Method and apparatus for increasing the dynamic range and accuracy of binding assays

Abstract

This invention relates to methods for increasing the dynamic range and accuracy of assays in which the presence, absence, activity or concentration of a target analyte is assayed by the emission or quenching of a light signal, or by a change (i.e., an evolution or loss) of a light signal in two or more time intervals. In preferred embodiments multiple digitized images are captured at varying times, and the images analyzed to identify captured images within the dynamic range of the assay. The invention further relates to apparati capable of implementing such methods.

1 . An apparatus for enhancing the dynamic range of an assay of the presence, absence, activity or concentration of two or more target analytes in one or more samples, wherein the presence, absence, activity or concentration of said target analytes is assayed by the emission or quenching of a light signal, said apparatus comprising: 
 (A) one or more containers for receiving a portion of said one or more samples, said containers additionally containing assay reagents comprising a compound that, in response to the presence of a target analyte causes a detectable light signal; and    (B) a computer system comprising a CCD camera detector, said computer system being specially adapted to detect said light signal and generate data corresponding to said detected signal; said computer system additionally processing a capability for comparing said generated data with data corresponding to the light signal generated by a known concentration of said target analyte in a known dynamic range of said assay and report the presence, absence, activity or concentration of said target analyte; wherein said computer system causes said CCD camera detector to independently detect sufficient light signal for each of said target analytes to ensure that said reported presence, absence, activity or concentration of each target analyte is determined using data corresponding to a light signal that is within said known dynamic range of said assay for that target analyte.    
     
     
         2 . An apparatus for enhancing the dynamic range of an assay of the presence, absence, activity or concentration of two or more target analytes in one or more samples, wherein the presence, absence, activity or concentration of said target analytes is assayed by the emission or quenching of a light signal, said apparatus comprising: 
 (A) one or more containers for receiving a portion of said one or more samples, said containers additionally containing assay reagents comprising a compound that, in response to the presence of a target analyte causes a detectable light signal; and    (B) a computer system comprising a CCD camera detector, said computer system being specially adapted to detect said light signal and generate data corresponding to said detected signal; said computer system additionally processing a capability for comparing said generated data with data corresponding to the light signal generated by a known concentration of said target analyte in a known dynamic range of said assay and report the presence, absence, activity or concentration of said target analyte; wherein said computer system causes said CCD camera detector to independently detect sufficient light signal for each of said target analytes to ensure that said reported presence, absence, activity or concentration of each target analyte is determined using data corresponding to a light signal that is within said known dynamic range of said assay for that target analyte; and wherein, for at least one of said target analytes, said computer system causes said CCD camera detector to detect light signal cumulatively until a total detected light signal is obtained that is within the known dynamic range of said assay for said target analyte; and wherein said total detected light signal is used to determine said presence, absence, activity or concentration of said target analyte.    
     
     
         3 . An apparatus for enhancing the dynamic range of an assay of the presence, absence, activity or concentration of two or more target analytes in one or more samples, wherein the presence, absence, activity or concentration of said target analytes is assayed by the emission or quenching of a light signal, said apparatus comprising: 
 (A) one or more containers for receiving a portion of said one or more samples, said containers additionally containing assay reagents comprising a compound that, in response to the presence of a target analyte causes a detectable light signal; and    (B) a computer system comprising a CCD camera detector, said computer system being specially adapted to detect said light signal and generate data corresponding to said detected signal; said computer system additionally processing a capability for comparing said generated data with data corresponding to the light signal generated by a known concentration of said target analyte in a known dynamic range of said assay and report the presence, absence, activity or concentration of said target analyte; wherein said computer system causes said CCD camera detector to independently detect sufficient light signal for each of said target analytes to ensure that said reported presence, absence, activity or concentration of each target analyte is determined using data corresponding to a light signal that is within said known dynamic range of said assay for that target analyte; and wherein, for at least one of said target analytes, said computer system causes said CCD camera detector to detect light signal discontinuously at more than one time interval so that a detected light signal is obtained that is within the known dynamic range of said assay for said target analyte;    and wherein said detected light signal within the known dynamic range of said assay for said target analyte is used to determine said presence, absence, activity or concentration of said target analyte.    
     
     
         4 . The apparatus of  claim 3 , wherein said computer system stores the cumulative change in said light signal in two or more of said time intervals.  
     
     
         5 . The apparatus of  claim 1 , wherein said apparatus simultaneously assays the presence, absence, activity or concentration of said more than one target analyte in the same sample.  
     
