WO2002027296A1 - Quantitative digital fluorography and related products and methods - Google Patents

Quantitative digital fluorography and related products and methods Download PDF

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Publication number
WO2002027296A1
WO2002027296A1 PCT/US2001/042219 US0142219W WO0227296A1 WO 2002027296 A1 WO2002027296 A1 WO 2002027296A1 US 0142219 W US0142219 W US 0142219W WO 0227296 A1 WO0227296 A1 WO 0227296A1
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Prior art keywords
receptor
analyte
image
solid support
antigen
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PCT/US2001/042219
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French (fr)
Inventor
Eduard J. Botz
Victoriano F. Rana
Kenneth R. Newton
Tsue-Ming Lin
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Hyperion, Inc.
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Priority to CA002424039A priority Critical patent/CA2424039A1/en
Priority to AU2001296885A priority patent/AU2001296885A1/en
Priority to EP01977795A priority patent/EP1328791A4/en
Publication of WO2002027296A1 publication Critical patent/WO2002027296A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2247—Sampling from a flowing stream of gas
    • G01N1/2252—Sampling from a flowing stream of gas in a vehicle exhaust
    • G01N15/1433—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2247—Sampling from a flowing stream of gas
    • G01N2001/2264—Sampling from a flowing stream of gas with dilution
    • G01N2015/019—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Electro-optical investigation, e.g. flow cytometers
    • G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
    • G01N2015/1452—Adjustment of focus; Alignment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators

Definitions

  • the present invention relates generally to quantitative digital fluorography
  • IFA immunofluorescent assay
  • IF A immunofluorescent assay
  • IFA is a visual binding assay and its currently most widely used application is the test for antinuclear antibodies (ANA), which is used as an indicator of autoimmune disorders, such as systemic lupus erythomatosus.
  • ANA antinuclear antibodies
  • a receptor specific for the analyte to be detected is immobilized on a solid support, generally a microscope slide.
  • this receptor can be a culture of human epithelium (HEp 2 cells) or a slice of mammalian tissue.
  • HEp 2 cells human epithelium
  • Patient serum is applied onto the receptor and left there to incubate for a duration specific to the test.
  • the analyte here the ANA
  • the analyte from the serum binds to sites on the receptor (in ANA to components of the cell nuclei, such as DNA or nucleoproteins).
  • the conjugate consisting of the probe and the reporter is applied to the receptor holding the analyte.
  • the probe is a compound which is specific to the analyte and binds to it (in ANA the probe is an antibody against ANA).
  • the reporter is a substance which emits a signal that can be registered by a detector. In IFA the fluorescent dye fluorescein isothiocyanate (FITC) is generally used as the reporter.
  • an immimocomplex consisting of receptor/analyte/probe/reporter in ANA: Nuclear Component/ANA/Anti-ANA/FITC
  • ANA Nuclear Component/ANA/Anti-ANA/FITC
  • the intensity of the fluorescence is an indicator for the concentration of analyte (here ANA) in the serum.
  • Reading of a traditional IFA test is performed in a dark chamber by a technician visualizing the cell culture in a fluorescent microscope and completing a manual report indicating whether the test was positive or negative and, if positive, what kind of binding pattern was recognized. If the referring physician also wants to know the titer, a second test run is carried out in which the sample is diluted serially.
  • the individual dilutions are visualized in the microscope to determine the highest dilution at which fluorescence can still be seen. That dilution factor is then reported to the referring physician as the titer.
  • Semi-quantitation is perfonned as discussed above, by serially diluting the sample and visually judging the highest dilution at which fluorescence can still be seen. The dilution factor is then used as the semi-quantitative parameter, usually referred to as the titer. Semi- quantitation increases the expense of the procedure as it requires a separate run and uses a substantial amount of reagents. Despite the significant and promising improvements made in the field of assays using fluorescent dyes, there remains a need in the art for additional and improved assays, as well as related apparatus and methods.
  • the present invention relates to an improved manual and automated method of performing visual binding tests such as IFA, referred to herein as Quantitative Digital
  • the invention also provides apparatus for performing such methods and methods of making such apparatus.
  • the invention may use dyes with specific advantageous properties, provides apparatus for performing automated and objective assays, and provides a method of recording and quantifying data to improve the system's accuracy and usefulness.
  • QDF uses a quantitative parameter referred herein to as fluorescence intensity units (FIU) which is derived from a digitized image and measures the average, fluorescence intensity of all pixels corrected for fluorescence background and instrument noise.
  • FIU fluorescence intensity units
  • One or more calibrators are preferably run with each assay. Their FIU values are plotted against known, for example ANA, titers to establish the calibration curve. The titers of the patient samples are taken from the calibration curve by interpolating their FIU values and are less subjective than the visual assessment of the serial dilutions by IFA.
  • QDF parameters provide substantially greater reading resolution since the calibration curve is continuous, whereas the semi-quantitation of IFA provides only as many distinct readings as there are dilutions.
  • QDF can be run manually like an IFA or fully automatically. In either case only one single run is required to obtain both visual and quantitative diagnostic infonnation.
  • the microscope image is broken into a two-dimensional pixel array, fluorescence intensity information is acquired for each pixel, digitized and stored in the computer. This same data array is used to both calculate the titer and generate the image to be displayed on the monitor.
