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    Cloning, expression and characterization of recombinant CagA protein of Helicobacter pylori using monoclonal antibodies: Its potential in diagnostics
    (Academic Press Ltd- Elsevier Science Ltd, 2020) Salih, Barik A.; Karakus, Cebrail; Ulupinar, Zeynep; Akbas, Fahri; Uslu, Merve; Yazici, Duygu; Bolek, Bora Kazim
    Helicobacter pylori CagA protein plays an important role in the severity of the gastric diseases. Our aims were to clone the cagA 5'conserved region of the gene, characterize the recombinant CagA (rCagA) protein by monoclonal antibodies (mAbs) and to use this protein for the detection of anti-CagA antibodies by an ELISA test. Our developed rCagA protein (67 kDa) showed an amino acid sequence homology of 83% and 80% with Western and East Asian type strains respectively. Two anti-rCagA (BS-53, CK-02) mAbs and 2 additional rCagA proteins of smaller sizes (60 kDa, 28 kDa) were developed for epitope determination. The BS-53 mAb recognized all 3 rCagA proteins while CK-02 mAb recognized only 2 of them indicating recognition of different epitopes. An in-house indirect ELISA using rCagA was developed to detect anti-CagA antibodies in sera of 59 patients. The ELISA results obtained when compared to those of the PCR gave a sensitivity, specificity and accuracy of 81%, 100% and 88% respectively. We have developed for the first time: a rCagA protein that showed high sequence homology with both Western and East Asian type strains and an indirect ELISA of high performance which can be used to detect anti-CagA antibodies in sera of infected patients worldwide.
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    Phylogenetic analysis of Helicobacter pylori cagA gene of Turkish isolates and the association with gastric pathology
    (BMC, 2013) Salih, Barik A.; Bolek, Bora Kazim; Yildiz, Mehmet Taha; Arikan, Soykan
    Background: The cagA gene is one of the important virulence factors of Helicobacter pylori. The diversity of cagA 5' conserved region is thought to reflect the phylogenetic relationships between different H. pylori isolates and their association with peptic ulceration. Significant geographical differences among isolates have been reported. The aim of this study is to compare Turkish H. pylori isolates with isolates from different geographical locations and to correlate the association with peptic ulceration. Methods: Total of 52 isolates of which 19 were Turkish and 33 from other geographic locations were studied. Gastric antral biopsies collected from 19 Turkish patients (Gastritis = 12, ulcer = 7) were used to amplify the cagA 5' region by PCR then followed by DNA sequencing. Results: The phylogenetic tree displayed 3 groups: A) a mix of 2 sub-groups Asian and African/Anatolian/Asian/European, B) Anatolian/European and C) American-Indian. Turkish H. pylori isolates clustered in the mixed sub-group A were mostly from gastritis patients while those clustered in group B were from peptic ulcer patients. A phylogenetic tree constructed for our Turkish isolates detected distinctive features among those from gastritis and ulcer patients. We have found that 2/3 of the gastritis isolates were clustered alone while 1/3 was clustered together with the ulcer isolates. Several amino acids were found to be shared between the later groups but not with the first group of gastritis. Conclusions: This study provided an additional insight into the profile of our cagA gene which implies a relationship in geographic locations of the isolates.