EP1410042A1 - Procedes et appareil de determination de groupes sanguins a l'aide de disques biologiques optiques - Google Patents
Procedes et appareil de determination de groupes sanguins a l'aide de disques biologiques optiquesInfo
- Publication number
- EP1410042A1 EP1410042A1 EP01994076A EP01994076A EP1410042A1 EP 1410042 A1 EP1410042 A1 EP 1410042A1 EP 01994076 A EP01994076 A EP 01994076A EP 01994076 A EP01994076 A EP 01994076A EP 1410042 A1 EP1410042 A1 EP 1410042A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- disc
- cells
- capture
- chamber
- blood
- 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- Withdrawn
Links
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Classifications
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- 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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- 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
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
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- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N35/00069—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
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- G01N15/02—Investigating particle size or size distribution
- G01N15/0272—Investigating particle size or size distribution with screening; with classification by filtering
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- G01N15/042—Investigating sedimentation of particle suspensions by centrifuging and investigating centrifugates
- G01N2015/045—Investigating sedimentation of particle suspensions by centrifuging and investigating centrifugates by optical analysis
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Definitions
- the present invention relates to the field of diagnostic assays and biological analysis and to identification of cell types in a biological sample and analyses related thereto.
- the invention is further related to the manufacture and use of optically readable discs for biological analysis.
- Medical diagnositcs are critical to the diagnosis and treatment of disease, as well as the general maintenance of good health. Particularly useful are the biological and chemical assays performed on whole blood or its components.
- a fourth main blood type, AB was found in 1902 by A. Decastrello and A. Sturli.
- genes encoding the antigenic determinants have also been identified. See for example United States Patent No. 5,326,857 to Yamamoto et al. (1994) entitled "ABO Genotyping" which discloses genes defining the ABO histo- blood groups and methods for the identification of histo-blood group ABO status.
- the methods described include the use of DNA probes or size separation of DNA fragments unique to a blood group status, DNA constructs, recombinant methods for providing histo-blood glycosyltransferases, methods for tumor suppression, purified histo-blood group glycosylfransferases, and antibodies produced therefrom which bind to protein epitopes.
- United States Patent No. 4,650,662 to Goldfinger et al. (1987) entitled "Portable Blood Typing Apparatus and Method” discloses a portable apparatus to enable rapid determination of an individual's ABO blood group and Rh blood type and a method of using such apparams.
- the apparams has a plurality of microtubes joined together which contain blood taken from an individual.
- the assembly of micro bes is connected during use to an assembly of reaction chambers containing blood typing reagents.
- the apparatus enables rapid visualization of the test reactions within the reaction chambers, and may be used in locations removed from a laboratory to determine the ABO blood group and Rh blood type of an individual.
- the analyzer comprises a rotatable plate carrying sample-bearing test-tubes and dilution test-tubes ananged along concentric circumferences; a dispensing needle which is movable by mechanical means between a washing position, a position for drawing a sample, a position for diluting the sample and a position for introducing the sample into a reading well; a station for washing said needle; a conveyor unit for conveying canier members which are provided with 12 reaction wells to a position for receiving diluted or the undiluted samples from the dispensing needle; an automatic feeder that feeds small balls into each of the wells during the forward motion along the conveyor unit; mechanical means for transferring the canier member to a reading zone; a unit that meters the specific antiserum or red cells into each one of the wells; and an optical reading device that horizontally reads the transmittance of each one of the wells, starting from the moment when antiserum or red cells are introduced; and a processor for functionally controlling the analyzer and for issuing an estimate of
- U.S. Patent No. 6,030,581 entitled “Laboratory In A Disc” (hereinafter “the '581 patent”) describes an apparatus that includes an optical disc, having a substantially self contained assay means for binding an analyte suspected of being in a sample.
- U.S. Patent No. 5,892,577 entitled “Apparatus and Method for Carrying Out Analysis of Samples” describes systems and methods for conducting an optical inspection of a biological, chemical or biochemical sample supported by an optical transparent disc.
- U.S. Patent No. 6,143,510 entitled “Measuring Method Using Whole Blood Sample” describes methods for quantitatively measuring analyte in an undiluted whole blood sample by contacting the sample with magnetic particles coated with a binding partner which binds to an analyte in the sample. There is no description of this assay being earned out on an optical bio-disc.
- U.S. Patent No. 5,993,665 entitled “Quantitative Cell Analysis Methods Employing Magnetic Separation” describes immobilization of microscopic entities into a defined region in a collection chamber such that analysis by automated means is possible. The '665 patent describes quantitative collection of magnetically labeled target entities.
- the invention provides, a method for determining a blood group type of an individual by direct typing on an optical bio-disc comprising applying red blood cells to at least one chamber in the optical bio-disc, the chamber surface including at least one capture field including a capture antibody, at least one positive control field, and at least one negative control field; incubating the samples in the disc to promote antigen-antibody interaction; placing the disc into an optical reader that supports it on a first side; rotating the disc about an axis substantially pe ⁇ endicular to the first side to separate non-captured cells from captured cells located on the chamber surface; obtaining a measurement for the test field, the positive control field, and the negative control field analyzing the measurement of the test field, the positive control field and the negative control field to determine blood group type of the individual.
- the capture antibody is an anti-IgG antibody or the capture antibody is an anti-IgM antibody.
- the capture antibody is an antibody specific for a red blood cell antigen.
- the red blood cell antigen is an ABO system blood group antigen
- the red blood cell antigen is an Rh system blood group antigen
- the red blood cell antigen is an MNSs system blood group antigen
- the red blood cell antigen is a P system blood group antigen
- the red blood cell antigen is a Lutheran system blood group antigen
- the red blood cell antigen is a Kell system blood group antigen
- the red blood cell antigen is a Lewis system blood group antigen
- the red blood cell antigen is a Duffy system blood group antigen
- the red blood cell antigen is a Kidd system blood group antigen
- the red blood cell antigen is a Fisher system blood group antigen or the red blood cell antigen is a blood group antigen from any other blood group.
- the optical bio-disc is a reflective disc or the optical bio-disc is a transmissive disc. In other embodiments of the first aspect, the optical bio-disc comprises a CD or a DVD.
- the optical bio-disc has software embedded therein and the analysis of the measurement profile is controlled by the software, resulting in a bar code displaying the blood group type of the individual.
- the capture antibody is biotinylated and is bound to the test field by streptavidin bound thereto, or the capture antibody is bound to the test field by a second antibody bound to thereto or capture the second antibody is biotinylated and is bound to the test field by streptavidin bound thereto.
- the positive control field has a molecule on its surface that binds all cells.
- the molecule is a lectin.
- the molecule is gold.
- the invention provides, a method for determining the presence of antibodies to an ABO blood group in individual's blood sample by reverse-typing on an optical bio-disc including purifying serum from a blood sample; creating at least one sample by mixing serum with cells of a known ABO blood group; injecting at least one sample into at least one channel in the optical bio-disc, thereby delivering the sample onto a capture field including a cell binding molecule; incubating the sample on the capture field to allow the cells to bind to the cell binding molecule; placing the disc into an optical reader that supports it on a first side; rotating the disc about an axis substantially pe ⁇ endicular to the first side; scanning the chamber with an incident beam of electromagnetic radiation by rotating the disc about an axis substantially pe ⁇ endicular to the first side by moving the incident beam in a direction radial to the axis; detecting a return beam of electromagnetic radiation formed by at least a part of the incident beam after interacting with the disc; converting the return beam into an output signal; analyzing the output
- the creating step includes the creation of two samples, a first sample utilizing Type Al cells and a second sample utilizing Type B cells.
- step (b) further comprises the creation of a sample with Type AB cells.
- the cell binding molecule is an anti-human immunoglobulin.
- the cell binding molecule is a lectin or the cell binding molecule is gold.
- the optical bio-disc is a reflective disc or the optical bio-disc is a transmissive disc. In certain embodiments of the second aspect, the optical bio-disc comprises a CD or a DND.
- the invention provides method for determining the presence of antibodies to an ABO blood group in an individual's blood sample by reverse- typing on an optical bio-disc comprising applying a blood sample to at least one microfluidic channel in the optical bio-disc including a separation chamber with at least one microfilter, at least one mixing chamber, and at least one capture chamber; spinning for a first time the disc at a first speed to effect separation of the blood sample into cells and serum in the separation chamber; spinning for a second time the disc at a second speed higher than the first, the second speed effecting movement of the semm through the microfluidic channel into a mixing chamber; adding cells of a known ABO blood group cells into the mixing chamber containing serum; spinning for a third time the disc in one direction and alternately in another direction at least once to effect mixing of the serum and the cells; incubating the cells in the semm for a sufficient period of time to allow antibody-antigen binding; spinning for a fourth time the disc at a third speed higher than the second, the third speed effecting movement of
- first mixing chamber connected to a first capture chamber and a second mixing chamber connected to a second capture chamber.
- Type Al cells are placed in the first mixing chamber and Type B cells are placed in the second mixing chamber.
- the method further comprising a third mixing chamber connected to a third capture chamber and AB cells are added to the third mixing chamber in which AB cells are added.
- the cell binding molecule is an anti-human immunoglobulin, the cell binding molecule is a lectin, or the cell binding molecule is gold.
- the optical bio-disc is a reflective disc or the optical bio-disc is a transmissive disc.
- the optical bio-disc comprises a CD or a DVD.
- the first speed is from about IX to about 3X, the second speed is greater than 3X but less than about 5X, and the third speed is greater than about 5X. (IX refers to the audio standard for speed).
- the invention provides a method for determining the presence of antibodies to a blood group type in an individual by reverse-typing on an optical bio-disc comprising purifying serum from a blood sample; creating at least one sample by mixing serum with cells of a known blood group phenotyype; injecting at least one sample into at least one channel in the optical bio-disc, thereby delivering the sample onto a capture field including a cell binding molecule; incubating the sample on the capture field to allow the cells to bind to the cell binding molecule; placing the disc into an optical reader that supports it on a first side; rotating the disc about an axis substantially pe ⁇ endicular to the first side; scanning the chamber with an incident beam of electromagnetic radiation by rotating the disc about an axis substantially pe ⁇ endicular to the first side by moving the incident beam in a direction radial to the axis; detecting a return beam of electromagnetic radiation formed by at least a part of the incident beam after interacting with the disc; converting the return beam into an output signal; analyzing
- the cell binding molecule is an anti-human immunoglobulin.
- the optical bio- disc is a reflective disc the optical bio-disc is a transmissive disc.
- the optical bio-disc comprises a CD or a DVD.
- the first speed is from about IX to about 3X, the second speed is greater than 3X but less than about 5X, and the third speed is greater than about 5X.
- the cells added are characterized as having at least one of the following: an ABO system blood group cell phenotype, an Rh system blood group cell phenotype, an MNSs system blood group cell phenotype, a P system blood group cell phenotype, a Lutheran system blood group cell phenotype, a Kell system blood group cell phenotype, a Lewis system blood group cell phenotype, a Duffy system blood group cell phenotype, a Kidd system blood group cell phenotype, a Fisher system blood group antigen or a red blood cell group antigen from any other group.
- the invention provides a method for determining the presence of antibodies to a blood group type in an individual's blood sample by antibody-typing on an optical bio-disc comprising applying a blood sample to at least one microfluidic channel in the optical bio-disc including a separation chamber with at least one microfilter, at least one mixing chamber, and at least one capture chamber; spinning for a first time the disc at a first speed to effect separation of the blood sample into cells and semm in the separation chamber; spinning for a second time the disc at a second speed higher than the first, the second speed effecting movement of the serum through the microfluidic channel into a mixing chamber; adding cells of a known blood group cell phenotype into the mixing chamber containing serum; spinning for a third time the disc in one direction and alternately in another direction at least once to effect mixing of the serum and the cells; incubating the cells in the serum for a sufficient period of time to allow antibody-antigen binding; spinning for a fourth time the disc at a third speed higher than the second, the third speed
- the cell binding molecule is a lectin or wherein the cell binding molecule is gold.
- the optical bio-disc is a reflective disc or the optical bio-disc is a transmissive disc.
- the optical bio-disc comprises a CD or a DVD.
- the first speed is from about IX to about 3X, the second speed is greater than 3X but less than about 5X, and the third speed is greater than about 5X.
- the cells added are characterized as having at least one of the following an ABO system blood group cell phenotype, an Rh system blood group cell phenotype, an MNSs system blood group cell phenotype, a P system blood group cell phenotype, a Lutheran system blood group cell phenotype, a Kell system blood group cell phenotype, a Lewis system blood group cell phenotype, a Duffy system blood group cell phenotype, a Kidd system blood group cell phenotype, a Fisher system blood group antigen, or a red blood cell group antigen from any other blood group.
- the invention provides an apparatus for determining a blood group type of an individual.
- the apparatus includes an optical bio-disc including at least one capture chamber including a layer including a first capture antibody, and a layer including a second capture antibody bound by the first capture antibody, the second capture antibody being specific for a blood group antigen; a disc drive assembly; an optical reader; and software for blood group analysis.
- the capture antibody is an anti-IgG antibody or the second capture antibody is an anti-IgM antibody.
- the capture antibody is an antibody specific for a red blood cell antigen.