  • the computer contains a library of typical images with known diagnosis.
  • a special algorithm, such as pattern recognition seeks out that image in the library, which is most similar to the image acquired from the patient sample.
  • a test report is then generated and optionally displayed and/or printed. The report lists the titer and displays the image from the sample next to the matched image from the library.
  • the QDF method may also be applied to other assay types within the intended scope of this invention, which can also benefit from combined visual and quantitative assessment. Examples are nucleic acid hybridization tests, such as fluorescence in situ hybridization (FISH), Dot Blot tests, and competitive receptor assays in which compounds to be screened for various purposes compete with a ligand for the binding site of a receptor.
  • FISH fluorescence in situ hybridization
  • Dot Blot tests and competitive receptor assays in which compounds to be screened for various purposes compete with a ligand for the binding site of a receptor.
  • One embodiment of the QDF method uses a series of steps to achieve optimal visual and quantitative information. This series of steps may be used to detect a particular target within a sample. The steps may proceed in a manual or automated fashion.
  • the sample which may be a fluid from a patient or other source, containing an analyte, binds to a receptor on a solid support to form an initial complex.
  • the analyte and receptor may be antibodies, antigens or nucleic acid molecules.
  • the solid support may be a microscope slide, a microliter plate, a suspension of microscopic spheres or beads or other support means. Any portion of the sample that remains unbound to the support may be removed by washing the solid support or other method.
  • the initial complex is then contacted via the analyte to an additional complex of a probe and fluorescent or luminescent . dye.
  • the probe may be an antibody or i muno globulin molecule.
  • the dye will have an absorption and an emission peak greater than 650 nm, the dye will be conjugated with moieties which reduce its non-specific binding and when excited at the appropriate wavelength, the dye will lose less fluorescence intensity than FITC currently loses in
  • This bound complex is an extended complex. Again, non-bound portions of the initial complex may be removed by washing the solid support. The dye is then excited and the resulting image is used in a QDF analysis.
  • an image of the luminescence may be obtained, either manually or automatically. This image may be digitized.
  • the solid support may automatically move into the field of view of the imagining apparatus and the image may be focused using only luminescent light.
  • the image may be obtained using transient-state fluorography.
  • a variety of tools may be used. These include an electronic camera, a CCD camera, a video camera and a digital camera.
  • the experimenter may use a scanning point detector, either a photomultiplier tube or photodiode.
  • a numerical value is calculated.
  • the calculation of the numerical value may be done by the FIU algorithm and fit to a calibration curve.
  • Calibrator samples may be used to then convert the numerical value to a parameter representing the amount of analyte within the sample.
  • the image is classified into one of a set of predefined patterns.
  • FIGURE 1 shows a typical QDF calibration curve.
  • FIGURE 2 shows a QDF patient report.
  • FIGURE 3 shows a schematic of a QDF instrument, showing the excitation light source, the means for obtaining an image, the position of the assay substrate/holder, the means for displaying the image and a calculation unit (computer) for performing the numerical procedures and displaying the results along with the image.
  • FIGURE 4 shows the two separate liquid cycles of the robotic assay processor,
  • HyPrep Plus as it would operate in the processing of a visual binding test such as IFA or QDF.
  • the present invention relates to an improved manual and automated method of performing immimofluorescen.ee assays (IFA) and other fluorographic (visual) binding assays, referred to herein as Quantitative Digital Fluorography (QDF).
  • IFA immimofluorescen.ee assays
  • QDF Quantitative Digital Fluorography
  • the invention also provides apparatus for performing such methods manually and automatically, methods of making such apparatus, and methods for recording and quantifying data to improve the system's accuracy and usefulness.
  • the dyes will preferably be low in or free of aggregation and serum binding and may be water soluble.
  • the dyes serve to label an analyte, antigen, antibody, nucleic acid, or other molecule, which is part of the extended complex.
  • the dyes may consist of a fluorophore moiety bound to one or more polyoxyhydrocarbyl moieties.
  • the dyes may also be coupled to two or more small, solubilizing axial ligands.
  • the dye contains two polyoxyhydrocarbyl moieties. Examples of these structures may be seen in U.S. Patents 5,919,922, 5,880,287,
  • this class of dyes is not indispensable to accomplish the objectives of the invention, but helps reduce background signal caused by autofluorescence (because these dyes have absorption peaks in the near infrared region), and nonspecific binding. In addition, these dyes have sufficient photostability for quantitative measurement.
  • LJB dyes preferably operate at near- IR excitation and emission wavelengths. They preferably, have excitation peaks above 680 nm and emission peaks between 685 and 800 nm, while FITC has corresponding peaks in the visible and near ultraviolet wavelengths. At the wavelengths where LJB dyes absorb and emit, autofluorescence is very low, and much lower than at the wavelengths of the FITC peaks. A decrease in autofluorescence leads to a higher signal-to-noise ratio. Scattering occurs to a lesser extent when the incident light is in the near-IR range, and therefore the use of LJB dyes allows a decrease in the background fluorescence and an increase in the signal- to-noise ratio.
  • FITC fluorescein isothiocyanate
  • LJB dyes Another advantage of LJB dyes is their reduced non-specific binding once conjugated with the probe, dyes should ideally not bind to any other molecules including the analyte, while the probe should bind to nothing but its target which is the analyte. Non-specific binding occurs when the dye binds to serum components, cellular constituents or even to the analyte. While many of the dyes developed to reduce autofluorescence suffer from increased non-specific binding, LJB dyes allow reductions in both autofluorescence and non-specific binding.