     
         6 . The apparatus of  claim 1 , wherein said apparatus sequentially assays the presence, absence, activity or concentration of said more than one target analyte in the same sample.  
     
     
         7 . The method of  claim 1 , wherein said step (C) is performed simultaneously for each target analyte being assayed.  
     
     
         8 . The method of  claim 1 , wherein said step (C) is performed sequentially for each target analyte being assayed.  
     
     
         9 . The apparatus of  claim 1 , wherein said one or more containers is a multi-well microtiter plate.  
     
     
         10 . The apparatus of  claim 1 , wherein said target analyte has an activity and wherein said computer system additionally calculates the rate of a target analyte activity in said sample.  
     
     
         11 . The apparatus of  claim 1 , wherein said target analyte is selected from the group consisting of an enzyme, a drug or metabolite, a co-factor, a receptor, a receptor ligand, a hormone, a cytokine, a blood factor, a virus, an antigen, a steroid, and an antibody.  
     
     
         12 . The apparatus of  claim 11 , wherein said assay assays the presence, absence, activity or concentration of an enzyme.  
     
     
         13 . The apparatus of  claim 12 , wherein said enzyme is selected from the group consisting of bone specific alkaline phosphatase, aldose reductase, myoglobin, and troponin I.  
     
     
         14 . The apparatus of  claim 11 , wherein said assay assays the presence, absence, activity or concentration of a drug or metabolite.  
     
     
         15 . The apparatus of  claim 14 , wherein said drug or metabolite is selected from the group consisting of: an anti-cancer drug, chemotherapeutic drug, anti-viral drug, non-steroidal anti-inflammatory drug (NSAID), steroidal anti-inflammatory drug, anti-fungal drug, detoxifying drug, analgesic, bronchodilator, anti-bacterial drug, antibiotic drugs, diuretic, digoxin, anti-metabolite, calcium channel blocker, drug for treatment of psoriasis, and a substance of abuse.  
     
     
         16 . The apparatus of  claim 11 , wherein said assay assays the presence, absence, activity or concentration of a co-factor.  
     
     
         17 . The apparatus of  claim 16 , wherein said co-factor is a vitamin, T 3 , or T 4 .  
     
     
         18 . The apparatus of  claim 11 , wherein said assay assays the presence, absence, activity or concentration of a cytokine.  
     
     
         19 . The apparatus of  claim 18 , wherein said cytokine is IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, TNFα, VEGF, GMCSF, FGFβ, INFγ, EGF, PDGF, MCSF, SCF, insulin, VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30, CD27, 4-1BB, LNGFR, OX40, TGFβR, or is a ligand of CCR1, CCR2α, β, CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2, or V28 receptor, or is a ligand of a receptor of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, or IL-13.  
     
     
         20 . The apparatus of  claim 11 , wherein said assay assays the presence, absence, activity or concentration of a receptor or receptor ligand.  
     
     
         21 . The apparatus of  claim 20 , wherein said receptor or receptor ligand is 4-1BB, Axl, BLR1, BLR2, CCR1, CCR2α, β, CCR3, CCR4, CCR5, CD27, CD30, CD4, CD4, CD40, CXCR1, CXCR2, CXCR3, CXCR4, EGFR, Eph, EPO receptor, Fas receptor, GCSFR, GHR, GMCSFRα, gp130, IFNgRα, IFNgRβ, IFNαR1, insulin-R, IL-1β, IL-2R β, IL-2Rγ chains, IL-4Rα, IL-3Rα, IL-5Rα, IL-6Rα, IL-7Rα, IL-9Rα, IL-10R, IL-11Rα, IL-12Rb1, IL-12Rb2, IL-13Rα, GMCSFRα, IL-3/IL-5/GM-CSF receptor common β-chain, LIFR β, LNGFR, MCSFR, Met, OBR, OSMRβ, OX40, PDGFR, PRL, Ron, SCFR, TPOR, TFR, TGFβR, TNFRI, TNFRII, TPOR, Trk, V28, VEGFR.  
     
     
         22 . The apparatus of  claim 11 , wherein said assay assays the presence, absence, activity or concentration of a hormone.  
     