- red blood cell antigen is an ABO system blood group antigen
- the red blood cell antigen is an Rh system blood group antigen
- the red blood cell antigen is an MNSs system blood group antigen
- the red blood cell antigen is a P system blood group antigen
- the red blood cell antigen is a Lutheran system blood group antigen
- the red blood cell antigen is a Kell system blood group antigen
- the red blood cell antigen is a Lewis system blood group antigen or the red blood cell antigen is a Duffy system blood group antigen
- the red blood cell antigen is a Kidd system blood group antigen
- the red blood cell antigen is a Fisher system blood group antigen, or a red blood cell group antigen from any other group.
- the red blood cell antigen is an ABO system blood group antigen
- the invention provides an optical-bio disc for performing a blood-typing assay.
- the bio-disc includes a substrate; a separation chamber associated with the substrate, the separation chamber including first inlet port; filter means associated with the separation chamber; a first mixing chamber in fluid communication with the separation chamber, the first mixing chamber including a second inlet port ; a second mixing chamber in fluid communication with the separation chamber, the second mixing chamber including a third inlet port; a first detection chamber in fluid communication with the first mixing chamber, the first detection chamber including a capture zone; and a second detection chamber in fluid communication with the second mixing chamber, the second detection chamber including a capture zone.
- the optical bio-disc does not contain a second inlet port leading to the mixing chamber.
- the filter means separates white blood cells, red blood cells, and platelets from the blood sample to provide a sample of serum.
- the sample of serum is directed into the first and second mixing chambers.
- the inlet port of the first mixing chamber is employed to direct cells of a first type into the first mixing chamber, and the inlet port of the second mixing chamber is employed to direct cells of a second type into the second mixing chamber.
- a mixture of serum and cells of the first type is directed into the first detection chamber, and a mixture of serum and cells of the second type is directed into the second detection chamber.
- Certain embodiments of the seventh aspect provide for disc rotation in a predetermined manner to mix the cells of the first type with semm in the first mixing chamber, and mix the cells of the second type with serum in the second mixing chamber.
- the predetermined manner of rotating the disc includes alternately rotating the disc in one direction and then an opposite direction to thereby create an agitation action to promote mixing of serum and cells.
- the capture zone in the first detection chamber includes a first type of capture agent implemented to capture specific cells having any affinity therefor.
- the capture zone in the second detection chamber includes a second type of capture agent implemented to capture specific cells having any affinity therefor.
- an incident beam of radiant energy is directed into the first detection chamber to determine whether any cells were captured by the first type of capture agent. In other embodiments, an incident beam of radiant energy is directed into the second detection chamber to determine whether any cells were captured by the second type of capture agent.
- the first type of capture agent is an anti-human immunoglobulin having an affinity for an antibody bound to the cells or a non-cell specific molecule that binds a molecule on the surface of all red blood cells.
- the second type of capture agent is an anti-human immunoglobulin having an affinity for an antibody bound to the cells or a non-cell specific molecule that binds a molecule on the surface of all red blood cells.
- the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in the substrate. In other certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in a cap bonded to the substrate. In yet other certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in a channel layer bonded between a cap portion and the substrate. In certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are partially formed in a cap portion and partially formed in the substrate, the cap portion and the substrate being bonded together in register to thereby fully form the chambers.
- the optical bio-disc further includes information encoded in an information layer readable by a disc drive.
- the encoded information is used to rotate the disc in a prescribed manner.
- the information layer is reflective.
- the information layer is semi-reflective.
- the invention provides a method for manufacturing a disc comprising: providing over a substrate of the disc an encoded informational layer; forming target areas; providing a capture layer in the target areas; attaching at least one capture agen.
- the encoded informational layer is .a reflective layer, annd the target areas are regions etched from the reflective layer.
- the encoded informational layer is a partially reflective and partially transmissive layer, and the target areas are regions adjacent the informational layer.
- Figure 1 is a pictorial presenting the integrated optical bio-disc with sample chambers, an optical disc drive for sample processing and data collection, a data processing unit, and a monitor.
- Figures 2A-2C are an exploded perspective view, top view, and partially cutaway perspective views, respectively, of a reflective disc according to an embodiment of the present invention.
- Figures 3A-3C are an exploded perspective view, top view, and partially cutaway perspective views, respectively, of a transmissive disc according to an embodiment of the present invention.
- Figure 4 is a pictorial and schematic diagram of an optical disc reading system according to an embodiment of the present invention.
- Figure 5 is a pictorial presenting a general schematic of the cell capture technologies of the invention.
- Figure 6 is a pictorial schematic presenting the biotin/streptavidin-based cell capture technologies of the invention.
- Figure 7A-F is a schematic presenting a series of cross sections demonstrating the preparation of one example of a bio-disc of the invention.
- Figure 8 is a pictorial flow diagram presenting one example of a forward ABO Rh blood typing method of the invention.
- Figure 9 is a plan view illustrating data output in the form of a bar code.
- Figure 10 is a pictorial flow diagram demonstrating the method of antibody typing for blood groups other than the ABO Rh bloods with sample preparation off- disc and sample analysis on disc.
- Figures 11 A-C is schematic presenting a series of side views that show red blood cells bound by antibodies (11 A), red blood cells with antibody bound in contact with the capture field (1 IB), and red blood cells bound by antibodies being captured by the capmre field (1 IC).
- Figure 1 IC depicts the incident beam aimed at the capture field.
- Figures 12 A-C is schematic presenting a series of side views that show red blood cells bound not recognized by antibodies (12A), red blood cells without antibodies bound thereto in contact with the capture field (12B), and red blood cells without antibodies bound thereto not being captured by the capture field (12C).
- Figure 12C depicts the incident beam aimed at the capture field.
- Figure 13 is a pictorial flow diagram presenting the method of antibody typing wherein sample preparation and processing are all done on the optical bio-disc.
- the schematic of the left capture chamber presents cells bound to the capture field as previously presented in Figure IIC, whereas the schematic of the right capmre chamber presents cells without antibody bound thereto not being bound to the capture field as previously presented in Figure 12C.
- Figure 14 is a plan view schematic representing one example of a microfluidic circuit containing two inlet ports, a separation chamber, two mixing chambers, two capture chambers, and the incident beam of electromagnetic radiation directed at the capture chamber.
- Figure 15 is a pictorial flow diagram illustrating a method of reverse typing for the ABO/Rh blood groups with sample preparation off-disc and sample analysis on disc.
- Figure 16 is a pictorial presenting the events of cell binding during the reverse typing test when antibodies to an ABO Rh blood group antigen are present.
- Figure 16A shows red blood cells agglutinated after contact with antibodies direct to an ABO Rh group antibody.
- Figure 16B shows the same red blood cells interacting the capture field.
- Figure 16C shows the capture of antibody-bound, agglutinated red blood cells by a lectin or antibody.
- Figure 16C depicts the incident beam aimed at the capture field.
- Figure 17 is a pictorial presenting the events of cell binding during the reverse typing test when no antibodies to an ABO/Rh blood group antigen are present.
- Figure 17 A shows red blood cells that are not agglutinated after contact with serum.
- Figure 17B shows the same red blood cells interacting the capture field.
- Figure 16C shows the capmre of single cells (i.e., non-agglutinated) red blood cells by a molecule, e.g., a lectin, that binds all cells inespective of antibody binding.
- An incident beam of electromagnetic radiation is striking the capture field.
- Figure 18 is a pictorial flow diagram presenting the method of reverse ABO/Rh typing wherein sample preparation and processing are all done on the optical bio-disc.
- the schematic of the left capture chamber presents cells bound to the capmre field as previously presented in Figure 16C, whereas the schematic of the right capture chamber presents cells bound to the capmre chamber as previously presented in Figure 17C.
- Figure 19 is a pictorial of a computer monitor screen shot presenting an output of an ABO blood typing test.
- the invention described herein provides diagnostic assays based on cell- capture technology adapted to an optical bio-disc and methods and compositions related thereto.
- the system utilizes an optical bio-disc 50, an optical detection system 52, a data processing unit 54, and a monitor 55 to display output results.
- the cost-benefit of the biological assays of the present invention is that the cost of an analysis of a sample can be as low as 7 cents, which is much less- expensive than a test run in a clinical setting.
- the methods described herein are capable of being canied out by a relatively unskilled person in almost any location. The person would only need to be able to obtain a biological sample, e.g., whole blood, from an individual. In addition, a series of tests can be performed on one sample on a single disc, which increases the cost and time efficiency of the analysis.
- the assays described herein can replace those now routinely carried out in clinical laboratories such as hospitals and service laboratories; those canied out at the point of patient care (e.g., in physician offices, in patient service centers, and in emergency vehicles/rooms).
- the methods of the present invention can be used as portable testing and detection systems. Important features of this technology are that it is (1) extremely low cost as to both the instrumentation and the necessary reagents, (2) it is fast, highly sensitive and reproducible, highly accurate, and (3) many assays can be carried out simultaneously.
- An optical bio-disc for use with embodiments of the present invention may have any suitable shape, diameter, or thickness, but preferably is implemented on a round disc with a diameter and a thickness similar to those of a compact disc (CD), a recordable CD (CD-R), CD-RW, a digital versatile disc (DVD), DVD-R, DVD-RW, or other standard optical disc format.
- the disc may include encoded information, preferably in a known format, for performing, controlling, and post-processing a test or assay, such as information for controlling the rotation rate of the disc, timing for rotation, stopping and starting, delay periods, multiple rotation steps, locations of samples, position of the light source, and power of the light source.
- Such encoded information is refened to generally as operational information. Alternatively, the operational information can be provided separately.
- the disc may be reflective, transmissive, or some combination of reflective and transmissive.
- a reflective disc an incident light beam is focused onto a reflective surface of the disc, reflected, and returned through optical elements to a detector on the same side of the disc as the light source.
- a transmissive disc light passes through the disc (or portions thereof) to a detector on the other side of the disc from the light source.
- a transmissive portion of a disc some light may also be reflected and detected as reflected light.
- a reflective disc 100 is shown with a cap 102, a channel layer 104, and a substrate 106.
- Cap 102 has inlet ports 110 for receiving samples and vent ports 112.
- Cap 102 may be formed primarily from polycarbonate, and may be coated with a reflective layer 116 on the bottom thereof. Reflective layer 116 is preferably made from a metal, such as aluminum or gold.
- Channel layer 104 defines fluidic circuits 128 by having desired shapes from channel layer 104.
- Each fluidic circuit 128 preferably has a flow channel 130 and a return vent channel 132, and some have a mixing chamber 134.
- a mixing chamber 136 can be symmetrically formed relative to the flow channel 130, while an off-set mixing chamber 138 is formed to one side of the flow channel 130.
- Fluidic circuits 128 can include other channels and chambers, such as preparatory regions or a waste region, as shown, for example, in U.S. Patent No. 6,030,581, which is inco ⁇ orated herein by reference.
- Channel layer 104 can include adhesives for bonding substrate to cap.
- Substrate 106 has polycarbonate layer 108, and has target zones 140 formed as openings in a reflective layer 148 deposited on the top of layer 108.
- Target zones 140 may be formed by removing portions of reflective layer 148 in any desired shape, or by masking target zone areas before applying reflective layer 148.
- Reflective layer 148 is preferably formed from a metal, such as aluminum or gold, and can be configured with the rest of the substrate to encode operational information that is read with incident light, such as through a wobble groove or through an anangement of pits. Light incident from under substrate 106 thus is reflected by layer 148, except at target zones 140, where it is reflected by layer 116.
- optical disc 100 is cut away to illustrate a partial cross-sectional view.
- An active capture layer 144 is formed over reflective layer 148.
- Capture layer 144 may be formed from nitrocellulose, polystyrene, polycarbonate, gold, activated glass, modified glass, or a modified polystyrene, for example, polystyrene-co-maleic anhydride.
- Channel layer 104 is over capture layer 144.
- Trigger marks 120 are preferably included on the surface of a reflective layer 148, and may include a clear window in all three layers of the disc, an opaque area, or a reflective or semi-reflective area encoded with information. These are discussed below.
- samples are introduced through inlet ports 110 of cap 102. When rotated, the sample moves outwardly from inlet port 110 along capture layer 144.
- detectable features may be present in the target zones. These features are refened to as investigational features. Examples of such processes are shown in the inco ⁇ orated U.S. Patent No. 6,030,581.
- the investigational features captured by the capture layer may be designed to be located in the focal plane coplanar with reflective layer 148, where an incident beam is typically focused in conventional readers; alternatively, the investigational features may be captured in a plane spaced from the focal plane.
- the former configuration is refened to as a "proximal” type disc, and the latter a “distal” type disc.
- a transmissive optical disc 150 has a cap 152, a channel layer 154, and a substrate 156.
- Cap 152 includes inlet ports 158 and vent ports 160 and is preferably formed mainly from polycarbonate.
- Trigger marks 162 similar to those for disc 100 may be included.
- Channel layer 154 has fluidic circuits 164, which can have structure and use similar to those described in conjunction with FIGS. 2A, 2B, and 2C.