  • LJB dyes have greater photostability than FITC or other near-IR dyes, meaning that the rate of decrease in the fluorescence intensity of LJB dyes is less. During the time interval when the fluorescence is being measured, the LJB dyes emit a stronger signal. The signal-to-noise ratio is thereby improved without losing the advantages of near-IR excitation and emission wavelengths.
  • the QDF assay represents an improvement over commonly used assays, such as IFA. While IFA is a primarily visual testing format with the option of deriving semi-quantitative information from a second run, QDF produces both visual and true- quantitative results by combining the two-run process of IFA into a single run. In the first run of a conventional IFA procedure, the test is read only visually and represents the investigator's judgment about the presence (positive result) or absence (negative result) of analyte. In the case of a positive result this first visual reading also includes information about the binding pattern which allows differential diagnosis of disease. If the reading of the first run is positive, a second testing run may be ordered by the referring physician to obtain semi-quantitative information which helps assessing the severity of the disease.
  • samples are serially diluted.
  • the experimenter tries to visually determine the highest dilution at which he believes signal fluorescence is still visible. This judgment is frequently difficult and inaccurate, if a substantial amount of background fluorescence is present that can be mistaken as signal.
  • the dilution so chosen is reported as the titer.
  • samples are mostly diluted 1:40 for the initial visual run. For semi-quantitation this base dilution is further diluted serially at the factor of 2 thus deriving dilutions of 1:80, 1:160 and so on.
  • QDF generates both visual and true-quantitative information in only one test run, which includes a pre-compiled set of calibrators with known titers.
  • the FIU values of the calibrators are plotted against their known titers to establish a calibration curve.
  • the titers of the patient samples are read from that calibration curve by interpolating their FIU readings.
  • the calibration curve enables continuous reading and thus much greater reading resolution than the few discrete data points of UFA'S serial dilution.
  • QDF also offers greater quantitative reliability, accuracy, and objectivity than conventional IFA.
  • QDF may use a robotic . assay processor, the HyPrep Plus or equivalent devices, to dispense and re-aspirate certain reagents and wash buffer to and from the receptor sites on the solid support (in an ANA test the cell cultures on the microscope slide), thus eliminating the need for manual performance of such operations.
  • Reading can be performed with one of three different types of instruments offering various degrees of reading automation.
  • the simplest version is an upgrade of the fluorescent microscopes which are currently used in the clinical laboratories for the reading of IFA tests (QDF-1).
  • the upgrade consists of a CCD-camera with frame grabber, a PC-computer with monitor and QDF software and an optional printer.
  • the QDF software enables automatic focusing, the upgrade may also include software driven motorized mechanics to perform this focus.
  • An advanced automated reading instrument (QDF- 1000) consists of an automated transport to move the solid supports (in ANA testing the slides with the cell cultures) in position under a magnification objective which is focused automatically.
  • the fluorescent dye on the immunocomplex is excited by a light source and the fluorescent image is captured by either an area detector or a scanning point detector, digitized and sent to the computer. The transport then automatically moves the next sample in position for reading.
  • Both QDF-1 and QDF-1000 may be used in conjunction with the HyPrep Plus or an equivalent robot capable of performing the washing of each test individually.
  • the system operates two liquid cycles, one for the dispensing and one for the aspiration, both passing through hollow tips which are close enough together to fit within one well.
  • This enables aspirating one liquid (e.g. wash buffer) completely through one tip with the cells remaining dry only for a moment, as the next liquid (e.g. conjugate) is dispensed immediately thereafter through the other tip.
  • washing the wells individually avoids any carry over of serum or reagent from one well to another.
  • Robots which operate only one liquid cycle cannot perform such operation as they would have to aspirate liquid sequentially from all wells in the test run before they could begin dispensing the next liquid, which then also would have to be performed sequentially for all wells in the same test run. As a result, the cells would be left dry for an extended period.
  • the third and most automated version represents an integration of the QDF- 1000 with the HyPrep Plus to provide a fully, automated "walk-away" system (QDF- 2000). If QDF- 1000 is used, the experimenter performs a total of one step manually
  • the slides are transferred automatically from the processing module to the reading module and the whole assay can run in a walk-away fashion beginning with the transfer of the patient samples from their receptacles to the wells on the slides and ending with the printing of the test report.
  • the software-driven automated focusing in all three QDF reading devices is unique, in that it uses the fluorescence light from the images, as opposed to white light from a Brightfield lamp. It was noticed that the FIU parameter reaches its highest value when the image is in focus. The focusing, algorithm therefore samples the FIU at various points until a change of height in either direction no longer raises the value.
  • Quantification of the assay in the three described QDF reading apparatuses occurs by the earlier described reading from a continuous calibration curve, rather than by subjective visual assessment of a discrete set of serial dilutions.
  • all three apparatuses could recognize binding patterns, further improving the accuracy and usefulness of positive/negative and concentration determinations.
  • traditional steady-state data collection would be sufficient to obtain the desired results.
