WO2010065797A2 - Analyses d'analytes immobilisés - Google Patents

Analyses d'analytes immobilisés Download PDF

Info

Publication number
WO2010065797A2
WO2010065797A2 PCT/US2009/066664 US2009066664W WO2010065797A2 WO 2010065797 A2 WO2010065797 A2 WO 2010065797A2 US 2009066664 W US2009066664 W US 2009066664W WO 2010065797 A2 WO2010065797 A2 WO 2010065797A2
Authority
WO
WIPO (PCT)
Prior art keywords
assay
analyte
cell
cells
iaa
Prior art date
Application number
PCT/US2009/066664
Other languages
English (en)
Other versions
WO2010065797A3 (fr
Inventor
David E. Kohne
Original Assignee
Kohne David E
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kohne David E filed Critical Kohne David E
Publication of WO2010065797A2 publication Critical patent/WO2010065797A2/fr
Publication of WO2010065797A3 publication Critical patent/WO2010065797A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to high sensitivity analyte assays utilizing immobilization of analytes.
  • IAA Immobilized Analyte Assays
  • ELISA Enzyme Linked Immunosorbent Assay
  • EIA Enzyme lmmuno Assay
  • sandwich assays which target a very wide variety of protein, nucleic acid, carbohydrate, lipid, or chemical analytes, where the target is immobilized on an assay surface
  • intact cell based assays where the target analyte is present on a cell surface or in a cell
  • assays in which the analyte is immobilized on a magnetic or non-magnetic particle surface and cytology assays where the analyte is associated with fixed cells or tissues.
  • IAA assay surface types including without limitation a flat or curved container surface (such as a test tube or microtiter plate surface), or a curved surface (such as a microparticle or nanoparticle surface or a curved surface on a surface protuberance, or an internal surface of a container surface hole or cavity, or a curved or other surface of a thread or fiber like structure).
  • a flat or curved container surface such as a test tube or microtiter plate surface
  • a curved surface such as a microparticle or nanoparticle surface or a curved surface on a surface protuberance, or an internal surface of a container surface hole or cavity, or a curved or other surface of a thread or fiber like structure.
  • An IAA generally consists of the following basic steps: i) Immobilize the sample analyte on a capture surface (for intact cell based assays and histology and histochemistry assays the analyte is naturally immobilized on or in an intact or essentially intact cell) and then wash away the analyte which is not immobilized.
  • the analyte molecule may be either specifically immobilized on an assay surface by a specific analyte capture molecule or immobilized or absorbed non-specifically to the surface. It is generally difficult to remove non- specifically bound analyte from the capture surface.
  • the analyte Before or after analyte immobilization the analyte may be specifically labeled with label molecules which produce signal directly (such as e.g. a fluorescent or radioactive molecule) or label molecules which produce signal indirectly (such as e.g. an enzyme molecule which is part of a signal generating system which produces signal generating molecules from a substrate or a biotin molecule or other hapten or receptor molecule which is later associated with a signal generating molecule).
  • label molecules used to label the analyte are typically in great excess over the analyte and these excess label molecules come directly into contact with the assay surface.
  • the labeling and/or immobilization step wash away all direct or indirect label which is not immobilized. It is generally difficult to remove non-specifically bound label from the capture surface, iii) Measure the direct or indirect signal from the label which remains immobilized on the surface after the assay wash steps.
  • the assay signal measured originates from label molecules specifically associated with surface immobilized analyte molecules and label molecules which are non-specifically bound to the assay surface.
  • Important factors which can determine the lowest number of analyte molecules detectable (i.e. the Lower Limit Of Detection, or LLOD of the analyte) for an IAA assay include the following. a) The lowest number of the direct or indirect assay label molecules which can be detected (i.e. the Lower Limit Of Detection or LLOD of the label).
  • the LLODs for different direct or indirect label types can differ by 10e4 or more and the lower the LLOD for the label used for the assay the lower the potential analyte detection LLOD for the assay.
  • the enzyme labels i.e.
  • the assay NSB signal represents the amount of active Non-Specifically immobilized or Bound (NSB) direct or indirect label on the assay capture surface at the end of the assay during the assay signal measurement step (i.e. label which is immobilized on the surface but is not specifically associated with immobilized analyte molecules).
  • NBS Non-Specifically immobilized or Bound
  • the presence and magnitude of assay NSB is detected by performing the assay in the absence of added analyte.
  • an assay label or signal generating molecule be immobilized on the assay surface only by associating directly with one or more surface immobilized analyte molecules.
  • a significant detectable amount of the input assay label virtually always becomes non-specifically immobilized on the assay surface and is not associated with immobilized analyte molecules.
  • the non-specific binding of the assay direct (e.g. radioactive, fluorescent, or nano- or micro- particle label) or indirect (e.g. enzyme molecules and/or biotin or other hapten molecules) labels occurs when the assay surface is contacted with the direct or indirect label during the assay.
  • NSB can be caused by hydrophobic, hydrophilic, and possibly covalent binding of the assay label molecules to some protein or other biological molecule or molecules which are non-specifically immobilized or adsorbed on the assay surface.
  • non-label immobilized proteins and other immobilized molecules may originate from the analyte sample being assayed or from an assay reagent such as a blocking reagent.
  • an assay reagent such as a blocking reagent.
  • IAAs and micro- or nano- particle based IAAs there are at least two different assay surfaces, the surface and interior of the cells and micro- or nano- particles and the surface of the container in which the cells reside.
  • Current assay blocking, labeling and washing reagents, procedures and stratagems are not adequate to prevent the NSB from occurring or to remove the NSB assay label from the assay surface or surfaces.
  • IAA NSB is almost always due to a combination of both hydrophobic and hydrophilic immobilization of the assay label to the assay surface or to proteins or other substances adsorbed to the assay surface, and current assay blocking, assay wash, and assay label reaction reagents are not adequate to prevent significant hydrophobic and/or hydrophilic non-binding of the assay label to the assay surface.
  • a variety of approaches are currently used for reducing NSB in IAAs.
  • One very commonly used approach is to reduce non-specific binding sites on the assay surface by pre-treating the assay surface with a blocking reagent which may contain protein and other blocking molecules which are known to bind to the assay surface or to other proteins immobilized on the assay surface.
  • a blocking reagent which may contain protein and other blocking molecules which are known to bind to the assay surface or to other proteins immobilized on the assay surface.
  • the assay surface is then thoroughly washed with a wash buffer to remove non- immobilized blocking reagents. It is commonly believed that the assay surface must be preblocked in order to prevent or reduce non-specific binding of assay label and other reactants, and that if this is not done the assay will suffer from high NSB. This step is done before contacting the assay label with the assay surface.
  • Blocking agents are generally empirically chosen. A variety of different blocking agents with different formulations are used for IAA assays and a particular blocking agent is often optimally useful for only particular analyte molecule types.
  • the main blocking agents used are proteins (such as bovine serum albumen, casein, fish gel, blood proteins, and others), and non-ionic detergents (such as Triton or Tween detergents), and at times synthetic polymers such as polyethylene glycol and polyvinylpyrrolidone.
  • a second approach to reducing the assay NSB is to react all specific binding agents (e.g., antibody) and/or assay label conjugates with the assay surface in the presence of one or more blocking reagents. Often the antibody and/or assay label conjugates are diluted into the blocking buffer used for the assay.
  • specific binding agents e.g., antibody
  • assay label conjugates are diluted into the blocking buffer used for the assay.
  • a third approach is to facilitate the removal of already immobilized NSB from the assay surface by adding protein (generally 1 % w/v or less) and/or relatively low concentrations of Triton X-100 or Tween 20 detergent (generally between 0.05% and 1 % w/v) to the wash buffer used in the assay.
  • protein generally 1 % w/v or less
  • Triton X-100 or Tween 20 detergent generally between 0.05% and 1 % w/v
  • the present invention is directed to methods for improving the detection of a wide variety of protein, nucleic acid, carbohydrate, lipid, or chemical analytes by immobilized analyte assays, by reducing non-specific binding in ways which significantly reduce the assay analyte lower limit of detection (LLOD), and to the resulting improved assay methods and reagents.
  • LLOD assay analyte lower limit of detection
  • IAAs include (but are not limited to), ELISA (Enzyme Linked Immunosorbent Assay), EIA (Enzyme lmmuno Assay) and sandwich assays where the target is immobilized on an assay surface, intact cell based assays where the target analyte is present on a cell surface or in a cell, assays in which the analyte is immobilized on a magnetic or non-magnetic particle surface, and cytology assays where the analyte is associated with fixed cells or tissues.
  • the invention includes the discovery of advantageous reagents and reagent concentrations which can dramatically improve assay detection sensitivity.
  • a first aspect of the invention concerns a method for reducing nonspecific binding (NSB) in an immobilized analyte assay, and involves binding signal generation conjugate with immobilized analyte in a labeling solution which includes a high concentration of non-ionic detergent having both hydrophilic and hydrophobic portions.
  • NBS nonspecific binding
  • Such high concentration of non-ionic detergent may be provided by one detergent, by each of multiple detergents, or by a combination of multiple detergents.
  • the labeling solution also includes a high concentration of at least one protein comprising both hydrophilic and hydrophobic portions, a cation (preferably a high concentration of cation), and a suitable buffer, and may also include a signal generation stabilizer.
  • the method also includes washing away non- bound signal generation conjugate with a wash solution which includes a high concentration of non-ionic detergent having both hydrophilic and hydrophobic portions, a high concentration of protein having both hydrophilic and hydrophobic portions, a high concentration of salt, a suitable buffer, and optionally a signal generation stabilizer.
  • a wash solution which includes a high concentration of non-ionic detergent having both hydrophilic and hydrophobic portions, a high concentration of protein having both hydrophilic and hydrophobic portions, a high concentration of salt, a suitable buffer, and optionally a signal generation stabilizer.
  • the labeling solution and wash solution are the same.
  • the labeling and/or wash solutions include blocking protein, high concentration of salt, and a non-ionic amphiphilic detergent; the labeling and/or wash solutions also includes an enzyme-stabilizing cation; the labeling and/or wash solutions include 0.3 to 6% (w/v) (e.g., 0.3 to 1 %, 0.5 to 1 %, 0.5 to 2%, 0.7 to 2%, 1 to 2%, 1 to 3%, 2 to 3%, 1 to 4%,2 to 4%, 3 to 4%, 1 to 5%, 2 to 5%, 3 to 5%, 1 to 6%, 2 to 6%, 3 to 6%, 4 to 6%) of a NSB-reducing protein or protein including both hydrophilic and hydrophobic portions (e.g., BSA, ovalbumin, casein, or other blocking protein indicated herein), 0.02 to 5 M (e.g., 0.02 to 0.2, 0.02 to 0.5, 0.02 to 1 , 0.02 to 2, 0.05 to
  • a label binding solution and/or wash solution may also include a molecule structurally related to the analyte or to a recognition moiety thereof, e.g., a nucleotide analog such as BrdU or IdU.
  • the method also involves immobilizing the analyte on a surface in the presence of a fixation reagent (fixation solution) or an immobilization solution; a fixation reagent including an alkaline compound, an ionic or non-ionic detergent, and a salt; a fixation regent includes 0.2 to 5 M (e.g., 0.2 to 2, 0.2 to 3, 0.2 to 5, 0.5 to 2, 0.5 to 3, 0.5 to 5, 1 to 3, 1 to 4, 1 to 5, 2 to 4 or 2 to 5 M) salt, 0.01 to 2% (w/v) (e.g., 0.01 to 0.1 , 0.01 to 0.2, 0.01 to 0.3, 0.01 to 0.5, 0.01 to 1 , 0.01 to 1.5, 0.05 to 0.1 , 0.05 to 0.2, 0.02 to 0.5, 0.02 to 1 , 0.01 to 2, 0.05 to 0.2, 0.05 to 0.3, 0.05 to 0.5, 0.05 to 1 , 0.05 to 1.5, 0.05 to 2,
  • the method also involves treating cells in a sample with a fixation reagent having a high concentration of salt (e.g., a fixation reagent as just specified in this paragraph or otherwise specified herein) and immobilizing the analyte from the cells on a surface in the presence of the fixation reagent.
  • a fixation reagent having a high concentration of salt e.g., a fixation reagent as just specified in this paragraph or otherwise specified herein
  • the method also includes treating the surface with a stringency wash, e.g., an acidic stringency wash, following the immobilizing of the analyte and before the binding of the label; such stringency wash includes an acid or acidic buffer or both at 0.01 to 2 M (e.g., 0.01 to 0.05, 0.01 to 0.10, 0.01 to 0.50, 0.01 to 1 , 0.1 to 2, 0, 0.05 to 0.1 , 0.05 to 0.5, 0.05 to 1 , 0.05 to 2, 0.05 to 5, 0.1 to 1 , 0.1 to 2, 0.1 to 3, 0.1 to 5, 0.5 to 2, 0.5 to 3, 0.5 to 5, 1 to 3, 1 to 4, or 1 to 5 M), salt at 0.05 to 5 M (e.g., at 0.05 to 0.2, 0.05 to 0.5, 0.05 to 1 , 0.05 to 2, 0.5 to 3, 0.5 to 2, 0.5 to 3, 0.5 to 5, 1 to 3, 1 to 4, 1 to 5, 2 to 4, or 2 to 5 M), a NSB-
  • a stringency wash
  • the signal generating conjugate is an antibody-enzyme conjugate, e.g., including an alkaline phosphatase.
  • the analyte species is a protein, a nucleic acid, a labeled nucleic acid, e.g., a nucleic acid with at least one detectable nucleotide analog incorporated, for example bromodeoxyuridine (BrdU) or iododeoxyuridine (IdU) or iodouracil (IU), a nucleoprotein, a carbohydrate, a lipid- containing species;
  • the analyte species is a nucleic acid and detection involves hybridization of complementary sequences;
  • the analyte species is a nucleic acid and detection involves specific binding to an incorporated nucleotide analog or portion thereof.
  • the protein is a cell surface protein or other cell surface marker, a signaling protein, or an enzyme.
  • the method involves treating cells in a sample and immobilizing an analyte species from the cells on a surface with a fixation reagent which includes 0.02 to 5 M salt, 0.01 to 2% ionic detergent, 0.01 to 1.0 M alkali base, and alcohol, if present, does not exceed 50% by weight (or other fixation reagent as specified above); treating the surface with an acidic stringency wash following the immobilizing and before the binding, where the stringency wash includes an acid or acidic buffer or both at 0.01 to 2 M, salt at 0.05 to 5 M, and a NSB-reducing protein (or other stringency wash as specified above); binding signal generation conjugate to target binding moiety (e.g., analyte or portion thereof, such as an analyte specified in embodiments above) in a labeling
  • target binding moiety e.g., analyte or portion thereof, such as an ana
  • the IAA is microparticle IAA (e.g., a nano- or microparticle based IAA), a microfluidics based IAA, a membrane based IAA, a slide based IAA.
  • microparticle IAA e.g., a nano- or microparticle based IAA
  • microfluidics based IAA e.g., a microfluidics based IAA
  • membrane based IAA e.g., a membrane based IAA
  • slide based IAA e.g., a slide based IAA.
  • the assay is a cell proliferation assay (CPA), a cytotoxicity assay, such as a Cell Proliferation Inhibition or Stimulation Assay(CPISA), a cell mediated cytotoxicity assay (CMCA), an antibody dependent cell mediated cytotoxicity assay (ADCMCA), a drug or chemical mediated cytotoxicity assay (DCMCA), a cell death assay (CDA), a cell DNA fragmentation assay (CDFA), a Cell Migration Assay (CMA), a cell chemosensitivity assay (CCSA), an oxidative stress assay(OSA), or a histochemistry assay (ICA), e.g., an ImmunoHCA (IHCA), a fixed or unfixed Whole Cell Assay (WCA), or an Elispot or Fluorospot Assay.
  • CCA cell proliferation assay
  • a cytotoxicity assay such as a Cell Proliferation Inhibition or Stimulation Assay(CPISA), a cell mediated cytotoxicity assay (CMCA), an antibody