- Substrate 156 may include target zones 170, and preferably includes polycarbonate layer 174. Substrate 156 may, but need not, have a thin semi-reflective layer 172 deposited on top of layer 174. Semi-reflective layer 172 is preferably significantly thinner than reflective layer 148 on substrate 106 of reflective disc 100 (FIGS. 1A-1C). Semi-reflective layer 172 is preferably formed form a metal, such as aluminum or gold, but is sufficiently thin to allow a portion of an incident light beam to penetrate and pass through layer 172, while some of the incident light is reflected back. A gold film layer, for example, is 95% reflective at a thickness greater than about 700 A, while the transmission of light through the gold film is about 50% transmissive at approximately 100 A.
- FIG. 3C is a cut-away perspective view of disc 150.
- the semi-reflective nature of layer 172 makes its entire surface available for target zones, including virtual zones defined by trigger marks or specially encoded data patterns on the disc.
- Target zones 170 may also be formed by marking the designated area in the indicated shape or alternatively in any desired shape. Markings to indicate target zone 170 may be made on semi-reflective layer 172 or on a bottom portion of substrate 156 (under the disc).
- Target zones 170 may be created by silk screening ink onto semi-reflective layer 172.
- Capture layer 180 is applied over semi-reflective layer 172.
- Capture layer 180 may be formed from the same materials as described above in conjunction with layer 144 (FIG. 2C) and serves substantially the same pu ⁇ ose when a sample is provided through an opening in disc 150 and the disc is rotated. In transmissive disc 150, there is no reflective layer comparable to reflective layer 116 in reflective disc 100 (HG. 2C).
- FIG. 4 shows an optical disc reader system 200.
- This system may be a conventional reader for CD, CD-R, DVD, or other known comparable format, a modified version of such a drive, or a completely distinct dedicated device.
- the basic components are a motor for rotating the disc, a light system for providing light, and a detection system for detecting light.
- a light source 202 provides light to optical components 212 to produce an incident light beam 204, a return beam 206, and a transmitted beam 208.
- return beam 206 is reflected from either reflective surface 148 or 116.
- Return beam 206 is provided back to optical components 212, and then to a bottom detector 210.
- a transmitted beam 208 is detected by a top detector 214.
- Optical components 212 can include a lens, a beam splitter, and a quarter wave plate that changes the polarization of the light beam so that the beam splitter directs a reflected beam through the lens to focus the reflected beam onto the detector.
- An astigmatic element such as a cylindrical lens, may be provided between the beam splitter and detector to introduce astigmatism in the reflected light beam.
- Data from detector 210 and/or detector 214 is provided to a computer 230 including a processor 220 and an analyzer 222.
- An image or output results can then be provided to a monitor 224.
- Computer 230 can represent a desktop computer, programmable logic, or some other processing device, and also can include a connection (such as over the Internet) to other processing and/or storage devices.
- a drive motor 226 and a controller 228 are provided for controlling the rotation and direction of disc 100 or 150. Controller 228 and the computer with processor 220 can be in communication or can be the same computer. Methods and systems for reading such a disc are also shown in Gordon, U.S. Patent No. 5,892,577, which is inco ⁇ orated herein by reference.
- a hardware trigger sensor 218 may be used with either a reflective or transmissive disc. Triggering sensor 218 provides a signal to computer 230 (or to some other electronics) to allow for the collection of data by processor 220 only when incident beam 204 is on a target zone. Alternatively, software read from a disc can be used to control data collection by processor 220 independent of any physical marks on the disc.
- the substrate layer may be impressed with a spiral track that starts at an innermost readable portion of the disc and then spirals out to an outermost readable portion of the disc.
- this track is made up of a series of embossed pits with varying length, each typically having a depth of approximately one-quarter the wavelength of the light that is used to read the disc.
- the varying lengths and spacing between the pits encode the operational data.
- the spiral groove of a recordable CD-like disc has a detectable dye rather than pits. This is where the operation information, such as the rotation rate, is recorded.
- the rotation rate may be variable with intervening or consecutive periods of acceleration, constant speed, and deceleration. These periods may be closely controlled both as to speed and time of rotation to provide, for example, mixing, agitation, or separation of fluids and suspensions with agents, reagents, antibodies, or other materials.
- the disc drive assembly is thus employed to rotate the disc, read and process any encoded operational information stored on the disc, analyze the liquid, chemical, biological, or biochemical investigational features in an assay region of the disc, to write information to the disc either before or after the material in the assay zone is analyzed by the read beam of the drive or deliver the information via various possible interfaces, such as Ethernet to a user, database, or anywhere the information could be utilized.
- the invention relates to clinical diagnostic assays based on cell-capture technology as employed on an optical bio-disc described herein.
- Various embodiments of this aspect of the invention are directed to blood-typing diagnostic assays.
- a sample is loaded into a chamber within the optical bio-disc, where a capture field having bio-active capmre agent is affixed thereto.
- the bio-disc is then subjected to conditions suitable for cell binding.
- the bio-disc is placed into a CD drive assembly and is spun radially at a speed sufficient to separate unbound cells from bound cells, e.g., about 1000 ⁇ m to about 4000 ⁇ m for about one to five minutes. This spinning causes the cells which are not bound by the capture agent to be removed from the capmre fields and collected in a separate part of the chamber (e.g., in a waste receptacle in the chamber).
- the term “capture field” encompasses target zone 170 of an optical bio-disc which has attached thereto, either directly or indirectly, a capture agent.
- the capture field is a discrete location on the surface having defined limits, metes and bounds.
- the term “capture agent” is a molecule A on the surface of the target zone 170 that recognizes a molecule B and binds with specificity thereto.
- the phrase "binds with specificity” is meant herein to refer to the binding of molecule A to molecule B by at least two fold, at least five fold, at least 10 fold, at least one hundred fold, at least 1000 fold, at least 10,000 fold or more when compared to other molecules that may be present in a biological sample.
- molecules that specifically recognize and bind to other molecules include antibodies, ligands, receptors, enzymes, substrates, biotin, avidin, and lectins
- the bioactive agent of the invention may be obtained from any source, including but not limited to viral, bacterial, fungal, plant, animal, in vitro or synthetically produced materials.
- the capture agent is a capmre antibody and the capture field has at least one capmre antibody bound thereto.
- antibody includes polyclonal, monoclonal, and recombinantly created antibodies. Antibodies of the invention can be produced in vivo or in vitro. Methods for the production of antibodies are well known to those skilled in the art. For example, see Antibody Production: Essential Techniques. Peter Delves (Ed.), John Wiley & Son Ltd, ISBN: 0471970107 (1997). Alternatively, antibodies may be obtained from commercial sources, e.g., Research Diagnostics Inc., Pleasant Hill Road, Flanders, NJ 07836, Ortho Diagnostic Systems, BioClone Anti-A, Immucor, etc.)
- a capture agent to be bound to a capture field is within the skill of those in the art.
- a receptor-specific ligand may be bound to a capture field for the pu ⁇ ose of binding cells expressing the receptor recognized by the ligand or a capture field may be bound by a lectin that binds specifically a sugar moiety expressed on the surface of a select population of cells for the pu ⁇ ose of binding those cells.
- the capture field may be bound by a capture antibody specific for a receptor on the surface of a cell.
- antibody is not meant to be limited to antibodies of any one particular species, e.g., human, mouse, rat, goat, etc., are all contemplated by the invention.
- the term “antibody” is also inclusive of any class or subclass of antibodies, as all antibody types may be used to bring about an agglutination reaction.
- the IgG antibody class may be used for agglutination pmposes or, if a higher antibody polyvalency is desired, the IgM class of antibodies may be utilized for the same pu ⁇ ose.
- Other types of immunoglobulins that bind specifically to cells are also included within the scope of the invention.
- Antibody fragments can also be utilized as a capmre agent of the invention.
- the capture field with the capture agent bound thereto can be structured in any way suitable to bind cells.
- one or more capmre agents can be directly linked to the capmre field.
- capture fields may be uniformly bound with a multiple copies of a single capmre agent or, alternatively, capture fields may be bound with multiple copies of two or more capmre agents to increase the specificity of the binding reaction.
- the capmre agent can be indirectly linked to the capture field.
- a capture field may be coated with a protein such as streptavidin and a capture agent such as an antibody can be linked to the streptavidin by way of a biotin moiety attached to the antibody.
- the capture field of the invention has a first capture agent bound thereto and the first capture agent binds a second capture agent.
- an anti-IgM IgG antibody can serve as a first capture agent bound to a capture field, which itself binds an IgM antibody, the second capture agent.
- the capture agent bound to a capture field can in certain embodiments comprise more than one capture agent linked to one another in tandem.
- the capture agents may be attached to the capture field in different ways. By way of non-limiting example, various constructions are presented in Table 1.
- IgX refers to any antibody inmrnunoglobulin, e.g., IgG, IgM, etc.
- the bioactive surface can consist of five total layers in which the antibodies that comprise the bioactive capture layer are biotinylated and bind directly to the layer of streptavidin (or any variant thereof, the third layer).
- Figure 5 demonstrates various capture agents attached to a capture field by various means.
- IgG, IgM, a lectin, or another type of cell binding molecule can be bound directly to a capture field.
- the capture agents of the invention can be linked in tandem to the capture field to facilitate capture agent availability and minimize steric hindrance for cell capture.
- IgG and IgM can serve as anchors for any one of capture agents IgG, IgM, a lectin, or another molecule that binds cells for the pu ⁇ ose of capture.
- capture technology of the invention the latter provided exemplary embodiments thereof.
- Figure 6 presents cell capture technology of the invention based on the strong recognition and binding of the streptavidin (or variants thereof) and biotin molecules.
- the bioactive agent streptavidin can be first applied to the capture field. This molecule can then be used to bind and hold capture various capture agents, e.g., biotinylated IgG or biotinylated IgM or biotinylated lectin or another cell binding molecule that is biotinylated.
- Figure 7 shows a representation of the capture chemistry of one embodiment of the forward typing assay on a reflective zone disk.
- Figure 7A shows the substrate 120 coated withreflective layer 142 and capmre zones 140 where the reflective layer has been removed through lithography.
- Figure 7B showts the layer of Streptavidin passively adsorbed to the capture zones 140.
- Figure 7C shows th e biotinylated first capture antibody 272 bound to the steptavidin 270 in the capture zones 140.
- Figure 7D shows the second capture antibodies 274, 276 and 278 with different specificities bound to the biotinylated first capture antibody 272.
- Figure 7E shows an assembled Bio-Disk with cap portion 116, reflective surface 146 and inlet port 122, adhesive member 118, channel 128 and capture chemistries 270, 272, 274 276 and 278 in the capmre zones 140 on the substrate 120.
- Figure 7F shows the specific cell capmre based on the antigens expressed on the surface of the red blood cell. It also demonstrates the method of detection by focusing an incident beam of electromagnetic radiation 280 passing through a capture field 140 to strike a reflective layer, thereby producing a return beam of electromagnetic radiation 282 which is delivered to a detector system 284.
- optical bio-disc encompasses a disc, such as a compact disc (CD), having thereon a means for carrying out a biological assay, method or analysis.
- the disc is capable of being subjected to intenogation by a beam from a light source and data may be detected from the biological assay means.
- the disc may have information encoded on the disc itself which is available for detection by a disc drive assembly. The optical bio-disc is described in greater detail herein below.
- the term "chamber" encompasses any three-dimensional space defined by at least one material which is affixed to or part of an optical bio-disc.
- the chamber is leak-proof so that a liquid sample may be loaded into the chamber and subjected to certain reaction conditions (such as antibody binding conditions) and to detection methods (such as beam intenogations).
- the chamber may be made of plastic, of metal, of glass or of any other material which is suitable for the biological assay for which the optical bio-disc is used.
- the chamber may hold from about 4 ⁇ l to about 50 ⁇ l.
- the chamber is in fluid communication with a second chamber which can be utilized as a waste repository following the biological assay.
- an antibody which "specifically binds” means an antibody that binds to an epitope which comprises a peptide sequence or a carbohydrate moiety or a lipid moiety or a combination thereof. Such an antibody will not promiscuously bind to other molecules that do not have that specific epitope. Such a specifically binding antibody will not bind (or cross react) with other molecules or compounds lacking such an epitope.
- the assay is performed within an optical bio-disc that includes a chamber (also, "flow chamber") having specific antibodies or other capmre molecules attached to the solid phase associated with that chamber.
- a chamber also, "flow chamber” having specific antibodies or other capmre molecules attached to the solid phase associated with that chamber.
- a method is described for the determination of the occunence of a specific cell type (e.g., a specific type of red blood cell) expressing cell-specific surface antigens (e.g., A or B antigens) captured by specific antibodies affixed to the capture field(s).
- a specific cell type e.g., a specific type of red blood cell
- cell-specific surface antigens e.g., A or B antigens
- An optical bio-disc may have multiple capmre fields within one chamber.
- a grouping of several capture fields is termed a "bar code" because the data resulting from cells binding to certain capture fields resembles the dark and light striped pattern known as a bar code.
- bar code also inco ⁇ orated within such a bar code are defined negative control areas and positive control areas.
- An optical bio-disc drive assembly is employed to rotate the disc, read and process any encoded information stored on the disc, and analyze the cell capture fields in the flow chamber of the bio-disc.
- the bio-disc drive is provided with a motor for rotating the bio-disc, a controller for controlling the rate of rotation of the disc, a processor for processing return signals from the disc, and analyzer for analyzing the processed signals.
