WO2011117202A1 - Capture de mycobactéries - Google Patents

Capture de mycobactéries Download PDF

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Publication number
WO2011117202A1
WO2011117202A1 PCT/EP2011/054267 EP2011054267W WO2011117202A1 WO 2011117202 A1 WO2011117202 A1 WO 2011117202A1 EP 2011054267 W EP2011054267 W EP 2011054267W WO 2011117202 A1 WO2011117202 A1 WO 2011117202A1
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WO
WIPO (PCT)
Prior art keywords
sample
mycobacteria
micro
organism
capture
Prior art date
Application number
PCT/EP2011/054267
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 WO2011117202A1 publication Critical patent/WO2011117202A1/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
    • G01N33/54313—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
    • G01N33/543—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
    • G01N33/569—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 capture to a surface of Mycobacteria spp and to subsequent processing such as assays for their presence or identification or drug susceptibility testing.
  • Mycobacteria are responsible for several severe infectious diseases in humans and animals.
  • the mycobacteria are characterised by a hydrophobic, waxy coat comprising mycolic acid or related compounds.
  • Mycolic acids are complex hydroxylated branched chain fatty acids,
  • hydrocarbon chains typically having hydrocarbon chains with a chain length in the range C77-80, which causes severe problems in sample handling, causing the bacteria to clump forming cords and to float on the surface of liquids and to be resistant to centrifugation .
  • the hydrocarbon chains may or may not contain sparse oxygenated groups such as hydroxyl, methoxy, keto or carboxyl .
  • Pathogenic mycobacteria include
  • Mycobacterium tuberculosis which is the causative agent of TB, the mycobacteria of the MAC complex (primarily M. avium and M. intracellulare) which are opportunistic pathogens in AIDS patients, M. paratuberculosis, which causes bowel inflammation, M. leprae causing leprosy, M. kansasii , M.
  • the mycobacteria there are also many other non-pathogenic mycobacteria, including M. smegmatis . Also, other members of the Mycolata family have similar hydrophobic coat components. In some, the chain length of the hydrophobic fatty acids is shorter than in the mycobacteria, at around 50 carbon atoms, and in others around 30.
  • microscopy can be done directly from the biological sample, it is more usual to first isolate and concentrate the mycobacteria from the biological specimens prior to analysis.
  • Biological samples can include sputum, urine, blood, bronchial lavage etc.
  • 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, purulent, and viscous. It can also be contaminated with other micro ⁇ organisms eg. Pseudomonas . In culture based methods, contaminating micro-organisms may out grow and mask
  • Treatment times can be 20-120 minutes. These treatments are designed to thin the sputum and kill the majority of contaminating organisms. Mycobacteria have a thick waxy coat and are more resistant to such treatments. Even so, it is estimated that up to 60% of Mycobacterium tuberculosis are killed or rendered non-viable by this treatment. In addition, because the Mycobacterium
  • the sample After treatment with the harsh decontaminants the sample is centrifuged to concentrate the mycobacteria which are then analysed by microscopy, culture or molecular amplification.
  • This centrifugation step introduces a risk of infection to the laboratory staff as the contents of any tube that cracks or breaks during the centrifugation may be aerosolised and contaminate the environment.
  • the centrifugation also introduces a bottle-neck in the sample processing as only a limited number of samples can be centrifuged at any one time.
  • the centrifugation pellets all material that was rendered denatured and insoluble by the harsh decontamination procedure and very large pellets can be obtained which pose problems for microscopy or molecular methods.
  • mycobacteria In other applications distinct from sample processing it might also be useful to bind the mycobacteria to a solid surface to allow easy concentration or manipulation of the organisms e.g. capture and washing of the mycobacteria from a phage solution to remove exogenous non-infecting phage or capture and transfer of the mycobacteria from one solution to another .
  • poly diallyldimethyl ammonium chloride -DADMAC binds mycobacteria to carboxylic acid micro-beads. Without being bound by the following theory, we believe that the backbone chain of the p-DADMAC hydrophobically interacts with the waxy coat of the
  • mycobacteria and the positive charge in the backbone of p- DADMAC can also interact with negative charges on the surface of the mycobacteria, the p-DADMAC then interacts ionically through its pendant quaternary ammonium groups with the carboxylic acids of the micro-beads.
  • p-DADMAC coated surfaces such as plastics and glass can bind mycobacteria directly.
  • mycobacteria can be either captured directly to p- DADMAC coated surfaces or can be captured to a surface indirectly.
  • sputum is decontaminated with NaOH and is then neutralised before being captured to DADMAC coated beads in the presence of a detergent at a relatively low ionic strength (50mM) .
  • the detergent is used to prevent clumping of the beads. In principle, it would be desirable to simplify this capture procedure .
  • the present invention now therefore provides in a first aspect a method for the capture of Mycobacterium from a sample comprising treating the sample with a reagent to raise the pH of the sample to at least 10, thereby to provide a high pH sample, and a) contacting said high pH sample with a solid surface, optionally bearing a capture agent, capable of binding
  • Mycobacteria at the pH of the high pH sample and separating said beads bearing captured Mycobacteria or b) contacting said high pH sample with a capture agent capable of binding Mycobacteria at the pH of said high pH sample to form a bound complex of said Mycobacteria and capture agent, and contacting said complex with a solid surface to bind said complex thereto.
  • pH of said high pH sample is preferably at least 12.
