WO2001020042A2 - Method for processing biological samples - Google Patents
Method for processing biological samples Download PDFInfo
- Publication number
- WO2001020042A2 WO2001020042A2 PCT/US2000/025423 US0025423W WO0120042A2 WO 2001020042 A2 WO2001020042 A2 WO 2001020042A2 US 0025423 W US0025423 W US 0025423W WO 0120042 A2 WO0120042 A2 WO 0120042A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- biological sample
- separation reagent
- adduct
- target species
- separation
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Definitions
- the present invention relates to methods, compositions and systems for isolating materials from biological samples.
- STR Multiplexed short tandem repeat
- epithelial cells tend to lysis more quickly with detergents than do sperm cell (under non-reducing conditions). There is no a priori reason to expect these protocols to yield a complete separation of the male and female fractions. If the sample contains far more of the victims DNA then the suspects, which is often the case, then STR analysis often reveals only the victim's genotype, leaving the suspect unidentified. Much effort has been invested in the generation of highly selective antibodies for binding to specific biological targets, such as human sperm. Hybridoma cell lines which produce antibodies which are found to be useful for in vitro assays (i.e. ELISA) and pharmaceutical applications (e.g.
- Photoaffinity labeling has become a popular technique for studying the binding interactions between biomolecules which accompany most biological events. According to this technique, one biomolecule expected to be involved in a binding event is decorated with a chemical group which will form covalent bonds to a second involved biomolecule during or after light activation.
- One popular photoaffinity labeling technique uses the arylazide group for the light activated attachment step. When light activated, arylazides lose diatomic nitrogen producing the reactive nitrene intermediate. Nitrenes are known to form covalent bonds to neighboring molecules by addition to unsaturated linkages or insertion into single covalent bonds (C-H or C-C).
- attachment of the arylazide group to one molecule allows it to be covalently coupled to a second molecule.
- This property of the arylazide group has been applied in the area of bioconjugation (e.g. biotin labeling) and protein crosslinking, as described in B. Lacey & W.N. Grant, Anal. Biochem. (1987) Vol. 163, p. 151 and U.C. Krieg, et al, Proc. Natl. Acad. Sci. USA (1986) Vol. 83, p. 8604.
- Active esters of photoaffinity labels such as arylazides are commercially available and protocols for their use in decorating proteins are well developed, as described in D.A. Geselowitz & R.D.
- the invention features a method of processing a biological sample.
- the method includes providing a separation reagent, reacting the biological sample with the separation reagent to capture a target species in the biological sample, creating an adduct of the target species and the separation reagent and separating the adduct from the biological sample.
- the separation reagent includes a microparticle and a receptor for a ligand on the target species
- Preferred embodiments of the invention include one or more of the following advantageous features.
- the covalent adduct is formed by activating a photoaffinity label coupled to the separation reagent. Separating the adduct includes capturing the microparticle magnetically, by filtration, or by centrifugation.
- the receptor includes at least one binding protein, which can be an antibody.
- the biological sample is a forensic sample and the target species is a sperm cell.
- the method includes releasing a DNA from the sperm cell, magnetically removing the adduct, and analyzing the DNA .
- the invention features a separation reagent for a biological sample.
- the separation reagent includes a microparticle, a receptor coupled to the microparticle, and a photoaffinity label coupled to the receptor.
- Preferred embodiments of the invention include one or more of the following advantageous features.
- the microparticle is a magnetic bead.
- the receptor includes at least one binding protein, which can be an antibody.
- the photoaffinity label includes an arylazide.
- the arylazide includes a nitroarylazide.
- the invention features an automated system for processing a biological sample.
- the automated system includes means for providing a separation reagent, means for reacting the biological sample with the separation reagent to capture a target species in the biological sample, means for creating an adduct of the target species and the separation reagent and means for separating the adduct from the biological sample.
- the separation reagent includes a microparticle and a receptor for a ligand on the target species
- the invention features an apparatus for processing a biological sample.
- the apparatus includes a first chamber for receiving the sample, a first capture means proximate to the first chamber for capturing a separation reagent, a second chamber in fluidic communication with the first chamber, and a second capture means proximate to the second chamber for capturing the separation reagent.
- the first and second capture means can include electromagnets. Advantages that can be seen in implementations of the invention include one or more of the following. Capturing the target species with a selective receptor provides a high degree of selectivity for the target species in the biological sample. The permanent attachment of the target species and separation reagent using a photoaffinity label allows for the complete separation of the target species from the biological sample.
- microparticles provides a large surface area for the permanent attachment of receptors, enabling the efficient capture of a large percentage, or all, of the target species molecules from the biological sample.
- the use of magnetic beads for sample separation and DNA isolation avoids the need for centrifugation steps, thereby enabling the full automation of sample processing and yielding highly reproducible results.
