Production of Polyclonal Antibody Against Alkyl Hydroperoxide Reductase ofHelicobacter pyloriand Its Antigenicity

Abstract

Stool-antigen detection kits for diagnosis of Helicobacter pylori infection have been widely used because of their full non-invasive nature. Because Helicobacter pylori strains show a distinctive genetic diversity, it is important to find a protein that is a common antigen of various strains and shows a strong immunogenicity for the development of a stool- antigen detection kit. Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori strongly reacts with the sera of patients with gastritis and peptic ulcer. Therefore, AhpC seems to be an excellent candidate as a target protein for this study. Accordingly, polyclonal antiserum against AhpC was produced in adult New Zealand white rabbits by using AhpC in the gel bands without adding Freund's adjuvant. In addition, isolation and purification of AhpC were perfomed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. In this study, a simple method was used for rapid production of polyclonal antibody against AhpC of H. pylori, which avoids both the long-term AhpC purification and the addition of Freund's adjuvant. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high-resolution capacity of this technique leads to a high level of purity of the protein and consequently to a very high specificity of the antibody. Moreover, this method avoids contamination by other non-specific proteins, which often appear during the purification process by column chromatographic techniques, which may also decrease the purity of the immunogen. The present method is simple, rapid and cost-effective, and also makes it possible to produce antibody for stool-antigen enzyme immunoassay in a short time and at low cost.

Antigenic Diversity Among Portuguese Clinical Isolates of Helicobacter pylori

“…However, autoimmunity problems should be considered, as amino acid sequence alignment revealed that H. pylori AhpC is more closely related to mammalian than to eubacterial homolog [40]. This favors the use of antibodies against H. pylori AhpC for H. pylori detection in stool samples, as proposed by others [42]. Spots 15a, 15b, and 15c having the same molecular weight but different pIs (between 6.5 and 5.9) were identified as AhpC (Table 2), corroborating the existence of different isoforms of this protein in result of post-translated modifications [34].…”

“…AhpC, a member of the thiol-dependent 2-Cys peroxiredoxin family, is a highly abundant cytosolic H. pylori protein [39] responsible for the reduction of toxic organic hydroperoxides to their corresponding alcohols [38]. AhpC may also function as a stress-induced molecular chaperone by forming high-molecular-weight complexes [40] and possesses mucin-binding activity once expressed at the external surface of H. pylori [41,42]. H. pylori AhpC appears to be essential for growth and colonization as AhpC-deficient mutant strains are highly sensitive to oxidative stress conditions and are not able to colonize mice [38].…”

Diagnosis of Helicobacter pylori Infection

“…Mohammadian et al. presented a simple, rapid, and cost‐effective production of a polyclonal antibody against alkyl hydroperoxide reductase (AhpC) of H. pylori for stool‐antigen enzyme immunoassay [23]. Nguyen et al.…”