WO2017046251A1 - A method and a device for isolation of pathogens - Google Patents

A method and a device for isolation of pathogens Download PDF

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Publication number
WO2017046251A1
WO2017046251A1 PCT/EP2016/071838 EP2016071838W WO2017046251A1 WO 2017046251 A1 WO2017046251 A1 WO 2017046251A1 EP 2016071838 W EP2016071838 W EP 2016071838W WO 2017046251 A1 WO2017046251 A1 WO 2017046251A1
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WO
WIPO (PCT)
Prior art keywords
matrix
pathogens
protein
coating
complex
Prior art date
Application number
PCT/EP2016/071838
Other languages
French (fr)
Inventor
Divya KHANDIGE SHARMA
Ragavendar MS
Nivedita MITRA
Ramya VUTUKURU
Original Assignee
Siemens Aktiengesellschaft
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 Siemens Aktiengesellschaft filed Critical Siemens Aktiengesellschaft
Publication of WO2017046251A1 publication Critical patent/WO2017046251A1/en

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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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • 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
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins

Definitions

  • the invention relates to a method for isolation of pathogens from a given sample.
  • the method comprises the steps of coat ⁇ ing a matrix with pathogen binding proteins to form an affin- ity matrix; contacting said sample with said affinity matrix forming a complex thereby; and isolating said complex.
  • the invention also relates to a device for isolation of path ⁇ ogens from a given sample.
  • the device comprises an affinity matrix including a matrix; and a protein coating at least partly coating said matrix.
  • the method disclosed therein involves a step of at least partly coating a matrix with a pathogen binding pro ⁇ tein to form an affinity matrix.
  • the coating has only a sin ⁇ gle type of pathogen binding protein.
  • the pathogen binding protein has an affinity towards certain type of bacteria, therefore binding to such bacteria when brought in contact.
  • the affinity matrix is brought in contact with a sample comprising pathogens. Once the affinity matrix contacts the pathogens in the sam ⁇ ple, a complex is formed.
  • the complex is isolated from the sample.
  • the device disclosed therein comprises of an affinity matrix which includes a ma ⁇ trix which is at least partly coated with a protein coating.
  • the protein coating is composed of a single type of pathogen binding protein that has an affinity towards the pathogens in the sample.
  • the object of the invention is therefore to have a method and a device of the aforementioned kind that can isolate greater varieties of pathogens that may be present in a given sample.
  • the invention achieves the object in that said protein coat ⁇ ing around said matrix is of a combination of two or more types of pathogen binding proteins.
  • the present invention relates to a method of isolation of pathogens from a given sample.
  • the method comprises a step of coating a matrix at least partly with a combination of two or more types of pathogen binding proteins.
  • a matrix along with the coating forms an affinity matrix.
  • a pathogen binding protein is a protein comprising a pathogen binding domain which has a specific af ⁇ finity towards pathogens.
  • the pathogen binding proteins may also be, for example, innate immune proteins like mannose binding lectin.
  • One type of pathogen binding protein can bind to a limited variety of pathogens. Therefore, to broaden the scope of isolation, a combination of two or more different types of pathogen binding proteins are used in the coating.
  • the affinity matrix in the next step of the method, is brought into contact with the sample comprising pathogens. Once brought in contact with the sam ⁇ ple, the affinity matrix binds to the pathogens in the sample to form a complex.
  • the proteins interact with the components in the cell membrane of the pathogens during the formation of the complex.
  • the complex formed is isolated. Therefore, the pathogens are isolated from the sample.
  • the complex is isolated using a magnet. In this case the matrix used is a paramagnetic bead. Thus, the complex gets attracted to the magnet thus resulting in its isolation.
  • the complex is isolated using a centrifuge.
  • the ma ⁇ trix is a glass bead.
  • the complex would settle down as residue on centrifugation .
  • the invention solves the problem by using a protein coating around said matrix which is of a combination of two or more types of pathogen binding proteins.