- FIG. 1 is a flowchart illustrating a method of isolating a target species from a biological sample.
- FIG. 2 illustrates the synthesis of a photoactivatable separation reagent according to the invention.
- FIG. 3 illustrates the use of a photoactivatable separation reagent to isolate DNA from a target species in a biological sample.
- FIG. 4 is a schematic diagram of an apparatus for isolating a target species from a biological sample according to the invention.
- the invention provides methods, compositions and systems for processing biological samples using microparticulate separation reagents to capture a target species from a biological sample.
- the methods begin with the preparation of a separation reagent (step 100).
- the separation reagent is formed by binding a receptor - for example, a binding protein having affinity for a target species in the biological sample - to a microparticle, such as a coated magnetic bead, as will be described in more detail in connection with FIG. 2 below.
- Microparticles for use in the invention generally have dimensions of from about 1 millimeter to about 1 nanometer and may be fabricated from a wide variety of materials, including latex polystyrene, colloidal metals or other appropriate substances using known techniques.
- the prepared separation reagent is reacted with a biological sample known or suspected to contain the target species (step 110), and an adduct of the separation reagent and the target species is formed (step 120), as will be described in more detail in connection with FIG. 3 below.
- This adduct is separated from the biological sample (step 130), and the target species is released for further analysis free of the biological sample (step 140).
- photoactivatable separation reagents for specific target species are prepared by decorating microparticles 200 with target-specific receptors 210 and attaching chemical species 220 to these receptors that will form covalent bonds between the receptor and the target species after photoactivation.
- the target species is human sperm and the receptor is a binding protein having an affinity for human sperm - for example, an anti-sperm antibody or mixture of anti-sperm antibodies such as those produced by cell lines ATCC HB-9762, HB-255 and HB-10039.
- the receptor should (1) not be crossreactive with other cell types found in the biological sample (or be minimally crossreactive); (2) bind to the target species with high affinity; and (3) and in this embodiment, bind selectively to the head of the sperm cell.
- the selective binding to the sperm head is important because the head contains the DNA targeted for isolation and the tails of the sperms are often found missing in case samples.
- Appropriate receptors can be identified by screening for affinity and the orientation of binding using known techniques ⁇ for example, microtiter plate based fluorescence assays and optical microscopy.
- fragments 230 which carry reactive sulf ydryl groups 240 on a portion of the antibody fragment which is a distance from the Fab region 250 of the antibody binding site.
- Fragments 230 are attached to microparticles such as coated magnetic beads 200, such as those available from Dynal Corporation of Oslo, Norway, or Bangs Laboratories, Inc., of Fishers, Indiana, by the reaction of sulfhydryl groups 240 with haloacetyl or maleimide groups on the microparticle surface, as described in K. Kato, et al., J. Immunology (1976) Vol. 116 (6), p. 1554.
- fragments 230 are attached to microparticles 200 through an additional linker such as a secondary antibody.
- Fab region 250 a distance from the surface of the microparticle 200 such that it can bind to the target antigen with out interference.
- photoaffinity label 220 is introduced by acylation with the NHS ester of a nitroarylazide, such as 5-azido-2-nitrobenzoyloxysuccinimide 270 (ANB- NOS, available from Pierce Chemicals) under known conditions.
- a nitroarylazide such as 5-azido-2-nitrobenzoyloxysuccinimide 270 (ANB- NOS, available from Pierce Chemicals) under known conditions.
- nitroarylazide group has a red shifted absorbance spectrum relative to other known arylazides, making its photoactivation possible with visible light, which is advantageous for many biological samples of interest, such as those collected in sexual assault cases, which may contain UV absorbing impurities.
- Separation reagent 280 is then isolated by magnetic capture, filtration, centrifugation or other separation techniques, and washed to remove impurities.
- the binding of an intact sperm cell to the modified antibody produces a permanent (covalent) antibody-sperm cell adduct 370.
- the capture of the microparticles, followed by rigorous washing will allow for the permanent separation of the sperm cells, and most importantly the DNA that they carry, from any other cells or cellular debris present in the sample.
- Separation reagent 300 is added to a solution of the biological sample to be analyzed 310, which is prepared by suspending the sample cells, collected, for example, from a sexual assault victim, in a suitable buffer (for example, containing surfactants for disruption of epithelial cells and the like).
- a suitable buffer for example, containing surfactants for disruption of epithelial cells and the like.
- low energy sonication is used to ensure that all target cells are extracted into solution 310.
- Separation reagent 300 captures target cell 320, forming receptor-ligand complex 330.
- complex 330 is separated from mixture 340 by capture of microparticles 350, by magnetic capture, filtration, centrifugation or other separation techniques, and is resuspended in fresh buffer.