  • the improved IAA has an analyte LLOD corresponding to 10,000 or fewer, 5000 or fewer, 2000 or fewer, 1000 or fewer, 500 or fewer, 100 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or fewer, or 3 or fewer cells; the improved IAA has an analyte LLOD which is no more than 0.50, 0.40, 0.30, 0.20, 0.10, 0.050, 0.010 or even less times the analyte LLOD of the prior standard IAA.
  • the assay is performed using an assay sample container (e.g., microwell plate) with both label binding and assay signal reading performed in that container, or with label binding performed in an assay sample container and assay signal is read in a separate signal reading container (e.g., a microwell plate).
  • an assay sample container e.g., microwell plate
  • label binding and assay signal reading performed in that container
  • label binding performed in an assay sample container and assay signal is read in a separate signal reading container (e.g., a microwell plate).
  • a related aspect concerns a method for determining the effect of a test condition on target cells, and involves determining the presence or amount of an analyte in cells exposed to the test condition in an assay as described in the preceding aspect, where the presence or amount of the analyte is expected to or may respond to the presence of the test condition.
  • the assay is run for samples respectively exposed to the test condition and not-exposed to the test condition, and the determining also involves comparing assay results for samples exposed to the test condition and not-exposed to the test condition.
  • the analyte is a nucleic acid, for example, a nucleic acid with an incorporated nucleotide analog, e.g., BrdU, IdU, or IU; a protein, for example, a protein with one or more incorporated halogen molecules, e.g., Cl, Br, or I, or one or more thiocyanate (SCN) molecules; a nucleic acid, protein, or other biomolecule containing one or more modifications caused by oxidative stress.
  • a nucleic acid for example, a nucleic acid with an incorporated nucleotide analog, e.g., BrdU, IdU, or IU
  • a protein for example, a protein with one or more incorporated halogen molecules, e.g., Cl, Br, or I, or one or more thiocyanate (SCN) molecules
  • SCN thiocyanate
  • the invention concerns a method for determining the presence or amount of an analyte in a biological sample by assaying the sample for that analyte using an IAA as described in the first aspect above or otherwise described herein for the present invention.
  • the analyte is a nucleic acid analyte; the analyte is a nucleic acid and the IAA involves detection of the nucleic acid by detecting the presence of a detectable nucleotide analog, e.g., BrdU, IdU, or IU; the IAA involves detection of a nucleic acid by specific nucleic acid hybridization; the analyte is a protein (e.g., a protein as identified herein) or modified protein (e.g., as identified herein).
  • a detectable nucleotide analog e.g., BrdU, IdU, or IU
  • the IAA involves detection of a nucleic acid by specific nucleic acid hybridization
  • the analyte is a protein (e.g., a protein as identified herein) or modified protein (e.g., as identified herein).
  • a related aspect of the invention concerns an assay kit which includes an amount of label conjugate (e.g., an amount selected to be sufficient for performing 1 -3, 1 -5, 1 -10, 1 -50, 1 -100, 5-10, 5-20, 5-100, 100-1000 assays); and a label conjugate binding solution which includes a high concentration of non-ionic detergent comprising both hydrophilic and hydrophobic portions, a high concentration of protein having both hydrophilic and hydrophobic portions, a high concentration of salt, a suitable buffer, and optionally a signal generation stabilizer (or other labeling or label binding solution as described for the first aspect above).
  • an amount of label conjugate e.g., an amount selected to be sufficient for performing 1 -3, 1 -5, 1 -10, 1 -50, 1 -100, 5-10, 5-20, 5-100, 100-1000 assays
  • a label conjugate binding solution which includes a high concentration of non-ionic detergent comprising both hydrophilic and hydrophobic portions, a high concentration of protein
  • the label conjugate binding solution includes about 0.8 M NaCI, about 0.2M tris-HCI pH7.4, about 3%w/v Pluronic F127, about 2% BSA, about 2x10e-3 M MgCI2, and may also contain glycerol, e.g., about 2% glycerol.
  • the assay kit also includes an assay wash solution which includes a high concentration of non-ionic detergent having both hydrophilic and hydrophobic portions, a high concentration of protein comprising both hydrophilic and hydrophobic portions, a high concentration of salt, a buffer, and optionally a signal generation stabilizer (or other wash solution as specified for the first aspect above), and which may be the same or different from the label conjugate binding solution; when the same the label conjugate binding solution and the wash solution may be packaged in the same or different containers; the wash solution includes about 0.8M NaCI, about 0.2M tris-HCI pH7.4, about 3%w/v Pluronic F127, about 2% BSA, and about 2x10e-3 M MgCI2, and may also contain glycerol, e.g., about 2% glycerol.
  • an assay wash solution which includes a high concentration of non-ionic detergent having both hydrophilic and hydrophobic portions, a high concentration of protein comprising both hydrophilic and hydrophobic portions, a high
  • the assay kit also includes a fixation solution, e.g., a high salt fixation solution, which may be a fixation solution which lyses mammalian cells; the fixation solution includes about 0.8M LiCI, about 0.045M NaOH, and about 0.2%w/v LiLS; the fixation solution includes about 2 M NaCI, about 0.1 M NaOH, and about 2%w/vNa Decylsulfate; the fixation solution is as specified for the first aspect above.
  • a fixation solution e.g., a high salt fixation solution, which may be a fixation solution which lyses mammalian cells
  • the fixation solution includes about 0.8M LiCI, about 0.045M NaOH, and about 0.2%w/v LiLS
  • the fixation solution includes about 2 M NaCI, about 0.1 M NaOH, and about 2%w/vNa Decylsulfate
  • the fixation solution is as specified for the first aspect above.
  • the assay kit also includes a stringency wash or solution, e.g., an acidic stringency wash; an included acidic stringency wash contains about 3M LiCI, about 0.05M glycine, about 0.02M HCI, and about 1 mg/ml Trypsin; an included stringency wash contains about 3M LiCI, about 0.05M glycine, about 0.02M HCI, and about 0.1 mg/ml Trypsin; an included stringency wash is as specified for the first aspect above.
  • a stringency wash or solution e.g., an acidic stringency wash
  • an included acidic stringency wash contains about 3M LiCI, about 0.05M glycine, about 0.02M HCI, and about 1 mg/ml Trypsin
  • an included stringency wash contains about 3M LiCI, about 0.05M glycine, about 0.02M HCI, and about 0.1 mg/ml Trypsin
  • an included stringency wash is as specified for the first aspect above.
  • the assay kit also includes an assay preparation solution for preparing an immobilized enzyme for reaction with its substrate, e.g., an assay preparation solution containing about about 0.8 M NaCI, about 0.2M tris- HCI pH7.4, about 3%w/v Pluronic F127, about 2% BSA, about 2x10e-3 M MgCI2, and may contain glycerol, e.g., about 2% glycerol.
  • the assay kit includes an enzyme substrate, e.g., an enzyme substrate which includes CDP-Star® with Sapphire IITM (from Applied Biosystems/Life Technologies).
  • the kit also includes at least one (e.g., 1 , 2, 3, 4, 5, 2 to 5, 2 to 10, at least 5, or at least 10) assay sample container (e.g., microwell plate such as a 96 well microwell plate) and/or at least one (e.g., 1 , 2, 3, 4, 5, 2 to 5, 2 to 10, at least 5, or at least 10) separate signal reading container (e.g., strip microwells).
  • assay sample container e.g., microwell plate such as a 96 well microwell plate
  • separate signal reading container e.g., strip microwells
  • another aspect of the invention concerns a method for producing an Immobilized Analyte Assay (IAA) having improved analyte detection lower limit of detection (LLOD), and involves identifying at least one improvement combination of assay reagents and assay procedures by determining whether a test combination of assay reagents and assay procedures used in a selected IAA for a selected analyte produces improved analyte detection LLOD compared to the analyte detection LLOD for standard combination of assay reagents and assay procedures, where the test combination is selected at least in part to provide increased competition for hydrophobic, hydrophilic, and ionic non-specific binding sites which could bind a direct or indirect label in the IAA, and where a test combination producing a lower analyte detection LLOD than the prior standard combination is a first stage improvement combination.
  • IAA Immobilized Analyte Assay
  • LLOD analyte detection lower limit of detection
  • the method includes repeating the determining with different test combinations until at least one first stage improvement combination is identified.
  • the method can include iteratively testing additional combinations of assay reagents assay procedures to identify one or more further improvement combinations and incorporating an improvement combination in the IAA, thereby providing an improved IAA having improved analyte LLOD.
  • the method involves selecting an analyte of interest and corresponding IAA of interest, where the IAA is performed with a standard combination of assay reagents and assay procedures; selecting an initial test combination of assay reagents and assay procedures expected to provide increased competition for hydrophobic, hydrophilic, and ionic non-specific binding sites which could bind a direct or indirect label used or proposed for use in the IAA; and then performing the IAA using the initial test combination of assay regents and assay procedures to produce Signal/Noise ratio assay results for the selected analyte; analyzing the assay results to determine whether the analyte detection LLOD for the selected analyte is improved relative to the analyte detection LLOD for the standard combination; and if needed, repeating the selecting of test combination, performing the assay, and analyzing assay result with different selections of test combinations of assay reagents and test conditions until at least one test combination is identified is a first stage improvement combination.
  • the method also includes iteratively determining whether further modified test combinations of assay reagents and assay procedures result in reduced assay LLOD compared to the standard IAA or the preceding improved IAA or both.
  • the test combinations include a label binding solution which contains a high salt concentration and a high concentration of at least one polymer having both hydrophobic and hydrophilic regions (for example, the at least one polymer can include a non-ionic detergent and a protein).
  • the test combinations include a high salt analyte binding or fixation solution;
  • the high salt analyte binding or fixation solution includes a blocking protein (e.g., BSA) and/or contains no more than a low level of alcohol (preferably less than 20%, 10%, 5%, 3%, 2%, or 1 % w/v or still more preferably is free or substantially free of alcohol)).
  • BSA blocking protein
  • a low level of alcohol preferably less than 20%, 10%, 5%, 3%, 2%, or 1 % w/v or still more preferably is free or substantially free of alcohol
  • the selected analyte is a protein analyte, a nucleic acid analyte (e.g., a labeled nucleic acid analyte, for example, a BrdU containing nucleic acid), a carbohydrate analyte, a lipid-containing analyte, a nucleoprotein analyte;
  • the selected analyte is a labeled DNA or RNA Nucleic Acid, e.g., a halogen labeled DNA or RNA Nucleic Acid such as BrdU, IdU, FU (fluorouracil or fluorodexyuracil) or IU (lodouracil or lododeoxy Uracil) labeled DNA or RNA;
  • the selected analyte is a labeled modified DNA or RNA which includes a modified RNA or DNA nucleotide or other compound having a modification caused by oxidative stress;
  • the current IAA is a CPA, or CMCA, or ADCMCA or CDA, or CCSA, or CMA, or CDFA, or CPISA, type IAA; the current IAA is a WCA type IAA; the current IAA is an HCA type WCA, e.g., an IHCA.
  • the method also involves transferring each of the control and experimental sample cells from their respective first assay containers to new separate control and experimental sample second assay containers; and determining the assay signal present in each of the control and experimental assay second containers, whereby signal noise due to NSB in the first assay containers is substantially reduced or substantially eliminated, providing a further improved WCA IAA.
  • nano- or micro-particle based IAA which involves binding of an assay label with specifically bound particle-immobilized analyte and at least one wash step of the bound assay label, after a first wash step following exposure of the assay label to the assay surfaces in first assay containers, transferring each of the control and experimental sample particles from their respective first assay containers to new separate control and experimental sample second assay containers; and determining the assay signal present in each of the control and experimental assay second containers, whereby signal noise due to NSB in the first assay containers is substantially reduced or substantially eliminated, providing a further improved nano- or micro-particle based IAA.
  • the present invention is directed to improvements in immobilized analyte assays (IAAs).
  • IAAs immobilized analyte assays
  • NAB non-specific binding
  • LLODs practical analyte lower limits of detection
  • the disparity between the practical analyte LLOD achieved for an IAA assay and the potential analyte LLOD of the IAA possible if the assay NSB were eliminated or significantly reduced is generally large and is greatest for the assay labels which have the lowest label LLODs.
  • the invention relates to methods for significantly decreasing the analyte LLODs for IAAs relative to the currently available LLODs for currently available IAA assays.
  • An important determinant of an IAA analyte LLOD is the ratio for the assay of [(the assay signal associated with a sample containing the analyte) ⁇ (the assay signal associated with a control sample which does not contain the analyte)]. This ratio is commonly known as the assay Signal/Noise ratio or Assay S/N ratio.
  • the lowest detectable amount of analyte by the assay is determined by assaying different dilutions of the analyte and determining the analyte concentration which gives an assay signal which can be detected with statistical significance above the assay Noise (i.e., the assay signal in the absence of analyte).
  • the invention further relates to methods for significantly decreasing the assay analyte LLOD by significantly decreasing the assay NSB of the IAA.
  • a very large fraction of the assay noise in currently available IAAs is due to NSB.
  • the practice of the invention must reduce the assay associated Noise (i.e. the signal associated with the assay NSB) signal to a significantly greater degree than the analyte associated assay signal is reduced.
  • This invention reduces the IAA NSB and thereby the assay analyte LLOD by the development and use of novel reagents, and modification of current assay procedure and/or stratagems.
  • the assay NSB is caused by the non-specific hydrophobic and hydrophilic immobilization of a significant amount of assay label to the assay surface when the assay label is put in contact with the assay surface(s). Further, once the assay label is surface-immobilized it is very difficult to efficiently remove the immobilized label from the surface during the wash steps.
  • Part of the strategy of the invention is to devise assay label reaction solutions which are significantly more effective at lowering the amount of assay label which is initially non-specifically immobilized (e.g., through hydrophobic and/or hydrophilic interactions) to the assay surface during the assay, as well as to assay wash buffers which are significantly more effective at removing the non- specifically immobilized label from the assay surface after it has become immobilized.
  • this has been done as follows: a) Reducing the NSB due to hydrophobic binding of assay label to the assay surface by utilizing an effective concentration of a block co-polymer non-ionic detergent and one or more proteins in the wash buffer and the assay labeling buffer, whose roles are to prevent (or at least reduce) hydrophobic and hydrophilic non-specific binding of the assay label to the assay surface.
  • the individual block co-polymer molecules and protein molecules used contain both hydrophobic and hydrophilic sites and both are effective at blocking the assay label from non-specifically binding to the assay surface.
  • the invention is advantageously practiced by utilizing protein and a non-ionic block co-polymer detergent which reduces hydrophobic and hydrophilic non-specific binding of the assay label to the assay surface.
  • the invention can use the assay stratagem of removing the assayed intact cells from the initial assay container to a new assay container before the assay signal measurement step in order to eliminate the assay NSB associated with the assay surface of the initial container in which binding steps were performed.
