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Immunoassay for H. Pylori in fecal specimens

a technology of fecal specimens and immunoassays, which is applied in the field of immunoassays for h. pylori in fecal specimens, can solve the problems of not exhibiting the specificity and sensitivity that are desired, preventing designing an assay around the use of a single antigen, and bacteria ordinarily cannot be cultured and isolated from fecal specimens

Inactive Publication Date: 2003-04-22
MERIDIAN BIOSCIENCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these assays have not exhibited the specificity and sensitivity that are desired in serodiagnosis.
One problem with of these immunoassays in cross-reactivity.
A second problem that has been encountered in designing immunoassays for H. pylori is strain variation.
These problems preclude designing an assay around the use of a single antigen.
There are certain disadvantages to using an ELISA which employs antigens to detect the presence of H. pylori antibodies.
Therefor, the antigen-based ELISA does not eliminate the need for the invasive procedure.
While ELISA's for detecting microorganisms such as C. difficile and adenovirus in fecal specimens are known, in studies of patients with gastric biopsies which are positive for H. pylori, the bacteria ordinarily can not be cultured and isolated from the fecal specimens.
This and the problems of cross reactivity and strain variation raised serious doubts that an ELISA could be designed that would be specific for H. pylori and sensitive enough to reliably detect H. pylori antigen directly from a fecal specimen.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of the Helicobacter pylori (H. pylori)

antigen

H. pylori (ATCC strain 43504) was streaked for isolation on Tryptic Soy Agar (TSA) supplemented with 5% defibrinated sheep blood. The plate was incubated at 37.degree. C. in a microaerophilic environment for 6-7 days. The resultant bacterial growth was evaluated by use of colony morphology, urease, catalase and oxidase reactions, and gram stain. Acceptable growth was subcultured to four TSA with sheep blood agar plates and grown at 37.degree. C. in a microaerophilic environment for 3-4 days.

Each plate was flooded with 5 ml of 0.85% NaCl and the bacterial growth was harvested by a plate spreader. The bacteria were centrifuged at 10,000 xg for 15 minutes at 2.degree.-8.degree. C. Each pellet was resuspended in 3 ml of 0.85% NaCl and combined to one centrifuge container. The bacterial suspension was centrifuged at 10,000 xg for 15 minutes at 2.degree.-8.degree. C. The pellet was resuspended and centrifuged as before. The final pe...

example 2

Production of Rabbit Polyclonal

The bacterial supplement obtained in Example 1 was diluted to equal parts with Freunds complete adjuvant (total immunogen is 1.0 ml) to provide 1.times.10.sup.8 cells per ml. This solution was mixed thoroughly and 0.2-0.5 ml of the solution was injected intramuscularly into the right hind leg and 0.1-0.25 ml of the solution was injected subcutaneously into each of eight to ten sites on the back. Subsequent injections were one month apart using Freunds incomplete adjuvant and the injection sites were limited to subcutaneous back.

A trial bleed was taken after three months. The bleed was taken from the central ear vein one week after the third injection. This bleed was incubated overnight at 2.degree.-8.degree. C. The next day the blood was centrifuged at 5,000 xg for 15 minutes at room temperature. The supernatant was collected and the pellet discarded. The supernatant was tested by an Indirect Fluorescent Assay (IFA). The IFA was performed by placing 10...

example 3

Horseradish Peroxidase Conjugation

The conjugation used 10 mg of DEAE purified rabbit anti-H. pylori antibody. The antibody was brought to a final volume of 2.5 ml by concentration or by the addition of 10 mM sodium bicarbonate pH 9.6. A PD-10 column (Pharmacia) was equilibrated with 10 mM sodium bicarbonate pH 9.6. The antibody was added to the column and nine fractions of 1.0 ml were taken. A protein concentration (OD.sub.280 E.O.=1.4) of each fraction was taken and those reading above 0.200 were pooled.

A separate PD-10 column was equilibrated with 1 mM sodium acetate trihydrate pH 4.3. The minimum amount of Horseradish Peroxidase (HRP) used was 1.172 mg HRP for each 1 mg of antibody. 11 / 2 times the calculated minimum HRP was weighted out and added to 1.0 ml of deionized water. A protein concentrated (OD.sub.403 E.O.=2.275) was performed and HRP diluted to 10 mg / ml with deionized water. 0.1M sodium m-periodate was added at a concentration of 0.2 ml for every 4 mg HRP. This reaction...

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Abstract

A process for the determination of H. Pylori in a fecal specimen comprising (a) dispersing a fecal specimen suspected of carrying H. pylori in a sample diluent; (b) contacting the fecal specimen in the diluent with a first polyclonal antibody for H. pylori antigen to form a complex of the antibody and the antigen; (c) separating said specimen and said complex; (d) exposing the complex to a second polyclonal antibody for said antigen and a portion of the antibody reacting with said complex, one of said first and second antibody being bound to a solid carrier and the other being labeled with a detecting agent; and (e) determining the amount of the labeled antibody and in turn determining the presence of H. pylori antigen in said fecal specimen.

Description

This invention relates to a method for detecting Helicobacter pylori in fetal specimens.H. pylori is a bacterium that is found in the upper gastrointestinal tract of humans which has been implicated in gastroduodenal diseases such as peptic ulcers, gastritis and other maladies. The bacterium was originally classified as a Campylobacter and then reclassified as a Heliobacter based on more detailed information regarding its ultrastructure and fatty acid composition.A number of different techniques, both invasive and noninvasive, have been used to detect H. pylori. The invasive techniques involve gastric biopsies and cultures. The noninvasive techniques include a urea breath test, in which the patient is given C-13 or C-14 labelled urea with a beverage, and the detection of H. pylori antibody in sera using antigens in enzyme-linked immunosorbent assays (ELISA). Examples of the latter techniques are found in U.S. Pat. No. 5,262,156 to Aleonohammad and European Patent Application 0 329 5...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): G01N33/569
CPCG01N33/56922G01N2333/205
Inventor LARKA, CHRISTOPHER VANCEYI, CHING SUI ARTHURKOZAK, KENNETH JAMES
Owner MERIDIAN BIOSCIENCE
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