- mixture 340 is carried directly to the irradiation stage, described next.
- the suspension containing complex 330 is irradiated with light from light source 360, producing covalent adduct 370 and permanently attaching target cell 320 to bead
- Adduct 370 is isolated by magnetic capture, filtration, centrifugation or other separation techniques, and purified by repeated washing, optionally including low energy sonication, until all traces of foreign cellular material (e.g., cellular material from a victim) are removed. Because adduct 370 (or optionally complex 330) can be separated from mixture
- Target cell DNA 380 is released from adduct 370, for example by chemical reduction (e.g. buffer containing 2-mercaptoethanol or dithiothreitol) and/or proteinase K digestion or other means, and the microparticles 390 are removed from the sample by magnetic capture, filtration, centrifugation or other separation techniques, if desired. Purified DNA 380 is then analyzed using known techniques, such as the prior art magnetic beadVPCR techniques described above.
- FIG. 4 One embodiment of an apparatus 400 for applying these techniques to the processing of the forensics samples is illustrated in FIG. 4.
- a forensics sample suspected to contain a biological target species e.g., sperm cells
- the sample can be of varying volume (e.g., from 1 to 100 milliliters or more) and composition (e.g., clothing, upholstery, etc.), and inlet 410 and chamber 420 are configured accordingly.
- a buffer solution is introduced into chamber 420 and the target species is suspended in the solution, for example by mixing using stirrer 430.
- the adduct can be captured prior to draining to prevent any loss of adduct (e.g., by magnetic interaction between electromagnet 450 and coated magnetic beads included in the separation reagent, by gravity, or other means as discussed above).
- the adduct can also be washed to remove all traces of contaminants by adding and draining additional solution through inlet 410 and outlet 430 respectively.
- the adduct is resuspended in clean buffer and valve 460 is opened to allow the mixture to flow to chamber 470 through tubing 480.
- the adduct is captured (e.g., by electromagnet 490). Chamber 470 is then sealed, and the target species is released from the captured adduct as described above. This results in a concentrated sample of the target species suitable for further analysis - for example, PCR amplification and analysis.
- the second chamber 470 can be omitted, in which case the target species is released for further analysis after processing in the first chamber 420.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002383415A CA2383415A1 (en) | 1999-09-15 | 2000-09-15 | Method for processing biological samples |
JP2001523811A JP2003509680A (en) | 1999-09-15 | 2000-09-15 | Methods for processing biological samples |
EP00961946A EP1212465A2 (en) | 1999-09-15 | 2000-09-15 | Method for processing biological samples |
AU73829/00A AU7382900A (en) | 1999-09-15 | 2000-09-15 | Method for processing biological samples |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15414899P | 1999-09-15 | 1999-09-15 | |
US60/154,148 | 1999-09-15 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001020042A2 true WO2001020042A2 (en) | 2001-03-22 |
WO2001020042A3 WO2001020042A3 (en) | 2001-11-22 |
Family
ID=22550197
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/025423 WO2001020042A2 (en) | 1999-09-15 | 2000-09-15 | Method for processing biological samples |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1212465A2 (en) |
JP (1) | JP2003509680A (en) |
AU (1) | AU7382900A (en) |
CA (1) | CA2383415A1 (en) |
WO (1) | WO2001020042A2 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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JP5773405B2 (en) * | 2010-07-09 | 2015-09-02 | 国立大学法人高知大学 | Method for detecting compounds that interact with molecules on cell membranes |
JPWO2017175523A1 (en) | 2016-04-06 | 2019-02-14 | コニカミノルタ株式会社 | Fluorescent immunostaining |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0156537A2 (en) * | 1984-03-02 | 1985-10-02 | Board Of Regents University Of Texas System | Biological magnetic fluids |
US4716122A (en) * | 1984-09-28 | 1987-12-29 | Organogen Medizinisch-Molekularbiologische Forschungsgesellschaft M.B.H. | Carrier material for use in immune determinations |
EP0332022A2 (en) * | 1988-03-10 | 1989-09-13 | BEHRINGWERKE Aktiengesellschaft | Magnetic protein conjugates, process for their production and their use |