- the rotation rate is variable and may be closely controlled both as to speed and time of rotation.
- the bio-disc may also be utilized to write information to the bio-disc either before, during or after the assay.
- the test material in the flow chamber and capture fields is intenogated by the read beam of the drive and analyzed by the analyzer.
- the bio-disc may include encoded information for controlling the rotation of the disc, providing processing information specific to the type of immunotyping assay to be conducted and for displaying the results on a monitor associated with the bio-drive.
- the methods encompass evaluation tests in CD, CD-R or DVD formats. Variations or alternative versions thereof according to the present invention include a robust capture chemistry that is stabilized on the optical bio-disc. Unbound nonspecific cells are spun off leaving behind specific target cells from the blood sample which are specifically bound to the capture field on the bio-disc. The read or intenogation beam of the drive detects the captured cells and generates images that can be analyzed.
- red blood cells contain large numbers of antigenic determinants that are classified into blood groups. These antigenic determinants represent red blood cell surface markers that consist of protein and/or carbohydrate moieties.
- blood group Antigen Factsbook Fractsbooks Series
- Dractsbooks Series by Marion E. Reid (Editor) and Christine Lomas-Francis (Editor) (January 1997) Academic Press; ISBN: 0125859651).
- the ABO blood grouping is perhaps the most important, serving as the basis for the determination of transfusion compatibility.
- Rhesus (Rh) blood grouping Another frequently relied upon red blood cell grouping, which is an important test during pregnancy.
- the subject for blood typing is a mammal, e.g., a mouse or a human.
- the subject is a non-human mammal or a non-human primate.
- methods are provided for ABO and/or Rh typing of blood.
- the specific antibodies and antigens relevant for the ABO blood typing system are presented in Table 2.
- Figure 16C depicts the incident beam aimed at the capture field.
- Figure 17 is a pictorial presenting the events of cell binding during the reverse typing test when no antibodies to an ABO/Rh blood group antigen are present.
- Figure 17A shows red blood cells that are not agglutinated after contact with semm.
- Figure 17B shows the same red blood cells interacting the capture field.
- Figure 16C shows the capture of single cells (i.e., non-agglutinated) red blood cells by a molecule, e.g., a lectin, that binds all cells inespective of antibody binding. An incident beam of electromagnetic radiation is striking the capture field.
- Figure 18 is a pictorial flow diagram presenting the method of reverse ABO/Rh typing wherein sample preparation and processing are all done on the optical bio-disc.
- the schematic of the left capture chamber presents cells bound to the capture field as previously presented in Figure 16C, whereas the schematic of the right capture chamber presents cells bound to the capture chamber as previously presented in Figure 17C.
- Figure 19 is a pictorial of a computer monitor screen shot presenting an output of an ABO blood typing test.
- the invention described herein provides diagnostic assays based on cell- capture technology adapted to an optical bio-disc and methods and compositions related thereto.
- the system utilizes an optical bio-disc 50, an optical detection system 52, a data processing unit 54, and a monitor 55 to display output results.
- the cost-benefit of the biological assays of the present invention is that the cost of an analysis of a sample can be as low as 7 cents, which is much less expensive than a test mn in a clinical setting.
- the methods described herein are capable of being canied out by a relatively unskilled person in almost any location.
- the assays described herein can replace those now routinely carried out in clinical laboratories such as hospitals and service laboratories; those canied out at the point of patient care (e.g., in physician offices, in patient service centers, and in emergency vehicles/rooms).
- the methods of the present invention can be used as portable testing and detection systems. Important features of this technology are that it is (1) extremely low cost as to both the instrumentation and the necessary reagents, (2) it is fast, highly sensitive and reproducible, highly accurate, and (3) many assays can be carried out simultaneously.
- An optical bio-disc for use with embodiments of the present invention may have any suitable shape, diameter, or thickness, but preferably is implemented on a round disc with a diameter and a thickness similar to those of a compact disc (CD), a recordable CD (CD-R), CD-RW, a digital versatile disc (DVD), DVD-R, DVD-RW, or other standard optical disc format.
- the disc may include encoded information, preferably in a known format, for performing, controlling, and post-processing a test or assay, such as information for controlling the rotation rate of the disc, timing for rotation, stopping and starting, delay periods, multiple rotation steps, locations of samples, position of the light source, and power of the light source.
- Such encoded information is refened to generally as operational information. Alternatively, the operational information can be provided separately.
- the disc may be reflective, transmissive, or some combination of reflective and transmissive.
- a reflective disc an incident light beam is focused onto a reflective surface of the disc, reflected, and returned through optical elements to a detector on the same side of the disc as the light source.
- a transmissive disc light passes through the disc (or portions thereof) to a detector on the other side of the disc from the light source.
- some light may also be reflected and detected as reflected light.
- a reflective disc 100 is shown with a cap 102, a channel layer 104, and a substrate 106.
- Cap 102 has inlet ports 110 for receiving samples and vent ports 112.
- Cap 102 may be formed primarily from polycarbonate, and may be coated with a reflective layer 116 on the bottom thereof. Reflective layer 116 is preferably made from a metal, such as aluminum or gold.
- Channel layer 104 defines fluidic circuits 128 by having desired shapes from channel layer 104.
- Each fluidic circuit 128 preferably has a flow channel 130 and a return vent channel 132, and some have a mixing chamber 134.
- a mixing chamber 136 can be symmetrically formed relative to the flow channel 130, while an off-set mixing chamber 138 is formed to one side of the flow channel 130.
- Fluidic circuits 128 can include other channels and chambers, such as preparatory regions or a waste region, as shown, for example, in U.S. Patent No. 6,030,581, which is inco ⁇ orated herein by reference.
- Channel layer 104 can include adhesives for bonding substrate to cap.
- Substrate 106 has polycarbonate layer 108, and has target zones 140 formed as openings in a reflective layer 148 deposited on the top of layer 108.
- Target zones 140 may be formed by removing portions of reflective layer 148 in any desired shape, or by masking target zone areas before applying reflective layer 148.
- Reflective layer 148 is preferably formed from a metal, such as aluminum or gold, and can be configured with the rest of the substrate to encode operational information that is read with incident light, such as through a wobble groove or through an anangement of pits. Light incident from under substrate 106 thus is reflected by layer 148, except at target zones 140, where it is reflected by layer 116.
- optical disc 100 is cut away to illustrate a partial cross-sectional view.
- An active capture layer 144 is formed over reflective" layer 148.
- Capture layer 144 may be formed from nitrocellulose, polystyrene, polycarbonate, gold, activated glass, modified glass, or a modified polystyrene, for example, polystyrene-co-maleic anhydride.
- Channel layer 104 is over capmre layer 144.
- Trigger marks 120 are preferably included on the surface of a reflective layer 148, and may include a clear window in all three layers of the disc, an opaque area, or
- samples are introduced through inlet ports 110 of cap 102. When rotated, the sample moves outwardly from inlet port 110 along capture layer 144.
- detectable features may be present in the target zones. These features are refened to as investigational features. Examples of such processes are shown in the inco ⁇ orated U.S. Patent No. 6,030,581.
- the investigational features captured by the capture layer may be designed to be located in the focal plane coplanar with reflective layer 148, where an incident beam is typically focused in conventional readers; alternatively, the investigational features may be captured in a plane spaced from the focal plane.
- the former configuration is refened to as a "proximal” type disc, and the latter a “distal” type disc.
- a transmissive optical disc 150 has a cap 152, a channel layer 154, and a substrate 156.
- Cap 152 includes inlet ports 158 and vent ports 160 and is preferably formed mainly from polycarbonate.
- Trigger marks 162 similar to those for disc 100 may be included.
- Channel layer 154 has fluidic circuits 164, which can have structure and use similar to those described in conjunction with FIGS. 2A, 2B, and 2C.
- Substrate 156 may include target zones 170, and preferably includes polycarbonate layer 174. Substrate 156 may, but need not, have a thin semi-reflective layer 172 deposited on top of layer 174. Semi-reflective layer 172 is preferably significantly thinner than reflective layer 148 on substrate 106 of reflective disc 100 (FIGS. 1 A-IC). Semi-reflective layer 172 is preferably formed form a metal, such as aluminum or gold, but is sufficiently thin to allow a portion of an incident light beam to penetrate and pass through layer 172, while some of the incident light is reflected back. A gold film layer, for example, is 95% reflective at a thickness greater than about 700 A, while the transmission of light through the gold film is about 50% transmissive at approximately 100 A.
- FIG. 3C is a cut-away perspective view of disc 150.
- the semi-reflective nature of layer 172 makes its entire surface available for target zones, including virtual zones defined by trigger marks or specially encoded data patterns on the disc.
- Target zones 170 may also be formed by marking the designated area in the indicated shape or alternatively in any desired shape. Markings to indicate target zone 170 may be made on semi-reflective layer 172 or on a bottom portion of substrate 156 (under the disc). Target zones 170 may be created by silk screening ink onto semi-reflective layer 172.
- Capture layer 180 is applied over semi-reflective layer 172.
- Capture layer 180 may be formed from the same materials as described above in conjunction with layer 144 (FIG. 2C) and serves substantially the same pu ⁇ ose when a sample is provided through an opening in disc 150 and the disc is rotated. In transmissive disc 150, there is no reflective layer comparable to reflective layer 116 in reflective disc 100 (FIG. 2C).
- FIG. 4 shows an optical disc reader system 200.
- This system may be a conventional reader for CD, CD-R, DVD, or other known comparable format, a modified version of such a drive, or a completely distinct dedicated device.
- the basic components are a motor for rotating the disc, a light system for providing light, and a detection system for detecting light.
- a light source 202 provides light to optical components 212 to produce an incident light beam 204, a return beam 206, and a transmitted beam 208.
- return beam 206 is reflected from either reflective surface 148 or 116.
- Return beam 206 is provided back to optical components 212, and then to a bottom detector 210.
- a transmitted beam 208 is detected by a top detector 214.
- Optical components 212 can include a lens, a beam splitter, and a quarter wave plate that changes the polarization of the light beam so that the beam splitter directs a reflected beam through the lens to focus the reflected beam onto the detector.
- An astigmatic element such as a cylindrical lens, may be provided between the beam splitter and detector to introduce astigmatism in the reflected light beam.
- -19- Data from detector 210 and/or detector 214 is provided to a computer 230 including a processor 220 and an analyzer 222. An image or output results can then be provided to a monitor 224.
- Computer 230 can represent a desktop computer, programmable logic, or some other processing device, and also can include a connection (such as over the Internet) to other processing and/or storage devices.
- a drive motor 226 and a controller 228 are provided for controlling the rotation and direction of disc 100 or 150. Controller 228 and the computer with processor 220 can be in communication or can be the same computer. Methods and systems for reading such a disc are also shown in Gordon, U.S. Patent No. 5,892,577, which is inco ⁇ orated herein by reference.
- a hardware trigger sensor 218 may be used with either a reflective or transmissive disc. Triggering sensor 218 provides a signal to computer 230 (or to some other electronics) to allow for the collection of data by processor 220 only when incident beam 204 is on a target zone. Alternatively, software read from a disc can be used to control data collection by processor 220 independent of any physical marks on the disc.
- the substrate layer may be impressed with a spiral track that starts at an innermost readable portion of the disc and then spirals out to an outermost readable portion of the disc.
- this track is made up of a series of embossed pits with varying length, each typically having a depth of approximately one-quarter the wavelength of the light that is used to read the disc.
- the varying lengths and spacing between the pits encode the operational data.
- the spiral groove of a recordable CD-like disc has a detectable dye rather than pits. This is where the operation information, such as the rotation rate, is recorded.
- the rotation rate may be variable with intervening or consecutive periods of acceleration, constant speed, and deceleration. These periods may be closely controlled both as to speed and time of rotation to provide, for example, mixing, agitation, or separation of fluids and suspensions with agents, reagents, antibodies, or other materials.
- the disc drive assembly is thus employed to rotate the disc, read and process any encoded operational information stored on the disc, analyze the liquid, chemical, biological, or biochemical investigational features in an assay region of the disc, to write information to the disc either before or after the material in the assay zone is analyzed by the read beam of the drive or deliver the information via various possible interfaces, such as Ethernet to a user, database, or anywhere the information could be utilized.
- the invention relates to clinical diagnostic assays based on cell-capture technology as employed on an optical bio-disc described herein.
- Various embodiments of this aspect of the invention are directed to blood-typing diagnostic assays.
- a sample is loaded into a chamber within the optical bio-disc, where a capture field having bio-active capture agent is affixed thereto.
- the bio-disc is then subjected to conditions suitable for cell binding.
- the bio-disc is placed into a CD drive assembly and is spun radially at a speed sufficient to separate unbound cells from bound cells, e.g., about 1000 ⁇ m to about 4000 ⁇ m for about one to five minutes. This spinning causes the cells which are not bound by the capture agent to be removed from the capture fields and collected in a separate part of the chamber (e.g., in a waste receptacle in the chamber).
- capture field encompasses target zone 170 of an optical bio-disc which has attached thereto, either directly or indirectly, a capture agent.
- the capture field is a discrete location on the surface having defined limits, metes and bounds.
- the term “capture agent” is a molecule A on the surface of the target zone 170 that recognizes a molecule B and binds with specificity thereto.
