[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

EP1117476A1 - Procede et dispositif de concentration de groupes selectionnes de micro-organismes - Google Patents

Procede et dispositif de concentration de groupes selectionnes de micro-organismes

Info

Publication number
EP1117476A1
EP1117476A1 EP99941184A EP99941184A EP1117476A1 EP 1117476 A1 EP1117476 A1 EP 1117476A1 EP 99941184 A EP99941184 A EP 99941184A EP 99941184 A EP99941184 A EP 99941184A EP 1117476 A1 EP1117476 A1 EP 1117476A1
Authority
EP
European Patent Office
Prior art keywords
beads
microorganisms
enclosure
size
target
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99941184A
Other languages
German (de)
English (en)
Other versions
EP1117476A4 (fr
Inventor
Ruth Eden
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centrus International Inc
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of EP1117476A1 publication Critical patent/EP1117476A1/fr
Publication of EP1117476A4 publication Critical patent/EP1117476A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • 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
    • 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
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia

Definitions

  • the present invention relates to products and processes used for the detection of microbes in a sample. More specifically, the present invention provides a method and device for aiding in the detection of the presence of specific microbial contamination in food samples, clinical specimens and other products.
  • This step is followed by a selective enrichment step where the bacteria of interest are allowed to grow while the indigenous microflora is suppressed.
  • the enrichment procedure is followed either by conventional plating methodology or a variety of more modern and rapid methods such as DNA amplification or immunoassay. It is therefore desired to separate at an early stage the target organisms from the other flora present in the product.
  • One such approach is the utilization of the immuno-magnetic separation technique, involving the utilization of immuno-magnetic particles specific for the target organisms. Magnetic beads with antibodies affixed to their surfaces are mixed with the sample containing the target organism. This organism will bind to the bead surfaces via the antibodies. The organism-bead complex is pulled out of the solution by a magnet, to concentrate the microorganisms.
  • U.S. patent 4,230,685 describes magnetically responsive microspheres having protein A associated with the outer surface.
  • the microspheres are reacted with antibodies selective to cells, bacteria or viruses to be separated from a mixed population.
  • the microorganism will attach to the antibody and thereby to the microspheres, and the microspheres are then used in a magnetic separation procedure.
  • the preferred microspheres are prepared from a mixture of albumin, Protein A, and magnetic particles.
  • the microspheres are prepared so that the Protein A is present in the exterior surface of the antibody binding.
  • U.S. patent 4,695,393 describes a process for the preparation of such magnetic beads, which can be used in separation of microorganisms.
  • U.S. patents 5,491 ,068 and 5,695,946 describe a method characterized by antibody capture of the organism of interest by the application of specialized magnetic beads. It entails the incubation of the capture cells to form colonies; removal of material from the colonies with colony lift membrane; and detection of the colony material on the membrane sheet by the use of labeled antibodies, PCR or nucleic acid probes.
  • the main problem with this method is the low sensitivity of one organism per gram. This low sensitivity is inherent in the methodology and is 50-100 fold lower than the desired sensitivity for most food pathogens.
  • U.S. patent 4,677,055 describes a process for concentrating bacteria utilizing magnetic gel to which anti-specific antigenic determinant antibodies are coupled. It involves the steps of obtaining medium containing the organisms possessing specific antigenic determinants and bringing them in contact with particles of the magnetic gel. This step is followed by the separation of the gel from the medium by magnetic means and inoculation into new medium. In general there are a number of problems associated with magnetic beads.
  • an object of the invention to provide a method and device that can be utilized with a large volume of media, to concentrate a target organism. It is another object of the invention to provide a method that is less labor intensive, more rapid and will lend itself to automation.
  • Fig. 1 shows a side view of the preferred device utilized to concentrate target organisms.
  • Fig. 2 shows a side view of another design of the device utilized to concentrate target organisms.
  • Figure 1 shows the preferred embodiment of a device for the separation of the target organisms from a suspension containing a mixture of organisms.
  • Beads 1 are made of materials such as nylon, polystyrene or glass.
  • the beads are coated with antibodies to specific microorganisms such as Salmonella, E. coli 0157:H7 and Listeria.
  • a cylindrical enclosure 2 is designed to contain the beads.
  • the enclosure is constructed from a frame 3 supporting a grid 4 covering the frame.
  • the grid's pore size is smaller than the size of the beads to assure that the beads stay within the enclosure 2. However, the pore size is large enough to allow bacteria to freely pass into the enclosure.