  • the device comprises an affinity matrix.
  • the affinity matrix includes a matrix and a protein coating that coats the matrix at least partly.
  • the matrix forms the base around which exists the protein coat ⁇ ing.
  • the protein coating comprises of pathogen binding pro ⁇ teins that bind to pathogens that may be present in the sam- pie.
  • the matrix may also be coated with immunoglobulin G to which pathogen binding domains of pathogen binding proteins are fused.
  • the protein coating coated around the matrix is a combination of two or more types of pathogen binding proteins.
  • a single type of pathogen binding protein would bind to a limited variety of pathogens.
  • a combination of two or more pathogen binding proteins is capable of binding to a plethora of path- ogens that may be present in the sample. Therefore, the scope of isolation is increased.
  • the ma ⁇ trix is a paramagnetic bead or a glass bead.
  • the isolation of the complex formed between the pathogen and the affinity ma ⁇ trix can be easily isolated if the matrix is a paramagnetic bead or a glass bead. If the matrix is a paramagnetic bead, a magnet may be used as the isolating means. Similarly, if the matrix is a glass bead, a centrifuge may be used as the iso ⁇ lating means.
  • said combination of two or more pathogen binding proteins is chosen from a group comprising of innate immune protein, acute phase pro ⁇ tein, and fusion protein containing pathogen binding domain.
  • the proteins that may be used but not limited to include toll like receptors (TLR) class of proteins, proteins of classical complement pathway, proteins of alternative complement path ⁇ way, collectins, ficolin, and acute phase protein.
  • TLR toll like receptors
  • FIG 1 illustrates a schematic diagram of an embodiment of a device according to the invention.
  • FIG 2 illustrates a flowchart of an embodiment of a meth- od according to the invention.
  • FIG 1 illustrates a schematic diagram of an embodiment of a device 1 for isolation of pathogen from a given sample.
  • the device 1 comprises of a matrix 2.
  • the matrix depicted in FIG 1 is a paramagnetic bead.
  • the paramagnetic bead 2 forms the base around which there exists a coating of antibody IgG 3 to which pathogen binding domains 4,5 are fused. Together with the coating, the matrix 2 forms an affinity matrix or device 1.
  • the pathogen binding domains 4,5 are chosen from two or more types of pathogen binding proteins.
  • the IgG 3 is fused with pathogen binding domain of ApoH 4 which is an acute phase protein and that of mannose binding lectin (MBL) 5.
  • ApoH 4 is a plasma glycoprotein expressed in human liver, intestine and tissues.
  • ApoH 4 binds to pathogens or their surface proteins with relatively high affinity.
  • the interaction of pathogen and ApoH 4 has ionic and hydrophobic components.
  • the posi ⁇ tively charged region of the protein interacts with anionic phospholipid head group and the flexible loop putatively in ⁇ serts into the lipid layer and positions Trp316 at the inter- face region between the acyl chains and phosphate head groups of the lipids, thereby anchoring the protein molecule to the membrane.
  • Mannose binding lectin 5 is a calcium dependent C-type serum protein that plays a central role in the innate immune response.
  • Mannose binding lectin 5 specifically recognizes mannose, fucose, glucose, N-acetylglucosamine, and N-acetylmannosamine in a Ca 2+ dependent manner.
  • mannose binding lectin 5 and ApoH 4 are known to bind a variety of pathogens, the binding efficiency may vary across pathogens. This limits the pathogen capture efficien ⁇ cy. Therefore, using a combination of both mannose binding lectin 5 and ApoH 4 increases the efficiency of pathogen binding .
  • FIG 2 provides an illustration of a method 6 of isolation of pathogens from a given sample, according to the invention.
  • the method 6 comprises a step 7 of coating the matrix 2 with antibodies 3 fused with pathogen binding domains 4,5 of two or more types of pathogen binding proteins to form an affini ⁇ ty matrix 1.
  • the affinity matrix 1 is brought in contact with the sample such that the pathogens are in the vicinity of the device 1.
  • the affinity matrix 1 forms a complex with the pathogens in the sample.
  • the complex formed between the affinity matrix 1 and the patho ⁇ gens is isolated from the sample using a magnet.
  • the matrix 2 being a paramagnetic bead gets attracted to the magnet, therefore isolating the pathogen along with it.

