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1.
J Immunol Methods ; 488: 112905, 2021 01.
Article in English | MEDLINE | ID: mdl-33129887

ABSTRACT

The CagA protein one of the key virulence factors of Helicobacter pylori plays an important role in the pathogenesis of peptic ulcer diseases. Unfortunately the cagA gene status can only be determined by PCR while serology is an alternative approach to detect antigens or antibodies. Our aim is to detect the CagA antigen in sera of infected subjects by the development of an in-house capture ELISA test. Gastric antral biopsies and serum samples were collected from 63 patients. PCR was used to determine the cagA status. Our previously developed recombinant CagA protein and monoclonal antibody were used for setting up the capture ELISA test. H. pylori positive [(38 gastritis, 14 duodenal ulcers (DU), 11 gastric ulcer (GU)] patients were determined by PCR. The cagA gene was detected in 21 (55%) of gastritis, 11 (78%) of DU and 7 (60%) of GU patients. The reagents used in setting up the capture ELISA test following optimization displayed high performance. This study showed that our developed in-house capture ELISA has the potential to detect the CagA antigen at very low concentrations even though not detected in our H. pylori infected patients sera but we are also intended to use it in saliva and stool samples.


Subject(s)
Antigens, Bacterial/blood , Bacterial Proteins/blood , Enzyme-Linked Immunosorbent Assay , Gastritis/diagnosis , Helicobacter Infections/diagnosis , Helicobacter pylori/immunology , Peptic Ulcer/diagnosis , Serologic Tests , Biomarkers/blood , Gastritis/blood , Gastritis/immunology , Gastritis/microbiology , Helicobacter Infections/blood , Helicobacter Infections/immunology , Helicobacter Infections/microbiology , Humans , Peptic Ulcer/blood , Peptic Ulcer/immunology , Peptic Ulcer/microbiology , Predictive Value of Tests , Reproducibility of Results
2.
Biologicals ; 68: 26-31, 2020 Nov.
Article in English | MEDLINE | ID: mdl-32943295

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Gene Expression Regulation, Bacterial/immunology , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Recombinant Proteins/immunology , Antibodies, Monoclonal/metabolism , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Helicobacter Infections/diagnosis , Helicobacter Infections/microbiology , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Humans , ROC Curve , Recombinant Proteins/metabolism
3.
J Chromatogr Sci ; 58(3): 217-222, 2020 Apr 22.
Article in English | MEDLINE | ID: mdl-31812997

ABSTRACT

The cagA gene of Helicobacter pylori that encodes an immunodominant CagA protein provokes severe mucosal damage and acts as a risk factor for the development of peptic ulcer disease and gastric cancer. Our aim is to develop an immunochromatographic test strip (ICTS) using our previously developed recombinant CagA (rCagA) protein and anti-rCagA monoclonal antibody (Mab) for the detection of anti-CagA antibodies in sera of infected patients. The rCagA was firstly conjugated to gold nanoparticle and placed into the conjugate pad. A nonconjugated rCagA and anti-rCagA Mab (CK-02) were immobilized on the test line and control line, respectively. Biopsy and serum samples from 30 H. pylori-infected patients were used. The presence of cagA gene in biopsy samples was first detected by PCR (Polymerase Chain Reaction), and 22 patients were found positive while 8 were negative. When serum samples were tested by our developed ICTS, 21 were positive for anti-CagA antibodies while 9 were negative. The serum samples were also tested by a commercial ELISA (Enzyme Linked Immunosorbent Assay), and when compared to the ICTS a sensitivity of 95% and a specificity of 100% were obtained. The ICTS can be used for rapid detection of CagA-positive H. pylori infection instead of expensive, time consuming and laborious invasive approaches.


Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Helicobacter Infections/blood , Immunoassay/methods , Antibodies, Bacterial/immunology , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Biopsy , Enzyme-Linked Immunosorbent Assay , Gold/chemistry , Helicobacter Infections/microbiology , Helicobacter Infections/pathology , Helicobacter pylori/immunology , Humans , Immunoassay/instrumentation , Metal Nanoparticles/chemistry , Polymerase Chain Reaction , Reagent Strips , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sensitivity and Specificity
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