  • a rod 5 is attached to the upper part of the enclosure.
  • the rod 5 allows the enclosure 2 to move in the solution and for subsequent removal of the device from the solution.
  • Figure 2 shows a different design of the device.
  • the beads 11 coated with antibodies are contained in the enclosure 12 made of a grid 13, shaped like a tea bag.
  • a non-wicking string 14 is attached to the upper part of the enclosure 12 allowing movement of the enclosure 12 in the solution, while disallowing the solution containing bacteria to wick up the string.
  • the grid's 13 pore size is smaller than the size of the beads to assure that the beads stay within the enclosure. However, the pore size is large enough to allow bacteria to freely pass into the enclosure.
  • the food sample to be tested for the presence of the target organism is mixed with the appropriate pre-enrichment broth.
  • the pre-enrichment broth is incubated at an appropriate temperature.
  • the enclosure 2 is immersed into the broth containing the sample thereby exposing the beads having immobilized thereon monoclonal or polyclonal antibodies to the selected bacteria of interest. This is accomplished by lowering the device 2 into the solution and agitating it for at least 30 minutes and up to several hours. This step allows cell capture by the beads, and the creation of bead-target microbial cell complexes.
  • the next step involves the separation of the beads with the bound target cells from the suspension containing the food particles and other mixed flora.
  • the device can be inserted into a new growth broth that includes a dye indicator and the changes in the dye characteristics can be utilized to determine presence or absence of the target organism.
  • the microorganisms do not need to be detached from the beads since attachment to the beads has no effect on their growth. Therefore cells can continue to multiply in the appropriate medium.
  • the beads can be removed from the enclosure and inoculated onto the surface of appropriate selective or differential agar.
  • Another approach is to utilize an immunoassay. Most immunoassays require lO O 5 cells ml " l, therefore the beads should contain enough cells to perform a direct immunoassay.
  • this method can be combined with DNA hybridization and amplification techniques such as PCR.
  • the method of the invention is particularly characterized by the use of immunological beads contained in an enclosure to select out target microorganisms from the sample.
  • the beads must be capable of effectively capturing the target microorganisms from the test sample, while not capturing significant numbers of other organisms that might be present at much higher numbers.
  • the antibody used need not be totally specific to the target organism since an additional selection step is available at the end of the assay.
  • the antibodies must be oriented with their binding sites outward to allow contact between the binding portion of the antibody and the target organism.
  • the size of the beads must be larger than the size of the microorganism, to remain contained in the enclosure, while allowing the target organism to enter the enclosure and attach to the beads.
  • the contact time between the beads and the target organism must be long enough to allow strong interaction. Several hours of interaction was found to yield the best results, i.e. the creation of strong interactions to produce high capture efficiency.
  • the beads are removed from the solution, by the removal of the enclosure in which they are contained. The enclosure and the beads are washed several times, and the beads are transferred into the detection system.
  • the new method and device can be utilized with a large volume of media, to concentrate a target organism, without the need to utilize only a portion of the pre-enrichment broth or a small volume of enrichment broth as required for magnetic beads.
  • the invention provides a method and device that is less labor intensive, more rapid and lends itself to automation. Many different designs, for containing the beads during the various steps of the assay, can be utilized.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Sustainable Development (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un procédé et un dispositif permettant de concentrer des organismes cibles à partir d'un mélange d'organismes. Des perles (1) fabriquées dans une matière telle que le nylon, le polystyrène ou le verre sont recouvertes d'anticorps de micro-organismes spécifiques. Ces perles (1) sont contenues dans une enceinte (2) entourée d'un matériau grillagée. La taille des pores de la grille est inférieure à la taille des perles afin de s'assurer que les perles restent à l'intérieur du matériau grillagé, et plus grande que la taille des micro-organismes pour permettre l'interaction des micro-organismes avec les perles. Une ficelle (5) est attachée à la partie supérieure de l'enceinte (2) pour permettre d'agiter le dispositif dans le milieu de croissance contenant les organismes cibles.
EP99941184A 1998-08-24 1999-08-23 Procede et dispositif de concentration de groupes selectionnes de micro-organismes Withdrawn EP1117476A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US9762798P 1998-08-24 1998-08-24
US97627P 1998-08-24
PCT/US1999/018618 WO2000010702A1 (fr) 1998-08-24 1999-08-23 Procede et dispositif de concentration de groupes selectionnes de micro-organismes