US4879220A (en) * | 1986-11-18 | 1989-11-07 | State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Crosslinking receptor-specific probes for electron microscopy |
EP0408078A2 (en) * | 1989-07-14 | 1991-01-16 | Roche Diagnostics GmbH | Method for the preparation of a solid phase coated with an immunologically active substance |
EP0475784A1 (en) * | 1990-09-14 | 1992-03-18 | Biosite Diagnostics Inc. | Antibodies to complexes of ligand receptors and ligands and their utility in ligand-receptor assays |
WO1994001773A1 (en) * | 1992-07-06 | 1994-01-20 | Biomira Inc. | Photoactivation of proteins for conjugation purposes |
WO1995016204A1 (en) * | 1993-12-07 | 1995-06-15 | University Court Of The University Of Glasgow | Surface-patterned device |
WO1995031429A1 (en) * | 1994-05-11 | 1995-11-23 | Trustees Of Boston University | Photocleavable agents and conjugates for the detection and isolation of biomolecules |
WO1996031781A1 (en) * | 1995-04-01 | 1996-10-10 | Boehringer Mannheim Gmbh | System for releasing and isolating nucleic acids |
-
2000
- 2000-09-15 JP JP2001523811A patent/JP2003509680A/en active Pending
- 2000-09-15 CA CA002383415A patent/CA2383415A1/en not_active Abandoned
- 2000-09-15 WO PCT/US2000/025423 patent/WO2001020042A2/en not_active Application Discontinuation
- 2000-09-15 AU AU73829/00A patent/AU7382900A/en not_active Abandoned
- 2000-09-15 EP EP00961946A patent/EP1212465A2/en not_active Withdrawn
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0156537A2 (en) * | 1984-03-02 | 1985-10-02 | Board Of Regents University Of Texas System | Biological magnetic fluids |
US4716122A (en) * | 1984-09-28 | 1987-12-29 | Organogen Medizinisch-Molekularbiologische Forschungsgesellschaft M.B.H. | Carrier material for use in immune determinations |
US4879220A (en) * | 1986-11-18 | 1989-11-07 | State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of The University Of Oregon | Crosslinking receptor-specific probes for electron microscopy |
EP0332022A2 (en) * | 1988-03-10 | 1989-09-13 | BEHRINGWERKE Aktiengesellschaft | Magnetic protein conjugates, process for their production and their use |
EP0408078A2 (en) * | 1989-07-14 | 1991-01-16 | Roche Diagnostics GmbH | Method for the preparation of a solid phase coated with an immunologically active substance |
EP0475784A1 (en) * | 1990-09-14 | 1992-03-18 | Biosite Diagnostics Inc. | Antibodies to complexes of ligand receptors and ligands and their utility in ligand-receptor assays |
WO1994001773A1 (en) * | 1992-07-06 | 1994-01-20 | Biomira Inc. | Photoactivation of proteins for conjugation purposes |
WO1995016204A1 (en) * | 1993-12-07 | 1995-06-15 | University Court Of The University Of Glasgow | Surface-patterned device |
WO1995031429A1 (en) * | 1994-05-11 | 1995-11-23 | Trustees Of Boston University | Photocleavable agents and conjugates for the detection and isolation of biomolecules |
WO1996031781A1 (en) * | 1995-04-01 | 1996-10-10 | Boehringer Mannheim Gmbh | System for releasing and isolating nucleic acids |
Non-Patent Citations (5)
Title |
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EBERLE A N: "PHOTO AFFINITY LABELING OF PEPTIDE HORMONE RECEPTORS" JOURNAL OF RECEPTOR RESEARCH, vol. 3, no. 1-2, 1983, pages 313-326, XP001009839 ISSN: 0197-5110 * |
KAUSCH ALBERT P ET AL: "Organelle isolation by magnetic immunoabsorption." BIOTECHNIQUES, vol. 26, no. 2, February 1999 (1999-02), pages 336-343, XP002170373 ISSN: 0736-6205 * |
MARGEL ET AL.: "Functional nanospheres: synthesis and biological applications" RECENT RES.DEVEL.IN POLYMER SCIENCE, vol. 1, 1997, pages 51-78, XP001009817 * |
MARGEL SHLOMO ET AL: "Synthesis, characterization, and use of new solid and hollow, magnetic and non-magnetic, organic-inorganic monodispersed hybrid microspheres." SCIENTIFIC AND CLINICAL APPLICATIONS OF MAGNETIC CARRIERS., 1997, pages 37-51, XP001009773 International Conference;Rostock, Germany; September 5-7, 1996, Plenum Press;Plenum Press 233 Spring Street, New York, New York, USA; London, England, UK ISBN: 0-306-45687-7 * |
SAFARK I ET AL: "Use of magnetic techniques for the isolation of cells" JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL APPLICATIONS,NL,ELSEVIER SCIENCE PUBLISHERS, vol. 722, no. 1-2, 5 February 1999 (1999-02-05), pages 33-53, XP004156204 ISSN: 0378-4347 * |
Also Published As
Publication number | Publication date |
---|---|
WO2001020042A3 (en) | 2001-11-22 |
JP2003509680A (en) | 2003-03-11 |
AU7382900A (en) | 2001-04-17 |
EP1212465A2 (en) | 2002-06-12 |
CA2383415A1 (en) | 2001-03-22 |
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