  • the non-ionic block co-polymer preferably used for the intact cell assays is also compatible with living and growing cells without appreciably affecting the antibody or labeling reactions.
  • an effective amount refers to a concentration or quantity of the particular reagent which is sufficient to produce an intended effect(s).
  • an effective amount is an amount which in combination with other reagents and for the relevant assay conditions, results in improved analyte LLOD.
  • the term "about” means the specified level or concentration plus or minus 50 percent. In particular embodiments, the variation may be less, e.g., plus or minus 40, 30, 20, 15, 10, 5, or 3%.
  • non- ionic detergent comprising both hydrophilic and hydrophobic portions
  • non- ionic amphiphilic detergent refer to uncharged compounds which exhibit detergent function in aqueous solution and which contain segments which are hydrophobic and segments which are hydrophilic in nature.
  • a "high concentration of" such a detergent refers to a concentration which is significantly higher than the concentration for a corresponding reagent in a prior reference standard assay, and/or that is a concentration of at least 0.2% (w/v) and often higher, e.g., at least 0.3, 0.4, 0.5, 0.7, 1 , or 1.5%.
  • signal generation conjugate and “label conjugate” refer to conjugates which include at least one specific binding moiety and at least one signal generation moiety.
  • a signal generation moiety is one which is involved in generation of the assay detectable signal, e.g., fluorescence or production of a detectable product of an enzymatic reaction.
  • a signal generation moiety may be a fluorescent dye or an enzyme such as an alkaline phosphatase.
  • non-specific binding refers to label which becomes bound or immobilized on an assay surface but is not specifically associated with analyte.
  • immobilized analyte refers to analyte species which is bound directly or indirectly to a solid phase surface and therefore prevented from moving freely in a liquid medium, usually an aqueous medium, under particular conditions. Indication that an analyte is immobilized does not mean that the analyte cannot be removed from the surface by a change of conditions, e.g., introduction of chaotropic agents or cleavage of a linker.
  • immobilized analyte assay refers to an assay in which the analyte species to be detected is directly or indirectly bound to an assay surface, typically a solid phase surface such as to a wall of a well in a plastic microwell plate or to a particle or a cell.
  • labeling solution refers to a solution (and/or to a reagent which is to be made up into a solution) which includes a label species which will directly or indirectly and specifically associate with analyte in a corresponding assay. Such label species will often be a signal generation conjugate.
  • protein comprising both hydrophilic and hydrophobic portions refers to a protein which has exposed hydrophilic and hydrophobic regions under the relevant assay conditions. Reference to a "high concentration” of such a protein refers to a concentration of at least 0.3% (w/v) in the solution.
  • NSB-reducing protein refers to a protein which is demonstrated to reduce NSB when used in a particular solution in a particular assay. In most cases, such an NSB-reducing protein will be a protein comprising both hydrophilic and hydrophobic portions.
  • the term "high concentration of cation” refers to a concentration of cation (usually contributed by an inorganic salt) which is at least 0.3 M (and often at least 0.4 M, 0.5 M, 0.5 M, 0.6 M, 0.7, 0.8, 0.9, 1.0 M or even higher) in the particular solution.
  • the term “high concentration” refers to a concentration which will contribute a high concentration of cation as just specified.
  • suitable buffer refers to a buffer selected to provide buffering action in a solution within an appropriate pH range considering the other species present in the solution and which is otherwise compatible with the other species present. For example, a suitable buffer will not cause undesired aggregation of other species or undesired inactivation of active proteins.
  • signal generation stabilizer refers to a compound or combination of compounds in an assay reagent which stabilizes signal output as compared to the signal output in the absence of the compound(s).
  • a signal generation stabilizer may stabilize an enzyme involved in the signal generation (e.g., divalent cations such as magnesium or zinc) or may reduce fluorescent quenching, or the like.
  • Reference to "immobilizing an analyte on a surface” and similar expressions refers to a process in which particular analyte molecules, complexes, or structures are bound directly or indirectly to a solid phase surface such that removal of the bound analyte without a change to more disruptive conditions is substantially prevented. Such binding may involve specific and/or non-specific binding.
  • fixation reagent and "fixation solution”) are used to refer to a solution (and/or to a dry reagent which is to be made up into a solution) which is used to immobilize or fix an analyte species to an assay surface.
  • a fixation reagent may also commonly cause or contribute to cell lysis, e.g., lysis which releases analyte from cells.
  • Fixation solutions which do not cause or contribute to cell lysis may also be referred to as "immobilization reagents" or “immobilization solutions”.
  • immobilization solutions are often used for assays involving protein analytes and other analytes which may be damaged or otherwise altered by exposure to conditions which would cause or significantly contribute to cell lysis. In many cases, an immobilization solution will not include a significant concentration of alkali base.
  • alkaline compound and "alkali base” are used to refer to basic compounds which contain an alkaline earth. In most cases, the compound releases hydroxide in aqueous solution, e.g., NaOH, KOH, and LiOH.
  • salt refers to an inorganic salt, e.g., LiCI or NaCI.
  • alcohol is used in its usual manner to refer to compounds in which a hydroxyl group is bound to an alkyl or substituted alkyl group. Common examples include methanol, ethanol, propanol, and butanol.
  • wash and “washing” are used to refer to a procedure in an assay in which a solution is used to remove unbound reagent from a particular environment and/or to replace a prior solution in that environment, e.g., a sample area of an assay container.
  • wash solution refers to a solution (and/or to a dry reagent which is to be made up into a solution) to be used in a wash procedure.
  • stringency wash and “acidic stringency wash” refer to solutions (and/or to dry reagents to be made up into a solution) used in some embodiments of the present IAA assays which are used following analyte immobilization and prior to label binding to reduce non-specific label binding.
  • the solution will be an acidic (i.e., an acidic stringency wash) and/or high salt concentration solution.
  • label LLOD is the minimum number of label molecules detectable in a particular signal generation and detection system
  • analyte LLOD and “assay LLOD” refers to the minimum number of analyte molecules detectable in a particular assay.
  • Reference to a LLOD corresponding to a number of cells means the number of cells needed to provide the level of analyte meeting the analyte LLOD.
  • test condition refers to environmental conditions to be tested or analyzed for effects of those conditions on an entity or entities. Normally the effects resulting from the test conditions are compared to the effects for one or more reference conditions and/or one or more other test conditions.
  • entity may, for example, be a molecular species, a cell (e.g., a mammalian cell), or a microorganism (e.g., a bacterium), among others.
  • biological sample refers to a sample obtained or derived from a biological source, e.g., from an organism or set of organisms, for use in an assay.
  • biological samples may, for example, be body fluids, tissues, cells, microorganisms, purified or enriched nucleic acids, or purified or enriched proteins, among others.
  • the term "assay sample container” refers to a container which is used or is intended to be used to hold a test and/or control sample at least during a label binding process.
  • the assay sample container may further be used for performing other steps, for example, subsequent wash steps, e.g., 0 to 1 to 2 subsequent wash steps.
  • the term “separate signal reading container” refers to a container which is used or is intended to be used to hold a test or control sample during reading of the assay signal but not during binding of label.
  • the separate signal reading container may also be used for wash steps, e.g., wash steps before reading of the signal, such as during 0 to 1 or 2 wash steps before the reading of assay signal.
  • the phrase "improvement combination of assay reagents and assay procedures" refers to a set of assay reagent and assay procedures selected in accordance with the present invention which has been demonstrated to provide improved analyte LLOD as compared to a reference set of assay reagent and assay procedures, usually a set corresponding to a reference assay or a prior iteration in the present assay improvement methods.
  • test combination of assay reagents and assay procedures refers to a set of assay reagents and assay procedures selected for testing in a present process for developing improved assays.
  • standard combination of assay reagents and assay procedures refers to a set of assay reagents and procedures normally used in a standard assay or an assay which is to be improved using the present invention.
  • the terms “Signal/Noise ratio”, “signal to noise ratio”, “S/N ratio”, “assay signal to noise ratio”, and like terms refer to the ratio of the assay signal divided by the assay background, generally calculated as: [(the assay signal associated with a sample containing the analyte) ⁇ (the assay signal associated with a control sample which does not contain the analyte)].
  • the invention improvement strategy involves a simultaneous surface saturation and binding competition strategy which is considerably more complex and comprehensive than current methods for reducing assay label NSB, and which relative to current methods, results in the following: a) reducing the number of assay label non-specific binding sites, and the non-specific binding strength of these assay label nonspecific binding sites which are associated with the assay surface and/or associated with proteins on the assay surface: b) significantly reducing the fraction of assay label molecules which are free in solution which can non-specifically bind to the assay surface; c) facilitating removal from the surface of assay label which has non-specifically bound to the surface; d) optionally including a non-target molecule structurally related to the target analyte; and e) preferably simplifying the IAA assay format. Items a) thru e) are discussed below.
  • this has been done as follows: a) Reducing the NSB due to hydrophobic binding of assay label to the assay surface and to proteins attached to the assay surface by utilizing an effective high concentration of: a block co-polymer non-ionic detergent which possesses both hydrophobic and hydrophilic chemical blocks; and an effective high concentration of one or more protein which also possess hydrophobic and hydrophilic chemical blocks which are different from the non- ionic detergent, as well as ionic chemical sites; and an effective high concentration of an inorganic salt; in the assay labeling buffer solution which can also be used as the assay wash buffer solution.
  • a block co-polymer non-ionic detergent which possesses both hydrophobic and hydrophilic chemical blocks
  • an effective high concentration of one or more protein which also possess hydrophobic and hydrophilic chemical blocks which are different from the non- ionic detergent, as well as ionic chemical sites
  • an effective high concentration of an inorganic salt in the assay labeling buffer solution which can also be used as the assay wash
  • the results of treating the assay surface with this wash-labeling reagent combination are: to thoroughly saturate the existing hydrophobic and hydrophilic and ionic label binding sites associated with the assay surface and the proteins present on the assay surface, in order to create a situation where there are fewer hydrophobic and hydrophilic and ionic label non-specific binding sites on the assay surface and the proteins present on the assay surface; to reduce the strength, relative to the untreated non-specific binding sites, of the non-specific binding sites which are present after treatment with the reagent so that the newly formed non-specific label binding sites cannot bind the assay label as tightly as the previous untreated surface binding sites.
  • Treating the assay label molecules with the invention improved wash-labeling reagent creates a situation where the high concentration of non-ionic detergent, protein, and salt present in the wash-label reagent competitively interacts: in solution with the existing hydrophobic and hydrophilic and ionic label binding sites associated with the free in solution assay label molecules; and on the assay surface with immobilized non-specific label binding sites.
  • the competitive effect in the invention improved wash-label reagent causes a situation where the assay surface immobilized hydrophobic and hydrophilic and ionic non-specific binding sites associated with the assay surface are, relative to standard methods, fewer and weaker, and the competitive effect reduces the likelihood that any particular immobilized non-specific label binding site will non- specifically bind to a non-specific binding site associated with a free in solution assay label molecule which collides with the assay surface.
  • Exposing the assay label to the assay surface creates a situation where the label which has non-specifically bound to the assay surface in the presence of the invention improved wash-label reagent is, relative to standard methods, less strongly non-specifically bound.
  • the invention improved wash-labeling solution can also include a molecule which is structurally related to the target recognized by a specific binding molecule.
  • a specific binding molecule For example, where anti-BrdU antibodies are used for recognizing and binding to +BrdU DNA, IdU can be included in the solution. Conversely, where anti-ldU antibodies are used, BrdU can be included in the solution.
  • such structurally related molecule will reduce both signal and noise, but will reduce noise proportionally more than the signal. Thus, such inclusion can be used to improve assay reproducibility, manipulate signal level, and/or improve signal to noise ratio.
  • the invention improved wash solution can also be used for the labeling solution the IAA assay can be, relative to standard methods, improved in simplicity.
  • the ultimate practical assay improvement parameter which identifies invention improved IAAs is the assay analyte of interest LLOD value.
  • the invention relates to methods for significantly decreasing the analyte LLODs for IAAs relative to the currently available LLODs for currently available IAA assays.
  • An important determinant of an IAA analyte LLOD is the ratio for the assay of [(the assay signal associated with a sample containing the analyte) ⁇ (the assay signal associated with a control sample which does not contain the analyte)]. This ratio is commonly known as the assay Signal/Noise ratio or Assay S/N ratio.
  • the analyte LLOD is typically determined by assaying different dilutions of the analyte and determining the analyte concentration which gives an assay signal which can be detected with statistical significance above the assay Noise or NSB signal (i.e. the assay signal in the absence of analyte).
  • the invention thus relates to methods for significantly decreasing the assay analyte LLOD by significantly decreasing the assay NSB of the IAA.
  • a very large fraction of the assay noise in currently available IAAs is due to NSB.
  • the practice of the invention reduces the assay associated Noise (i.e. the signal associated with the assay NSB) signal to a significantly greater degree than the analyte associated assay signal is reduced.
  • This invention reduces the IAA NSB and thereby the assay analyte LLOD by the development and use of novel assay reagents and modification of current assay procedure and/or stratagems.
  • invention improved IAAs The above described general strategy for producing invention improved IAAs is implemented by using experimentation to identify the reagents and combinations of reagents and combinations of reagents and procedures, which produce invention improved IAAs which have improved NSBs and S/N ratios relative to current standard IAA methods. Following is a brief description of the general process used to follow the above described basic strategy for producing invention improved IAAs, and for further improving and optimizing the initially identified invention improved IAAs.
