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WO1997034149A1 - Method of diagnosing a mycobacterial disease and immunoassay kit - Google Patents

Method of diagnosing a mycobacterial disease and immunoassay kit Download PDF

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Publication number
WO1997034149A1
WO1997034149A1 PCT/EP1997/001037 EP9701037W WO9734149A1 WO 1997034149 A1 WO1997034149 A1 WO 1997034149A1 EP 9701037 W EP9701037 W EP 9701037W WO 9734149 A1 WO9734149 A1 WO 9734149A1
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lam
mycobacterial
patient
urine
diagnosing
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PCT/EP1997/001037
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French (fr)
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Stefan Svenson
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Priority to AU20942/97A priority Critical patent/AU2094297A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS

Definitions

  • the present invention relates to a method of diagnosing a mycobacterial disease, a method of monitoring the effects of therapeutic treatment of a mycobacterial disease and an immunoassay kit for diagnosing a mycobacterial disease in a patient.
  • mycobacteria causing diseases in man and the most important ones, which are pathogenic to humans, belong to the group Mycobacterium tuberculosis (TBC) and Mycobacterium avium complex (MAC). These bacteria have different, but closely related carbohydrate antigens on their cell walls, namely lipoarabinomannans (LAM), and arabinomannans (AM).
  • LAM lipoarabinomannans
  • AM arabinomannans
  • One method of detecting a mycobacterial infection is based on detection of antibodies against LAM in a blood or serum sample form a patient ( Theuer CP, Chaisson RE, Bias D (1989), Am. Rev. Respir. Dis. 139 (4, Part 2) : A 395).
  • antibodies against a bacterial antigen are used for diagnosing it is possible that a past infection and/or vaccination rather than an on-going infection or disease is detected.
  • Mycobacterial diseases can be therapeutically treated by administration of certain antibiotics, such as Rifampicin, Etambutol, and Isoniazid.
  • lipoarabinomannans and arabinomannans (AM), derived from Mycobacterium tuberculosis (TBC) and Mycobacterium avium complex (MAC)
  • LAM lipoarabinomannans
  • AM arabinomannans
  • TBC Mycobacterium tuberculosis
  • MAC Mycobacterium avium complex
  • the present invention is directed to a method of diagnosing a mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), wherein, in a sample of feces, sputum or urine from said patient, the presence of a mycobacterial antigen selected from the group consisting of lipoarabino- mannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined.
  • TBC Mycobacterium tuberculosis
  • MAC Mycobacterium avium complex
  • Said determination is preferably performed by the use of polyclonal or monoclonal antibodies directed against said mycobacterial antigen, and of an assay detecting antigen/antibody complexes formed.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoblotting immunoblotting.
  • the sample of feces, sputum or urine is pretreated by heat sterilization.
  • the invention is further directed to an immunoassay kit, which comprises optionally labeled polyclonal or monoclonal antibodies directed against a mycobacterial antigen selected from the group consisting of lipoarabino- mannans (LAM), arabinomannans (AM), and fragments of LAM and AM.
  • LAM lipoarabino- mannans
  • AM arabinomannans
  • the labels of the antibodies are selected in agreement with the diagnostic method to be used, e.g. an enzyme label when ELISA is to be used etc.
  • said antibodies directed against said mycobacterial antigen has been coupled to a carrier.
  • the carrier may be a solid support such as a plastic or glass surface, a membrane of e.g. derivatized carboxy cellulose, filter of e.g. Nylon or the like.