  • QDF- 1000 and QDF-2000 could also use transient-state measurements, thus further improving both the visual and quantitative accuracy of the reading
  • the three QDF reader instruments may include any combination of the following features: a. Automated motion of microscope slides or similar objects into the instrument and positioning of the cell cultures for reading. b. Opeiation of the HyPrep Plus and QDF-reader from the same PC- computei with automatic transfer of patient samples from the processing software to the reading software. c. Acquisition of magnified fluorescent images of the cells on the slides with sufficient numbers of cells in the image to obtain good fluorescence intensity statistics and with sufficient resolution to distinguish the binding patterns in the assay. d. Automated focusing of the cell images. e. Quantitation of the fluorescence intensity from the cellular fluorescence images. f Normalization of the quantitative parameter obtained per "e" to reporting units such as titer. g. Pattern recognition to name specific bmding pattern types m the acquired images h. Potential for transient state measurement in a QDF instrument

Abstract

The present invention thus relates to marker components, fluorescent probes, oligonucleotides, hybridization assays, and binding assays such as immunoassays, DNA assays and receptor assays using such products and methods for making such products. According to the present invention, delectably labeled marker components are provided thus comprise a fluorophore moiety coupled to two or more small solubilizing axial ligands, which preferably reduce or remove the problems of solvent sensibility and non-specific binding.

Description

QUANTITATIVE DIGITAL F UOROGRAPHY AND RELATED PRODUCTS AND METHODS
INTRODUCTION The present invention relates generally to quantitative digital fluorography
(QDF) and related products and methods.
BACKGROUND OF THE INVENTION
The following description of the background of the invention is intended to aid in the understanding of the invention, but is not admitted to describe or constitute prior art to the invention.
An example of a fluorographic binding assay is the immunofluorescent assay (IF A), frequently also called indirect fluorescent antibody assay. IFA is a visual binding assay and its currently most widely used application is the test for antinuclear antibodies (ANA), which is used as an indicator of autoimmune disorders, such as systemic lupus erythomatosus. In IFA, a receptor specific for the analyte to be detected is immobilized on a solid support, generally a microscope slide. In the ANA test this receptor can be a culture of human epithelium (HEp 2 cells) or a slice of mammalian tissue. Patient serum is applied onto the receptor and left there to incubate for a duration specific to the test. During this time interval the analyte (here the ANA) from the serum binds to sites on the receptor (in ANA to components of the cell nuclei, such as DNA or nucleoproteins).
After the incubation period unbound serum components are removed by washing. Next, the conjugate consisting of the probe and the reporter is applied to the receptor holding the analyte. The probe is a compound which is specific to the analyte and binds to it (in ANA the probe is an antibody against ANA). The reporter is a substance which emits a signal that can be registered by a detector. In IFA the fluorescent dye fluorescein isothiocyanate (FITC) is generally used as the reporter. After another incubation period, followed by another removal of unbound fractions by another wash step, an immimocomplex consisting of receptor/analyte/probe/reporter (in ANA: Nuclear Component/ANA/Anti-ANA/FITC) remains on the slide and is visualized in a fluorescent microscope. The intensity of the fluorescence is an indicator for the concentration of analyte (here ANA) in the serum. The advantage of this visual type of testing versus purely quantitative assay formats is that distinct patterns of binding of the analyte to components of the receptor (ANA to nuclear components) can be distinguished. These patterns can be used as indicators for distinct subsets of disease.
Reading of a traditional IFA test is performed in a dark chamber by a technician visualizing the cell culture in a fluorescent microscope and completing a manual report indicating whether the test was positive or negative and, if positive, what kind of binding pattern was recognized. If the referring physician also wants to know the titer, a second test run is carried out in which the sample is diluted serially.
The individual dilutions are visualized in the microscope to determine the highest dilution at which fluorescence can still be seen. That dilution factor is then reported to the referring physician as the titer.
While the ability of differential diagnosis is a clear advantage, visual binding tests also have a number of shortcomings. They fail to offer complete automation in the processing and the reading stages of an assay. In addition, reading a visual binding test has traditionally been subjective, thereby leading to increased expense and decreased reproducibility of findings. IFA in particular has also traditionally suffered from the disadvantages of high background fluorescence (i.e., fluorescence that originates from a source other than the analyte being measured). High background fluorescence decreases the signal-to-noise ratio, obscures the visualization of binding patterns, and renders the interpretation of the results even more subjective. IFA does not enable true quantitation but only an approximation called semi-quantitation and thus has limited diagnostic sensitivity. Semi-quantitation is perfonned as discussed above, by serially diluting the sample and visually judging the highest dilution at which fluorescence can still be seen. The dilution factor is then used as the semi-quantitative parameter, usually referred to as the titer. Semi- quantitation increases the expense of the procedure as it requires a separate run and uses a substantial amount of reagents. Despite the significant and promising improvements made in the field of assays using fluorescent dyes, there remains a need in the art for additional and improved assays, as well as related apparatus and methods.
SUMMARY OF THE INVENTION The present invention relates to an improved manual and automated method of performing visual binding tests such as IFA, referred to herein as Quantitative Digital
Fluorography (QDF). The invention also provides apparatus for performing such methods and methods of making such apparatus. The invention may use dyes with specific advantageous properties, provides apparatus for performing automated and objective assays, and provides a method of recording and quantifying data to improve the system's accuracy and usefulness.