  • improved IAA modify one or more of the reagents or procedures of the improved IAA according to the basic strategy and perform the assay and determine whether the assay NSB and/or S/N ratio of the modified assay is significantly improved relative to the progenitor IAA.
  • an exemplary description of one embodiment of the invention is presented primarily in terms of a well known specific type of IAA in which the analyte to be detected in the assay is a modified DNA or RNA precursor (i.e., nucleotide analog) which has been incorporated in vivo or in vitro into the DNA or RNA of a cell or virus or a modified DNA or RNA nucleotide (i.e., modified nucleotide) which has been produced by the modification of an already existing nucleotide present in in vivo or in vitro DNA or RNA of a cell or virus by oxidative stress, radiation, and/or some other mechanism.
  • a modified DNA or RNA precursor i.e., nucleotide analog
  • modified nucleotide i.e., modified nucleotide
  • DNA or RNA precursors include but are not limited to BU (BromUracil), BrdU (BromodeoxyUridine), IdU (IododeoxyUridine), and IU (IodoUracil), IdU (fluorodeoxyUridine), and FU (fluoroUracil) or a radioactively labeled DNA or RNA precursor or a hapten labeled DNA or RNA precursor.
  • BU BinaryUracil
  • BrdU BromodeoxyUridine
  • IdU IododeoxyUridine
  • IU IodoUracil
  • IdU fluorodeoxyUridine
  • FU fluoroUracil
  • Methods for the in vivo or in vitro incorporation of one or more of these precursors into cellular or viral DNA or RNA are well known and therefore will not be further described herein.
  • oxidative stress of modified nucleotides in cell DNA or RNA include, but are not limited to, 8-OH-Guanosine, 8-OH- deoxyguanosine, Thymidine glycol, 2-OH-adenine, 5-OH-cytosine, 5-OH-uracil, and formyluracil, among others.
  • further embodiments of the invention can be produced which detect the presence of oxidative modifications of proteins and other biomolecules in cells and animal fluids. These oxidative modifications produce halogenated and SCN (thiocyanate) modified proteins and other cellular components, protein modifications resulting from protein carbonylation (e.g., by reaction with lipid-derived aldehydes and ketones), as well as other types of modifications.
  • this exemplary description primarily involves a description of the use of the invention for the improved Elisa detection of cellular DNA containing BrdU by detecting in vivo synthesized BrdU labeled cell DNA analyte using an antiBrdU antibody-alkaline phosphatase conjugate label and an enzyme generated chemiluminescent signal for detection in a particular type of IAA, in an Enzyme linked immunosorbent assay (Elisa) format (also referred to as an enzyme linked immuno assay (EIA). Numerous tests currently are marketed which are designed to detect cell DNA analyte containing BrdU and other DNA labels using the Elisa test format.
  • Elisa Enzyme linked immunosorbent assay
  • EIA enzyme linked immuno assay
  • CCA cell proliferation assays
  • CMCA cell mediated cytotoxicity assays
  • ADCMCA antibody dependent cell mediated cytotoxicity assays
  • CDA cell death assays
  • CDFA cell DNA fragmentation assays
  • CMA Cell Migration Assays
  • CCSA cell chemosensitivity assays
  • the invention can be utilized to produce, for example, BrdU DNA analyte CPA, CPISA, CMCA, ADCMCA, CMA, CDA, and CDFA assays with significantly improved performance characteristics.
  • these tests rely on the ability of cells to incorporate a direct or indirect signal generating molecule into cellular DNA during cell DNA synthesis.
  • a variety of direct or indirect signal generating molecule types are used for these assays.
  • nucleic acid bases or nucleosides or nucleotides which are DNA precursor molecules which are associated with: one of many different types of halogen (such as BromdeoxyUridine, IododeoxyUridine, and others); one of many different types of hapten or ligand or receptor molecules(such as Digoxigenin, biotin, and others); one of many different types of radioactivity(such as tritium, carbon 14, phosphorous 32 or 33, and others); one of many different types of fluorescent molecules (such as fluorescein and others).
  • halogen such as BromdeoxyUridine, IododeoxyUridine, and others
  • hapten or ligand or receptor molecules such as Digoxigenin, biotin, and others
  • radioactivity such as tritium, carbon 14, phosphorous 32 or 33, and others
  • fluorescent molecules such as fluorescein and others.
  • an exemplary application of the invention is presented in detail by describing an invention improved Elisa IAA for detecting mammalian cell BrdU DNA which has significantly improved performance characteristics relative to the currently available IAA Elisa tests which detect cell BrdU DNA.
  • the invention improved test is significantly faster in performance than currently available IAA Elisa tests; has a significantly Lower Limit Of Detection (LLOD) of BrdU cell DNA than currently available IAA Elisa mammalian cell BrdU DNA detection tests; can detect smaller numbers of BrdU labeled cells even in the presence of large numbers of cells which have not incorporated BrdU into their DNA than currently available IAA Elisa tests; and can be done with fewer cells than current tests.
  • LLOD Limit Of Detection
  • This improved test utilizes an antiBrdU Antibody-Alkaline Phosphatase conjugate label to produce a chemiluminescent assay signal.
  • IAA Elisa cell BrdU DNA detection test which have significantly improved performance characteristics relative to currently available IAA Elisa cell BrdU DNA detection tests and which vary from each other in assay reagent composition and/or time of assay performance and/or BrdU cell DNA and/or the number of recommended input cells and/or the number of non- BrdU labeled cells which may be present.
  • These embodiments include CPA, CPISA, and various cytotoxicity assays.
  • the performance characteristics of the currently available cell BrdU DNA assays are generally described in the following terms. i) The lowest number of BrdU labeled cells (i.e. the +BrdU cell Lower Limit Of Detection or +BrdU cell LLOD) which can be detected in the assay. For currently available BrdU cell DNA assays this +BrdU cell LLOD value reflects the lowest number of +BrdU cells which can be detected in the absence of non-dividing cells or non-proliferating cells.
  • the gold standard for these tests is generally considered to be the tritiated thymidine incorporation test and the currently available tests with the lowest BrdU DNA cell LLODs often make the claim that the test has the same BrdU DNA cell LLOD as the tritiated thymidine incorporation test.
  • the most sensitive of the current BrdU cell DNA assays and the tritiated thymidine incorporation assay can detect a lower limit of about 200 to 500 +BrdU cells in the absence of cells which are not dividing.
  • the available information indicates that the BrdU cell LLODs are much higher when the assay is done in the presence of a significant number (i.e. 10e3 to 10e5 -BrdU cells). ii).
  • the time necessary to perform the assay The fastest currently available BrdU cell DNA assays take 1.5 to 3 hours to get from the beginning of the fixation step to the point where the assay signal results have been measured. iii) The number of cells necessary to perform the assay. The recommended lower limit of input cells for the most sensitive current assays is about 500 to 5000 cells.
  • the invention improved cell BrdU cell DNA detection assay has significantly improved performance characteristics which include but are not limited to the following. i) A significantly lower +BrdU cell LLOD than any of the currently available cell BrdU cell DNA detection assays or the gold standard tritiated thymidine incorporation assay.
  • a large number of different embodiments of the invention can be practiced and which differ in their performance characteristics and their +BrdU cell LLODs. Multiple different preferred embodiments of the invention are associated with very low + BrdU LLODs values and can detect the presence of 1 to 5 BrdU cells.
  • the most sensitive currently available BrdU cell DNA detection assays can detect a lower limit of about 200 to 300 +BrdU cells in the absence of significant numbers of cells which have not incorporated BrdU into their DNA.
  • Preferred assay embodiments of the invention which have a +BrdU cell LLOD of 1 to 50 cells have been developed which take as little as 30 minutes to perform in contrast to a time of about 90 minutes for the shortest of the current assays.
  • An effective period of time can vary depending on the nucleic acid synthesis rate of the cells and the degree of incorporation desired, but will be a time sufficient for sufficient BrdU to be incorporated for properly conducting the assay. In most cases the BrdU exposure lasts 2-24 hours or more. The number of cells to be assayed is generally predetermined before the assay.
  • Step 1 Preparing the Cells for the Assay Fixation and Cell DNA
  • Control and experimental cells in suspension in cell culture media or blood or some other aqueous liquid solution or buffer do not need to be processed further before fixation.
  • Step 2 Fixation of the Cells and Immobilization of the Cell DNA on the Assay Surface
  • fixation the cell
  • DNA is released from the cells and, in most applications, converted to single strand DNA.
  • a fix + cell sample mix is not already in a MPW
  • a MPW such as a Delta Nunc 96 well white Microtiter Plate.
  • an effective temperature generally done at room temperature
  • an effective time generally done for 10 minutes or longer but can be done for 1 -2 minutes
  • the improved fixation and cell DNA immobilization reagent contains a preferred high concentration of salt (generally 0.2 to 5 M salt, e.g., LiCI or NaCI), a preferred moderate concentration (generally 0.01 to 0.5% w/v), of an ionic detergent (e.g., Lithium Lauryl Sulfate or LLS, Sodium Dodecyl Sulfate or SDS, or Sodium Decyl Sulfate), and alkali base (e.g., NaOH or KOH) at a preferred concentration (e.g., 0.1 % or less (w/v)), and does not contain alcohol (or at least does not contain a high concentration of alcohol).
  • salt generally 0.2 to 5 M salt, e.g., LiCI or NaCI
  • a preferred moderate concentration generally 0.01 to 0.5% w/v
  • an ionic detergent e.g., Lithium Lauryl Sulfate or LLS, Sodium Dodecyl Sulfate or SDS, or Sodium
  • the fixation solution also includes IdU or BrdU (e.g., at 10e-3 to 10e-8 M).
  • Step 3 Stringency treatment of Assay Surface to Lower NSB Add an effective composition and volume of stringency solution to the MPW or container and incubate at an effective temperature for an effective time and then remove the stringency solution from the MPW or container. Treatment of the assay surface with stringency solution generally results in less assay label NSB, and greater +B cell sample assay signals for the assay.
  • the assay surface is further briefly washed one to two times with an effective volume of stringency solution to remove unincorporated BrdU and the stringency solution is immediately removed from the assay surface and while optionally but preferably the assay surface is then washed with Wash buffer 1.
  • the stringency step solution contains an acid (a preferred acid is HCI at a concentration from 0.01 to 0.1 M) or an acidic buffer (a preferred buffer is a glycine-HCI buffer at a concentration of 0.02 to 0.1 M), high salt (a preferred salt is LiCI at a concentration of 0.5 to 5M), and a protein (preferred proteins include pepsin or trypsin at 0.05 to 1 mg/ml).
  • the stringency solution also includes IdU or BrdU (e.g., at 10e-3 to 10e-8 M).
  • Step 4 Contact Assay Label Conjugate (in this case the label is antiBrdU antibody-Alkaline Phosphatase conjugate) with the Immobilized cell DNA Dilute an effective concentration of Antibody-Enzyme conjugate label into Wash Solution 2 and add an effective amount of this dilution to the MPW or container and incubate at an effective temperature (generally done at 15 to 30 " C) for an effective time (generally done for 10 minutes but shorter and longer times can be effective) to label the assay surface immobilized BrdU cell DNA.
  • the conjugate labeling solution also includes IdU or BrdU (e.g., at 10e-3 to 10e-8 M). Then remove the label conjugate solution from the well or container. Effective concentrations of Antibody-Enzyme conjugate and effective amouints of the dilution can be determined empirically to provide desirable assay results, e.g., optimal or near optimal.
  • a high concentration of protein proteease free Bovine Serum Albumin is preferred at a concentration of 0.3% to 4% (w/v)
  • a high salt concentration a preferred salt is LiCI or NaCI at a concentration
  • Wash Solution 2 also includes IdU or BrdU (e.g., at 10e-3 to 10e-8 M.
  • the conjugate dilution and reaction solution is designed to minimize the non-specific immobilization of the label conjugate to the assay surface during the conjugate reaction step, and to facilitate the removal of non- specifically immobilized assay label from the assay surface.
  • Step 5 Post Assay Labeling Wash Steps Add an effective composition and volume of post conjugate labeling wash solution (Wash Solution 2) to the MPW or other container and incubate from 0 to 10 minutes or more. Then remove the Wash Solution 2 from the MPW or other container. Do this wash step an effective number of times (generally 3 to 5 times).
  • Wash Solution 2 an effective composition and volume of post conjugate labeling wash solution
  • Improved Wash Solution 2 (WS 2) Reagent
  • protein proteease free Bovine Serum Albumin is preferred at a concentration of 0.3% to 4% (w/v)
  • a high salt concentration a preferred salt is LiCI or NaCI at a concentration of 0.2 to 5M
  • a buffer
  • Wash Solution 2 also includes IdU or BrdU (e.g., at 10e-3 to 10e-8 M).
  • the improved WS 2 is designed to minimize the non-specific immobilization of the label conjugate to the assay surface during the conjugate reaction step and to facilitate the removal from the assay surface of label conjugate which does become non-specifically immobilized, and to stabilize the assay immobilized alkaline phosphatase enzyme during the wash steps.
  • Step 6 Preparing the MPW or Container Assay Surface for the Enzyme Substrate Assay Signal Generation
  • the Wash Solution 1 used here as Preparation Solution, also includes IdU or BrdU (e.g., at 10e-3 to 10e-8 M). Do this wash step twice.
  • Step 7 Determine the Assay Signal produced by the MPW or other Container Put the MPW or other container in a signal measuring device and determine the assay signal for each MPW or other container associated with experimental cells which were exposed to BrdU, or control cells which were not exposed to BrdU, as well as any other control aspect.
  • the preferred time of signal measurement is 1 second. Utilize the results to determine the (Total Assay Signal Associated with the +BrdU cells/Noise ratio i.e. the assay S/N ratio) for each MPW or other container associated with cells which were exposed to BrdU.
  • Step 8 Determine the +BrdU Cell Lower Limit Of Detection (+BrdU Cell LLOD) for the Assay
  • Z Z
  • the +BrdU cell LLOD [(Z - 1 ) ⁇ (assay S/N -1 )] (the number of +BrdU cells assayed in the MPW or other container).
  • Aspect Two represents a more flexible format where the analyzed cells are grown and exposed to BrdU in one MPW or container and then transferred to another MPW or container to be assayed by the BrdU cell DNA detection assay.
  • Nalgene Delta Nunc or Corning Co-Star tissue culture white microtiter plates are currently preferred for these assays.
  • BrdU cell LLOD in the presence of few or no non-dividing cells ⁇ « 1 -5 +BrdU cells BrdU cell LLOD in the presence of 10e2 to 10e5 non dividing cells ⁇ 1 -5 +BrdU cells
  • the cells of interest can be BrdU labeled either during, before, or after, the experimental treatment of the cells.
  • the target cells can be BrdU labeled before or after treatment of the cells with a potential or actual cytotoxic condition, e.g., chemical, drug, or cells.
  • Step 1 Preparing the Cells for the Assay Fixation and Cell DNA Immobilization to the MPW Assay Surface
  • Step 2 Fixation of the Cells and Immobilization of the Cell DNA on the Assay Surface
  • fixation and immobilization reagent 0.8M LiCI, 0.045M NaOH, 0.2% Lithium Lauryl Sulfate
  • Step 3 Stringency treatment of Assay Surface to Lower NSB Add 400 ⁇ l of stringency solution (3m LiCI, 0.05 M Glycine, 0.02m HCI, 1 mg/ml of Trypsin) to the MPW and incubate for 10 minutes at room temperature and then decant to remove the stringency solution from the MPW.
  • stringency solution 3m LiCI, 0.05 M Glycine, 0.02m HCI, 1 mg/ml of Trypsin
  • the assay surface is washed and decanted and blotted once or twice more to remove unincorporated BrdU.
  • Step 4 Contact Assay Label (in this case the label is antiBrdU antibody- Alkaline Phosphatase conjugate) with the Immobilized Cell DNA Note that it is important that essentially all free BrdU be eliminated from the assay container before this step. This means that an adequate number of washes must be done before this step in order to dilute out and remove the free BrdU. One of skill in the art will know how to determine the adequate number of wash steps to accomplish this. However if an antildU antibody is used in the antibody-label conjugate to detect the immobilized BrdU analyte this is not necessary or at least can be less important.