- the phrase "binds with specificity” is meant herein to refer to the binding of molecule A to molecule B by at least two fold, at least five fold, at least 10 fold, at least one hundred fold, at least 1000 fold, at least 10,000 fold or more when compared to other molecules that may be present in a biological sample.
- molecules that specifically recognize and bind to other molecules include antibodies, ligands, receptors, enzymes, substrates, biotin, avidin, and lectins
- the bioactive agent of the invention may be obtained from any source, including but not limited to viral, bacterial, fungal, plant, animal, in vitro or synthetically produced materials.
- the capmre agent is a capture antibody and the capture field has at least one capmre antibody bound thereto.
- antibody includes polyclonal, monoclonal, and recombinantly created antibodies. Antibodies of the invention can be produced in vivo or in vitro. Methods for the production of antibodies are well known to those skilled in the art. For example, see Antibody Production: Essential Techniques, Peter Delves (Ed.), John Wiley & Son Ltd, ISBN: 0471970107 (1997). Alternatively, antibodies may be obtained from commercial sources, e.g., Research Diagnostics Inc., Pleasant Hill Road, Flanders, NJ 07836, Ortho Diagnostic Systems, BioClone Anti-A, frnmucor, etc.)
- a capture agent to be bound to a capture field is within the skill of those in the art.
- a receptor-specific ligand may be bound to a capture field for the pu ⁇ ose of binding cells expressing the receptor recognized by the ligand or a capture field may be bound by a lectin that binds specifically a sugar moiety expressed on the surface of a select population of cells for the pu ⁇ ose of binding those cells.
- the capmre field may be bound by a capture antibody specific for a receptor on the surface of a cell.
- antibody is not meant to be limited to antibodies of any one particular species, e.g., human, mouse, rat, goat, etc., are all contemplated by the
- antibody is also inclusive of any class or subclass of antibodies, as all antibody types may be used to bring about an agglutination reaction.
- the IgG antibody class may be used for agglutination pu ⁇ oses or, if a higher antibody polyvalency is desired, the IgM class of antibodies may be utilized for the same pu ⁇ ose.
- Other types of immunoglobulins that bind specifically to cells are also included within the scope of the invention.
- Antibody fragments can also be utilized as a capture agent of the invention.
- the use of antibodies in the art of medical diagnostics is well known to those skilled in the art. For example, see Diagnostic and Therapeutic Antibodies (Methods in Molecular Medicine), Andrew J. T.
- the capture field with the capture agent bound thereto can be structured in any way suitable to bind cells.
- one or more capture agents can be directly linked to the capmre field.
- capmre fields may be uniformly bound with a multiple copies of a single capmre agent or, alternatively, capture fields may be bound with multiple copies of two or more capmre agents to increase the specificity of the binding reaction.
- the capture agent can be indirectly linked to the capture field.
- a capture field may be coated with a protein such as streptavidin and a capmre agent such as an antibody can be linked to the streptavidin by way of a biotin moiety attached to the antibody.
- the capture field of the invention has a first capture agent bound thereto and the first capture agent binds a second capture agent.
- an anti-IgM IgG antibody can serve as a first capture agent bound to a capture field, which itself binds an IgM antibody, the second capture agent.
- the capture agent bound to a capture field can in certain embodiments comprise more than one capture agent linked to one another in tandem.
- the capture agents may be attached to the capture field in different ways.
- various constructions are presented in Table 1.
- IgX refers to any antibody inmmunoglobulin, e.g., IgG, IgM, etc.
- the bioactive surface can consist of five total layers in which the antibodies that comprise the bioactive capmre layer are biotinylated and bind directly to the layer of streptavidin (or any variant thereof, the third layer).
- Figures 5 and 6 demonstrates various capture agents attached to a capmre field by various means.
- IgG, IgM, a lectin, or another type of cell binding molecule can be bound directly to a capture field.
- the capture agents of the invention can be linked in tandem to the capture field to facilitate capture agent availability and minimize steric
- IgG and IgM can serve as anchors for any one of capmre agents IgG, IgM, a lectin, or another molecule that binds cells for the p pose of capture.
- capmre agents IgG, IgM, a lectin, or another molecule that binds cells for the p pose of capture Many variations are possible for the capture technology of the invention, the latter provided exemplary embodiments thereof.
- Figure 6 presents cell capture technology of the invention based on the strong recognition and binding of the streptavidin (or variants thereof) and biotin molecules.
- the bioactive agent streptavidin can be first applied to the capture field. This molecule can then be used to bind and hold capture various capmre agents, e.g., biotinylated IgG or biotinylated IgM or biotinylated lectin or another cell binding molecule that is biotinylated.
- Figure 7 shows a representation of the capmre chemistry of one embodiment of the forward typing assay on a reflective zone disk.
- Figure 7A shows the substrate 120 coated withreflective layer 142 and capture zones 140 where the reflective layer has been removed through lithography.
- Figure 7B showts the layer of Streptavidin passively adsorbed to the capture zones 140.
- Figure 7C shows th e biotinylated first capture antibody 272 bound to the steptavidin 270 in the capture zones 140.
- Figure 7D shows the second capture antibodies 274, 276 and 278 with different specificities bound to the biotinylated first capture antibody 272.
- Figure 7E shows an assembled Bio-Disk with cap portion 116, reflective surface 146 and inlet port 122, adhesive member 118, channel 128 and capmre chemistries 270, 272, 274276 and 278 in the capture zones 140 on the substrate 120.
- Figure 7F shows the specific cell capture based on the antigens expressed on the surface of the red blood cell. It also demonstrates the method of detection by focusing an incident beam of electromagnetic radiation 280 passing through a capmre field 140 to strike a reflective layer, thereby producing a return beam of electromagnetic radiation 282 which is delivered to a detector system 284.
- optical bio-disc encompasses a disc, such as a compact disc (CD), having thereon a means for carrying out a biological assay, method or analysis.
- the disc is capable of being subjected to intenogation by a beam from a light source and data may be detected from the biological assay means.
- the disc may have information encoded on the disc itself which is
- optical bio-disc available for detection by a disc drive assembly.
- the optical bio-disc is described in greater detail herein below.
- the term "chamber" encompasses any three-dimensional space defined by at least one material which is affixed to or part of an optical bio-disc.
- the chamber is leak-proof so that a liquid sample may be loaded into the chamber and subjected to certain reaction conditions (such as antibody binding conditions) and to detection methods (such as beam inte ⁇ ogations).
- the chamber may be made of plastic, of metal, of glass or of any other material which is suitable for the biological assay for which the optical bio-disc is used.
- the chamber may hold from about 4 ⁇ l to about 50 ⁇ l.
- the chamber is in fluid communication with a second chamber which can be utilized as a waste repository following the biological assay.
- an antibody which "specifically binds” means an antibody that binds to an epitope which comprises a peptide sequence or a carbohydrate moiety or a lipid moiety or a combination thereof. Such an antibody will not promiscuously bind to other molecules that do not have that specific epitope. Such a specifically binding antibody will not bind (or cross react) with other molecules or compounds lacking such an epitope.
- the assay is performed within an optical bio-disc that includes a chamber (also, "flow chamber") having specific antibodies or other capture molecules attached to the solid phase associated with that chamber.
- a chamber also, "flow chamber” having specific antibodies or other capture molecules attached to the solid phase associated with that chamber.
- a method is described for the determination of the occunence of a specific cell type (e.g., a specific type of red blood cell) expressing cell-specific surface antigens (e.g., A or B antigens) captured by specific antibodies affixed to the capture field(s).
- a specific cell type e.g., a specific type of red blood cell
- cell-specific surface antigens e.g., A or B antigens
- An optical bio-disc may have multiple capture fields within one chamber.
- a grouping of several capture fields is termed a "bar code" because the data resulting from cells binding to certain capture fields resembles the dark and light striped pattern known as a bar code.
- bar code also inco ⁇ orated within such a bar code are defined negative control areas and positive control areas.
- An optical bio-disc drive assembly is employed to rotate the disc, read and process any encoded information stored on the disc, and analyze the cell capture fields in the flow chamber of the bio-disc.
- the bio-disc drive is provided with a motor for rotating the bio-disc, a controller for controlling the rate of rotation of the disc, a processor for processing return signals from the disc, and analyzer for analyzing the processed signals.
- the rotation rate is variable and may be closely controlled both as to speed and time of rotation.
- the bio-disc may also be utilized to write information to the bio-disc either before, during or after the assay.
- the test material in the flow chamber and capture fields is intenogated by the read beam of the drive and analyzed by the analyzer.
- the bio-disc may include encoded information for controlling the rotation of the disc, providing processing information specific to the type of immunotyping assay to be conducted and for displaying the results on a monitor associated with the bio-drive.
- the methods encompass evaluation tests in CD, CD-R or DVD formats. Variations or alternative versions thereof according to the present invention include a robust capture chemistry that is stabilized on the optical bio-disc. Unbound nonspecific cells are spun off leaving behind specific target cells from the blood sample which are specifically bound to the capture field on the bio-disc. The read or intenogation beam of the drive detects the captured cells and generates images that can be analyzed.
- red blood cells contain large numbers of antigenic determinants that are classified into blood groups. These antigenic determinants represent red blood cell surface markers that consist of protein and/or carbohydrate moieties.
- blood group Antigen Factsbook Fractsbooks Series
- Dractsbooks Series by Marion E. Reid (Editor) and Christine Lomas-Francis (Editor) (January 1997) Academic Press; ISBN: 0125859651).
- the ABO blood grouping is perhaps the most important, serving as the basis for the determination of transfusion compatibility.
- Rhesus (Rh) blood grouping Another frequently relied upon red blood cell grouping, which is an important test during pregnancy.
- the subject for blood typing is a mammal, e.g., a mouse or a human.
- the subject is a non-human mammal or a non-human primate.
- methods are provided for ABO and/or Rh typing of blood.
- the specific antibodies and antigens relevant for the ABO blood typing system are presented in Table 2.
- Rh blood typing system there are three genes making up Rhesus antigens: C, D, and E, all found on chromosome 1. There are two possible alleles at each locus: c or C; D; and e or E. If an individual's Rh genotype contains at least one of the C, D, E antigens, they are Rhesus positive. Only individuals with the genotype cde/cde (n) are Rhesus negative.
- the invention provides a system for blood typing or the detection of antibodies directed against a particular blood type.
- the system includes methods for the isolation and processing of whole blood, introduction of the sample into an optical bio-disc having at least one chamber with a target zone 170 and capture fields contained therein, an optical disc reader, and a system for the data processing and a display for data presentation.
- Bio-assay methods of the invention can be conveniently designed into a "bar code" format for sample testing and data presentation.
- this particular embodiment for blood typing is illustrated in Figures 10.
- one optical bio-disc would contain thereon several capmre fields, each of which has affixed thereto a capture agent, e.g., antibody, which is specific for a particular determinant on the surface of a red blood cell.
- a capture agent e.g., antibody
- a direct benefit of this type of approach is that the characteristic pattern of a particular blood-type would be predetermined as a barcode readout. Therefore, the subsequent analysis of blood samples from subjects would then be compared to the known barcode result in order to determine the blood-type of that subject immediately.
- one strip of capture fields on a bio-disc includes separate capture fields which are a ⁇ anged in a row and are in fluid communication with one another and which have affixed thereto
- the optical bio-disc and disc reader assembly would allow a person to carry out blood typing analysis in the field (that is, not in a clinical setting) expeditiously.
- the invention provides a methods for antibody typing a blood sample, i.e., assaying a patients semm for the occunence of antibodies directed to an antigen of a blood group other than that of the ABO system.
- the invention provides a method for antibody typing wherein the sample undergoes processing prior to being loaded onto a bio-disc ( Figure 10 and 11A-C, 12A-C).
- the invention provides a method for antibody typing wherein the sample is loaded onto a bio-disc without significant processing (Figure 13).
- whole blood is first separated from semm prior to utilizing the serum in the bio-disc blood grouping assay.
- Whole blood can be separated into semm and cells by light centrifugation.
- the semm which contains a patient's antibodies, is then mixed with one or more O type ABO cells having a known phenotype for a blood group type other than that of ABO.
- the sample is incubated for a period of time, e.g., about fifteen to thirty minutes, at 37o C to allow the patients antibodies to interact with these cells. After incubation, the cells are washed several times and loaded into one or more chambers in the bio-disc.
- the red blood cells will have the antibodies bound thereto.
- These antibody-bound cells can then be captured on a capture field by an appropriate capture agent, e.g., an anti-human IgG.
- an appropriate capture agent e.g., an anti-human IgG.
- the capture field is examined for the occunence of cells. The capture field can then be examined by the optical reader to determine whether cells being tested are present in the capture field, and thereby determine the antibody status of the individual.
- the method provides for the separation of blood cells and semm by passage through a separation chamber 250 in the optical bio-disc. Separation of the fluid and cellular components of whole blood is effected by spinning the disc at a first speed, thereby moving the sample through at least one microfilter designed to separate red blood cells, white blood cells and platelets from the semm. Seram is then moved to at least one mixing chamber 252 by spinning the disc at a second speed, which is higher than the first speed. Cells of a known blood group phenotype are then added through a separate entry port 256 into at least one mixing chamber of the bio-disc.
