METHOD FOR PROCESSING BIOLOGICAL SAMPLES

Abstract: Methods, compositions and systems for processing biological samples employ microparticulate separation reagents to capture a target species from a biological sample. Photoactivation of a photoaffinity label coupled to the separation reagent creates a covalent adduct of the separation reagent and target species. The adduct is captured and DNA from the target species is analyzed using known techniques. Capture is accomplished using magnetic beads, filtration, centrifugation or other separation techniques. The method of the invention is amenable to complete automation.

Description: METHOD FOR PROCESSING BIOLOGICAL SAMPLES BACKGROUND OF THE INVENTION The present invention relates to methods, compositions and systems for isolating materials from biological samples. Multiplexed short tandem repeat ("STR") analysis of human DNA has been found to be a very useful technique for the identification of persons for law enforcement purposes. In order for the data generated to be accepted in a court of law, the STR method requires the use of DNA from a single individual that has been isolated, purified and quantified using a highly reproducible protocol. When applied to the analysis of sexual assault evidence, difficulties are encountered because the samples which are collected often contain cellular material from both the victim and the suspect. Extraction of the total DNA from the cellular mixture yields DNA from both persons in an unknown ratio, often with more of the victims DNA than the suspect's. Current protocols call for the separation of the male (sperm) and female (e.g. epithelial) components before the DNA isolation step by using a single differentiating feature of these cell types: 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. birth control) are commercially available (e.g. ATCC HB-9762, HB-255 and HB- 10039). 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). Thus, 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. Neumann, Bioconjugate Chem. (1995) Vol. 6, p. 502 and A.C. Forster, et al, Nucleic Acids Research (1985) Vol. 13, p. 745. NHS activated esters, for example, will react with primary amino groups of proteins (e.g. lysine residues) producing stable amide bonds. Photoactivation of the decorated protein, after binding to a receptor, will produce a covalent adduct between the photoaffinity label and the receptor. The automated isolation of DNA for PCR amplification is a topic of current interest. The development of magnetic bead methods for DNA isolation is seen as being a generally useful activity, increasing the throughput and reproducibility of the PCR method as a whole. Many new technologies of this type have appeared recently which have not been tested for STR amplification. For example, Dynal Corporation has introduced a magnetic bead protocol which has become quite popular in molecular biology research and is finding applications in the clinical laboratory. Hardware for the automation of the Dynal method is commercially available (Biomek 2000, Beckman Coulter, Inc., Fullerton, California). Automated assays employing magnetic beads, such as the Isolex 300i system for CD34 cell isolation available from Nexell Therapeutics, Inc., of Irvine, California, have reported and are commercially available. SUMMARY OF THE INVENTION In general, in one aspect, 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 . In general, in another aspect, 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. In general, in another aspect, 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 In general, in still another aspect, 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. In preferred embodiments, 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. The use of 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. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will become apparent from the description, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS 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. Like reference numbers and designations in the various drawings indicate like elements. DETAILED DESCRIPTION The invention provides methods, compositions and systems for processing biological samples using microparticulate separation reagents to capture a target species from a biological sample. As shown in FIG. 1 , 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). Referring to FIG. 2, in one embodiment 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. In the described embodiment, 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. Ideally, 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. While the invention is described in this embodiment, those skilled in the art will recognize that the principles of the invention can be applied advantageously to other targets and receptors, such as detection of pathogens in food, profiling organisms present in environmental samples, separating fetal blood cells from maternal blood cells for cytogenetic analysis, isolation of lymphocytes or other cells from whole blood, etc. As shown in FIG. 2, selective reduction of the disulfides which connect the heavy chains of an antibody 210 (e.g. with 2-aminoethanethiol) produces antibody 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. Optionally, fragments 230 are attached to microparticles 200 through an additional linker such as a secondary antibody. Because of the location of sulfhydryl groups 240, the chemical bonding of sulfhydryl groups 240 to the surface of the microparticles will thus place Fab region 250 a distance from the surface of the microparticle 200 such that it can bind to the target antigen with out interference. After purification of adduct 260 by magnetic capture, filtration, centrifugation or other separation techniques, 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. See U.C. Krieg, et al, Proc. Natl. Acad. Sci. USA (1986) Vol. 83, p. 8604. The 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. Referring to FIG. 3, the binding of an intact sperm cell to the modified antibody, after light activation, 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). Optionally, 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. In one embodiment, 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. In another embodiment, 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 350. 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 340 by magnetic capture, no centrifugation step is necessary to isolate the target cells from the biological sample. As a result, the isolation of target cells, and analysis of target DNA. can be completely automated, for example using known techniques and hardware such as the Biomek 2000 available from Beckman Coulter, Inc. This automation results in increased efficiency and reproducibility as compared to previously known methods. 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. 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) is introduced through inlet 410 into a chamber 420 loaded with a separation reagent or reagents as described above. In this embodiment, 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. After the target species is captured by and attached to the separation reagent (to form an adduct as described above), the remaining solution is drained through outlet 440. Optionally, depending on the relative size of the separation reagent and outlet 440, 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. Optionally, the adduct is resuspended in clean buffer and valve 460 is opened to allow the mixture to flow to chamber 470 through tubing 480. As the mixture flows through chamber 470, 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. In other embodiments, 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. The invention has been described in terms of particular embodiments. Those skilled in the art will recognize that other embodiments are within the scope of the following claims.