QDF uses a quantitative parameter referred herein to as fluorescence intensity units (FIU) which is derived from a digitized image and measures the average, fluorescence intensity of all pixels corrected for fluorescence background and instrument noise. One or more calibrators are preferably run with each assay. Their FIU values are plotted against known, for example ANA, titers to establish the calibration curve. The titers of the patient samples are taken from the calibration curve by interpolating their FIU values and are less subjective than the visual assessment of the serial dilutions by IFA. Also, QDF parameters provide substantially greater reading resolution since the calibration curve is continuous, whereas the semi-quantitation of IFA provides only as many distinct readings as there are dilutions.
QDF can be run manually like an IFA or fully automatically. In either case only one single run is required to obtain both visual and quantitative diagnostic infonnation. The microscope image is broken into a two-dimensional pixel array, fluorescence intensity information is acquired for each pixel, digitized and stored in the computer. This same data array is used to both calculate the titer and generate the image to be displayed on the monitor. The computer contains a library of typical images with known diagnosis. A special algorithm, such as pattern recognition, seeks out that image in the library, which is most similar to the image acquired from the patient sample. A test report is then generated and optionally displayed and/or printed. The report lists the titer and displays the image from the sample next to the matched image from the library. The diagnosis of the matched image is indicated as assistance for the referring physician in diagnosing the patient whose sample had been tested. While IFA is used as an example in the above paragraphs, the QDF method may also be applied to other assay types within the intended scope of this invention, which can also benefit from combined visual and quantitative assessment. Examples are nucleic acid hybridization tests, such as fluorescence in situ hybridization (FISH), Dot Blot tests, and competitive receptor assays in which compounds to be screened for various purposes compete with a ligand for the binding site of a receptor. One embodiment of the QDF method uses a series of steps to achieve optimal visual and quantitative information. This series of steps may be used to detect a particular target within a sample. The steps may proceed in a manual or automated fashion. The sample, which may be a fluid from a patient or other source, containing an analyte, binds to a receptor on a solid support to form an initial complex. The analyte and receptor may be antibodies, antigens or nucleic acid molecules. The solid support may be a microscope slide, a microliter plate, a suspension of microscopic spheres or beads or other support means. Any portion of the sample that remains unbound to the support may be removed by washing the solid support or other method. The initial complex is then contacted via the analyte to an additional complex of a probe and fluorescent or luminescent . dye. The probe may be an antibody or i muno globulin molecule. Preferably, the dye will have an absorption and an emission peak greater than 650 nm, the dye will be conjugated with moieties which reduce its non-specific binding and when excited at the appropriate wavelength, the dye will lose less fluorescence intensity than FITC currently loses in
IFA tests. This bound complex is an extended complex. Again, non-bound portions of the initial complex may be removed by washing the solid support. The dye is then excited and the resulting image is used in a QDF analysis.
Once the extended complex has been created, an image of the luminescence may be obtained, either manually or automatically. This image may be digitized.
There are a number of ways to automate the process of obtaining the image. The solid support may automatically move into the field of view of the imagining apparatus and the image may be focused using only luminescent light. The image may be obtained using transient-state fluorography. In actually capturing the image, a variety of tools may be used. These include an electronic camera, a CCD camera, a video camera and a digital camera. Alternatively, the experimenter may use a scanning point detector, either a photomultiplier tube or photodiode.
From the image data, a numerical value is calculated. The calculation of the numerical value may be done by the FIU algorithm and fit to a calibration curve. Calibrator samples may be used to then convert the numerical value to a parameter representing the amount of analyte within the sample. In addition to calculating a numerical value from the image data, the image is classified into one of a set of predefined patterns.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 shows a typical QDF calibration curve. FIGURE 2 shows a QDF patient report. FIGURE 3 shows a schematic of a QDF instrument, showing the excitation light source, the means for obtaining an image, the position of the assay substrate/holder, the means for displaying the image and a calculation unit (computer) for performing the numerical procedures and displaying the results along with the image. FIGURE 4 shows the two separate liquid cycles of the robotic assay processor,
HyPrep Plus, as it would operate in the processing of a visual binding test such as IFA or QDF.
The drawings are not necessarily to scale. Certain features of the invention may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness. Detailed Description Of The Invention
The present invention relates to an improved manual and automated method of performing immimofluorescen.ee assays (IFA) and other fluorographic (visual) binding assays, referred to herein as Quantitative Digital Fluorography (QDF). The invention also provides apparatus for performing such methods manually and automatically, methods of making such apparatus, and methods for recording and quantifying data to improve the system's accuracy and usefulness.
The following description includes preferred modes of carrying out the invention and is made for the general purpose of illustrating the general principles of the invention rather than limiting the invention in any manner.
I. PREFERRED DYES
Various types of dyes may be used with the present invention. Applications previously filed with the U.S. Patent and Trademark Office claim dyes useful in the present invention. The dyes will preferably be low in or free of aggregation and serum binding and may be water soluble. The dyes serve to label an analyte, antigen, antibody, nucleic acid, or other molecule, which is part of the extended complex. The dyes may consist of a fluorophore moiety bound to one or more polyoxyhydrocarbyl moieties. However, the dyes may also be coupled to two or more small, solubilizing axial ligands. Preferably, the dye contains two polyoxyhydrocarbyl moieties. Examples of these structures may be seen in U.S. Patents 5,919,922, 5,880,287,
5,707,813, 5,677,199, 5,641,878, 5,606,045, 5,403,928 and U.S. Patent Application No. 09/440,313; which are all incorporated herein by reference in their entirety, including any drawings.