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

Abstract

To provide a method (6) of isolation of pathogens from a sample, the method (6) comprising the steps of coating a matrix (2) at least partly with pathogen binding proteins (4,5) to form an affinity matrix (1); contacting said sample with said affinity matrix (1) forming a complex thereby; and isolating said complex, and to provide a device (1) for isolation of pathogens from a sample, the device (1) comprising an affinity matrix (1) including a matrix (2); and a protein coating at least partly coating said matrix (2); it is suggested that the protein coating around said matrix (2) is of a combination of two or more types of pathogen binding proteins (4,5).

Description

Title of the invention
A method and a device for isolation of pathogens Description
The invention relates to a method for isolation of pathogens from a given sample. The method comprises the steps of coat¬ ing a matrix with pathogen binding proteins to form an affin- ity matrix; contacting said sample with said affinity matrix forming a complex thereby; and isolating said complex.
The invention also relates to a device for isolation of path¬ ogens from a given sample. The device comprises an affinity matrix including a matrix; and a protein coating at least partly coating said matrix.
Such a method and device have already been disclosed in EP 2505666 Al . The method disclosed therein involves a step of at least partly coating a matrix with a pathogen binding pro¬ tein to form an affinity matrix. The coating has only a sin¬ gle type of pathogen binding protein. The pathogen binding protein has an affinity towards certain type of bacteria, therefore binding to such bacteria when brought in contact. In the next step of the disclosed method, the affinity matrix is brought in contact with a sample comprising pathogens. Once the affinity matrix contacts the pathogens in the sam¬ ple, a complex is formed. In the next step of the method, the complex is isolated from the sample. The device disclosed therein comprises of an affinity matrix which includes a ma¬ trix which is at least partly coated with a protein coating. The protein coating is composed of a single type of pathogen binding protein that has an affinity towards the pathogens in the sample.
However, such a method and device can isolate only a limited variety of bacteria as the affinity of a single type of path¬ ogen binding protein is limited. Therefore, if the sample comprises of a variety of bacteria that does not have any af¬ finity towards the pathogen binding protein used in the coat¬ ing, such variety of bacteria would not be isolated from the sample and would be lost.
The object of the invention is therefore to have a method and a device of the aforementioned kind that can isolate greater varieties of pathogens that may be present in a given sample. The invention achieves the object in that said protein coat¬ ing around said matrix is of a combination of two or more types of pathogen binding proteins.
The present invention relates to a method of isolation of pathogens from a given sample. According to the invention, the method comprises a step of coating a matrix at least partly with a combination of two or more types of pathogen binding proteins. Such a matrix along with the coating forms an affinity matrix. A pathogen binding protein is a protein comprising a pathogen binding domain which has a specific af¬ finity towards pathogens. The pathogen binding proteins may also be, for example, innate immune proteins like mannose binding lectin. One type of pathogen binding protein can bind to a limited variety of pathogens. Therefore, to broaden the scope of isolation, a combination of two or more different types of pathogen binding proteins are used in the coating.
According to the invention, in the next step of the method, the affinity matrix is brought into contact with the sample comprising pathogens. Once brought in contact with the sam¬ ple, the affinity matrix binds to the pathogens in the sample to form a complex. The proteins interact with the components in the cell membrane of the pathogens during the formation of the complex. In the next step of the method according to the invention, the complex formed is isolated. Therefore, the pathogens are isolated from the sample. According to an embodiment of the invention, the complex is isolated using a magnet. In this case the matrix used is a paramagnetic bead. Thus, the complex gets attracted to the magnet thus resulting in its isolation.
According to yet another embodiment of the invention, the complex is isolated using a centrifuge. In this case the ma¬ trix is a glass bead. Thus the complex would settle down as residue on centrifugation .
Based on the aforementioned device the invention solves the problem by using a protein coating around said matrix which is of a combination of two or more types of pathogen binding proteins. According to the invention, the device comprises an affinity matrix. The affinity matrix includes a matrix and a protein coating that coats the matrix at least partly. The matrix forms the base around which exists the protein coat¬ ing. The protein coating comprises of pathogen binding pro¬ teins that bind to pathogens that may be present in the sam- pie. The matrix may also be coated with immunoglobulin G to which pathogen binding domains of pathogen binding proteins are fused.