  • suitable pH raising reagents comprises an alkali, which may for instance be sodium hydroxide, potassium hydroxide, or lithium hydroxide, sodium hydroxide being preferred .
  • 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.
  • Said capture reagent is preferably polymeric and 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 at the pH of said high pH sample. Suitably these are quaternary ammonium groups.
  • 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
  • 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 may be a solid or semi-solid sample such as faeces or 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.
  • 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 be sufficiently hydrophobic in character to bind
  • microplates usually employed to bind proteins
  • the solid is such that its surface by itself has the ability to bind the Mycobacteria 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 accordance with e.g.
  • said pH raising reagent further comprises a sputum thinning reagent.
  • a sputum thinning reagent is generally N-acetyl cysteine .
  • reagents for thinning sputum that can be used include sodium dichloroisocyanurate, sodium hypochlorite, mercaptoethanol , and 1 , 4-dithioerythritol or 1 , 4-dithiothreitol .
  • Said capture of the Mycobacteria is preferably carried out at an ionic strength of at least 0.3M, e.g. from 0.5 to 1.0M. This contrasts with what is shown in WO2008/065047. It is found that beads bearing captured Mycobacteria are not prone to clumping at high ionic strength and high pH, even in the absence of detergent, which is preferably not present. Non-Mycobacteria organisms that may be present will generally be rendered non-viable by the alkali treatment described above and in addition, not being sufficiently hydrophobic will not bind to the capture reagent, e.g. to p-DADMAC coated surfaces, thus giving a degree of selectivity of the type of organism captured.
  • said surface is suitably provided by beads. These may be of micro or nano dimensions. Suitably they are paramagnetic for easy
  • They may have a carboxylic acid polymer surface or a surface characterised by sulphate or phosphate groups.
  • the captured Mycobacterium may be any of those referred to above.
  • the invention includes a method for the detection of a Mycobacterium, comprising capturing said Mycobacterium to a surface by a method as described, optionally washing said captured Mycobacterium, and detecting said captured
  • the detection method used may be any appropriate method.
  • M. tuberculosis for mycobacteria in general and M. tuberculosis in general.
  • these will microscopic detection, e.g. by acid fast staining, optionally preceded by culturing to multiply said Mycobacteria, PCR - polymerase chain reaction, TMA - transcription mediated amplification, SDA - strand
  • LAM lipoarabinomannan
  • LAM lipoarabinomannan
  • lyse captured cells may be lyse captured cells to increase accessibility of cell components to the detection system, such as by mechanical lysis disruption methods or by ultrasonic treatment. Mechanical disruption may be carried out using lysis beads in a Disruptor GenieTM or the like.
  • the invention includes a Mycobacterium assay kit comprising either (a) a soluble capture reagent having both a hydrophobic character whereby the capture reagent is capable of binding a
  • Mycobacterium to be detected by hydrophobic interaction therewith and a polyionic character
  • a substrate having a surface for capturing said Mycobacteria to said surface by binding said capture reagent to said surface by polar
  • said surface and said capture reagent or (b) a capture reagent coated on and thus immobilised upon a solid surface, said capture reagent having both a hydrophobic and polyionic character whereby the capture reagent is capable of binding a Mycobacterium to be detected, or else a solid surface capable of directly binding Mycobacteria under high pH conditions, a strong base for raising the pH of a Mycobacterium sample to at least 10,
  • an antibody for binding said Mycobacterium such as an anti- LAM antibody or binding fragment thereof;
  • -a detection reagent for use in detecting a metabolite produced upon culture of said Mycobacterium.
  • the sample may also be a gaseous, e.g. air, sample having
  • Such a sample may be bubbled into a capture reagent solution to bind the Mycobacteria to the capture reagent.
  • a solid surface may be a microscope slide or beads as described above.
  • the captured Mycobacteria are not harmed by this capture and remain viable.
  • the invention can be used for drug susceptibility testing of the Mycobacteria.
  • the Mycobacteriacan be exposed to a drug in such a way as to allow the drug to affect the Mycobacteria.
  • the Mycobacteriacan be captured in any of the ways described herein and then can be investigated for viability, optionally after neutralisation, using any number of previously
  • Mycobacteriacan then be investigated for viability using any number of described methods which might include microscopy using viability stains, phage based methods, culture-based methods or PCR-based methods .
  • the drugs used may include those commonly used to treat tuberculosis such as rifampicin, streptomycin, isoniazid, ethambutol, pyrazinamide, and ciprofloxacin.
  • a kit as described above may include one or more drugs for drug susceptibility testing.
  • M. tuberculosis was used as a representative model organism for the mycobacterium genus .
  • EXAMPLE 1 Concentration of Mycobacterium tuberculosis from sputum under conditions of high pH.
  • the Mycobacterium tuberculosis was concentrated from sputum in high pH conditions using beads (BioMag Amine beads, Bangs Laboratories, catalogue number BM546) coated with poly diallyl dimethyl ammonium chloride (pDADMAC) . The presence of TB on the beads was then
  • the sputum was thinned following standard laboratory procedure by adding an equal volume of 0.5 M NaOH, 2% (w/v) N-acetyl cysteine.
  • the beads were resuspended in 1 ml PBS containing 0.75M NaCl and again collected on a magnet.
  • the slide was stained with auramine 0 for acid fast mycobacteria using a standard protocol.
  • Example 2 Further investigation of detection of TB in sputum by p-DADMAC-coated magnetic beads
  • Sample 5 is of interest as microscopy and culture were negative but the ELISA was positive; the patient was designated as clinical TB.