The use of this class of dyes is not indispensable to accomplish the objectives of the invention, but helps reduce background signal caused by autofluorescence (because these dyes have absorption peaks in the near infrared region), and nonspecific binding. In addition, these dyes have sufficient photostability for quantitative measurement.
The preferred class of dyes used in the present invention referred to herein as La Jolla Blue (LJB) dyes, offers several advantages over commonly used fluorescent dyes such as fluorescein isothiocyanate (FITC). LJB dyes preferably operate at near- IR excitation and emission wavelengths. They preferably, have excitation peaks above 680 nm and emission peaks between 685 and 800 nm, while FITC has corresponding peaks in the visible and near ultraviolet wavelengths. At the wavelengths where LJB dyes absorb and emit, autofluorescence is very low, and much lower than at the wavelengths of the FITC peaks. A decrease in autofluorescence leads to a higher signal-to-noise ratio. Scattering occurs to a lesser extent when the incident light is in the near-IR range, and therefore the use of LJB dyes allows a decrease in the background fluorescence and an increase in the signal- to-noise ratio.
Another advantage of LJB dyes is their reduced non-specific binding once conjugated with the probe, dyes should ideally not bind to any other molecules including the analyte, while the probe should bind to nothing but its target which is the analyte. Non-specific binding occurs when the dye binds to serum components, cellular constituents or even to the analyte. While many of the dyes developed to reduce autofluorescence suffer from increased non-specific binding, LJB dyes allow reductions in both autofluorescence and non-specific binding.
In addition, LJB dyes have greater photostability than FITC or other near-IR dyes, meaning that the rate of decrease in the fluorescence intensity of LJB dyes is less. During the time interval when the fluorescence is being measured, the LJB dyes emit a stronger signal. The signal-to-noise ratio is thereby improved without losing the advantages of near-IR excitation and emission wavelengths.
II. PREFERRED QUANTITATIVE DIGITAL FLUOROGRAPHY APPARATUS AND METHODS
The QDF assay represents an improvement over commonly used assays, such as IFA. While IFA is a primarily visual testing format with the option of deriving semi-quantitative information from a second run, QDF produces both visual and true- quantitative results by combining the two-run process of IFA into a single run. In the first run of a conventional IFA procedure, the test is read only visually and represents the investigator's judgment about the presence (positive result) or absence (negative result) of analyte. In the case of a positive result this first visual reading also includes information about the binding pattern which allows differential diagnosis of disease. If the reading of the first run is positive, a second testing run may be ordered by the referring physician to obtain semi-quantitative information which helps assessing the severity of the disease. For this purpose the samples are serially diluted. The experimenter tries to visually determine the highest dilution at which he believes signal fluorescence is still visible. This judgment is frequently difficult and inaccurate, if a substantial amount of background fluorescence is present that can be mistaken as signal. The dilution so chosen is reported as the titer. As an example, in the ANA test, samples are mostly diluted 1:40 for the initial visual run. For semi-quantitation this base dilution is further diluted serially at the factor of 2 thus deriving dilutions of 1:80, 1:160 and so on. If, as an example, the experimenter believes that signal fluorescence is still seen at a dilution of 1:160 but no longer at 1:320, the titer reported is 160. Since background fluorescence from autofluorescence, non-specific binding and light scattering renders such judgment complicated and since the neighboring dilutions are 1:80 and 1:320, this type of reading is seen as relatively crude and inaccurate.
QDF, on the other hand, generates both visual and true-quantitative information in only one test run, which includes a pre-compiled set of calibrators with known titers. The FIU values of the calibrators are plotted against their known titers to establish a calibration curve. The titers of the patient samples are read from that calibration curve by interpolating their FIU readings. The calibration curve enables continuous reading and thus much greater reading resolution than the few discrete data points of UFA'S serial dilution. As a result, in addition to avoiding a second run, QDF also offers greater quantitative reliability, accuracy, and objectivity than conventional IFA.
In addition, QDF may use a robotic . assay processor, the HyPrep Plus or equivalent devices, to dispense and re-aspirate certain reagents and wash buffer to and from the receptor sites on the solid support (in an ANA test the cell cultures on the microscope slide), thus eliminating the need for manual performance of such operations. Reading can be performed with one of three different types of instruments offering various degrees of reading automation. The simplest version is an upgrade of the fluorescent microscopes which are currently used in the clinical laboratories for the reading of IFA tests (QDF-1). The upgrade consists of a CCD-camera with frame grabber, a PC-computer with monitor and QDF software and an optional printer. As the QDF software enables automatic focusing, the upgrade may also include software driven motorized mechanics to perform this focus. An advanced automated reading instrument (QDF- 1000) consists of an automated transport to move the solid supports (in ANA testing the slides with the cell cultures) in position under a magnification objective which is focused automatically. The fluorescent dye on the immunocomplex is excited by a light source and the fluorescent image is captured by either an area detector or a scanning point detector, digitized and sent to the computer. The transport then automatically moves the next sample in position for reading.