- the microfluidic circuit has inlet ports 256 for the addition of cells of a known blood group phenotype to the mixing chamber(s).
- the inlet ⁇ ort(s) 256 are not necessary, since the mixing chamber has been preloaded with a microparticle coated with a specific antigen of a red blood cell blood type group, e.g., A antigen or B antigen.
- a specific antigen of a red blood cell blood type group e.g., A antigen or B antigen.
- Such particles may be conveniently prepared by purifying the red blood cell antigen, e.g., through recombinant gene expression and subsequent biochemical isolation, absorbing it onto the particles. These particles may then be loaded into the mixing chamber during construction of the bio-disc, e.g., in freeze-dried form.
- a bio- disc of this construction would be particularly useful in countries and areas where access to red blood cells of a known blood type phenotype is difficult.
- Mixing of the seram and cells is accomplished by spinning the disc at least once one-half a rotation counter clockwise and then clockwise one-half a rotation.
- the samples are then allowed to incubate in the mixing chamber for a sufficient time to allow antibody-antigen interaction.
- the cells are then moved to a capmre chamber 254 with a capture field by spinning the disc at a third speed, which is higher than the second speed.
- the cells are allowed to interact with the capture field, which has bound to it anti-human IgG, for a sufficient time to allow antibody-antigen interaction.
- the disc is then spun again to remove unbound cells (e.g., 400 ⁇ m-4000 ⁇ m) Data is then collected from the capture fields to determine if cells are bound to the capture field.
- the occunence of cells in a capture field indicates that the individual's seram has antibodies directed to an antigen on the surface of the particular red blood cell blood type phenotype being tested.
- the invention provides a method of antibody typing a blood sample on an optical bio-disc.
- blood may be conveniently isolated by finger-stick, diluted and loaded into the bio-disc for processing.
- multiple capture fields are exposed to the sample being tested.
- the multiple capture fields serve the pmpose of providing separate test areas in a target zone 170, each bound by a unique capture agent.
- unique capmre agents in a test designed for the ABO blood system would include anti-A antibody and anti-B antibody, each loaded onto separate capture fields on the disc.
- Antibodies with the appropriate specificity for antigens of different blood groupings are available commercially (e.g., anti-A and anti-B antibodies may be obtained from Fisher Scientific, Los Angles, CA, Catalogue Nos. 23287247 and 23287248, respectively).
- Positive and negative controls for the test would include a positive control capture field in which the capture agent is a molecule that binds all cells, e.g., a lectin isolated from Lycopersicon esculentum that binds ⁇ -D-glucosamine oligomers (Sigma Aldrich Chemical, Catalogue No. L-0651), and a negative control capmre field having no capture agent bound thereto.
- the capture agent is a molecule that binds all cells, e.g., a lectin isolated from Lycopersicon esculentum that binds ⁇ -D-glucosamine oligomers (Sigma Aldrich Chemical, Catalogue No. L-0651), and a negative control capmre field having no capture agent bound thereto.
- Various embodiments of this method of the invention may be similarly designed for the pmpose of blood typing according to any other blood typing system, e.g., for testing the Rh system blood group, the MNSs system blood group, the Lutheran system blood group, the Kell system blood group, the Lewis system blood group, the Duffy system blood group, the Kidd system blood group, the Fisher system blood group, or any other blood group.
- any other blood typing system e.g., for testing the Rh system blood group, the MNSs system blood group, the Lutheran system blood group, the Kell system blood group, the Lewis system blood group, the Duffy system blood group, the Kidd system blood group, the Fisher system blood group, or any other blood group.
- one or more blood type systems may be simultaneously tested on a single bio-disc.
- the remaining blood groups may complicate a blood typing but are not as important.
- antibodies to the antigens not expressed on red blood cells are non-red-cell stimulated (or naturally occuning), due to the similarity between the blood group antigens structure and environmental agents.
- the antibodies may be of the IgM, IgA or IgG class.
- the IgM antibodies can cause direct agglutination when mixed with cells bearing the antigen the antibody is directed against.
- the IgG antibodies can cross the placenta and cause hemolytic disease of the newborn in subsequent pregnancies.
- the ABO and Rh blood groups are the most important groups in transfusion medicine.
- Naturally occurring antibodies to the ABO and Rh antigens are of the IgM class.
- Antibodies to the ABO and Rh antigens are readily available and direct agglutination tests can be performed on red blood cell samples.
- agglutination of the red cells is not the test; the test in this instance is cell capture based on antigen-antibody interactions. The interaction is specific and accurate and indicates which antigens are present on the red blood cell surface.
- agglutination of cells captured on the optical disk is looked for and analyzed by software algorithm(s).
- the invention provides a methods for detecting specific antibodies to an ABO/Rh blood group antigen, e.g., assaying a patients serum for the occunence of anti-A or anti-B antibodies.
- the invention provides a method for reverse typing wherein the sample undergoes processing prior to being loaded onto a bio-disc ( Figure 15 and 16 A-C and 17A-C).
- the invention provides a method for reverse typing wherein the sample is loaded onto a bio-disc without significant processing (Figure 18).
- whole blood is first separated from seram prior to utilizing the seram in the bio-disc blood grouping assay.
- Whole blood can be separated into serum and cells by light centrifugation.
- the serum which contains a patient's antibodies, is then mixed with one or more cells having an ABO cell phenotype.
- the sample is incubated for a period of time, e.g., about one to five minutes, at room temperature to allow the patient's antibodies to interact with these cells.
- an anti-human antibody is used as the capture agent
- the cells are washed several times and loaded into one or more chambers in the bio-disc; if a lectin capture agent is used, washing is unnecessary.
- red blood cells will be agglutinated as a result of the antibodies bound thereto. These agglutinated cells can then be captured on a capture field by an appropriate capmre
- -33- agent e.g., a lectin that binds all cells.
- the capture field is examined for the occunence of agglutinated cells. The capture field can then be examined by the optical reader to determine whether cells being tested are agglutinated, and thereby determine that antibodies to an ABO Rh blood group antigen were present in the individual's blood.
- whole blood is loaded directly onto the bio-disc into a microfluidic circuit ( Figure 14).
- the method provides for the separation of blood cells and seram by passage through a separation chamber 250 in the optical bio-disc. Separation of the fluid and cellular components of whole blood is effected by spinning the disc at a first speed, moving the sample through at least one microfilter designed to separate red blood cells, white blood cells and platelets from the seram. Seram is then moved to at least one mixing chamber 252 by spinning the disc at a second speed, which is higher than the first speed. Cells of a specific ABO group phenotype are then added through a separate port entry into at least one mixing chamber.
- Mixing of the seram and cells is accomplished by spinning the disc at least once one-half a rotation counter clockwise and then clockwise one-half a rotation.
- the samples are then allowed to incubate in the mixing chamber for a sufficient time to allow antibody-antigen interaction.
- the cells are then moved to a capmre chamber 254 with a capture field by spinning the disc at a third speed, which is higher than the second speed.
- the cells are allowed to interact with the capture field which has bound to it anti-human immunoglobulin or another capmre agent for a sufficient time to allow capture agent interaction with the cells.
- the disc is then spun again to remove unbound cells, e.g., 400 ⁇ m to 4000 ⁇ m.
- Data is then collected from the capmre fields to determine if cells bound thereto are agglutinated.
- the occunence of agglutinated cells in a capture field indicates that the individual's serum has antibodies directed to an antigen on the surface of the particular red blood cell blood type phenotype being tested.
- the invention provides an optical-bio disc for performing a blood-typing assay, the disc comprising: a substrate; a separation chamber associated with the substrate, the separation chamber including an inlet port; filter means associated with the separation chamber; a first mixing chamber in fluid
- the first mixing chamber including an inlet port ; a second mixing chamber in fluid communication with the separation chamber, the second mixing chamber including an inlet port; a first detection chamber in fluid communication with the first mixing chamber, the first detection chamber including a capmre zone; and a second detection chamber in fluid communication with the second mixing chamber, the second detection chamber including a capture zone.
- the filter means separates white blood cells, red blood cells, and platelets from the blood sample to provide a sample of seram.
- the sample of seram is directed into the first and second mixing chambers.
- the inlet port of the first mixing chamber is employed to direct cells of a first type into the first mixing chamber
- the inlet port of the second mixing chamber is employed to direct cells of a second type into the second mixing chamber.
- a mixture of seram and cells of the first type is directed into the first detection chamber
- a mixture of seram and cells of the second type is directed into the second detection chamber.
- Certain embodiments of the seventh aspect provide for disc rotation in a predetermined manner to mix the cells of the first type with serum in the first mixing chamber, and mix the cells of the second type with serum in the second mixing chamber.
- the predetermined manner of rotating the disc includes alternately rotating the disc in one direction and then an opposite direction to thereby create an agitation action to promote mixing of serum and cells.
- the capture zone in the first detection chamber includes a first type of capture agent implemented to capture specific cells having any affinity therefor.
- the capture zone in the second detection chamber includes a second type of capture agent implemented to capture specific cells having any affinity therefor.
- an incident beam of radiant energy is directed into the first detection chamber to determine whether any cells were capmred by the first type of capture agent. In other embodiments, an incident beam of radiant energy is directed into the second detection chamber to determine whether any cells were captured by the second type of capture agent.
- the first type of capture agent is an anti-human immunoglobulin having an affinity for an antibody bound to the cells or a non-cell specific molecule that binds a molecule on the surface of all red blood cells.
- the second type of capture agent is an anti-human immunoglobulin having an affinity for an antibody bound to the cells or a non-cell specific molecule that binds a molecule on the surface of all red blood cells.
- the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in the substrate. In other certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in a cap bonded to the substrate. In yet other certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in a channel layer bonded between a cap portion and the substrate. In certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are partially formed in a cap portion and partially formed in the substrate, the cap portion and the substrate being bonded together in register to thereby fully form the chambers.
- the optical bio-disc further includes information encoded in an information layer readable by a disc drive.
- the encoded information is used to rotate the disc in a prescribed manner.
- the information layer is reflective. In other embodiments, the information layer is semi-reflective.
- the invention provides a method for manufacturing a disc comprising: providing over a substrate of the disc an encoded informational layer;
- the encoded informational layer is .a reflective layer, annd the target areas are regions etched from the reflective layer. In certain embodiments, the encoded informational layer is a partially reflective and partially transmissive layer, and the target areas are regions adjacent the informational layer.
- the tracking of the bio-disc of the present invention is a forward Wobble Set FDL21 : 13707 or FDL21 : 1270 coating with 300 nm of gold.
- FDL21 13707
- FDL21 1270 coating with 300 nm of gold.
- oval data windows of size 2x1 mm are etched out by Lithography.
- "U" shaped channels are used to create chambers that are 25 um in height. It takes about 7 uls of sample to fill the entire chamber including the inlet and outlet ports.
- a 8-window/4-channel format to be preferentially used.
- a semi-reflective transmissive disc (FDL 20/21:00708) is used which allows the entire surface of a transmissive disk may be used for capmre zones, without the use of lithography to form data windows.
- Fraylock "U” shaped adhesive DBL 201 Rev C 3M94661 or straight channels are used to create the chambers.
- the cover disc utilized is a gold disk, fully reflective with 48 sample inlets with a diameter of 0.040 inches location equidistant at radius 26mm.
- This first layer may be a polycarbonate layer or a metallized polycarbonate layer in a optical disc such as a CD, CD-ROM, DVD or DVD-ROM. Prior to subsequent treatment, the first layer is cleaned with isopropanol.
- the second layer consists of either polystyrene or polycarbonate. This layer may be formed by injection molding of bulk plastic or spin or spray coating of the plastic in a volatile solvent on a solid substrate.
- the primary capture layer is formed by abso ⁇ tion of the protein streptavidin (Sigma, St. Louis, MO, Catalogue No. S-4762) (or any variant thereof) on the second layer.
- the adso ⁇ tion process is accomplished by exposure of
- -37- the second layer to a first solution (1 mg/ml solution of streptavidin (or any variant thereof) at neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris buffer, ionic strength (varied by addition of NaCl, KCl or MgC12) between 50 and 200 mM). Exposure times may range between 30 seconds and 12 hours. After exposure of the second layer to the first solution, the excess streptavidin (or any variant thereof) is washed away with water.
- the secondary capture layer consists of biotin-labeled antibody (the first capmre antibody) that recognize and bond to other antibodies from a certain animal source (e.g., mouse or human) (e.g., biotinylated anti-mouse IgG (raised in sheep), Vector Laboratories, lot # L0602, Catalog # BA-9200).
- a solution of the first capture antibody (the second solution) is exposed to the third layer for between 10 minutes and 3 hours.
- the second solution comprises a 1 mg/ml solution of the first capture antibody at neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris buffer, ionic strength (varied by addition of NaCl, KCl or MgC12) between 50 and 200 mM.
- the biotin moiety on the surface of the first capture antibody is bound by the streptavidin (or any variant thereof) which comprises the third layer. After exposure of this layer to the second solution, the excess first capmre antibody is washed away with water.
- the bioactive capture layer the fifth layer, consists of the second capmre antibody, which recognizes and binds to a specific type of biological cell based on some antigen on the surface of that cell.
- the animal source of the second capture antibody must match the specificity of the first capture antibody.
