J Bacteriol Virol.  2018 Mar;48(1):14-22. 10.4167/jbv.2018.48.1.14.

Characterization of Specific IgA Response to Antigenic Determinants of Helicobacter pylori Urease Encoded by ureA and ureB in Children

• Shin MK
• Jun JS
• Kwon SW
• Lee DH
• Ha JH
• Park JS
• Song DH
• Jung MH
• Kang HL
• Baik SC
• Park JS
• Youn HS
• Cho MJ
• Seo JH
• Lee WK

• KMID: 2409050
• DOI: http://doi.org/10.4167/jbv.2018.48.1.14

Abstract

Helicobacter pylori (H. pylori), a causative agent of chronic gastritis and gastric cancer, has several virulent factors for own survival and progression toward gastric diseases in human stomach. Of those, H. pylori produces mainly urease (10~15% total protein weight) that neutralize the gastric acid for survival. Here, we identified the antigenic epitope of urease and then developed an ELISA using the antigen including the epitope of urease. We identified the antigenic epitope of urease that induces IgA antibodies in human using truncated mutants. Eight kinds of serially-truncated mutant of UreA and UreB were prepared and subjected to immunoblot using pooled sera of patients with gastric disorders. UreBEnd protein containing UreB epitope was produced and investigated its diagnostic value via ELISA in children. As a result, mutants having last 24 amino acid residues of UreB carboxyl terminus deleted did not show IgA-reactive band. The clones that contained the downstream of 448(th) amino acid in UreB showed IgA-reactive band. The serodiagnostic value of the UreBEnd recombinant protein including identified epitope was confirmed via IgA ELISA and shown to have 97% sensitivity and 100% specificity. These results demonstrated that carboxyl terminal region of UreB carries an antigenic epitope for IgA response in human. It may be useful for detecting H. pylori infection with improved test accuracy and minimum use of endoscopy.

Keyword

Helicobacter pylori; Urease; Epitope; IgA; ELISA; Children

MeSH Terms

Antibodies
Child*
Clone Cells
Endoscopy
Enzyme-Linked Immunosorbent Assay
Epitopes*
Gastric Acid
Gastritis
Helicobacter pylori*
Helicobacter*
Humans
Immunoglobulin A*
Sensitivity and Specificity
Stomach
Stomach Diseases
Stomach Neoplasms
Urea*
Urease*
Antibodies
Epitopes
Immunoglobulin A
Urea
Urease

Figure

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  Figure 1. Schematic diagram of truncated mutant clones of UreA and UreB

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  Figure 2. Reactivities of human antisera to truncated mutant proteins of UreA (A) and UreB (B). The truncated mutant proteins of UreA and UreB were expressed from pET15b clones. Reactivity of the proteins were analyzed by patient's sera. Lane M, molecular weight markers; 1, UreA; 2, UreA142; 3, UreA85; 4, UreB; 5, UreB546; 6, UreB495; 7, UreB412; 8, UreB244; 9, UreB134; 10, UreB448.

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  Figure 3. Production of recombinant UreBEnd protein. The recombinant UreBEnd protein was purified from pEGexs/UreBEnd clone by Glutathione column. Reactivity of the UreBEnd protein was analyzed with patients' pooled sera. Lane M, molecular weight markers; 1, Non-induction; 2, the whole cell lysate after IPTG induction; 3, the supernatant of whole cell lysate after IPTG induction; 4, the pellets of whole cell lysate after IPTG induction; 5, elution; 6, thrombin treated elution; 7, vector control;8~11, elution 1~4.

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  Figure 4. Location and sequence of antigenic determinant of UreB. Colored underlined sequence denotes the epitope sequence. Underlined numbers indicate 3′-termini's amino acid site of truncated mutant proteins.

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  Figure 5. IgA distribution of sera from the patients with/without gastric symptoms measured by ELISA using purified recombinant UreBEnd. (A) Dashed line indicates the average of absorbance value: negative, 0.35; strong, 0.96; weak reactivity to CLO test, 0.95. (B) Receiver operating characteristic (ROC) curves of UreBEnd ELISA for the diagnosis of H. pyloriinfection. The area under the ROC of UreBEnd ELISA was 0.997 (95% confidence interval; p< 0.001).

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