Both QDF-1 and QDF-1000 may be used in conjunction with the HyPrep Plus or an equivalent robot capable of performing the washing of each test individually. In
IFA, cell cultures (wells) for 6 to 96 samples are located on the same microscope slide. Removal of unbound sample components and conjugate after the incubations is performed by rinsing the slides and dipping them into a water bath. This treatment has substantial shortcomings as it carries the risk of transferring serum constituents and reagents from one well to another. In addition, wash buffer is not completely removed from the cell cultures prior to the application of the conjugate. This causes a dilution of the conjugate by residual wash buffer and results in less intense fluorescence seen in the microscope. Drying the slides prior to the application of the conjugate is not an option, as this would interfere with the performance of the assay. HyPrep Plus washes each well individually and thus avoids these shortcomings. This is accomplished by a unique mechanism. As shown in FIGURE 4, the system operates two liquid cycles, one for the dispensing and one for the aspiration, both passing through hollow tips which are close enough together to fit within one well. This enables aspirating one liquid (e.g. wash buffer) completely through one tip with the cells remaining dry only for a moment, as the next liquid (e.g. conjugate) is dispensed immediately thereafter through the other tip. In addition, washing the wells individually avoids any carry over of serum or reagent from one well to another. Robots which operate only one liquid cycle cannot perform such operation as they would have to aspirate liquid sequentially from all wells in the test run before they could begin dispensing the next liquid, which then also would have to be performed sequentially for all wells in the same test run. As a result, the cells would be left dry for an extended period.
The third and most automated version represents an integration of the QDF- 1000 with the HyPrep Plus to provide a fully, automated "walk-away" system (QDF- 2000). If QDF- 1000 is used, the experimenter performs a total of one step manually
(i.e. the transfer of the slides from the HyPrep Plus to the reader) after the processing of the samples is completed; if QDF-2000 is used, the slides are transferred automatically from the processing module to the reading module and the whole assay can run in a walk-away fashion beginning with the transfer of the patient samples from their receptacles to the wells on the slides and ending with the printing of the test report.
The software-driven automated focusing in all three QDF reading devices is unique, in that it uses the fluorescence light from the images, as opposed to white light from a Brightfield lamp. It was noticed that the FIU parameter reaches its highest value when the image is in focus. The focusing, algorithm therefore samples the FIU at various points until a change of height in either direction no longer raises the value.
Quantification of the assay in the three described QDF reading apparatuses, occurs by the earlier described reading from a continuous calibration curve, rather than by subjective visual assessment of a discrete set of serial dilutions. Using a variety of possible methods, such as pattern recognition, all three apparatuses could recognize binding patterns, further improving the accuracy and usefulness of positive/negative and concentration determinations. For all three apparatuses traditional steady-state data collection would be sufficient to obtain the desired results. However, QDF- 1000 and QDF-2000 could also use transient-state measurements, thus further improving both the visual and quantitative accuracy of the reading
The three QDF reader instruments may include any combination of the following features: a. Automated motion of microscope slides or similar objects into the instrument and positioning of the cell cultures for reading. b. Opeiation of the HyPrep Plus and QDF-reader from the same PC- computei with automatic transfer of patient samples from the processing software to the reading software. c. Acquisition of magnified fluorescent images of the cells on the slides with sufficient numbers of cells in the image to obtain good fluorescence intensity statistics and with sufficient resolution to distinguish the binding patterns in the assay. d. Automated focusing of the cell images. e. Quantitation of the fluorescence intensity from the cellular fluorescence images. f Normalization of the quantitative parameter obtained per "e" to reporting units such as titer. g. Pattern recognition to name specific bmding pattern types m the acquired images h. Potential for transient state measurement in a QDF instrument
Other advantages of the compositions and methods of the present invention will be apparent to those m the art upon review of the examples provided herein. EXAMPLES
To assist in understanding the present invention, the following Examples of uses of the invention are included. The following Examples of uses of the invention should not, of course, be construed in specifically limiting the invention and such variations of the invention, now know or later developed, which would be within the purview of one sldlled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed. EXAMPLE 1:
Testing for antinuclear antibodies as an adjunct in the diagnosis of autoimmune disorders, such as systemic lupus erythematosus, rheumatoid arthritis, etc. EXAMPLE 2:
Testing for anti-neutrophil cytoplasmic antibodies (ANCA) as an adjunct in the diagnosis of granulomatosis. EXAMPLE 3:
Testing for infectious diseases, such as Lyme disease, AIDS, chlamydia, toxoplasmosis, rubella, cytomegalovirus, herpes, etc. EXAMPLE 4:
Various types of nucleic acid hybridization assays which are currently being performed only visually but could greatly benefit from a combined visual and quantitative assessment, such as fluorescence in situ hybridization (FISH). EXAMPLE 5:
Various blotting assays, such as dot blots, western, southern and Northern blots. CONCLUSION
The above example applications, relating to the present invention, should not, of course, be construed as limiting the scope of the invention. Such variations of the invention, now known-or later developed, which would fall within the purview of those skilled in the art are to be considered as falling within the scope of the invention as hereinafter claimed.