  • the immunoassay kit may additionally comprise an optionally labeled second monoclonal or polyclonal antibody, a positive control, a negative control, and optionally buffer solution(s) and/or washing solution(s).
  • the present invention is further directed to a method of monitoring the effects of therapeutic treatment of a mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), wherein, in a sample of feces, sputum or urine from said patient, the presence and amount of a mycobacterial antigen selected from the group consisting of lipoarabinomannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined at appropriate stages of said therapeutic treatment.
  • LAM lipoarabinomannans
  • AM arabinomannans
  • fragments of LAM and AM fragments of LAM and AM
  • a sandwich ELISA is used for the detection of mycobacterial antigens in a sample of urine from a tuberculosis patient.
  • the wells of a microtiter plate were coated with 100 ⁇ l of (mono or polyclonal) antibodies against lipoarabinomannan (LAM) per well. After overnight incubation at room temperature, the unbound antibody was removed and the remaining free binding sites of the wells were blocked by 200 ml 0.5% casein for 1 h at 37°C. Excess casein was removed, the plate was washed 3 times with washing buffer (0.05% Tween 20 in PBS) and 100 ⁇ l of urine samples were added.
  • washing buffer 0.05% Tween 20 in PBS
  • biotinylated anti-LAM IgG (5 ⁇ g/ml in PBS), incubate the membrane for 30 min at room temperature.
  • mice were given phosphate buffered saline (PBS). Urine samples were collected from the mice on the next day and analyzed by both catch-up ELISA 6
  • Urine samples from 20 patients with active tuberculosis and from 3 patients with both HIV and Mycobacterium avium complex (MAC) were analyzed; all became positive in the test. 18 healthy control patients were also analyzed and the results were negative. Matched patients with other diseases (30 patients) were also included in these studies, and interestingly some of these control patients (5 patients, positive in the assay of the invention) who initially were clinically asserted to be none TB showed upon follow-up to either have TB or a more or less recent history of TB.
  • LAM lipoarabinomannan
  • Purified LAM may be used as a positive control in the immunoassay of the invention, and as starting material for the preparation of monospecific polyclonal antibodies against LAM (which will be exemplified below).
  • Dry cell wall (5 g) from Mycobacterium tuberculosis is sonicated in Na-acetate buffer, pH 4.7, 5X3 min, followed by extraction with 80% phenol for 1 h at 70°C. After centrifugation for 30 min at 3500 rpm the phenolic phase is reextracted with water, the phenolic phase is discarded, and the aqueous phase is pooled with the aqueous phase from the centrifugation. The pooled aqueous phase is dialyzed against 3 x 5 liters of water overnight. Chromatography on octyl- Sepharose ® (Pharmacia , Sweden) yields 5 mg (0.1%) of LAM.
  • Both monoclonal and polyclonal antibodies may be use in the immunoassay of the invention.
  • the preparation of polyclonal antibodies starting with the bacterial cell wall, and monospecific polyclonal antibodies starting with LAM are exemplified.
  • LAM (6.5 mg) is oxidized with 0.01 M NalO 4 for 7 min at 4°C in the dark. Excessive NalO 4 is eliminated by addition of ethylene glycol. Fragments of LAM are separated by gel chromatography. Coupling of the fragments to aminoethyl Bio-Gel P-2 ( Bio-Rad, USA) through reductive amination at room temperature, pH 8, for 5 days results in a 70% yield. Blockage of excessive amino groups was performed by acetylation with Na-acetate using a water soluble carbodiimide such as EDAC at room temperature, pH 4.5, for 17 h, followed by washing and subsequent equilibration of the column with PBS. Affinity-LAM column yields purified monospecific polyclonal anti-LAM IgG, which can be used instead of monoclonal antibodies in the immunoassay of the invention.

