(-12) mol/L. CONCLUSION: The mAbs specially against Hp have been obtained, which may facilitate further study of detection and vaccine development of Hp.