  • the effective concentration of total enzyme-antibody conjugate protein to be used is determined empirically for each different preparation of enzyme-conjugate. For the lot used in the current example of enzyme-antibody conjugate, the effective amount is 0.05 ⁇ g per ml. Then decant and blot to remove the label conjugate solution from the well or container.
  • Step 6 Preparing the MPW Assay Surface for the Enzyme Substrate Assay Signal Generation
  • Wash Solution 1 400 ⁇ l of Wash Solution 1 to the MPW then immediately decant and blot to remove it from the MPW. Do this wash and decanting and blotting step twice.
  • 50 ⁇ l of the enzyme substrate CDP- Star 50 ⁇ l of the enzyme substrate CDP- Star to each MPW and incubate at room temperature for 10 to 30 minutes. Alternatively the incubation can take place in the signal measuring device.
  • Step 7 Determine the Assay Signal produced in the MPW or other
  • Container Put the MPW in a signal measuring device and determine the assay signal for each MPW associated with experimental cells which were exposed to BrdU, or control cells which were not exposed to BrdU, as well as any other control MPW.
  • the preferred time of signal measurement is 1 second. Utilize the results to determine the (Total Assay Signal/Total Noise) Ratio (i.e. the assay S/N ratio) for each MPW associated with cells which were exposed to BrdU.
  • Step 8 Determine the +BrdU Cell Lower Limit Of Detection (+BrdU Cell LLOD) for the Assay
  • Z Z
  • the +BrdU cell LLOD [(Z - 1 ) ⁇ (assay S/N -1 )] (the number of +BrdU cells assayed in the MPW).
  • BrdU cell LLOD in the presence of few or no non-dividing cells ⁇ « 1 -5 +BrdU cells BrdU cell LLOD in the presence of 10e2 to 10e5 non dividing cells ⁇ 1 -5 +BrdU cells
  • the cells of interest can be BrdU labeled either during, before, or after, the experimental treatment of the cells.
  • the target cells can be BrdU labeled before or after treatment of the cells with a potential or actual cytotoxic condition, e.g., chemical, drug, or cells.
  • Step 1 Preparing the Cells for the Assay Fixation and Cell DNA Immobilization to the MPW Assay Surface Control and experimental cells of interest which are in suspension in cell culture media or blood or some other aqueous liquid solution or buffer do not need to be processed further before fixation. If desired such cells may be washed or fractionated and resuspended in fresh cell growth media or buffer to remove free unincorporated BrdU from the sample or for some other reason. Adhered cells may be resuspended and used in this protocol. Cells in solid masses may be separated into an individual cell suspension and used in this assay.
  • Option 1 Add 0.24 volume of suspended cells to a container or MPW (25- 50 ⁇ l is recommended for a MPW) and then add 0.76 volume of fixation and immobilization reagent (1.1 M LiCI, 0.06M NaOH, 0.27% Lithium Lauryl Sulfate). Alternatively, add an effective volume of suspended cells to a container or MPW (5-20 ⁇ l is recommended for a MPW) and then add 3-5 volumes of fixation and immobilization reagent (2M NaCI, 0.I mNaOH, 2% Sodium Decyl Sulfate, 0.001 M EDTA) to the MPW or container to lyse the cells and shake or vortex the MPW.
  • fixation and immobilization reagent 1.1 M LiCI, 0.06M NaOH, 0.27% Lithium Lauryl Sulfate.
  • an effective volume of suspended cells to a container or MPW (5-20 ⁇ l is recommended for a MPW) and then add 3-5 volumes of fixation and immobilization reagent (2M NaCI
  • Option 2 Add one volume of suspended cells and one to 3 volumes of fixation and immobilization solution to a glass or plastic container and thoroughly mix the solution. Then pipette 50-100 ⁇ l of the mixture into a MPW or container and incubate for 10 minutes at room temperature to facilitate the immobilization of the cell DNA. Then decant and blot to remove the fixation solution.
  • Step 3 Stringency treatment of Assay Surface to Lower NSB Add 400 ⁇ l of stringency solution (3m LiCI, 0.05 M Glycine, 0.02m HCI, 0.05 to 1 mg/ml of Trypsin) to the MPW or container and incubate for 10 minutes at room temperature and then decant and blot to remove the stringency solution from the MPW. Optionally, the well can then be washed with Wash solution 1. When the assayed cells are in suspension in a solution containing a high concentration of free BrdU the assay surface is washed and decanted and blotted enough times to remove unincorporated BrdU.
  • stringency solution 3m LiCI, 0.05 M Glycine, 0.02m HCI, 0.05 to 1 mg/ml of Trypsin
  • Step 6 Preparing the MPW Assay Surface for the Enzyme Substrate Assay Signal Generation
  • Wash Solution 1 400 ⁇ l of Wash Solution 1 to the MPW then immediately decant and blot to remove it from the MPW or other container. Do this wash and decanting and blotting step twice.
  • Step 7 Determine the Assay Signal produced by the MPW or other Container Put the MPW in a signal measuring device and determine the assay signal for each MPW associated with experimental cells which were exposed to BrdU, or control cells which were not exposed to BrdU, as well as any other control MPW. The preferred time of signal measurement is 1 second. Utilize the results to determine the assay S/N ratio) for each MPW or other container associated with cells which were exposed to BrdU.
  • Step 8 Determine the +BrdU Cell Lower Limit Of Detection (+BrdU Cell LLOD) for the Assay
  • Z Z
  • (the +BrdU cell LLOD) [(Z - 1 ) ⁇ (assay S/N -1 )] [the number of +BrdU cells assayed in the well or container).
  • CMCA antibody dependent cell mediated cytotoxicity assays
  • ADCMCA antibody dependent cell mediated cytotoxicity assays
  • CDFA cell DNA fragmentation assays
  • CCSA cell chemosensitivity assays
  • a variety of direct or indirect signal generating molecule types are used for these assays. These include but are not limited to nucleic acid bases or nucleosides or nucleotides which are DNA precursor molecules which are associated with: one of many different types of halogen (such as BromdeoxyUridine, IododeoxyUridine, and others); one of many different types of hapten or ligand or receptor molecules(such as Digoxigenin, biotin and others); one of many different types of radioactivity (such as tritium, carbon 14, phosphorous 32 or 33, and others); one of many different types of fluorescent molecules(such as fluorescein and others).
  • halogen such as BromdeoxyUridine, IododeoxyUridine, and others
  • hapten or ligand or receptor molecules such as Digoxigenin, biotin and others
  • radioactivity such as tritium, carbon 14, phosphorous 32 or 33, and others
  • fluorescent molecules such as fluorescein and others.
  • Proliferating cells produce newly synthesized cellular DNA as part of the cell proliferation process.
  • BrdU can be incorporated into the DNA of proliferating cells.
  • BrdU is present in the growth medium, then as the number of proliferating cells increases the amount of BrdU labeled cell DNA in the cell sample increases.
  • the detection of BrdU cell DNA in a cell sample indicates that the assayed cell sample contains proliferating cells.
  • Migrating cells can be isolated, detected and quantitated by using a CMA.
  • a CMA To perform a CMA a two chamber device is commonly used where the two chambers are separated by a cell permeable barrier that can be crossed by cells which can actively migrate. A population of cells is placed in a first chamber and the second chamber is monitored for the appearance of migrating cells. If the cells placed in the first chamber are labeled with BrdU then the cells which have migrated to the second chamber can be detected by detecting the BrdU cell DNA associated with the migrated cells.
  • cells which have not been labeled with BrdU may be placed in the first chamber, and cells which have migrated can be detected by labeling them with BrdU after the migration has taken place, and then detecting the migrated cells BrdU DNA.
  • the above-described preferred Aspect One and Aspect Two embodiment protocols, as well as many other invention improved embodiments can be used essentially unchanged for detecting the migration and/or proliferation of a wide variety of different cells, even when the ratio of migrating and/or dividing cells to non-migrating and/or non- dividing cells for the assayed cell sample is very small.
  • Many biological or chemical compounds either inhibit or stimulate cell DNA synthesis and the proliferation of cells.
  • BrdU is present in the cell growth media
  • the inhibition or stimulation of cell proliferation by a drug or chemical or biological compound can be detected by comparing the amount of BrdU cell DNA present in experimentally treated cells, e.g., drug or other chemical treated cells, relative to the amount of BrdU cell DNA present in a control sample of cells, where the control cell sample is identical to the experimentally treated cell sample, e.g., drug or other chemical or biological compound treated cell sample except for the absence of experimental condition, e.g., the drug, or other chemical or biological compound.
  • control and experimentally treated cells in the assay may be BrdU labeled during the experimental treatment period or at the end of the experimental treatment period.
  • Observed smaller experimental cell proliferation stimulation relative to the control cells may be due to either an increased rate of division of experimental cells relative to control cells, or a lower rate of loss of experimental cells by cell death relative to control cells, or both.
  • Natural cell death generally occurs by apoptosis or necrosis. In the process of natural cell death dying cells become porous and disintegrate, and the target cell DNA and other target cell debris is dispersed into the blood or culture media the target cells reside in.
  • a variety of factors can induce natural cell death in a cell. These factors include but are not limited to specific cells such as T cells and natural killer(NK) cells, specific antibodies and antigens, particular chemicals or biochemicals, and particular physical treatments (such as heat or cold or light).
  • the control and experimentally treated cells in the assay may be BrdU labeled during the experimental treatment period or at the end of the experimental treatment period. Assays to detect and monitor cell death are currently widely used and include Elisa-based chemiluminescent assays designed to detect BrdU cell DNA.
  • the occurrence of cell death in a cell population after a treatment can be detected by monitoring the amount of experimental cell sample DNA which is present in intact experimental sample cells, and the amount of experimental cell DNA which is extracellular and dispersed into the blood or culture medium of the experimental cell sample. This can be done by as follows.
  • the fraction of the experimental cell BrdU which is in the extracellular fraction at the end of the treatment due to cell death is then equal to 1 - [(the amount of BrdU cell DNA present in the intact experimental cells) ⁇ (the amount of BrdU cell DNA present in the intact control cells)]. Since the amount of cell BrdU DNA present in both the intact cells of the experimental and control cell samples at time zero of treatment is the same, then the greater the fraction of the target cell BrdU DNA which is in the experimental cell extracellular lysate fraction and the smaller the fraction of the target cell BrdU DNA which is in the experimental intact cell fraction, the greater the extent of cell death in the experimental cell sample.
  • This method of measuring cell death is herein termed the Intact Cell Comparison Method or the ICCM.
  • the occurrence of cell death in a cell population after a treatment can also be detected by using cells pre-labeled with BrdU as described above, and after the experimental treatment directly determining for the experimental cell sample the amount of experimental cell sample DNA which is associated with intact experimental sample cells, and the amount of experimental cell DNA which is extracellular and dispersed into the blood or culture medium of the experimental cell sample.
  • the fraction of experimental sample cells which have undergone cell death during the experimental treatment is equal to the ratio [(the amount of extracellular BrdU cell DNA present in the experimental sample) ⁇ (the amount of BrdU cell DNA associated with intact experimental cells)].
  • the occurrence of cell death and cytotoxic effects on treated cells can also be detected by monitoring the amount of cell DNA which is synthesized in intact experimental sample Target cells, relative to the amount of cell DNA synthesized in the control cells.
  • This can be done by: a) simultaneously labeling the experimental and control (untreated) Target cells with BrdU during the experimental treatment period, and then determining the amount of BrdU labeled cell DNA synthesized in the experimental Target cells relative to the amount of BrdU labeled cell DNA synthesized in the control Target cells; b) alternatively this can be done by first treating the experimental Target cells, then at or near the middle or end of the experimental treatment period, labeling the experimentally treated and control Target cells, and then determining the amount of BrdU labeled cell DNA synthesized in the experimental Target cells relative to the amount of BrdU labeled cell DNA synthesized in the control Target cells.
  • the BrdU labeling can be done in the presence of the treatment agent if desired.
  • the intact cells in both the experimental and control cell samples contain the same number of Target cells and the same amount of unlabeled cell DNA.
  • CDA 1 CDA 2
  • CDA 3 CDA 4
  • CDA 4 can be utilized in most cell death and cytotoxicity assay types, including those discussed below.
  • CMCA Cell Mediated Cytotoxicity Assay
  • ADCMCA Antibody Dependent Cell Mediated Cytotoxicity Assay
  • cytotoxic T cells cytotoxicT or cT cells
  • NK natural killer cells
  • the occurrence of cell death in a cell population after treating the target cells with cT or NK can be detected by determining whether the target cell DNA is extracellularly dispersed into the blood or culture medium, or present in intact cells, after the treatment. This can be done by utilizing one of the above described CDA Formats.
  • CCSA Cell ChemoSensitivitv Assay
  • Chemical treatment of mammalian and other cells often causes inhibition or stimulation of cell growth or cell death.
  • the occurrence of cell death in a cell population after treating the target cells with a chemical or drug can be detected by determining whether the chemical or drug treated target cell DNA becomes extracellular (i.e. dispersed into the blood or culture medium) or present in intact cells after the treatment.
  • the occurrence of cell death in a cell population after treating the target cells with a drug or chemical can be detected by determining whether after treatment the target cell DNA is extracellularly dispersed into the blood or culture medium which the cells reside in, or present in intact cells. This can be done by utilizing one of the above described CDA Formats.
  • CDFA Cell DNA Fragmentation Assay
  • Cell death by apoptosis results in the fragmentation of cellular DNA and the dispersal of the fragmented DNA into the extracellular medium the cells reside in. This DNA fragmentation can be detected by using either the above described CDA Format 1 or Format 2.
  • CDA Format 1 either the Aspect One or Aspect Two invention improved assay can be used, and it is expected that for the above described CDA Format 2 either the Aspect One or Aspect Two invention improved assay can be used to determine the extent of cell death due to drug or chemical treatment in an experimental sample.
  • the invention relates to methods for significantly decreasing the analyte LLODs for IAAs relative to the LLODs for currently available IAA assays, by using improved invention methods to reduce the NSB for the IAA.
  • improved practices of the invention to other IAAs is described below.
  • Histology or cytology is the study of the microscopic anatomy of cells and tissues and is performed by examining a thin preparation of fixed and immobilized tissue or cells under a microscope. The ability to visualize or differentially identify microscopic structures or the presence of particular analyte molecules in a cell is frequently enhanced through the use of histological stains which include nucleic acid probes and antibodies, and other specific binding entities. Histopatholoqy is the microscopic study of fixed and immobilized diseased tissue and cells. [00162] Histology Assays (HA) and Histopathology Assays (HPA) are widely used to detect and characterize a wide variety of different target analytes which are associated with fixed and immobilized cells or tissue.
  • HA Histology Assays
  • HPA Histopathology Assays
  • the target analyte may be a structure or component associated with the fixed and immobilized cells or tissue, or a molecule or macromolecule associated with the fixed and immobilized cell or tissue.