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  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
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Abstract

A method of diagnosing of mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), is disclosed. In a sample of feces, sputum or urine from said patient, the presence of a mycobacterial antigen selected from the group consisting of lipoarabinomannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined, e.g. by the use of polyclonal or monoclonal antibodies directed against said mycobacterial antigen, and an assay detecting antigen/antibody complexes formed. The sample of feces, sputum or urine can be pretreated by heat sterilization. Further, an immunoassay kit for use in the method of diagnosing, and a method of monitoring the effects of therapeutic treatment of a mycobacterial disease, are described.

Description

Method of diagnosing a mycobacterial disease and immunoassav kit
The present invention relates to a method of diagnosing a mycobacterial disease, a method of monitoring the effects of therapeutic treatment of a mycobacterial disease and an immunoassay kit for diagnosing a mycobacterial disease in a patient.
Background
There are several types of mycobacteria causing diseases in man, and the most important ones, which are pathogenic to humans, belong to the group Mycobacterium tuberculosis (TBC) and Mycobacterium avium complex (MAC). These bacteria have different, but closely related carbohydrate antigens on their cell walls, namely lipoarabinomannans (LAM), and arabinomannans (AM). One method of detecting a mycobacterial infection is based on detection of antibodies against LAM in a blood or serum sample form a patient ( Theuer CP, Chaisson RE, Bias D (1989), Am. Rev. Respir. Dis. 139 (4, Part 2) : A 395). However, when antibodies against a bacterial antigen are used for diagnosing it is possible that a past infection and/or vaccination rather than an on-going infection or disease is detected.
The ability to diagnose an on-going mycobacterial infection or disease at an early stage could be life-saving for especially immunosupprimized patients, e.g. HIV infected patients. Mycobacterial diseases can be therapeutically treated by administration of certain antibiotics, such as Rifampicin, Etambutol, and Isoniazid.
It is also important to be able to monitor the effects of therapeutic treatment of a mycobacterial disease in a patient, especially since it is known that there are several mycobacterial strains which are resistant to antibiotics.
CONFIRMATION COW An immunoassay kit for diagnosing a mycobacterial disease in a patient will be a useful tool in this context
Description of the invention
It has now surprisingly been found that lipoarabinomannans (LAM), and arabinomannans (AM), derived from Mycobacterium tuberculosis (TBC) and Mycobacterium avium complex (MAC), can be detected in samples of feces, sputum or urine from infected patients. It will now be possible to directly detect components of such bacteria and to determine the relative amounts of these mycobacterial antigens in samples of body fluids excreted by a patient, thus reflecting an on-going infection or disease in said patient.
Since LAM and AM are stabile at normal temperatures for sterilization ( e.g. 60-100°C) the samples of feces, sputum or urine can be heat sterilized as a pretreatment, thus avoiding undeliberate contact with e.g. HIV infected body fluid, which is a great advantage for laboratory personnel.
Thus, the present invention is directed to a method of diagnosing a mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), wherein, in a sample of feces, sputum or urine from said patient, the presence of a mycobacterial antigen selected from the group consisting of lipoarabino- mannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined.
Said determination is preferably performed by the use of polyclonal or monoclonal antibodies directed against said mycobacterial antigen, and of an assay detecting antigen/antibody complexes formed.
Examples of suitable assays which can be used are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunoblotting. In an advantageous embodiment of the invention the sample of feces, sputum or urine is pretreated by heat sterilization.
The invention is further directed to an immunoassay kit, which comprises optionally labeled polyclonal or monoclonal antibodies directed against a mycobacterial antigen selected from the group consisting of lipoarabino- mannans (LAM), arabinomannans (AM), and fragments of LAM and AM.
The labels of the antibodies are selected in agreement with the diagnostic method to be used, e.g. an enzyme label when ELISA is to be used etc.
In a preferred embodiment of the invention said antibodies directed against said mycobacterial antigen has been coupled to a carrier. The carrier may be a solid support such as a plastic or glass surface, a membrane of e.g. derivatized carboxy cellulose, filter of e.g. Nylon or the like.
The immunoassay kit may additionally comprise an optionally labeled second monoclonal or polyclonal antibody, a positive control, a negative control, and optionally buffer solution(s) and/or washing solution(s).
Instructions for use will be provided with a kit according to the invention.
The present invention is further directed to a method of monitoring the effects of therapeutic treatment of a mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), wherein, in a sample of feces, sputum or urine from said patient, the presence and amount of a mycobacterial antigen selected from the group consisting of lipoarabinomannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined at appropriate stages of said therapeutic treatment. The monitoring will be performed by using the method of diagnosing a mycobacterial disease in a patient according to the invention at several, appropriate stages of therapeutic treatment of a patient, such as prior to administration of e.g. an antibiotic, and then 3, 5, 10 and 20 days after the first administration etc. It will then be possible to evaluate the effects of the therapeutic treatment by comparing the amounts of detected mycobacterial antigen at the different stages of treatment.