Publications (2)

Publication Number Publication Date
EP1117476A1 true EP1117476A1 (fr) 2001-07-25
EP1117476A4 EP1117476A4 (fr) 2007-08-22

Family

ID=22264348

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99941184A Withdrawn EP1117476A4 (fr) 1998-08-24 1999-08-23 Procede et dispositif de concentration de groupes selectionnes de micro-organismes

Country Status (6)

Country Link
EP (1) EP1117476A4 (fr)
CN (1) CN1320060A (fr)
AU (1) AU773645B2 (fr)
CA (1) CA2342108A1 (fr)
MX (1) MXPA01001873A (fr)
WO (1) WO2000010702A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060105349A1 (en) * 2004-11-12 2006-05-18 Promega Corporation Device and method for purification of biological materials
CN101215598B (zh) * 2008-01-11 2010-11-17 中华人民共和国吉林出入境检验检疫局 免疫富集丝检测细菌的方法及免疫富集刷
US9964474B2 (en) * 2009-04-03 2018-05-08 3M Innovative Properties Company Microorganism concentration process and device
CN110093261B (zh) * 2019-04-28 2022-11-25 陕西科技大学 一种用于沉积物中厌氧菌原位分离的装置及使用方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3970518A (en) * 1975-07-01 1976-07-20 General Electric Company Magnetic separation of biological particles
EP0016552A1 (fr) * 1979-02-28 1980-10-01 Kendrew Biosystems, Inc. Procédé de séparation magnétique de cellules et analogues et microsphères à utiliser dans ce procédé
WO1989002449A1 (fr) * 1986-05-26 1989-03-23 Karsten Thies Reactif et procede de liaison de polymeres a des micro-organismes dans des solutions aqueuses
US4910148A (en) * 1987-02-10 1990-03-20 Dynal A. S. Magnetic separation of magnetized particles from biological fluids
US5175093A (en) * 1989-11-07 1992-12-29 Lehigh University Bioactive cells immobilized in alginate beads containing voids formed with polyethylene glycol

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3840345A (en) * 1972-06-23 1974-10-08 Co Ind De Procedes & D Applic Self agglomerating fluidized bed reacting apparatus
JPS635263A (ja) * 1986-06-24 1988-01-11 Yasunobu Tsukioka 血液等の検査方法
FR2624401A1 (fr) * 1987-12-11 1989-06-16 Charbonnages De France Grille de fluidisation refroidie
US4931401A (en) * 1988-09-01 1990-06-05 La Societe De Recherche Snc Inc. Bioreactor
US5186824A (en) * 1991-09-04 1993-02-16 Large Scale Biology Corporation System for solid phase reactions
US5342790A (en) * 1992-10-30 1994-08-30 Becton Dickinson And Company Apparatus for indirect fluorescent assay of blood samples