  • assays are often described as histochemistry assays (HCA). These include but are not limited to HCA for detecting cell associated target analytes such as nucleic acids, protein, carbohydrates, lipids, structures, organelles, viruses, bacteria, parasites, and other fixed and immobilized cell associated target analytes.
  • IHCA ImmunoHCA
  • Other common HCAs utilize nucleic acid probes directly or indirectly labeled with a signal generating molecule to detect specific nucleic acid analytes in fixed cells.
  • improved HA and HPA will be discussed in terms of IHCAs.
  • the invention improvement aspects of this discussion on the application of the invention to produce improved IHCAs are also generally applicable to other HAs and HPAs and regardless of the analyte of interest, alone or in combination.
  • An IHCA generally involves the following steps. i) Fix and immobilize the cell and the cell associated analyte of interest on a surface and then wash away the cell components which are not immobilized. ii) After analyte immobilization the analyte may be specifically labeled with label molecules which produce signal directly (such as e.g. a fluorescent or radioactive molecule) or label molecules which produce signal indirectly (such as e.g. an enzyme molecule which is part of a signal generating system which produces signal generating molecules from a substrate).
  • label molecules which produce signal directly such as e.g. a fluorescent or radioactive molecule
  • label molecules which produce signal indirectly such as e.g. an enzyme molecule which is part of a signal generating system which produces signal generating molecules from a substrate.
  • the label molecules used to label the analyte are in great excess over the analyte and these excess label molecules come directly into contact with the assay surface which is the fixed cell in which the immobilized analyte of interest resides.
  • the labeling step a significant fraction of the input label is immobilized non-specifically on the assay cell surface.
  • the labeling and/or immobilization step wash away all direct or indirect label which is not immobilized. It is generally difficult to remove non-specifically bound label from the capture surface.
  • the assay signal measured originates from label molecules specifically associated with immobilized analyte molecules and label molecules which are non- specifically bound to the assay cell surface.
  • Hydrophobic or hydrophilic binding of assay label occurs in virtually all IAA assays including HAs and HPAs and IHCAs. These assays are associated with a significant amount of NSB caused by hydrophobic or hydrophilic binding of assay label and therefore the practical analyte LLODs of current IHCAs are very significantly limited by NSB.
  • the disparity between the practical analyte LLOD achieved for an IHCA assay and the potential analyte LLOD of the IHCA possible if the assay NSB were eliminated or significantly reduced is generally large and is greatest for the assay labels which have the lowest label LLODs.
  • the invention thus also relates to methods for significantly decreasing the analyte LLODs for IHCAs relative to the currently available LLODs for currently available IHCA assays.
  • An important determinant of an IHCA analyte LLOD is the ratio for the assay of: [(the assay signal associated with a sample containing the analyte) ⁇ (the assay signal associated with a control sample which does not contain the analyte)]. This ratio is commonly known as the assay Signal/Noise ratio or Assay S/N ratio.
  • the invention relates to improved methods for significantly decreasing the hydrophobic and hydrophilic NSB of assay label for IAAs in general.
  • the invention further relates to the application of these improved methods for significantly decreasing the hydrophobic and hydrophilic NSB of assay label for IHCAs.
  • a very large fraction of the assay noise in currently available IHCAs is due to NSB.
  • the practice of the invention must reduce the assay associated Noise (i.e. the signal associated with the assay NSB) signal to a significantly greater degree than the analyte associated assay signal is reduced.
  • the practice of the invention reduces the IHCA NSB and thereby the assay analyte LLOD by the development and use of novel assay reagents, and modification of current assay procedure and/or stratagems.
  • Lowering the analyte LLOD for an IHCA effectively enhances the ability of the IHCA to differentially identify the presence of the analyte in a target cell and just as importantly to differentially visualize microscopic structures defined by the analyte in a target cell.
  • the assay NSB is caused by the non-specific hydrophobic and hydrophilic immobilization of a significant amount of assay label to the assay surface when the assay label is put in contact with the assay surface. Further, once the assay label is cell surface immobilized it is very difficult to efficiently remove the immobilized label from the surface during the wash steps.
  • Part of the strategy of the invention is to devise assay label reaction solutions which are significantly more effective at lowering the amount of assay label which is initially non-specifically hydrophobically and/or hydrophilically immobilized to the assay surface during the assay, as well as assay wash buffers which are significantly more effective at removing the non-specifically immobilized label from the assay surface after it has become immobilized.
  • the following practices can result in a significant improvement in IHCA NSB values and a significant decrease in the analyte LLOD for the IHCAs and HAs and HPAs in general.
  • the individual block co-polymer molecules and protein molecules used contain both hydrophobic and hydrophilic sites and both are effective at blocking the assay label from non-specifically binding to the assay surface.
  • WCA Whole Cell Assays aim to qualitatively and/or quantitatively detect and characterize individual target cell types and the biochemical constituents of the target cells by detecting one or more particular analytes in the target cells of interest.
  • WCA refers to an assay which assays fixed or unfixed cells.
  • One type of WCA relies on detecting and analyzing the assay signal from a single fixed or unfixed cell in order to detect the presence of the cell associated target analyte. Such an approach is used in flow cytometry assays and in assays which analyze individual cells which are immobilized on a surface.
  • Another type of WCA assay relies on the detection and analysis of the integrated assay signal from a population of cells to detect the analyte associated with the cells.
  • WCAs are currently used for detecting a variety of cell associated target analytes including but not limited to nucleic acids, protein, receptors, carbohydrates, lipids, structures, organelles, viruses, bacteria, parasites, and other fixed and immobilized cell associated target analytes.
  • Unfixed cells are suitable for assaying for cell surface analytes, but are usually not suitable for assaying for intracellular analytes.
  • Fixed cells can usually be used to assay for either intracellular or cell surface analytes. The choice of the type of WCA to use depends on the purpose of the assay and the cell system to be assayed.
  • WCA which utilizes antibodies directly or indirectly labeled with a signal generating molecule to detect one or more specific analyte associated with the cells of interest.
  • Other common WCAs utilize nucleic acid probes directly or indirectly labeled with a signal generating molecule to detect specific nucleic acid analytes in fixed cells. It is expected that one or more invention improvement aspect or combination of invention improvement aspects of this discussed here will be generally applicable to essentially all WCAs regardless of the analyte of interest.
  • a WCA generally involves the following steps. i) Prepare fixed or unfixed cells in suspension. It is important for the WCA that the sample cells be in a single cell state and not be clumped. ii) The cell associated analyte in the cell suspension is then specifically labeled with label molecules which produce signal directly (such as e.g. a fluorescent signal molecule) or indirectly (such as enzyme molecules). The amount of label molecule used to label the analyte is in great excess over the analyte, and the excess label molecules come directly into contact with the assay surface which is the cell internal or external surface which the immobilized target analyte of interest is associated with.
  • label molecules which produce signal directly (such as e.g. a fluorescent signal molecule) or indirectly (such as enzyme molecules).
  • the amount of label molecule used to label the analyte is in great excess over the analyte, and the excess label molecules come directly into contact with the assay surface which is the cell internal or external surface which the immobilized target ana
  • the assay cell surface For a flow cytometer WCA there is effectively just one assay surface, the assay cell surface.
  • the assay cell surface For the WCA where the integrated label signal from a population of cells is determined there are two assay surfaces which contain label NSB and which contribute to the integrated cell population NSB signal, the assay cell surface and the assay surface of the container the cells are assayed in. After the labeling and/or immobilization step wash away all label which is not immobilized. It is generally difficult to remove non-specifically bound label from a capture surface.
  • iii) Measure the direct or indirect signal from the label which remains immobilized on the cell surface after the assay wash step.
  • the assay signal measured originates from label molecules specifically associated with immobilized analyte molecules and label molecules which are non- specifically bound to the assay cell surface, and the surface of the cells in the assay.
  • the invention relates to methods for significantly decreasing the analyte LLODs for WCAs relative to the currently available LLODs for currently available WCAs.
  • An important determinant of a WCA analyte LLOD is the ratio for the assay of, [(the assay signal associated with a sample containing the analyte) ⁇ (the assay signal associated with a control sample which does not contain the analyte)]. This ratio is commonly known as the assay Signal/Noise ratio or Assay S/N ratio.
  • the invention further relates to methods for significantly decreasing for a WCA the assay analyte LLOD by significantly decreasing the assay NSB of the WCA.
  • the practice of the invention must reduce the assay associated Noise (i.e. the signal associated with the assay NSB) signal to a significantly greater degree than the analyte associated assay signal is reduced.
  • the practice of the invention reduces the WCA NSB and thereby the assay analyte LLOD by the development and use of novel assay reagents, and modification of current assay procedure and/or stratagems. Lowering the analyte LLOD for a WCA effectively enhances the ability of the WCA to differentially identify the presence of the analyte in a target cell.
  • the individual block co-polymer molecules and protein molecules used contain both hydrophobic and hydrophilic sites and both are effective at blocking the assay label from non-specifically binding to the assay surface and are effective at eliminating cell clumping.
  • An added significant advantage is that live or fixed cells can be assayed in the presence of high concentrations of block copolymers such as the Pluronic non-ionic detergents without affecting the viability or integrity of the cells.
  • Block copolymers such as the Pluronic non-ionic detergents
  • IAAs are currently used to detect a large number of different protein types. These include without limitation protein analyte assays based on Elisa, microdot, microfluidic, Western, and other formats.
  • the above described experimental process for using the basic invention strategy to produce invention improved IAAs can be used to produce invention improved protein analyte IAAs of all kinds which are improved in analyte LLOD relative to current IAAs.
  • the development of an ultrasensitive, low cost, easy to perform, assay technology for detecting and quantitating the presence of a wide variety of specific biomarkers on the surface of live or fixed cells would represent an important advance in diagnostic assays. The most developed technology to date is flow cytometry.
  • the ideal assay would need to be rapid, simple, specific, quantitative, ultrasensitive and broadly applicable to a wide variety of cell surface markers, including proteins, polysaccharides, and any other cell surface moiety to which an antibody or other specific binding agent can specifically bind. Furthermore the capability of the assay to be performed in either sophisticated high technology laboratories or in resource limited laboratories would make it attractive as a commercially viable product worldwide.
  • the present invention provides specific, ultrasensitive diagnostic assays equal to or lower in sensitivity to flow cytometry and other currently used methods, but in simple to use ELISA formats that are low-cost and require minimal technical expertise to perform, so that the assay can be performed in either sophisticated high technology laboratories or in resource- limited laboratories in developing countries around the world.
  • chemilumenscent signal generation and detection systems e.g., detection systems that have been used to detect 5 enzyme molecules in 5 microliters in 20 minutes using a simple inexpensive luminometer
  • a detection system has been developed which can be applied to detect a wide variety of either protein or nucleic acid analytes.
  • An example is the ZivaTM Ultrasensitive Brdu Cell proliferation Kit (Jaden Biosciences, Inc.).
  • this kit has been able to detect 1 -4 proliferating cells in the presence of a background of 100,000 non- proliferating cells, a claim others in the field have not been able to approach, a level not even approached by other available methods.
  • the present invention as applied in the Ziva technology has been utilized for the ultrasensitive detection of cells containing surface biomarkers as well.
  • the capability to detect a low number of live or fixed cells which have one or more specific biomarkers present on the cell's surface, even in the presence of a large number of cells which do not have the specific biomarker targets present on their cell surface has great practical utility, i) It greatly simplifies the assay sample collection step because it is necessary to obtain only a small number of research or medical biological sample cells for.
  • a skin prick blood sample from a human, mouse, or other animal can provide enough sample cells for use in the present ultrasensitive assays, ii) It also simplifies the assay steps and greatly reduces the amount of reagents required to run a test iii) It is substantially lowers the cost relative to current technology.
  • the reagents utilized as discussed above are specially designed for a WCA to: a) preserve the integrity of the unfixed cells and the cell surface bound analyte(s) of interest, during the assay procedure; b) minimize the non-specific assay signal and maximize the analyte specific assay signal associated with the fixed and unfixed cell in order to facilitate ultrasensitive assay detection.
  • the invention improved WCA minimizes: a) the signal associated with the non-specific binding of assay signal generating molecules to cell and assay container surfaces; b) endogenous cell associated non-specific assay signal (i.e. cell associated assay signal which is not related to the presence of the analyte of interest such as assay signal caused by endogenous cell enzymes.
  • the signal generation system used for the Ziva assay and also used for initial versions of cell surface marker assays employs Calf Intestine Alkaline Phosphatase (AP) enzyme and a dioxetane substrate.
  • the assay was designed so that the number of cells represented in each assay is statistically accurate to ⁇ ⁇ 10-20%. ⁇ Note that the Ziva assay RLU/cell signals obtained (7000—11 ,000 RLU/cell) are quite large and can be made much larger, and that the Negative sample background signal is very low.
  • the present invention accordingly provides ultrasensitive assays to detect cell surface markers on the membranes of either or both of fixed and unfixed cells.
  • the present invention also concerns and applies to high sensitivity detection of polypeptide cell surface markers, including CD3, CD4, CD8, ROR1 , among others.
  • detection sensitivities of about 2 x e-3 (one target cell in 500), e.g., 10e-3, 5 x 10e-4, 2 x 10e-4, 5 x 10e-5, 2 x 10e-5, or 10e-5 or better are achieved, or the detection sensitivity is in a range between any two of the specified values.
  • sample sizes of about 10O ⁇ l, 50 ⁇ l, 20 ⁇ l, 10 ⁇ l, or even 5 ⁇ l of blood or other biological sample fluid are sufficient to conduct the assay.
  • Assays such as the cell surface marker assays described herein may be utilized in a number of different ways, e.g., in a number of different associated methods beyond simple quantitation of marker. These include, for example, a method for determining proliferation of a sub-population or sub-group of cells, where the sub-population or sub-group bears a cell marker, e.g., a cell surface marker, rendering the sub-population or sub-group distinct from other cells in the original population or group.