According to a preferred embodiment of the invention, the protein coating coated around the matrix is a combination of two or more types of pathogen binding proteins. A single type of pathogen binding protein would bind to a limited variety of pathogens. However, a combination of two or more pathogen binding proteins is capable of binding to a plethora of path- ogens that may be present in the sample. Therefore, the scope of isolation is increased.
According to a preferred embodiment of the invention, the ma¬ trix is a paramagnetic bead or a glass bead. The isolation of the complex formed between the pathogen and the affinity ma¬ trix can be easily isolated if the matrix is a paramagnetic bead or a glass bead. If the matrix is a paramagnetic bead, a magnet may be used as the isolating means. Similarly, if the matrix is a glass bead, a centrifuge may be used as the iso¬ lating means.
According to an embodiment of the invention, said combination of two or more pathogen binding proteins is chosen from a group comprising of innate immune protein, acute phase pro¬ tein, and fusion protein containing pathogen binding domain. The proteins that may be used but not limited to include toll like receptors (TLR) class of proteins, proteins of classical complement pathway, proteins of alternative complement path¬ way, collectins, ficolin, and acute phase protein.
The present invention is further described hereinafter with reference to illustrated embodiments shown in the accompany- ing drawings, in which:
FIG 1 illustrates a schematic diagram of an embodiment of a device according to the invention.
FIG 2 illustrates a flowchart of an embodiment of a meth- od according to the invention.
Hereinafter, embodiments for carrying out the present inven¬ tion are described in detail. The various embodiments are de¬ scribed with reference to the drawings, wherein like refer- ence numerals are used to refer to like elements throughout. In the following description, for purpose of explanation, nu¬ merous specific details are set forth in order to provide a thorough understanding of one or more embodiments. It may be evident that such embodiments may be practiced without these specific details.
FIG 1 illustrates a schematic diagram of an embodiment of a device 1 for isolation of pathogen from a given sample. The device 1 comprises of a matrix 2. The matrix depicted in FIG 1 is a paramagnetic bead. The paramagnetic bead 2 forms the base around which there exists a coating of antibody IgG 3 to which pathogen binding domains 4,5 are fused. Together with the coating, the matrix 2 forms an affinity matrix or device 1. The pathogen binding domains 4,5 are chosen from two or more types of pathogen binding proteins. In FIG 1, the IgG 3 is fused with pathogen binding domain of ApoH 4 which is an acute phase protein and that of mannose binding lectin (MBL) 5. ApoH 4 is a plasma glycoprotein expressed in human liver, intestine and tissues. It is an acute phase protein found circulating in an inactive ring conformation. In the activat¬ ed form, ApoH 4 binds to pathogens or their surface proteins with relatively high affinity. The interaction of pathogen and ApoH 4 has ionic and hydrophobic components. The posi¬ tively charged region of the protein interacts with anionic phospholipid head group and the flexible loop putatively in¬ serts into the lipid layer and positions Trp316 at the inter- face region between the acyl chains and phosphate head groups of the lipids, thereby anchoring the protein molecule to the membrane. Mannose binding lectin 5, on the other hand, is a calcium dependent C-type serum protein that plays a central role in the innate immune response. It binds to carbohydrates (specifically mannose residues) on the surface of a wide range of pathogens where it can activate lectin complement pathway. The lectin part of mannose binding lectin 5 has a carboxy terminal carbohydrate recognition domain that recog¬ nizes the carbohydrate patterns on the microbial surface. Mannose binding lectin 5 specifically recognizes mannose, fucose, glucose, N-acetylglucosamine, and N-acetylmannosamine in a Ca2+ dependent manner.
Although mannose binding lectin 5 and ApoH 4 are known to bind a variety of pathogens, the binding efficiency may vary across pathogens. This limits the pathogen capture efficien¬ cy. Therefore, using a combination of both mannose binding lectin 5 and ApoH 4 increases the efficiency of pathogen binding .
FIG 2 provides an illustration of a method 6 of isolation of pathogens from a given sample, according to the invention. The method 6 comprises a step 7 of coating the matrix 2 with antibodies 3 fused with pathogen binding domains 4,5 of two or more types of pathogen binding proteins to form an affini¬ ty matrix 1. In the next step 8, the affinity matrix 1 is brought in contact with the sample such that the pathogens are in the vicinity of the device 1. The affinity matrix 1 forms a complex with the pathogens in the sample. The patho¬ gen binding domains 4, 5 of the pathogen binding proteins in¬ teract with the cellular membrane of the pathogens and are therefore linked. In the next step 9 of the method 6, the complex formed between the affinity matrix 1 and the patho¬ gens is isolated from the sample using a magnet. The matrix 2 being a paramagnetic bead gets attracted to the magnet, therefore isolating the pathogen along with it.