     
         23 . The apparatus of  claim 22 , wherein said hormone is adrenaline, adrenocorticotropic hormone, testosterone, angiotensinogen, antidiuretic hormone, atrial-natriuretic peptide, calcitonin, calcitriol, cholecystokinin, chorionic gonadotropin, cortisol, dopamine, erythropoietin, estradiol, follicle-stimulating hormone, gastrin, glucagon, gonadotropin-releasing hormone, gorticotropin-releasing hormone, growth hormone, growth hormone-releasing hormone, insulin, insulin-like growth factor-1, leptin, luteinizing hormone, melatonin, aldosterone, neuropeptide Y, noradrenaline, oxytocin, parathyroid hormone, progesterone, prolactin, renin, secretin, somatostatin, theophylline, thiiodothyronine, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, or thyroxine.  
     
     
         24 . The apparatus of  claim 11 , wherein said assay assays a binding activity of an antigen or an antibody.  
     
     
         25 . The apparatus of  claim 24 , wherein said assay assays a binding activity of an antigen characteristic of  Chlamydia, Streptococcus pyogenes  Group A bacteria,  H. pylori , or  M. tuberculosi , hepatitis virus, rubella, CMV, HIV, FIV, or prostate specific antigen, or an antibody elicited in response to any of such antigens.  
     
     
         26 . The apparatus of  claim 11 , wherein said assay assays a binding activity of an autoimmune immunoglobulin, thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM immunoglobulin.  
     
     
         27 . The apparatus of  claim 11 , wherein said assay assays a binding activity of a tumor marker.  
     
     
         28 . The apparatus of  claim 1 , wherein said light signal is an evolution or loss of a fluorescent light signal.  
     
     
         29 . The apparatus of  claim 1 , wherein said light signal is an evolution or loss of a chemiluminescent light signal.  
     
     
         30 . The apparatus of  claim 1 , wherein said light signal is an evolution or loss of an ultraviolet light signal.  
     
     
         31 . The apparatus of  claim 1 , wherein said light signal is an evolution or loss of a visible wavelength light signal.  
     
     
         32 . A method for enhancing the dynamic range of an assay of the presence, absence, activity or concentration of two or more target analytes in one or more samples wherein the presence, absence, activity or concentration of said target analytes is assayed by the emission or quenching of a light signal, wherein said method comprises the steps: 
 (A) conducting an assay for the presence, absence, activity or concentration of each of said target analytes in said one or more samples wherein said assays each cause light signals to be emitted or quenched;    (B) employing a computer system comprising a CCD camera detector to detect said light signals, and to generate data corresponding to said detected signals; and    (C) causing said computer system to compare said generated data using data corresponding to the light signal generated by a known concentration of said target analyte in a known dynamic range of said assay and report the presence, absence, activity or concentration of said target analyte; wherein said computer system causes said CCD camera detector to independently detect sufficient light signal for each of said target analytes to ensure that said reported presence, absence, activity or concentration of each target analyte is determined using data corresponding to a light signal that is within said known dynamic range of said assay for that target analyte; and wherein, for at least one of said target analytes, said computer system causes said CCD camera detector to detect light signal cumulatively until a total detected light signal is obtained that is within the known dynamic range of said assay for said target analyte; and wherein said total detected light signal is used to determine said presence, absence, activity or concentration of said target analyte.    
     
     
         33 . A method for enhancing the dynamic range of an assay of the presence, absence, activity or concentration of two or more target analytes in one or more samples wherein the presence, absence, activity or concentration of said target analytes is assayed by the emission or quenching of a light signal, wherein said method comprises the steps: 
 (A) conducting an assay for the presence, absence, activity or concentration of each of said target analytes in said one or more samples wherein said assays each cause light signals to be emitted or quenched;    (B) employing a computer system comprising a CCD camera detector to detect said light signals, and to generate data corresponding to said detected signals; and    (C) causing said computer system to compare said generated data using data corresponding to the light signal generated by a known concentration of said target analyte in a known dynamic range of said assay and report the presence, absence, activity or concentration of said target analyte; wherein said computer system causes said CCD camera detector to independently detect sufficient light signal for each of said target analytes to ensure that said reported presence, absence, activity or concentration of each target analyte is determined using data corresponding to a light signal that is within said known dynamic range of said assay for that target analyte; and wherein, for at least one of said target analytes, said computer system causes said CCD camera detector to detect light signal discontinuously at more than one time interval so that a detected light signal is obtained that is within the known dynamic range of said assay for said target analyte; and wherein said detected light signal within the known dynamic range of said assay for said target analyte is used to determine said presence, absence, activity or concentration of said target analyte.    
     
     
         34 . The method of  claim 33 , wherein said computer system stores the cumulative change in said light signal in two or more of said time intervals.