  • the high sensitivity provided by the present methods allows the proliferation of the sub-population or sub-group to be assayed over time and/or over different conditions to determine the increase or decrease in cell number using the cell marker as analyte.
  • the assay signal level is indicative of the number of cells in the sample volume.
  • the different conditions may, for example, include exposure to selected molecules such as potential therapeutic molecules or known or potential growth stimulators.
  • a method for determining the number or relative number of copies of a particular cell marker(s), e.g., cell surface marker(s), per cell.
  • the number of copies of the selected marker can be assayed using the present methods over time and/or over a set of different conditions.
  • Such different conditions may, for example, include exposure to selected molecules and/or other conditions which may induce or inhibit expression of the selected cell marker.
  • a number of different signal generation techniques may be used in the present assays. While advantageous assays may be provided using alkaline phosphatase as the signal generation moiety in a binding agent-signal generation moiety (e.g, Ab-enzyme conjugate), a variety of other labels may also be used, e.g., horseradish peroxidase.
  • alkaline phosphatase e.g., Ab-enzyme conjugate
  • other labels e.g., horseradish peroxidase.
  • Target Surface Cell Marker Human T Cell Receptor (TCR) CD3 present on the external cell surface of in vitro cultured Jurkat human T cells.
  • Target Cells 1 ) Human in vitro cultured Jurkat T cell line JBI-JK 1 (+) cells which have approximately 4000 CD3 T Cell Receptor (TCR) molecules per cell on the surface of an average JK(+) cell. 2)Human White Blood Cells (WBC) from human whole blood.
  • TCR T Cell Receptor
  • Control Cells 1 ) Human in vitro cultured Jurkat T cell line JBI-JK 1 (-) cells which do not possess detectable human CD3 TCR molecules.
  • the JK 1 (-) cell line was derived from the JK 1 (+) cell line;
  • Target Detection Antibodies 3 different Anti CD3 Monoclonal AntiBody (MAb) clones
  • Control MAbs for Monitoring Non-Specific Binding to Cell and Assay Surfaces Anti BrdU MAb
  • Target Detection MAB-AP Conjugate CD3 MAb-Alkaline Phosphatase (AP) conjugate
  • Control MAb-AP Conjugate - for Monitoring Non-Specific Binding to Cell and Assay Surfaces Anti BrdU MAb-AP conjugate
  • Luminometer Instrument for measuring chemiluminescence: 1 )
  • Assay design, development and optimization of ideal signal to noise ratios involves the selection of the appropriate monoclonal antibody, antibody conjugation, optimization of reagent formulations, substrates, methodologies and assay parameters to either reduce or enhance the following assay development parameters.
  • Total Assay Signal Detection Parameter Total Assay Signal (TAS) was measured in terms of Luminescent Counts/sec (LC/s)
  • T-NSAS Total Non-Specific Assay Signal
  • T-NSAS Non-Specific Assay Signal
  • NSAS Non-Specific Assay Signal
  • the various sources of NSAS include the non-specific binding of signal generating detection MAb-AP conjugate molecules to cell and assay container surfaces and the generation of non-conjugate specific chemiluminescent signal in the assay by enzyme (e.g. AP) endogenous to the sample being assayed, as well as other factors.
  • enzyme e.g. AP
  • the Target cell detection parameter was measured in terms of the number of LC/s observed per Target Cell or LC/TC.
  • the assay Target cell LC/TC value is equal to [(the TAS) - (the T-NSAS)I ⁇ [the number of target cells in the assay] for the assay.
  • CD3 Receptor Target Receptor Molecule or TRM
  • the TRM detection parameter was measured in terms of the number of LC/s observed per Target Receptor Molecule or LC/TRM. Determining this parameter requires knowing the average number of receptors per CD3 positive cell. 5.
  • Signal Generating Enzyme Molecule Detection Parameter The
  • Enzyme molecule detection parameter was measured in terms of the number of LC generated by one Enzyme molecule in a standard time period under defined reactions conditions.
  • the MAB-Enzyme molecule detection parameter was measured in terms of the number of LC generated by one Enzyme molecule in the MAB-Enzyme conjugate preparation in a standard time period under defined reactions conditions.
  • the CAM complex stability is measured in terms of the stability over time of one or more concentrations of CAM complex.
  • MAB-Enzyme conjugate Specificity Parameter The CAM Specificity parameter is measured in terms of the specificity of detection of the desired analyte by the CAM in the assay of interest.
  • NTC Non- Target Cell
  • Non-Target Cell or NTC Non-Specific Assay Signal (NSAS) Detection Parameter Which Specifically Reflects the NSAS Associated Only With The NTCs.:This parameter contributes to the T-NSAS of the assay. This parameter measures only the portion of the T-NSAS which is associated with the Non-Target cells in the assay 11.
  • E-NSAS ENDOGENOUS Non-Specific Assay Signal
  • This parameter contributes to the T-NSAS of the assay.
  • This Cell E- NSAS detection parameter is measured in terms of the E-NSAS per cell in the assay or LC/C and is equal to [(the E-NSAS) ⁇ (the number of cells present in the assay)].
  • the cell population for an assay can be comprised of one or more of the following cell types, T cells, B cells, RBCs, other blood cells, or other cell types.
  • Non-enzymatically generated Non-Specific Assay Signal From the Chemiluminescent Substrate: This parameter contributes to the T-NSAS of the assay and can be measured in terms of (the number of LC/ volume of substrate utilized for an assay), or in terms of [(LC signal due to the non-enzymatic generation of the signal) ⁇ (number of NTCs in the assay)] i.e. (the number of LC/NTC).
  • NSAS Associated With Any of the Basic Components or Reagents of the Assay (Includes But is Not Limited to, the Cell Growth Medium and Its Components, the Assay Wash and Processing Reagents and Their Components, the Unprocessed Biological Sample, and Any Other Pertinent Assay Component): This parameter contributes to the T-NSAS of the assay. This source of NSAS contributes to the T-NSAS of the assay is measured in terms of total LCs per assay.
  • AWS Assay Wash Solution
  • MAB-AB conjugate + cell CD3 T Cell Receptor reaction solution and the Cell Elisa assay wash solution (here termed the assay wash solution or AWS).
  • AWS Assay Wash Solution
  • This JBI proprietary AWS solution is designed to stabilize intact cells, to promote and stabilize the specific interaction of the CD 3 MAB-AP conjugate with cell surface CD3 receptors, to minimize the non-specific binding of the MAB-AP conjugate to assay surfaces, and to facilitate the mechanical aspects of the assay wash and decant steps.
  • JK(+CD3 cells which possess around 3000-4000 CD3 TCR molecules per cell on average) and JK(-CD3 cells which possess no detectable CD3 TCR molecules) cells were separately assayed as described above and the chemiluminescent assay signal from an aliquot of each sample was measured in a JBI Insight microtiter plate luminometer.
  • This luminometer is specially designed to maximize the linear dynamic range of photon detection range (>10e8) and greatly minimize the luminometer background (0 to 2 LC/sec) and the effect of light activated autoluminescence associated with white microter plates.
  • Such autoluminescence can be very large but is typically 500 to 2000 LC/sec right after loading the plate.
  • the autoluminescent signal decreases with time but can still be significant after 20 min or so. After 5 min or so the autoluminescent signal is near 1 LC/sec in the Jaden Instrument.
  • the +CD3 cell Total Assay Signal (TS) 2.9 x 10e6 LC/sec ⁇ 1032 LC per Target +CD3 Cell b)
  • the aliquot of -CD3 cells contained 2800 -CD3 cells The measured signal for 2800 -CD3 cells
  • the -CD3 cell Total Assay Signal (TS) 4844 LC/sec ⁇ 1.7 LC per -CD3 cell c) Lower Limit of Detection (LLOD) of +CD3 cells by the CD3 POP assay, assuming that a 2/1 Assay Signal/Assay Noise ratio is statistically significant.
  • the present invention has demonstrated an exemplary assay with the capability of detecting less than 10 +CD3 cells in about one hour.
  • This exemplary CD3 intact cell surface biomarker detection assay can undergo further refining or optimizing. Both the present configuration and such refinements can be applied to assays for other cell surface markers.
  • From the CD3 PROP assay data one can infer that if one +CD3 cells were mixed with 560 -CD3 cells and assayed, a 2/1 S/N ratio would result. It can then be said that the assay is capable of detecting 1 +CD3 cell in the presence of about 560 -CD3 cells, a detection sensitivity of -1.7 x 1 Oe-3.
  • the present methods also concern CD4 detection.
  • the invention as described above for CD3 can also be applied to CD4 (as well as CD8 and others), using the corresponding antibody or other specific binding agent in the conjugate.
  • the World Health Organization recommends that CD4 cell counts be used as a measure of immune function and in meeting set criteria to determine initiation of antiretroviral therapy (ART) in people infected with HIV.
  • ART antiretroviral therapy
  • flow cytometry the most commonly used method to determine CD4 cell counts is flow cytometry, bit it has been noted that flow cytometry with its multiple labels constitutes overkill for this application It is estimated that each flow cytometry test costs between $15 to $30 to perform, which in most cases is more than the monthly salary of those needing the test. Governments are forced to supplement testing costs adding to the overall financial burden of countries the least able to afford it.
  • flow cytometery has to be conducted in sophisticated laboratory facilities that have highly trained technicians to perform the assay.
  • the analysis typically requires 1 - 2 ml_s of blood sample collected by a phlebotomist. Due to the scarcity of sophisticated laboratories in developing countries, specimens are often shipped for long distances. Performing venipuncture in rural settings or obtaining that amount of blood from sick infants is also problematic.
  • the present methods and kits also include detection of ROR1 (embryonic tyrosine kinase receptor), e.g., in connection with treatment monitoring for B-cell chronic lymphocytic leukemia (CLL).
  • CLL chronic Lymphocytic Leukemia
  • CLL is a clinically heterogeneous blood cancer; the most common leukemia of the 4 major types of leukemia diagnosed in adults.
  • CLL itself can be categorized into three lymphocyte types, B, T, and NK cells, with B-CLL being the most common.
  • B-CLL chronic lymphocytic leukemia
  • CLL chronic myeloma
  • the disease progression of CLL generally takes two forms, slow and rapid progression.
  • the proliferation of the B-cells proceeds at a slower rate with a typical average survival rate of >10 years (ref Baskar).
  • the slower form of CLL treatment may not be necessary for years.
  • the survival rate for the CLL aggressive form is an average of 2 years.
  • the detection lower limit of B-CLL cells with: a) the standard two color FACS method is -1 % of the total leucocytes (Rawstron).i.e. detect ⁇ 1 B-CLL cell in the presence of 100 normal blood WBCs or 7800 B-CLL cells in 780,000 normal blood WBCs; (b) the standard 4 color FACS methods for B-CLL cell is ⁇ 1 B-CLL cell in -333 normal blood WBCs (Rawstron) or -2400 B-CLL cells per 780,000 normal WBCs.
  • the most sensitive flow cytometry method is a more sensitive multiple color method which detects ⁇ 1 BCLL cell in -10,000 other blood WBCs or -78 B-CLL cells in 780,000 normal WBCs.
  • the improved MRD Flow method detection sensitivity was made possible by the use of a more specific set of BCLL detection antibodies ( Kay 2008) (Bottcher 2009).
  • the most sensitive BCLL cell detection method is the allele specific oligonucleotide (ASO) primer PCR test which is about 10 fold more sensitive than the MRD Flow method, but is not routinely used in clinical practice(Kay 2008) (Bottcher 2009).
  • ASO allele specific oligonucleotide
  • MRD negative Minimum Residual Disease
  • the DNA from 5- 10 x 10e6 patient leucocytes must be used in the PCR reaction in order to ensure that -50 to 100 starting copies of the BCLL cell associated gene or genes will be present in the PCR reaction mix.
  • the ASO PCR method is further complicated by mutations in the immunoglobulin-H (IGH) gene of CLL patients(RAWSTRON 2001 ). Clearly the inability to detect a specific marker in a small numbers of cells remains a significant challenge to observing the initial emergence of reoccurrence or onset of disease.
  • ROR1 is an embryonic tyrosine kinase receptor involved in organogenesis. Monoclonal antibodies specific for ROR1 has been produced and is now commercially available. It has been reported that there are approximately 4,000-7,000 ROR1 molecules expressed on the surface of malignant B cells from CLL patients (Baskar 2008). Jurkat CD3+ cells have -3000 CD3 receptor molecules per cell.
  • ROR1 The discovery of ROR1 provided an opportunity the apply the present ultrasensitive detection technology to B-CLL diagnosis to provide an ultrasensitive assay to detect and quantitate ROR1 , for the detection of B-CLL cells in human blood using both fixed and unfixed cells. While further refinements are possible, the assay substantially as described for CD3 can be applied to ROR1 , of course using antiRORI antibody-signal moiety conjugate. Once again, because relatively few cells are necessary for analysis using the present technology, small blood volumes such as those provided by finger prick or heel prick techniquescan be used.
  • IAAs are currently used to detect DNAs and/or RNAs from a large number of different sources. These include without limitation nucleic acid analyte assays based on Elisa, microdot, microarray, microfluidic, Northern, and other formats. It is expected that the above described experimental process for using the basic invention strategy to produce invention improved IAAs can be used to produce invention improved nucleic acid analyte IAAs of all kinds which are improved in analyte LLOD relative to current IAAs. E. Application of the Invention to Different Direct and Indirect Labeling Formats
  • a wide variety of direct or indirect labels are utilized in current IAAs for the purpose of detecting the presence of the analyte of interest in the assay. These include without limitation: indirect labels such as enzymes, biotin, or streptavidin, other ligands or receptors or haptens, and others; and direct labels such as radioactive isotopes or molecules or conjugates bearing such radioactive isotopes, fluorescent, nano-or micro-particles, colorimetric or fluorescent or chemiluminescent enzyme substrates, and others. It is expected that the above described experimental process for using the basic invention strategy to produce invention improved IAAs can be used to produce invention improved analyte IAAs of all kinds which use any one or more of these direct or indirect labels.
  • the present assay improvements are applicable to a very broad range of protein, nucleic acid, carbohydrate, lipid, or chemical analytes. Some examples are pointed out below, but this list should be recognized as presenting only examples, and not as limiting in any manner. Assays for the indicated analytes will, in many cases, be assays of types mentioned above.
  • EXAMPLE 1 Determination of the LLOD of Proliferating Mouse P815 Cells As Measured by the Standard Tritiated Thymidine Method and an Aspect One Invention Improved IAA Method
  • EXAMPLE 2 Determination of the LLOD of Proliferating Mouse P815 Cells As Measured by the Standard Tritiated Thymidine Method and an Aspect Two Invention Improved IAA Method
  • Mouse P815 cells were grown in one container for about 18 hours under standard culture conditions in 200 ⁇ l of RP-10 cell culture media: a) in the presence of 10e-5 M BrdU, or: b) in the presence of a standard amount of tritiated thymidine, or: c) in the absence of either BrdU or tritiated thymidine.
  • EXAMPLE 3 Determination of the LLOD of Proliferating Mouse P815 Cells In the Presence of Large Numbers of Non-Proliferating Mouse Cells an Aspect Two Invention Improved IAA Method
  • Mouse P815 cells were grown in one container for about 18 hours under standard culture conditions in 200 ⁇ l of RP-10 cell culture media: a) in the presence of 10e-5 M BrdU, or: b) in the absence of BrdU.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention porte sur des analyses d'analytes immobilisés améliorées, qui utilisent des procédés pour la réduction de liaison non spécifique. Les analyses améliorées apportent des améliorations significatives à la sensibilité, et, souvent, à la vitesse d'analyse.
PCT/US2009/066664 2008-12-03 2009-12-03 Analyses d'analytes immobilisés WO2010065797A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US11972008P 2008-12-03 2008-12-03
US61/119,720 2008-12-03
US23814309P 2009-08-29 2009-08-29
US61/238,143 2009-08-29