All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

Other embodiments are within the following claims.WHAT IS CLAIMED IS:
1. A method for generating both visual and quantitative information from one single set of data derived from the reading of a test comprising the following steps:
A. contacting a receptor immobilized on a solid support with a sample to be tested for the presence and amount of an analyte, wherein during an incubation period said analyte binds to said receptor, thereby forming a initial complex comprising said analyte linked to said receptor;
B. removing sample components not bound to the receptor after the completion of said incubation period;
C. contacting said initial complex with a conjugate comprising a probe that specifically binds to said analyte and is linked to a luminescent compound; wherein, during a second incubation period said probe binds to said analyte, thereby extending said initial complex to an extended complex comprising said receptor bound to said analyte bound to said probe bound to said luminescent compound;
D. removing fractions of said conjugate not bound to the analyte after completion of said incubation period;
E. obtaining an image of the luminescence from a source of excitation, using a means to select the emitted light and a means to detect and digitize an image;
F. displaying the image for viewing; and G. calculation of a numerical value from the image data and using one or more calibrator samples to convert such numerical value to a parameter representing the amount of said analyte in the sample.
2. The method of claim 1, wherein steps A-D are automatically processed on a robotic assay processor operating two separate liquid cycles, one cycle performing the dispensing of liquid and the other performing the aspiration of liquid, thus enabling the complete removal of one type of liquid immediately followed by the dispensing of the subsequent type of liquid without the testing areas remaining dry for any length of time; when there are testing areas for more than one sample on the same unit of said solid support, the operation of said two liquid cycles further enables said removal of unbound sample components and unbound conjugate to be carried out by washing each testing area individually.
3. The method of claim 1 where obtaining the image is automated by use of one or more of the following improvements: A. automated motion of said solid support into the field of view of the imaging apparatus; and/or
B. automated focusing of the image using only luminescent light; and/or
C . automated generation of a test report.
4. The method of claim 1 where, in addition to calculation of a numerical value from the image data, the image is classified into one of a set of predefined patterns.
5. The method of claim 1, wherein said receptor is an antigen and said analyte and said probe are antibodies.
6. The method of claim 1, wherein said analyte is an antinuclear antibody.
7. The method of claim 1, wherein said receptor is an antigen in a mammalian cell that is immobilized on said solid support.
8. The method of claim 1, wherein said receptor is an antigen in a mammalian tissue section that is immobilized on said solid support.
9. The method of claim 1, wherein said receptor is an antigen in a prokaryotic cell that is immobilized on said solid support.
10. The method of claim 1, wherein said receptor is a viral antigen in a cell infected with a virus and immobilized on said solid support, and wherein the analyte is an antibody against the viral antigen.
11. The method of claim 1, wherein said receptor is an antigen in a deactivated pathogen that is immobilized on said solid support, and wherein the analyte is an antibody against such antigen.
12. The method of claim 1, wherein said receptor is a dot blot of an isolated antigen that is immobilized on said solid support, and the analyte is an antibody against such antigen.
13. The method of claim 1, wherein said receptor is a structured microarray of multiple antigens (microarray) that is immobilized on said object, and wherein the analyte is an antibody against one of such antigens.
14. The method of claim 1 , wherein said probe is an immunoglobulin molecule.
15. The method of claim 1, wherein said receptor is a first nucleic acid molecule and said probe is a second nucleic acid molecule.
16. The method of claim 1, wherein said receptor is a structured array of multiple strands of nucleic acids (microarray) that is immobilized on said object, and wherein the analyte is a nucleic acid strand complementary to one of the strands in the receptor.
17. The method of claim 1, wherein said sample comprises fluid from a patient.
18. The method of claim 1, wherein said removal of said sample components not bound to said receptor and said removal of said conjugate not bound to said analyte are accomplished by washing the area of said solid support holding said initial complex and extended complex, respectively.
19. The method of claim 1, wherein said obtaining an image of the luminescence is accomplished with an area detector.
20. The method of claim 19, wherein said area detector is a CCD camera, a video camera or a digital camera.
21. The method of claim 1, wherein said obtaining an image of the luminescence is accomplished with a scanning point detector.
22. The method of claim 21, wherein said scanning point detector is a photomultiplier tube or a photodiode.
23. The methods of claim 19, 20 and 21, wherein said image is a digitized image of said luminescence.
24. The method of claim 1, wherein said obtaining an image comprises calculating the fluorescence intensity unit (FIU) of said image and using said FIU to read a titer from a calibration curve.
25. The method of claim 1, wherein said solid support is a microscope slide.
26. The method of claim 1, wherein said solid support is a microtiter plate.
27. The method of claim 1, wherein said solid support is a suspension of microscopic spheres or beads.
28. The method of claim 1, wherein said fluorescent dye has an absorption and emission peak above 650 nanometers, thus substantially reducing background from autofluorescence.
29. The method of claim 1, wherein said fluorescent dye is conjugated with moieties that substantially reduce its non-specific binding.
30. The method of claim 1, wherein said fluorescent dye, when excited at the appropriate wavelength, loses less fluorescence intensity than FITC currently uses in IFA tests.
31. The method of claim 1, wherein said obtaining an image of the luminescence is-performed by transient-state fluorography.
32. The method of claim 1, wherein said calculation of a numerical value is the FIU algorithm (difference of the means of the above and below average pixels), followed by fit to a calibration curve obtained by least squares fitting procedure to the calibrators.
33. The method of claim 1, wherein said automated focusing is done by maximizing the value of the numerical measure used.
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