Claims: WHAT IS CLAIMED: 1. A method of processing a biological sample, comprising: providing a separation reagent comprising a microparticle and a receptor for a ligand on a target species in the biological sample ; reacting the biological sample with the separation reagent to capture the target species; creating a covalent adduct of the target species and the separation reagent ; and separating the adduct from the biological sample. 2. The method of claim 1 , wherein the covalent adduct is formed by activating a photoaffinity label coupled to the separation reagent. 3. The method of claim 1, wherein separating the adduct comprises magnetically capturing the microparticle. 4. The method of claim 1 , wherein separating the adduct comprises capturing the microparticle by filtration. 5. The method of claim 1, wherein separating the adduct comprises capturing the microparticle by centrifugation. 6. The method of claim 1, wherein the receptor comprises at least one binding protein. 7. The method of claim 1 , wherein the receptor comprises at least one antibody. 8. The method of claim 7, wherein the biological sample is a forensic sample and the target species is a sperm cell. 9. The method of claim 8, further comprising: releasing a DNA from the sperm cell; magnetically removing the adduct; and analyzing the DNA. 10. A separation reagent for a biological sample comprising: a microparticle; a receptor coupled to the microparticle; and a photoaffinity label coupled to the receptor. 11. The separation reagent of claim 10. wherein the microparticle is a magnetic bead. 12. The separation reagent of claim 10, wherein the receptor comprises at least one binding protein. 13. The separation reagent of claim 10, wherein the receptor comprises at least one antibody. 14. The separation reagent of claim 10, wherein: the photoaffinity label comprises an arylazide. 15. The separation reagent of claim 14, wherein: the arylazide comprises a nitroarylazide. 16. An automated system for processing a biological sample, comprising: means for providing a separation reagent comprising a microparticle and a receptor for a ligand on a target species in the biological sample ; means for reacting the biological sample with the separation reagent to capture the target species; means for creating an adduct of the target species and the separation reagent ; and means for separating the adduct from the biological sample. 17. An apparatus for processing a biological sample, comprising: a first chamber for receiving a biological 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. 18. The apparatus of claim 17. wherein the first and second capture means comprise electromagnets.

Inventors: CHAPMAN WILLIAM H JR, KLEVAN LEONARD, CHAPMAN, WILLIAM, H., JR., KLEVAN, LEONARD
Applicants: CHAPMAN WILLIAM H JR, KLEVAN LEONARD, MIRAIBIO INC, CHAPMAN, WILLIAM, H., JR., KLEVAN, LEONARD, MIRAIBIO INC.
Application year: 2000
Main classification: C12Q1
IPC codes: C12M1, C12Q1, G01N1, G01N33