Claims

We claim: 1. A method (6) of isolation of pathogens from a given sam¬ ple, said method (6) comprising the steps of:
a. coating a matrix (2) at least partly with a combina¬ tion of two or more types of pathogen binding pro¬ teins (4,5) to form an affinity matrix (1);
b. contacting said sample with said affinity matrix (1) forming a complex thereby; and
c. isolating said complex.
2. The method (6) of claim 1 wherein:
isolating said complex using a magnet.
3. The method (6) of claim 1 wherein:
isolating said complex using a centrifuge.
4. A device (1) for isolation of pathogens from a given
sample, said device comprising:
a. an affinity matrix (1) including:
i. a matrix (2); and
ii. a protein coating at least partly coating said matrix (2 ) ;
characterized in that:
b. said protein coating around said matrix is of a com¬ bination of two or more types of pathogen binding proteins (4,5) .
5. The device (1) of claim 4 wherein:
said matrix (2) is a paramagnetic bead or glass bead.
6. The device (1) of claims 4 and 5 wherein:
said combination of pathogen binding proteins (4,5) are selected from a group comprising of innate immune pro¬ tein, acute phase protein, and fusion protein containing pathogen binding domain.
PCT/EP2016/071838 2015-09-15 2016-09-15 A method and a device for isolation of pathogens WO2017046251A1 (en)

Applications Claiming Priority (2)

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IN983KO2015 2015-09-15
IN983/KOL/2015 2015-09-15

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110262925A1 (en) * 2010-04-21 2011-10-27 Nanomr, Inc. Methods for isolating a target analyte from a heterogeneous sample
EP2505666A1 (en) 2011-04-02 2012-10-03 New England Biolabs, Inc. Methods and compositions for enriching either target polynucleotides or non-target polynucleotides from a mixture of target and non-target polynucleotides
WO2012145018A1 (en) * 2011-04-21 2012-10-26 Nanomr, Inc. Compositions for isolating a target analyte from a heterogeneous sample
US20130316355A1 (en) * 2010-04-21 2013-11-28 Nanomr, Inc. Methods for isolating a target analyte from a heterogeneous sample
US20140170652A1 (en) * 2012-12-19 2014-06-19 Nanomr, Inc. Target capture system

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110262925A1 (en) * 2010-04-21 2011-10-27 Nanomr, Inc. Methods for isolating a target analyte from a heterogeneous sample
US20130316355A1 (en) * 2010-04-21 2013-11-28 Nanomr, Inc. Methods for isolating a target analyte from a heterogeneous sample
EP2505666A1 (en) 2011-04-02 2012-10-03 New England Biolabs, Inc. Methods and compositions for enriching either target polynucleotides or non-target polynucleotides from a mixture of target and non-target polynucleotides
WO2012145018A1 (en) * 2011-04-21 2012-10-26 Nanomr, Inc. Compositions for isolating a target analyte from a heterogeneous sample
US20140170652A1 (en) * 2012-12-19 2014-06-19 Nanomr, Inc. Target capture system

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