Publications (2)

Publication Number Publication Date
WO2010065797A2 true WO2010065797A2 (fr) 2010-06-10
WO2010065797A3 WO2010065797A3 (fr) 2016-04-21

Family

ID=42233876

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/066664 WO2010065797A2 (fr) 2008-12-03 2009-12-03 Analyses d'analytes immobilisés

Country Status (1)

Country Link
WO (1) WO2010065797A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140356885A1 (en) * 2013-05-30 2014-12-04 Siemens Healthcare Diagnostics Inc. Reducing Non-Specifically Bound Molecules on Supports
WO2010065797A3 (fr) * 2008-12-03 2016-04-21 Kohne David E Analyses d'analytes immobilisés

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2459893C (fr) * 2001-09-10 2014-01-21 Meso Scale Technologies, Llc Procedes et appareils permettant de realiser de multiples mesures sur un echantillon
US7175992B2 (en) * 2002-04-10 2007-02-13 Response Biomedical Corporation Sensitive immunochromatographic assay
US8592219B2 (en) * 2005-01-17 2013-11-26 Gyros Patent Ab Protecting agent
US7056473B2 (en) * 2004-04-29 2006-06-06 Response Biomedical Corp. Method and apparatus of quantitative assays
US20070048747A1 (en) * 2005-09-01 2007-03-01 Leslie Thomas M Methods for assaying analytes
WO2010065797A2 (fr) * 2008-12-03 2010-06-10 Kohne David E Analyses d'analytes immobilisés

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010065797A3 (fr) * 2008-12-03 2016-04-21 Kohne David E Analyses d'analytes immobilisés
US20140356885A1 (en) * 2013-05-30 2014-12-04 Siemens Healthcare Diagnostics Inc. Reducing Non-Specifically Bound Molecules on Supports

Also Published As

Publication number Publication date
WO2010065797A3 (fr) 2016-04-21

Similar Documents

Publication Publication Date Title
Corkum et al. Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient
Arrigucci et al. FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry
RU2666989C2 (ru) Мультиплексные анализы на основе аптамеров
JP4151986B2 (ja) リンパ球機能の測定方法
Bosak et al. Aptamer–gold nanoparticle conjugates for the colorimetric detection of arboviruses and vector mosquito species
US20100248257A1 (en) Compiled Methods for Analysing and Sorting Samples
US20090081689A1 (en) Reagents and methods to enrich rare cells from body fluids
US20170322204A1 (en) Simultaneous detection of biomolecules in biological entities
JP2010523995A (ja) 血液型抗体スクリーニング
JPH08511340A (ja) 細胞決定用イムノアッセイ
Lehmann et al. LEGENDplex™: bead-assisted multiplex cytokine profiling by flow cytometry
US20210405033A1 (en) Analyte detection and methods therefor
CN106841603B (zh) 一种利用血糖仪高灵敏定量检测黄曲霉毒素b1的方法
WO2015109020A1 (fr) Utilisation d'agents d'acides nucléiques pour détection et quantification numériques ultrasensibles de molécules cibles
JP6979408B2 (ja) 標的検体の細胞内局在
Alsohaimi Analytical detection methods for diagnosis of COVID-19: developed methods and their performance
US20200166528A1 (en) Methods for Improving Assays of Biological Samples
CN104726606A (zh) 一种pcr酶联双杂交法检测病原微生物检测方法
US20160258938A1 (en) Method of detecting an analyte in a sample
JP2018506020A (ja) 核酸増幅と組合せたイムノアッセイによる固体支持体上の被検体検出
WO2010065797A2 (fr) Analyses d'analytes immobilisés
US20230160887A1 (en) Flow Cytometry System and Methods for the Diagnosis of Infectious Disease
WO2008013975A2 (fr) Procédés et nécessaire de mesure de la fonction lymphocytaire
US20040197836A1 (en) Measurement of F-actin in whole blood cellular subsets
CN115786349B (zh) 一种用于外周血中杀伤性t淋巴细胞无痕分选的核酸适体、互补序列及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09831157

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09831157

Country of ref document: EP

Kind code of ref document: A2