WO2011117201A1 - Détection de mycobactéries - Google Patents

Détection de mycobactéries Download PDF

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Publication number
WO2011117201A1
WO2011117201A1 PCT/EP2011/054266 EP2011054266W WO2011117201A1 WO 2011117201 A1 WO2011117201 A1 WO 2011117201A1 EP 2011054266 W EP2011054266 W EP 2011054266W WO 2011117201 A1 WO2011117201 A1 WO 2011117201A1
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WO
WIPO (PCT)
Prior art keywords
components
capture reagent
capture
cell wall
binding
Prior art date
Application number
PCT/EP2011/054266
Other languages
English (en)
Inventor
Christopher John Stanley
Stuart Mark Wilson
Original Assignee
Microsens Medtech Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Microsens Medtech Limited filed Critical Microsens Medtech Limited
Publication of WO2011117201A1 publication Critical patent/WO2011117201A1/fr

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Classifications

    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the present invention relates to the detection of
  • Pathogenic mycobacteria are responsible for several severe infectious diseases in humans and animals, they include
  • Mycobacterium tuberculosis which is the causative agent of TB, the mycobacteria of the MAC complex (primarily M. avium and M. intracellular ⁇ e) which are opportunistic pathogens in AIDS patients, M. paratuberculosis, which causes bowel inflammation, M. leprae causing leprosy, M. kansasii , M.
  • the Mycobacteria are characterised by a cell membrane that contains a number of components, including lipoarabinomannan (LAM) and mycolic acids.
  • LAM is a complex branched chain carbohydrate linked to a lipid.
  • Biological samples in which Mycobacteria can be found include sputum, urine, blood, bronchial lavage, milk, and faeces.
  • sputum One of the most common specimen types delivered for diagnosis is sputum.
  • Sputum presents unique problems for bacteriology. Sputum is heterogeneous in nature and can be bloody,
  • sputum is thinned and at the same time decontaminated by the use of various pre-treatments . These treatments include the use of 0.25-0.5 M sodium hydroxide for decontamination with or without N-acetyl L-cysteine, sodium dodecyl sulphate, oxalic acid or trisodium phosphate. Treatment times can be 20-120 minutes . After treatment with the harsh decontaminants the sample is generally centrifuged to concentrate the Mycobacteria which are then analysed by microscopy, culture or molecular amplification . In the past the use of immunoassay detection methods for
  • LAM ELISA LAM ELISAs have also been developed for use in other sample types such as sputum or blood but these are not widely used.
  • binding agents including poly diallyldimethyl ammonium chloride (p- DADMAC) bind Mycobacteria and they can be either captured directly to p-DADMAC coated surfaces or can be captured to a surface indirectly. It is believed that p-DADMAC binds to the hydrophobic mycolic acids in the outer membrane of the Mycobacterial cell.
  • p- DADMAC poly diallyldimethyl ammonium chloride
  • the present invention provides a method for the capture from a sample of Mycobacteria cell wall components, which method comprises contacting a sample containing said
  • the cell wall components may be insoluble (e.g. able to be separated by centrifugation) or may be soluble.
  • the capture reagent is non-immunological .
  • the capture reagent may be a soluble reagent and preferably has both a hydrophobic character and a polar character.
  • the hydrophobic character may assist its binding to lipid containing
  • binding to cell wall materials may also be through a mixture of hydrophobic and ionic interactions or purely ionic
  • Its polar character may participate in binding to solid surfaces, as may the hydrophobic character,
  • the capture reagent may itself be a solid surface having the requisite cell wall component binding ability.
  • the invention includes a method for the binding of components of the Mycobacterial cell wall by polyionic compounds or polyionic surfaces.
  • Said capture reagent is preferably polymeric and
  • said capture reagent preferably comprises a hydrocarbon chain or chains bearing multiple polar sites, which may be spaced along said chain or may connect hydrocarbon chains.
  • said capture reagent comprises cationic groups which remain positively charged even at high pH such as may be used to decontaminate a sample (e.g. pH ⁇ 10, for instance 12-14) .
  • cationic groups which remain positively charged even at high pH such as may be used to decontaminate a sample (e.g. pH ⁇ 10, for instance 12-14) .
  • pH ⁇ 10 e.g. pH ⁇ 10, for instance 12-14
  • a preferred capture reagent is poly- diallyldimethyl ammonium chloride (pDADMAC) .
  • the molecular weight of the poly-DADMAC may be in the range of less than 100,000 (very low), 100,000 - 200,000 (low), 200,000 - 400,000 or 500,000 (medium) or over 500,000 (high) .
  • An alternative said capture agent is polybrene, i.e. 1 , 5-dimethyl-l , 5-diazaundecamethylene polymethobromide (or hexadimethrine bromide)
  • Both of these reagents have a methylene chain of 4-6 units bearing spaced quaternary ammonium groups.
  • the sample may be a fluid sample such as sputum
  • urine including blood components such as plasma or serum
  • blood including blood components such as plasma or serum
  • bronchial lavage etc.
  • a solid sample such as a tissue biopsy, e.g. a skin sample, which may be homogenised and which preferably is treated to extract or disperse micro-organisms into a liquid to produce a fluid sample.
  • the capture reagent may preferably be sufficiently hydrophobic in character to bind hydrophobically to plastics, e.g. to the polystyrene
  • microplates usually employed to bind proteins
  • the solid is such that its surface by itself has the ability to bind the Mycobacteria cell wall components without the use of a separate capture agent coated on the surface.
  • suitable ionic groups such as quaternary ammonium groups into the polymer structure of a plastics material, as in quaternary ammonium ion exchange beads, which may be produced in
  • the preferred compounds are polycationic
  • quaternary amines such as p-DADMAC or hexadimethrine bromide that remain positively charged at the high pHs (>12) used for sample decontamination (and thinning of sputum samples) .
  • polyanionic compounds such as dextran sulphate can be used.
  • the polyionic compounds may be bound to the surface of a magnetic bead, or the bead itself may have a surface that is positively or negatively charged.
  • the beads may also be non-magnetic and of high density to promote rapid settling in solution.