- a solution of the second capture antibody is exposed to the fourth layer for between 10 minutes and 3 hours.
- This third solution comprises a 1 mg/ml solution of the second capmre antibody at neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris buffer, ionic strength (varied by addition of NaCl, KCl or MgC12) between 50 and 200 mM.
- excess second capmre antibody is washed away with a buffer similar to that described above.
- the discs of the invention are leak checked to make certain that none of the chambers leak during spinning of the disc with the sample in situ.
- Each channel is filled with a blocking agent. Blocking is done for least 1 hour.
- the discs are then spun at 5000 ⁇ m for 5 minutes and examined. After checking for leaks and removing the blocking solution, the disc is placed in a vacuum chamber for 2-48 hours. After vacuum treatment, discs are placed in a vacuum pouch and stored at 2-8°C until use.
- the disc can be heat or ultrasonically bonded to make certain no fluid escapes from the chamber.
- the forward blood typing assay is conducted on a bio-disc comprising: (1) gold reflective base disc, treated with photo-lithography to remove the gold in specific capmre zones, with appropriate chemistry placed over the capture zones, (2) 25 um thick channel layer, and (3) gold reflective cover disc, assembled into a functional bio-disk.
- Results are presented in Figure 19, which is a representation of a graphical output for ABO blood typing.
- Example 3 Antibody Typing Assay On Bio-Disc (Sample Preparation Off-Disc)
- the reverse blood typing assay is conducted on a bio- disc comprising: (1) gold reflective base disc, treated with photo-lithography to remove the gold in specific capture zones, with appropriate chemistry placed over the
- Whole blood is centrifuged at an appropriate speed and time to result in a pellet of cells and non-hemolyzed serum or plasma.
- the semm or plasma is separately mixed with Type Ay and Type B Reagent Red Blood Cells (Ortho Clinical Diagnostics).
- the mixture of the cells and semm or plasma may take place in test tubes or directly on the disk in a mixing chamber. If the mixing occurs in a test tube, each mixture is then placed in separate channels of the disk. After a short, room temperature incubation (2 to 5 minutes) to allow the seram or plasma to interact with the reagent red blood cells, the disk is placed into the drive.
- the automated agglutination-detection software developed in-house centrifuges the disk, causing the agglutinated and/or non-agglutinated cells to travel over the capture zone and be non- specifically captured. Excess cells will be centrifuged to the outer edge of the flow channel.
- the disk is scanned with the standard 780nm laser of the optical drive using the bottom detector and the software registers
- the program algorithm determines which reagent red blood cells were agglutinated and assigns an ABO phenotype, based on reverse typing to the plasma or seram sample. The entire process takes about 10 minutes from insertion of disk into the drive and receiving the reverse blood typing. The forward and reverse typings of an individual should be in agreement, signifying the conect typing of that individual.
- a method for determining a blood group type of an individual by direct typing on an optical bio-disc comprising: applying red blood cells to at least one chamber in the optical bio-disc, the chamber surface including at least one capmre field including a capture antibody, at least one positive control field, and at least one negative control field; incubating the samples in the disc to promote antigen-antibody interaction; placing the disc into an optical reader that supports it on a first side; rotating the disc about an axis substantially pe ⁇ endicular to the first side to separate non-captured cells from capmred cells located on the chamber surface; obtaining a measurement for the test field, the positive control field, and the negative control field analyzing the measurement of the test field, the positive control field and the negative control field to determine blood group type of the individual.
- a method for determining the presence of antibodies to an ABO blood group of an individual's blood sample by reverse-typing on an optical bio-disc including: purifying seram from a blood sample; creating at least one sample by mixing serum with cells of a known ABO blood group; injecting at least one sample into at least one channel in the optical bio-disc, thereby delivering the sample onto a capture field including a cell binding molecule; incubating the sample on the capmre field to allow the agglutinated and non- agglutinated cells to bind to the cell binding molecule; placing the disc into an optical reader that supports it on a first side; rotating the disc about an axis substantially pe ⁇ endicular to the first side;
- a method for determining the presence of antibodies to an ABO blood group of an individual's blood sample by reverse-typing on an optical bio-disc comprising: applying a blood sample to at least one microfluidic channel in the optical bio- disc including a separation chamber with at least one microfilter, at least one mixing chamber, and at least one capture chamber; spinning for a first time the disc at a first speed to effect separation of the blood sample into cells and seram in the separation chamber; spinning for a second time the disc at a second speed higher than the first, the second speed effecting movement of the seram through the microfluidic channel into a mixing chamber; adding cells of a known ABO blood group cells into the mixing chamber containing seram; spinning for a third time the disc in one direction and alternately in another direction at least once to effect mixing of the seram and the cells; incubating the cells in the serum for a sufficient period of time to allow antibody-antigen binding; spinning for a fourth time the disc at a third speed higher than the second, the third speed effecting movement of the cells into
- Rh blood typing system there are three genes making up Rhesus antigens: C, D, and E, all found on chromosome 1. There are two possible alleles at each locus: c or C; D; and e or E. If an individual's Rh genotype contains at least one of the C, D, E antigens, they are Rhesus positive. Only individuals with the genotype cde/cde (n) are Rhesus negative.
- the invention provides a system for blood typing or the detection of antibodies directed against a particular blood type.
- the system includes methods for the isolation and processing of whole blood, introduction of the sample into an optical bio-disc having at least one chamber with a target zone 170 and capture fields contained therein, an optical disc reader, and a system for the data processing and a display for data presentation.
- Bio-assay methods of the invention can be conveniently designed into a "bar code" format for sample testing and data presentation.
- this particular embodiment for blood typing is illustrated in Figures 10.
- one optical bio-disc would contain thereon several capture fields, each of which has affixed thereto a capture agent, e.g., antibody, which is specific for a particular determinant on the surface of a red blood cell.
- a capture agent e.g., antibody
- a direct benefit of this type of approach is that the characteristic pattern of a particular blood-type would be predetermined as a barcode readout. Therefore, the subsequent analysis of blood samples from subjects would then be compared to the known barcode result in order to determine the blood-type of that subject immediately.
- one strip of capmre fields on a bio-disc includes separate capture fields which are ananged in a row and are in fluid communication with one another and which have affixed thereto
- the optical bio-disc and disc reader assembly would allow a person to carry out blood typing analysis in the field (that is, not in a clinical setting) expeditiously.
- the invention provides a methods for antibody typing a blood sample, i.e., assaying a patients serum for the occunence of antibodies directed to an antigen of a blood group other than that of the ABO system.
- the invention provides a method for antibody typing wherein the sample undergoes processing prior to being loaded onto a bio-disc ( Figure 10 and 11 A-C, 12A-C).
- the invention provides a method for antibody typing wherein the sample is loaded onto a bio-disc without significant processing ( Figure 13).
- Cells of a known blood group phenotype other than that of the ABO system e.g., Kell, Duffy Kidd, etc., are available commercially for testing pu ⁇ oses, (Immucor, Inc. Norcross, GA, PANOSCREEN®).
- whole blood is first separated from serum prior to utilizing the seram in the bio-disc blood grouping assay.
- Whole blood can be separated into seram and cells by light centrifugation.
- the seram which contains a patient's antibodies, is then mixed with one or more O type ABO cells having a known phenotype for a blood group type other than that of ABO.
- the sample is incubated for a period of time, e.g., about fifteen to thirty minutes, at 37o C to allow the patients antibodies to interact with these cells. After incubation, the cells are washed several times and loaded into one or more chambers in the bio-disc.
- the red blood cells will have the antibodies bound thereto.
- These antibody-bound cells can then be captured on a capture field by an appropriate capture agent, e.g., an anti-human IgG.
- an appropriate capture agent e.g., an anti-human IgG.
- the capture field is examined for the occurrence of cells. The capture field can then be examined by the optical reader to determine whether cells being tested are present in the capture field, and thereby determine the antibody status of the individual.
- the method provides for the separation of blood cells and serum by passage through a separation chamber 250 in the optical bio-disc. Separation of the fluid and cellular components of whole blood is effected by spinning the disc at a first speed, thereby moving the sample through at least one microfilter designed to separate red blood cells, white blood cells and platelets from the seram. Serum is then moved to at least one mixing chamber 252 by spinning the disc at a second speed, which is higher than the first speed. Cells of a known blood group phenotype are then added through a separate entry port 256 into at least one mixing chamber of the bio-disc.
- the microfluidic circuit has inlet ports 256 for the addition of cells of a known blood group phenotype to the mixing chamber(s).
- the inlet port(s) 256 are not necessary, since the mixing chamber has been preloaded with a microparticle coated with a specific antigen of a red blood cell blood type group, e.g., A antigen or B antigen.
- a specific antigen of a red blood cell blood type group e.g., A antigen or B antigen.
- Such particles may be conveniently prepared by purifying the red blood cell antigen, e.g., through recombinant gene expression and subsequent biochemical isolation, absorbing it onto the particles. These particles may then be loaded into the mixing chamber during construction of the bio-disc, e.g., in freeze-dried form.
- a bio- disc of this construction would be particularly useful in countries and areas where access to red blood cells of a known blood type phenotype is difficult.
- Mixing of the seram and cells is accomplished by spinning the disc at least once one-half a rotation counter clockwise and then clockwise one-half a rotation.
- the samples are then allowed to incubate in the mixing chamber for a sufficient time to allow antibody-antigen interaction.
- the cells are then moved to a capmre chamber 254 with a capture field by spinning the disc at a third speed, which is higher than the second speed.
- the cells are allowed to interact with the capture field, which has bound to it anti-human IgG, for a sufficient time to allow antibody-antigen interaction.
- the disc is then spun again to remove unbound cells (e.g., 400 ⁇ m-4000 ⁇ m) Data is then collected from the capture fields to determine if cells are bound to the capmre field.
- the occurrence of cells in a capture field indicates that the individual's semm has antibodies directed to an antigen on the surface of the particular red blood cell blood type phenotype being tested.
- the invention provides a method of antibody typing a blood sample on an optical bio-disc.
- blood may be conveniently isolated by finger-stick, diluted and loaded into the bio-disc for processing.
- multiple capmre fields are exposed to the sample being tested.
- the multiple capture fields serve the pmpose of providing separate test areas in a target zone 170, each bound by a unique capture agent.
- unique capture agents in a test designed for the -ABO blood system would include anti-A antibody and anti-B antibody, each loaded onto separate capture fields on the disc.
- Antibodies with the appropriate specificity for antigens of different blood groupings are available commercially (e.g., anti-A and anti-B antibodies may be obtained from Fisher Scientific, Los Angles, CA, Catalogue Nos. 23287247 and 23287248, respectively).
- Positive and negative controls for the test would include a positive control capture field in which the capture agent is a molecule that binds all cells, e.g., a lectin isolated from Lycopersicon esculentum that binds ⁇ -D-glucosamine oligomers (Sigma Aldrich Chemical, Catalogue No. L-0651), and a negative control capmre field having no capture agent bound thereto.
- the capture agent is a molecule that binds all cells, e.g., a lectin isolated from Lycopersicon esculentum that binds ⁇ -D-glucosamine oligomers (Sigma Aldrich Chemical, Catalogue No. L-0651), and a negative control capmre field having no capture agent bound thereto.
- Various embodiments of this method of the invention may be similarly designed for the pu ⁇ ose of blood typing according to any other blood typing system, e.g., for testing the Rh system blood group, the MNSs system blood group, the Lutheran system blood group, the Kell system blood group, the Lewis system blood group, the Duffy system blood group, the Kidd system blood group, the Fisher system blood group, or any other blood group.
- any other blood typing system e.g., for testing the Rh system blood group, the MNSs system blood group, the Lutheran system blood group, the Kell system blood group, the Lewis system blood group, the Duffy system blood group, the Kidd system blood group, the Fisher system blood group, or any other blood group.
- one or more blood type systems may be simultaneously tested on a single bio-disc.
- the remaining blood groups may complicate a blood typing but are not as important.
- antibodies to the antigens not expressed on red blood cells are non-red-cell stimulated (or naturally occuning), due to the similarity between the blood group antigens stmcture and environmental agents.
- the antibodies may be of the IgM, IgA or IgG class.
- the IgM antibodies can cause direct agglutination when mixed with cells bearing the antigen the antibody is directed against.
- the IgG antibodies can cross the placenta and cause hemolytic disease of the newborn in subsequent pregnancies.
- the ABO and Rh blood groups are the most important groups in transfusion medicine.
- Naturally occuning antibodies to the ABO and Rh antigens are of the IgM class.
- Antibodies to the ABO and Rh antigens are readily available and direct agglutination tests can be performed on red blood cell samples.
- agglutination of the red cells is not the test; the test in this instance is cell capture based on antigen-antibody interactions. The interaction is specific and accurate and indicates which antigens are present on the red blood cell surface.
- agglutination of cells captured on the optical disk is looked for and analyzed by software algorithm(s).
- the invention provides a methods for detecting specific antibodies to an ABO/Rh blood group antigen, e.g., assaying a patients seram for the occunence of anti-A or anti-B antibodies.
- the invention provides a method for reverse typing wherein the sample undergoes processing prior to being loaded onto a bio-disc ( Figure 15 and 16A-C and 17A-C).
- the invention provides a method for reverse typing wherein the sample is loaded onto a bio-disc without significant processing (Figure 18).