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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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Selon l'invention, la mycobactérie Mycobacterium est capturée à partir d'un échantillon par traitement dudit échantillon avec un réactif qui élève le pH de l'échantillon jusqu'à au moins 10, pour obtenir ainsi un échantillon à pH élevé, et par a) mise en contact dudit échantillon à pH élevé avec une surface solide telle que des billes d'échange d'ions ammonium quaternaire, capables de fixer Mycobacteria au pH de l'échantillon à pH élevé, et séparation de ladite surface portant les Mycobacteria capturées dudit échantillon ou par b) mise en contact dudit échantillon à pH élevé avec un agent de capture tel que pDADMAC, capable de fixer les Mycobacteria au pH dudit échantillon à pH élevé pour former un complexe lié desdites Mycobacteria et dudit agent de capture, et mise en contact dudit complexe avec une surface solide pour y fixer ledit complexe.
PCT/EP2011/054267 2010-03-22 2011-03-21 Capture de mycobactéries WO2011117202A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1004709.0A GB201004709D0 (en) 2010-03-22 2010-03-22 Capture of mycobacteria
GB1004709.0 2010-03-22

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WO2011117202A1 true WO2011117202A1 (fr) 2011-09-29

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Cited By (1)

* 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

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 (4)

* Cited by examiner, † Cited by third party
Title
GLATMAN-FREEDMAN ET AL., JNL CLIN MICROBIOL, November 1996 (1996-11-01), pages 2795 - 2802
GRANT I. R. ET AL., APPL ENVIRON MICROBIOL., vol. 64, no. 9, September 1998 (1998-09-01), pages 3153 - 8
HETLAND G. ET AL., IMMUNOLOGY, vol. 82, 1994, pages 445 - 449
STRATMANN ET AL., J CLIN MICROBIOL., vol. 40, no. 11, November 2002 (2002-11-01), pages 4244 - 4250

Cited By (1)

* 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

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