The invention will now be further illustrated by the following specific, but not limiting, experiments.
Sandwich enzyme-linked immunosorbent assay (sandwich-ELISA)
A sandwich ELISA is used for the detection of mycobacterial antigens in a sample of urine from a tuberculosis patient.
The wells of a microtiter plate were coated with 100 μl of (mono or polyclonal) antibodies against lipoarabinomannan (LAM) per well. After overnight incubation at room temperature, the unbound antibody was removed and the remaining free binding sites of the wells were blocked by 200 ml 0.5% casein for 1 h at 37°C. Excess casein was removed, the plate was washed 3 times with washing buffer (0.05% Tween 20 in PBS) and 100 μl of urine samples were added. After incubation for 1 h at 37°C, the plate was then washed and incubated with 100 μl of corresponding Biotin-labeled antibody (Method of biotinylation of IgG -see J.Cell Biol. Z2.783-788). The plate was then washed and incubated with 100 μl of Extravidin-alkaline phosphatase conjugate (1/5000 dilution) for 1 h at 37°C. Excess of conjugate was removed, the plate was washed and developed by adding 100 μl substrate solution, and the absorbance at 405 nm was recorded. Detection of LAM in urine using Immunodyne ABC (dip-stick test)
1- Spot load 1 μl of capture antibody (anti LAM IgG) in PBS onto the membrane and air dry for 10 min.
2- Block the membrane with e.g. 0.5% casein in PBS for 30 min at room temperature.
3- Incubate the membrane with the urine sample for 30 min at room temperature.
4- Wash the membrane in 2x changes of wash solution (0.1% Triton X-100 in PBS)
5 min per wash. Rinse the membrane with PBS.
5- Add biotinylated anti-LAM IgG (5 μg/ml in PBS), incubate the membrane for 30 min at room temperature.
6- Repeat wash procedure as in step 4.
7- Add Extravidin-alkaline phosphatase (1/10,000 dilution), incubate for 30 min at room temperature.
8- Rewash the membrane as in step 4.
9- Develop the membrane by the addition of substrate solution.
To test the hypothesis that LAM is excreted with urine a group of mice was injected with a cell wall homogenate (1 mg/mouse) extracted from Mycobacterium tuberculosis.
Control mice were given phosphate buffered saline (PBS). Urine samples were collected from the mice on the next day and analyzed by both catch-up ELISA 6
(sandwich ELISA) and dip-stick test. Only animals injected with the cell wall homogenate became positive in the test, and none of the controls. Both polyclonal and monoclonal antibodies against LAM have been used in this assay.
Urine samples from 20 patients with active tuberculosis and from 3 patients with both HIV and Mycobacterium avium complex (MAC) were analyzed; all became positive in the test. 18 healthy control patients were also analyzed and the results were negative. Matched patients with other diseases (30 patients) were also included in these studies, and interestingly some of these control patients (5 patients, positive in the assay of the invention) who initially were clinically asserted to be none TB showed upon follow-up to either have TB or a more or less recent history of TB.
Purification of lipoarabinomannan (LAM) from Mycobacterium tuberculosis
Purified LAM may be used as a positive control in the immunoassay of the invention, and as starting material for the preparation of monospecific polyclonal antibodies against LAM ( which will be exemplified below).
Dry cell wall (5 g) from Mycobacterium tuberculosis is sonicated in Na-acetate buffer, pH 4.7, 5X3 min, followed by extraction with 80% phenol for 1 h at 70°C. After centrifugation for 30 min at 3500 rpm the phenolic phase is reextracted with water, the phenolic phase is discarded, and the aqueous phase is pooled with the aqueous phase from the centrifugation. The pooled aqueous phase is dialyzed against 3 x 5 liters of water overnight. Chromatography on octyl- Sepharose® (Pharmacia , Sweden) yields 5 mg (0.1%) of LAM.
Preparation and purification of antibodies against LAM (anti-LAM IgG)
Both monoclonal and polyclonal antibodies may be use in the immunoassay of the invention. The preparation of polyclonal antibodies starting with the bacterial cell wall, and monospecific polyclonal antibodies starting with LAM are exemplified.
Polyclonal antibodies Bacterial cell wall (0.5 g) from Mycobacterium tuberculosis is sonicated to homogeneity in PBS. A rabbit is immunized with 50 μg homogenate in FCA (Freund's Complete Adjuvant) per lymph node at an interval of 2 weeks. The rabbit is then bled, and the antiserum is collected. Purification of anti-cell wall IgG is achieved with Protein-A Sepharose® (Pharmacia, Sweden). Purification of the anti-LAM IgG is achieved by affinity-LAM column chromatography yielding purified polyclonal anti-LAM IgG.
Monospecific polyclonal antibodies
LAM (6.5 mg) is oxidized with 0.01 M NalO4 for 7 min at 4°C in the dark. Excessive NalO4 is eliminated by addition of ethylene glycol. Fragments of LAM are separated by gel chromatography. Coupling of the fragments to aminoethyl Bio-Gel P-2 ( Bio-Rad, USA) through reductive amination at room temperature, pH 8, for 5 days results in a 70% yield. Blockage of excessive amino groups was performed by acetylation with Na-acetate using a water soluble carbodiimide such as EDAC at room temperature, pH 4.5, for 17 h, followed by washing and subsequent equilibration of the column with PBS. Affinity-LAM column yields purified monospecific polyclonal anti-LAM IgG, which can be used instead of monoclonal antibodies in the immunoassay of the invention.