- whole blood is first separated from serum prior to utilizing the seram in the bio-disc blood grouping assay.
- Whole blood can be separated into seram and cells by light centrifugation.
- the seram which contains a patient's antibodies, is then mixed with one or more cells having an ABO cell phenotype.
- the sample is incubated for a period of time, e.g., about one to five minutes, at room temperature to allow the patient's antibodies to interact with these cells.
- an anti-human antibody is used as the capmre agent
- the cells are washed several times and loaded into one or more chambers in the bio-disc; if a lectin capture agent is used, washing is unnecessary.
- red blood cells will be agglutinated as a result of the antibodies bound thereto. These agglutinated cells can then be captured on a capture field by an appropriate capture
- the capmre field is examined for the occunence of agglutinated cells.
- the capture field can then be examined by the optical reader to determine whether cells being tested are agglutinated, and thereby determine that antibodies to an ABO Rh blood group antigen were present in the individual's blood.
- whole blood is loaded directly onto the bio-disc into a microfluidic circuit ( Figure 14).
- the method provides for the separation of blood cells and serum by passage through a separation chamber 250 in the optical bio-disc. Separation of the fluid and cellular components of whole blood is effected by spinning the disc at a first speed, moving the sample through at least one microfilter designed to separate red blood cells, white blood cells and platelets from the serum. Seram is then moved to at least one mixing chamber 252 by spinning the disc at a second speed, which is higher than the first speed. Cells of a specific ABO group phenotype are then added through a separate port entry into at least one mixing chamber.
- Mixing of the seram and cells is accomplished by spinning the disc at least once one-half a rotation counter clockwise and then clockwise one-half a rotation.
- the samples are then allowed to incubate in the mixing chamber for a sufficient time to allow antibody-antigen interaction.
- the cells are then moved to a capmre chamber 254 with a capture field by spinning the disc at a third speed, which is higher than the second speed.
- the cells are allowed to interact with the capture field which has bound to it anti-human immunoglobulin or another capture agent for a sufficient time to allow capture agent interaction with the cells.
- the disc is then spun again to remove unbound cells, e.g., 400 ⁇ m to 4000 ⁇ m.
- Data is then collected from the capture fields to determine if cells bound thereto are agglutinated.
- the occunence of agglutinated cells in a capture field indicates that the individual's seram has antibodies directed to an antigen on the surface of the particular red blood cell blood type phenotype being tested.
- the invention provides an optical-bio disc for performing a blood-typing assay, the disc comprising: a substrate; a separation chamber associated with the substrate, the separation chamber including an inlet port; filter means associated with the separation chamber; a first mixing chamber in fluid
- the first mixing chamber including an inlet por ; a second mixing chamber in fluid communication with the separation chamber, the second mixing chamber including an inlet port; a first detection chamber in fluid communication with the first mixing chamber, the first detection chamber including a capmre zone; and a second detection chamber in fluid communication with the second mixing chamber, the second detection chamber including a capture zone.
- the filter means separates white blood cells, red blood cells, and platelets from the blood sample to provide a sample of seram.
- the sample of seram is directed into the first and second mixing chambers.
- the inlet port of the first mixing chamber is employed to direct cells of a first type into the first mixing chamber
- the inlet port of the second mixing chamber is employed to direct cells of a second type into the second mixing chamber.
- a mixture of seram and cells of the first type is directed into the first detection chamber
- a mixture of serum and cells of the second type is directed into the second detection chamber.
- Certain embodiments of the seventh aspect provide for disc rotation in a predetermined manner to mix the cells of the first type with seram in the first mixing chamber, and mix the cells of the second type with seram in the second mixing chamber.
- the predetermined manner of rotating the disc includes alternately rotating the disc in one direction and then an opposite direction to thereby create an agitation action to promote mixing of seram and cells.
- the capture zone in the first detection chamber includes a first type of capture agent implemented to capture specific cells having any affinity therefor.
- the capture zone in the second detection chamber includes a second type of capture agent implemented to capture specific cells having any affinity therefor.
- an incident beam of radiant energy is directed into the first detection chamber to determine whether any cells were captured by the first type of capture agent. In other embodiments, an incident beam of radiant energy is directed into the second detection chamber to determine whether any cells were captured by the second type of capmre agent.
- the first type of capmre agent is an anti-human immunoglobulin having an affinity for an antibody bound to the cells or a non-cell specific molecule that binds a molecule on the surface of all red blood cells.
- the second type of capture agent is an anti-human immunoglobulin having an affinity for an antibody bound to the cells or a non-cell specific molecule that binds a molecule on the surface of all red blood cells.
- the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in the substrate. In other certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in a cap bonded to the substrate. In yet other certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are formed in a channel layer bonded between a cap portion and the substrate. In certain embodiments of the seventh aspect, the separation chamber, the first and second mixing chambers, and the first and second detection chambers are partially formed in a cap portion and partially formed in the substrate, the cap portion and the substrate being bonded together in register to thereby fully form the chambers.
- the optical bio-disc further includes information encoded in an information layer readable by a disc drive.
- the encoded information is used to rotate the disc in a prescribed manner.
- the information layer is reflective. In other embodiments, the information layer is semi-reflective.
- the invention provides a method for manufacturing a disc comprising: providing over a substrate of the disc an encoded informational layer;
- the encoded informational layer is .a reflective layer, annd the target areas are regions etched from the reflective layer. In certain embodiments, the encoded informational layer is a partially reflective and partially transmissive layer, and the target areas are regions adjacent the informational layer.
- the tracking of the bio-disc of the present invention is a forward Wobble Set FDL21.13707 or FDL2L1270 coating with 300 nm of gold.
- oval data windows of size 2x1 mm are etched out by Lithography.
- "U" shaped channels are used to create chambers that are 25 um in height. It takes about 7 uls of sample to fill the entire chamber including the inlet and outlet ports.
- a 8-window/4-channel format to be preferentially used.
- a semi-reflective transmissive disc (FDL 20/21:00708) is used which allows the entire surface of a transmissive disk may be used for capmre zones, without the use of lithography to form data windows.
- Fraylock "U” shaped adhesive DBL 201 Rev C 3M94661 or straight channels are used to create the chambers.
- the cover disc utilized is a gold disk, fully reflective with 48 sample inlets with a diameter of 0.040 inches location equidistant at radius 26mm.
- This first layer may be a polycarbonate layer or a metallized polycarbonate layer in a optical disc such as a CD, CD-ROM, DVD or DVD-ROM. Prior to subsequent treatment, the first layer is cleaned with isopropanol.
- the second layer consists of either polystyrene or polycarbonate. This layer may be formed by injection molding of bulk plastic or spin or spray coating of the plastic in a volatile solvent on a solid substrate.
- the primary capture layer is formed by abso ⁇ tion of the protein streptavidin (Sigma, St. Louis, MO, Catalogue No. S-4762) (or any variant thereof) on the second layer.
- the adso ⁇ tion process is accomplished by exposure of
- -37- the second layer to a first solution (1 mg/ml solution of streptavidin (or any variant thereof) at neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris buffer, ionic strength (varied by addition of NaCl, KCl or MgC12) between 50 and 200 mM). Exposure times may range between 30 seconds and 12 hours. After exposure of the second layer to the first solution, the excess streptavidin (or any variant thereof) is washed away with water.
- the secondary capture layer consists of biotin-labeled antibody (the first capture antibody) that recognize and bond to other antibodies from a certain animal source (e.g., mouse or human) (e.g., biotinylated anti-mouse IgG (raised in sheep), Vector Laboratories, lot # L0602, Catalog # BA-9200).
- a solution of the first capture antibody (the second solution) is exposed to the third layer for between 10 minutes and 3 hours.
- the second solution comprises a 1 mg/ml solution of the first capture antibody at neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris buffer, ionic strength (varied by addition of NaCl, KCl or MgC12) between 50 and 200 mM.
- the biotin moiety on the surface of the first capture antibody is bound by the streptavidin (or any variant thereof) which comprises the third layer. After exposure of this layer to the second solution, the excess first capture antibody is washed away with water.
- the bioactive capmre layer the fifth layer, consists of the second capmre antibody, which recognizes and binds to a specific type of biological cell based on some antigen on the surface of that cell.
- the animal source of the second capture antibody must match the specificity of the first capture antibody.
- a solution of the second capture antibody is exposed to the fourth layer for between 10 minutes and 3 hours.
- This third solution comprises a 1 mg/ml solution of the second capmre antibody at neutral pH (+/- 0.5 pH) in either phosphate (sodium or potassium) or Tris buffer, ionic strength (varied by addition of NaCl, KCl or MgC12) between 50 and 200 mM.
- excess second capture antibody is washed away with a buffer similar to that described above.
- the discs of the invention are leak checked to make certain that none of the chambers leak during spinning of the disc with the sample in situ.
- Each channel is filled with a blocking agent. Blocking is done for least 1 hour.
- the discs are then spun at 5000 ⁇ m for 5 minutes and examined. After checking for leaks and removing the blocking solution, the disc is placed in a vacuum chamber for 2-48 horns. After vacuum treatment, discs are placed in a vacuum pouch and stored at 2-8°C until use.
- the disc can be heat or ultrasonically bonded to make certain no fluid escapes from the chamber.
- the forward blood typing assay is conducted on a bio-disc comprising: (1) gold reflective base disc, treated with photo-lithography to remove the gold in specific capture zones, with appropriate chemistry placed over the capture zones, (2) 25 um thick channel layer, and (3) gold reflective cover disc, assembled into a functional bio-disk.
- Results are presented in Figure 19, which is a representation of a graphical output for ABO blood typing.
- Example 3 Antibody Typing Assay On Bio-Disc (Sample Preparation Off -Disc)
- the reverse blood typing assay is conducted on a bio- disc comprising: (1) gold reflective base disc, treated with photo-lithography to remove the gold in specific capture zones, with appropriate chemistry placed over the
- Whole blood is centrifuged at an appropriate speed and time to result in a pellet of cells and non-hemolyzed seram or plasma.
- the seram or plasma is separately mixed with Type At, and Type B Reagent Red Blood Cells (Ortho Clinical Diagnostics).
- the mixture of the cells and seram or plasma may take place in test tubes or directly on the disk in a mixing chamber. If the mixing occurs in a test tube, each mixture is then placed in separate channels of the disk. After a short, room temperature incubation (2 to 5 minutes) to allow the seram or plasma to interact with the reagent red blood cells, the disk is placed into the drive.
- the automated agglutination-detection software developed in-house centrifuges the disk, causing the agglutinated and/or non-agglutinated cells to travel over the capmre zone and be non- specifically captured. Excess cells will be centrifuged to the outer edge of the flow channel.
- the disk is scanned with the standard 780nm laser of the optical drive using the bottom detector and the software registers
- the program algorithm determines which reagent red blood cells were agglutinated and assigns an ABO phenotype, based on reverse typing to the plasma or serum sample. The entire process takes about 10 minutes from insertion of disk into the drive and receiving the reverse blood typing. The forward and reverse typings of an individual should be in agreement, signifying the conect typing of that individual.
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Abstract
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US24947700P | 2000-11-17 | 2000-11-17 | |
US249477P | 2000-11-17 | ||
US25272500P | 2000-11-22 | 2000-11-22 | |
US252725P | 2000-11-22 | ||
PCT/US2001/043250 WO2002059622A1 (fr) | 2000-11-17 | 2001-11-19 | Procedes et appareil de determination de groupes sanguins a l'aide de disques biologiques optiques |
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EP1410042A1 true EP1410042A1 (fr) | 2004-04-21 |
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EP01994076A Withdrawn EP1410042A1 (fr) | 2000-11-17 | 2001-11-19 | Procedes et appareil de determination de groupes sanguins a l'aide de disques biologiques optiques |
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WO (1) | WO2002059622A1 (fr) |
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WO2003043403A2 (fr) * | 2001-11-19 | 2003-05-30 | Burstein Technologies, Inc. | Methode et appareil de typage sanguin a l'aide de bio-disques |
EP1600778B1 (fr) * | 2003-02-19 | 2013-07-24 | Japan Science and Technology Agency | Dispositif d'analyse de sang et procede d'analyse de sang |
EP1883815A4 (fr) * | 2005-05-20 | 2008-10-22 | Univ Rmit | Dispositif d'essai |
KR20150111696A (ko) | 2014-03-26 | 2015-10-06 | 삼성전자주식회사 | 혈액 검사기 및 그에 따른 혈액 검사 방법 |
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GB9418981D0 (en) * | 1994-09-21 | 1994-11-09 | Univ Glasgow | Apparatus and method for carrying out analysis of samples |
US5585069A (en) * | 1994-11-10 | 1996-12-17 | David Sarnoff Research Center, Inc. | Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis |
US5631166A (en) * | 1995-03-21 | 1997-05-20 | Jewell; Charles R. | Specimen disk for blood analyses |
AP9901660A0 (en) * | 1997-02-28 | 1999-09-30 | Burstein Lab Inc | Laboratory in a disk. |
-
2001
- 2001-11-19 WO PCT/US2001/043250 patent/WO2002059622A1/fr not_active Application Discontinuation
- 2001-11-19 EP EP01994076A patent/EP1410042A1/fr not_active Withdrawn
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