  • a sample containing intact Mycobacterial cell wall material, e.g. fragments of cells, or soluble components derived from such cells is incubated with the polyionic compound-coated beads. This procedure can preferably be used to concentrate cell components from large sample volumes e.g. 1 to 10 ml of thinned sputum or 1 to 100 ml (e.g. 1 - 10 ml) of urine, thus greatly increasing the sensitivity of the detection method.
  • Capture may take place under near neutral pH conditions described in WO2008/065047, preferably in the presence of detergent as described there, or may take place under high pH conditions as generated by a decontaminating agent like sodium hydroxide. Generally, no detergent need then be used. Suitable high pH decontaminating agents include other
  • alkalis such as KOH and LiOH.
  • the beads can be washed to remove sample contaminants prior to detection and, when high pH capture conditions are used, to adjust the pH to something more suited to the intended detection method.
  • the bound material can be released into solution from the bead surface by standard techniques such as heating, addition of detergents or the addition of chaotropic salts.
  • the beads can be disrupted by a mechanical lysis process or ultrasound treatment for example to release the surface coating.
  • the supernatant containing released material can then be detected using an immunoassay such as a specific ELISA or a lateral flow device or mass spectrometry or GC-MS .
  • An ELISA method based on microplates, e.g. 96 well microplates, or adapted for a lateral flow format is preferred as this is most suited to use in the routine TB testing laboratory.
  • the bead coated with cells fragments or soluble components can be used directly in an ELISA or lateral flow device, in the latter case the bead diameter may need to be smaller than the pore size of the porous material from which the device is constructed to allow unimpeded flow of the beads .
  • a mechanical lysis disruption method to release the bound material increases the signal in the subsequent ELISA detection assay. It is believed that the mechanical disruption process lyses intact cells, disperses the cellular components and breaks up structures such as lipid rafts. This process may make the cellular components more accessible to binding by polyionic compounds or
  • polyionic surfaces and also increases the effective molar concentration of components in the sample.
  • the invention includes a micro-organism assay kit comprising either (a) a soluble capture reagent capable of binding Mycobacteria cell wall components, a substrate having a surface for capturing said cell wall components to said surface by binding said capture reagent to said surface, or (b) a capture reagent coated on and thus immobilised upon a solid surface, said capture reagent being capable of binding Mycobacteria cell wall components to be detected, and an immunological binding partner for binding said cell wall components.
  • the assay kit may comprise at the least a Mycobacterial capture reagent immobilised upon a solid surface, preferably para-magnetic beads. Kits may further contain decontamination reagents, wash buffers and a detection system which is preferably an ELISA or more preferably a lateral flow device that detects the cellular components directly or after elution from the solid surface.
  • a detection system which is preferably an ELISA or more preferably a lateral flow device that detects the cellular components directly or after elution from the solid surface.
  • Example 1 Demonstration of binding of the Mycobacterial cellular component LAM present in sputum by p-DADMAC coated beads and subsequent detection with a commercially available LAM ELI SA
  • Sputum samples were scored by direct fluorescent microscopy.
  • the samples were thinned using a mixture of NaOH and N- acetylcysteine (NALC) and the thinned sputum was extracted using p-DADMAC coated magnetic beads, which are further described in WO 2008/065047.
  • 80 ⁇ 1 of the pDADMAC coated beads (about 5 mg of beads) was used for 1 ml of the thinned sputum at a pH of 14 at a high ionic strength provided by addition of an equal volume of 3 M NaCl and incubated for 10 min.
  • the beads were collected using a magnet, washed in 1ml PBS buffer containing 0.5% Tween 20, collected using the magnet again and then resuspended in 0.1 ml PBS containing 0.5% Tween 20.
  • the magnetic beads were boiled for 5 min, then the extracts were eluted by adding the magnetic beads to 50 ⁇ 1 of lysis beads (lysis matrix B beads, Fischer Scientific Inc.) and disruption for 5 min at 2800 rpm on a Disruptor Genie (Scientific Industries) . 100 ⁇ of each eluate was analysed directly by a commercially available microplate LAM ELISA (Clearview, Invernesss Medical Innovations Inc.). Results Microscopy Absorbance
  • the ELISA performed on the sputum extracted by the p-DADMAC- coated beads was able to differentiate smear positive from smear negative sputum.
  • a smear-positive and a smear-negative sputum were thinned and centrifuged at 13,000 x g for 5 min to remove intact cells and larger fragments.
  • the supernatants and the resuspended pellets were extracted using p-DADMAC-coated beads, generally as in Example 1.
  • Extracted antigens were eluted from the beads by a lysis disruption process as described in Example 1 and analysed by the LAM-specifc ELISA.
  • the p-DADMAC-coated beads are able to extract LAM antigens from both the soluble supernatant and the pellet of smear- positive sputum.
  • a BCG culture was centrifuged at 13,000 x g for 5 min and
  • the p-DADMAC-coated magnetic beads were able to extract the soluble BCG derived LAM antigen that had been spiked into urine .
  • a BCG culture was centrifuged at 13,000 x g for 5 min and 50 ⁇ 1 of the supernatant spiked into 4 ml urine. 2.
  • the spiked urines and non-spiked control urines were extracted at neutral pH for 10 min using variously coated or non-coated paramagnetic beads.
  • the beads were captured and washed with 1 ml PBS.
  • the beads were resuspended in ⁇ PBS, 5% (w/v) BSA, 0.5% (v/v) Tween20.
  • Coating the beads with either p-DADMAC or dextran sulphate improved the signal compared to non-coated amine beads.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon le procédé de l'invention, les composants des parois cellulaires des mycobactéries sont capturés par contact avec un réactif de capture, par exemple, chlorure de poly-diallyldiméthylammonium (pDADMAC) ou polybrène, qui les lie et est lui-même soit lié à une surface solide, soit présent en solution, ledit procédé consistant ensuite à capturer, en outre, lesdits composants sur une surface par liaison dudit réactif de capture à ladite surface.
PCT/EP2011/054266 2010-03-22 2011-03-21 Détection de mycobactéries WO2011117201A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1004710.8 2010-03-22
GBGB1004710.8A GB201004710D0 (en) 2010-03-22 2010-03-22 Detection of mycobacteria