Claims

1. A method of diagnosing a mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), c h a r a c t e r i z e d in that in a sample of feces, sputum or urine from said patient, the presence of a mycobacterial antigen selected from the group consisting of lipoarabinomannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined.
2. A method according to claim 1 , wherein said determination is performed by the use of polyclonal or monoclonal antibodies directed against said mycobacterial antigen, and of an assay detecting antigen/antibody complexes formed.
3. A method according to claim 2, wherein said assay is selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunoblotting.
4. A method according to any one of claims 1-3, wherein said sample of feces, sputum or urine is pretreated by heat sterilization.
5. An immunoassay kit for diagnosing a mycobacterial disease in a patient, which comprises optionally labeled polyclonal or monoclonal antibodies directed against a mycobacterial antigen selected from the group consisting of lipoarabinomannans (LAM), arabinomannans (AM), and fragments of LAM and AM.
6. An immunoassay kit according to claim 5, wherein said antibodies directed against said mycobacterial antigen has been coupled to a carrier.
7. An immunoassay kit according to claim 6 or 7, which additionally comprises an optionally labeled second monoclonal or polyclonal antibody, a positive control, a negative control, and optionally buffer solution(s) and/or washing solution(s).
8. A method of monitoring the effects of therapeutic treatment of a mycobacterial disease in a patient typically caused by Mycobacterium tuberculosis (TBC) or Mycobacterium avium complex (MAC), c h a r a c t e r i z e d in that in a sample of feces, sputum or urine from said patient, the presence and amount of a mycobacterial antigen selected from the group consisting of lipoarabinomannans (LAM), arabinomannans (AM), and fragments of LAM and AM, is determined at appropriate stages of said therapeutic treatment.
PCT/EP1997/001037 1996-03-12 1997-03-03 Method of diagnosing a mycobacterial disease and immunoassay kit Ceased WO1997034149A1 (en)

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Cited By (17)

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WO2001027613A3 (en) * 1999-10-12 2001-10-18 Connex Ges Zur Optimierung Von Improved method for the detection of acid resistant microorganisms in a stool
EP1158991A4 (en) * 1999-03-15 2003-05-14 Malaghan Inst Of Medical Res TREATMENT OF ASTHMA
EP1329718A3 (en) * 2002-01-10 2004-06-02 Becton, Dickinson and Company Methods and devices for collecting and preparing specimens for detection of mycobacteria and their antigens
EP0952849A4 (en) * 1996-12-31 2004-09-08 Univ New York EARLY SCREENING FOR MYCOBACTERIAL DISEASES
WO2006012413A1 (en) 2004-07-20 2006-02-02 Chemogen, Inc. Enriched antibody for detecting mycobacterial infection, methods of use and diagnostic test employing same
EP1710584A4 (en) * 2003-12-22 2007-11-14 Seikagaku Kogyo Co Ltd Method of measuring lipoarabinomannan and application thereof
EP1882939A1 (en) * 2006-07-27 2008-01-30 The Jordanian Pharmaceutical Manufacturing Co. Urinary immunochromatographic antigen detection cup
WO2009117462A1 (en) * 2008-03-18 2009-09-24 Wisconsin Alumini Research Foundation Mycobacterial culture screening test for mycobacterium avium complex bacteria
US7807182B2 (en) 1996-12-31 2010-10-05 Colorado State University Research Foundation Early detection of mycobacterial disease using peptides
US8158371B2 (en) 2006-09-08 2012-04-17 Wisconsin Alumni Research Foundation Assay for antibodies to Mycobacterium paratuberculosis
JP2014232117A (en) * 2012-04-05 2014-12-11 株式会社ビーエル Immunological detection method and detection kit for mycobacterium tuberculosis complex
WO2016012449A1 (en) * 2014-07-22 2016-01-28 Tbdiadirect Ab Monoclonal antibody, method, kit and use
US9315566B2 (en) 2011-01-24 2016-04-19 National University Of Singapore Pathogenic mycobacteria-derived mannose-capped lipoarabinomannan antigen binding proteins
WO2016130638A1 (en) * 2015-02-10 2016-08-18 University Of Utah Research Foundation Methods of detecting analytes and diagnosing tuberculosis
WO2019186486A1 (en) 2018-03-29 2019-10-03 Foundation Of Innovative New Diagnostics Antibody or antibody combination and method using same for detection of an antigen related to mycobacterium in a urine sample of a subject
CN111337665A (en) * 2020-01-16 2020-06-26 卢氏实验室公司 Immunochromatographic test strip for detecting tuberculosis infection and preparation method thereof
WO2021127096A1 (en) * 2019-12-17 2021-06-24 National Jewish Health Methods of detecting lipoarabinomannan and diagnosing nontuberculosis mycobacterial infection

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Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0952849A4 (en) * 1996-12-31 2004-09-08 Univ New York EARLY SCREENING FOR MYCOBACTERIAL DISEASES
US7807182B2 (en) 1996-12-31 2010-10-05 Colorado State University Research Foundation Early detection of mycobacterial disease using peptides
EP1158991A4 (en) * 1999-03-15 2003-05-14 Malaghan Inst Of Medical Res TREATMENT OF ASTHMA
WO2001027613A3 (en) * 1999-10-12 2001-10-18 Connex Ges Zur Optimierung Von Improved method for the detection of acid resistant microorganisms in a stool
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