Diagnostic methods

An antigen, Helicobacter pylori technology, applied in the field of diagnosis of Helicobacter pylori infection, can solve the problems of infection risk, dislike of handling stool samples, etc.

Inactive Publication Date: 2005-06-01
比恩诺尔股份公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Similarly, staff may dislike handling stool samples and preparing samples for such analysis due to the inherent risk of infection

Method used

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Embodiment 1

[0043] General procedure for preparing the carrier substrate of the invention

[0044] To prepare the carrier substrate, Fab' fragments were first prepared from specific monoclonal anti-H. pylori antibodies as follows. First, use ImmunoPure F(ab′) 2 Preparation kit (PIERCE, USA) for preparation of F(ab') from monoclonal anti-H. pylori antibodies such as monoclonal anti-H. 2 fragment. Other known commercially available kits and methods may likewise be used. F(ab') was then dithiothreitol (DTT, Merck) in HEPES / EDTA buffer 2 Fragments were split into Fab' fragments with HEPES / EDTA buffer containing 150 mM NaCl, 10 mM HEPES, 5 mM EDTA, pH 6.0 as described by Ishikawa [Ishikawa, E., J. Immunoassay 4 (1983) 209- 320] typically performed overnight in microdialysis tubing. Briefly, F(ab') at a concentration of 0.2-0.5 mg / ml 2 Fragments were mixed with HEPES / EDTA buffer and 6.25 mM DTT solution in microdialysis tubing. The dialysis tubing was immersed in 250 ml of argon-purged H...

Embodiment 2

[0048] General procedure for measuring Helicobacter pylori antigen by SPR

[0049] H. pylori antigens in biological samples can be measured using the following general SPR procedure. The surface of the carrier substrate prepared in Example 1 was rinsed with PBS (pH 7.2). Negative samples (blanks) were measured first. The negative sample used varies with the biological sample to be measured. Thus, for example, when measuring a stool sample, a negative sample is a stool sample negative for H. pylori; when measuring a urine sample, a negative sample is a urine sample from a subject without H. pylori infection; when measuring a serum sample , negative samples were serum samples obtained from subjects without H. pylori infection. Then, the surface of the carrier substrate was sequentially brought into contact with the standard solution, the positive and negative control solutions and the sample, the flow cell of the measuring device was filled with each solution to be measured f...

Embodiment 3

[0051] Preparation of standard curve and measurement reproducibility

[0052] H. pylori antigens used as standards were extracted from bacterial pellets of H. pylori strain ATCC 49503 using the glycine-acid extraction procedure described by Rautelin and Kosunen [J. Clin. Microbiol. 25(10) (1987) 1944-1951]. Protein concentration was determined by Bradford assay [Bradford, Anal. Biochem. 72 (1976) 248]. Standards were diluted in 0.1 M PBS (pH 7.2) to concentrations of 0.001, 0.01, 0.1, 1, 10, 100 and 270 μg / ml and measured following the general procedure described in Example 2. Three separate measurements were performed on subsequent days to analyze reproducibility.

[0053] The results are shown in Figure 3A. The standard curve shown in Figure 3A demonstrates that responses and antigen concentrations are directly comparable on a semi-log scale. Table 1 shows the standard deviation of the three standard curve measurements over subsequent days (Figure 3B). Excellent reproduc...

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Abstract

The present invention relates to a new method for the diagnosis of Helicobacter pylori infection. In particular, the present invention relates to novel non-invasive methods for detecting the presence in biological samples of H. pylori antigens or metabolites produced by said bacteria using biosensor-based measurements. The present invention also relates to the use of a biosensor comprising a Helicobacter pylori-specific antibody or an antigen-binding fragment thereof immobilized thereon and a biomolecule-repelling polymer that prevents non-specific binding. The invention also relates to detection kits used in the method.

Description

field of invention [0001] The present invention relates to a new method for diagnosing Helicobacter pylori infection. In particular, the present invention relates to new non-invasive methods for detecting the presence in biological samples of H. pylori antigens or metabolites produced by said bacteria using biosensor-based measurements. The invention also relates to the use of a biosensor comprising specific antibodies to Helicobacter pylori or antigen-binding fragments thereof immobilized on the biosensor together with a biomolecule-repelling polymer preventing non-specific binding. The invention also relates to detection kits used in the method. Background of the invention [0002] Helicobacter pylori is a curved gram-negative bacterium found in the upper gastrointestinal tract of humans. Since the bacterium was first isolated in 1982, a large body of evidence has concentrated on the association of H. pylori with a variety of gastric conditions, including dyspepsia (hear...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/205G01N21/27G01N33/543G01N33/553G01N33/569
CPCG01N33/54393G01N33/56922Y10S435/808Y10S436/805Y10S436/806
Inventor V·克莱莫拉E·埃罗拉M·维安德尔
Owner 比恩诺尔股份公司
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