Publications (1)

Publication Number Publication Date
WO2011117201A1 true WO2011117201A1 (fr) 2011-09-29

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8603771B2 (en) 2010-07-02 2013-12-10 Microsens Medtech Limited Capture of micro-organisms
CN104176945A (zh) * 2014-09-02 2014-12-03 安徽信灵检验医学科技有限公司 一种阳离子膜化修饰剂及其防脱玻片的制备方法
WO2015171786A1 (fr) * 2014-05-06 2015-11-12 The Johns Hopkins University Systèmes polymères cationiques pour capture bactérienne sélective

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4207398A (en) 1976-07-02 1980-06-10 Rohm And Haas Company Process for preparing physically stable quaternary ammonium anion exchange resins by chloromethylation and amination in the absence of additional organic solvent
WO1992021975A1 (fr) * 1991-05-30 1992-12-10 Abbott Laboratories Procedes et reactifs avec capture d'ions permettant de determiner la teneur en digoxine
WO2008065047A1 (fr) 2006-11-29 2008-06-05 Microsens Medtech Ltd Capture de microorganismes de type mycobactéries

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4207398A (en) 1976-07-02 1980-06-10 Rohm And Haas Company Process for preparing physically stable quaternary ammonium anion exchange resins by chloromethylation and amination in the absence of additional organic solvent
WO1992021975A1 (fr) * 1991-05-30 1992-12-10 Abbott Laboratories Procedes et reactifs avec capture d'ions permettant de determiner la teneur en digoxine
WO2008065047A1 (fr) 2006-11-29 2008-06-05 Microsens Medtech Ltd Capture de microorganismes de type mycobactéries

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
REITHER KLAUS ET AL: "Low sensitivity of a urine LAM-ELISA in the diagnosis of pulmonary tuberculosis.", BMC INFECTIOUS DISEASES, vol. 9, no. 141, 2009, pages 1 - 10, XP002634778, ISSN: 1471-2334 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8603771B2 (en) 2010-07-02 2013-12-10 Microsens Medtech Limited Capture of micro-organisms
WO2015171786A1 (fr) * 2014-05-06 2015-11-12 The Johns Hopkins University Systèmes polymères cationiques pour capture bactérienne sélective
CN104176945A (zh) * 2014-09-02 2014-12-03 安徽信灵检验医学科技有限公司 一种阳离子膜化修饰剂及其防脱玻片的制备方法

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