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3970518A (en) * 1975-07-01 1976-07-20 General Electric Company Magnetic separation of biological particles
EP0016552A1 (fr) * 1979-02-28 1980-10-01 Kendrew Biosystems, Inc. Procédé de séparation magnétique de cellules et analogues et microsphères à utiliser dans ce procédé
WO1989002449A1 (fr) * 1986-05-26 1989-03-23 Karsten Thies Reactif et procede de liaison de polymeres a des micro-organismes dans des solutions aqueuses
US4910148A (en) * 1987-02-10 1990-03-20 Dynal A. S. Magnetic separation of magnetized particles from biological fluids
US5175093A (en) * 1989-11-07 1992-12-29 Lehigh University Bioactive cells immobilized in alginate beads containing voids formed with polyethylene glycol

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO0010702A1 *

Also Published As

Publication number Publication date
WO2000010702A1 (fr) 2000-03-02
CN1320060A (zh) 2001-10-31
CA2342108A1 (fr) 2000-03-02
EP1117476A4 (fr) 2007-08-22
MXPA01001873A (es) 2002-09-02
AU773645B2 (en) 2004-05-27
AU5488699A (en) 2000-03-14

Similar Documents

Publication Publication Date Title
US5587286A (en) Methods and kits for detection of cells in food materials
JP5480505B2 (ja) 生物学的成分の濃縮ユニット及び濃縮方法
CA2112603C (fr) Methode de detection de bacteries
Grant et al. Isolation of Mycobacterium paratuberculosis from milk by immunomagnetic separation
Brewster et al. Filtration capture and immunoelectrochemical detection for rapid assay of Escherichia coli O157: H7
US10352834B2 (en) Method for treating at least one biological sample containing a target microorganism
JPS59141067A (ja) 汚染された生物学的媒質中の病原性微生物の免疫細菌学的検出方法
CN101971032B (zh) 在液体培养基中通过凝集实时检测微生物的方法
WO2009122216A1 (fr) Système d'essai basé sur la séparation immunomagnétique pour détecter des micro-organismes dans un échantillon d'aliments
Huang et al. Evaluation of culture enrichment procedures for use with Salmonella detection immunoassay
Rodrigues et al. Rapid detection of salmonellas in raw meats using a fluorescent antibody‐microcolony technique
Hanai et al. Comparison of commercially available kits with standard methods for detection of Salmonella strains in foods
US20200300737A1 (en) SAMPLE PREPARATION AND SPECIFIC CAPTURE FOR MULTIPLEX DETECTION OF TARGET ANALYTES (i.e., BACTERIA, VIRUSES, ETC.)
US20130344477A1 (en) Methods of capturing bindable targets from liquids
CN101375163A (zh) 用噬菌体检测微观活生物体的装置与方法
AU773645B2 (en) Method and device for concentrating selected groups of microorganisms
US7071005B1 (en) Method and device for concentrating selected groups of microorganisms
US6159689A (en) Methods of capture and assay procedures
JP3773633B2 (ja) 大腸菌ｏ１５７の分析方法及び分析用試薬
WU et al. RAPID PROTOCOL (5.25 H) FOR THE DETECTION OF ESCHERICHIA COLI O157: H7 IN RAW GROUND BEEF BY AN IMMUNO‐CAPTURE SYSTEM (PATHATRIX) IN COMBINATION WITH COLORTRIX AND CT‐SMAC 1
US20150079597A1 (en) Method for capturing and concentrating a microorganism in a biological sample
Todd et al. Assessment of an enzyme immunoassay for the detection of salmonellas in foods and animal feeding stuffs
JP2001004631A (ja) 磁気ビーズ固定化抗体を用いた微生物の迅速検定法
Blivet et al. Rapid detection methods for pathogens
Porter et al. Magnetic particle-based separation techniques for monitoring bacteria from natural environments

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010323

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RBV Designated contracting states (corrected)

Designated state(s): DE ES FR GB IT LU

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: CENTRUS INTERNATIONAL, INC.

RIN1 Information on inventor provided before grant (corrected)

Inventor name: EDEN, RUTH F.

REG Reference to a national code

Ref country code: DE

Ref legal event code: 8566

A4 Supplementary search report drawn up and despatched

Effective date: 20070724

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20071023