Immobilised-Bead Immunomultiplex Cytokine Assay

Abstract

Embodiments of this invention include image-based systems and methods for detection of one or more biological molecules. A matrix has identifiable analyte-specific capture particle(s) suspended therein, not all being in the same detection plane. Analyte-specific binding moieties (e.g., antibodies) capture analytes. Analyte-specific detector molecules with conjugated detection moieties are then attached to the analyte-specific binding moiety forming particle complexes. A computer assisted, image-based detection system captures images of the particle complexes. Using different sets of analyte-specific detector molecules, each set having a characteristic identifiable feature; it is now possible to implement rapid multiplex assays of cytokines. These image-based systems can be used to aid in diagnosis of disease, evaluation of therapy for disease, or laboratory investigation.

What is claimed is: 
     
         1 . A non-flow based multi-analyte detection system not requiring remobilisation of capture particles, comprising:
 (1) a first type of analyte-specific capture particle immobilised on a detection surface, said capture particle having:
 (i) at least one capture particle identification parameter that distinguishes said first type of said analyte-specific capture particle from another type of capture particle, 
 (ii) said first type of analyte-specific capture molecule having a binding moiety specific for a first analyte, said binding moiety attached to said capture particle; 
   (2) a first type of analyte-specific detector molecules, each of said detector molecules having:
 (i) an analyte-specific binding moiety; and 
 (ii) a luminescent moiety having an identification parameter different from that of said first type of capture particle; 
   (3) a second type of analyte-specific capture particle immobilised said detection surface, said second type of capture particle having:
 (i) at least one capture particle identification parameter that is different from the capture particle identification parameter of said first type of analyte-specific capture particle; 
 (ii) said second type of analyte-specific capture particle having a binding moiety specific for a second analyte, said binding moiety bound to said capture particle, where 
   (4) upon binding of a first analyte to said first type of analyte-specific capture particle, upon binding of said first type of analyte-specific detector molecule to said first analyte bound to said first type of analyte-specific capture molecule, and upon binding of a second analyte to said second type of analyte-specific capture particle, and upon binding of said second type of analyte-specific detector molecule to said second analyte bound to said second type of analyte-specific capture molecule, forming:   a first complex of said first type of analyte-specific detector molecule, said first analyte, and said first type of analyte-specific capture particle immobilised on said detection surface and   a second complex of said second type of analye-specific detector molecule, said second analyte, and said second type of analyte-specific capture particle molecule immobilised on said detection surface; and   (5) capturing a composite image comprising said first and said second complexes.   
     
     
         2 . The system of  claim 1 , further comprising:
 (a) a multi-well plate holder compatible with at least one type of well-plate,   (b) a multi-well plate having at least one well,   (c) at least one lens,   (d) an image capture device,   (e) at least one light source, and   (f) an image capture device being operably linked a computer.   
     
     
         3 . The system of  claim 1  where immobilisation of said capture particle takes place before, during or after said analyte-specific capture molecules and said analyte-specific detection molecules have interacted with said analytes. 
     
     
         4 . The system of  claim 1 , where at least one type of capture particle has a shape selected from the group consisting of cylindrical, conical, spherical, elliptical, ribbon-like, ovoid, spiral, amoeba-like, tube-like, and flat-sided comprising 4 or more flat sides. 
     
     
         5 . The system of  claim 1 , wherein upon illumination, each of said types of capture particle emits electromagnetic radiation within the range of ultraviolet to infrared. 
     
     
         6 . The system of  claim 1 , where said capture particle is distinguished from debris, where said debris has a surface selected from the group excluding cylindrical, conical, spherical, elliptical, ribbon-like, ovoid, spiral, amoeba-like, tube-like and flat sided comprising 4 or more flat sides. 
     
     
         7 . The system of  claim 1 , where at least one type of said capture particle comprises a material selected from the group consisting of polymers, composites, inorganics, and natural products. 
     
     
         8 . The system of  claim 7 , where at least one type of said capture particle is a polymer selected from the group consisting of polypropylene, polyethylene, polyacetylene, polypyrrole, and conducting polymers. 
     
     
         9 . The system of  claim 7 , where at least one type of said capture particle is a composite selected from the group consisting of glass fibre composites and carbon fibre composites. 
     
     
         10 . The system of  claim 7 , where at least one type of said capture particle is a natural product chosen from the group consisting of silk, wax, rubber, and a resin. 
     
     
         11 . The system of  claim 1  where at least one type of said capture particles have moieties facilitating conjugation of said capture molecules. 
     
     
         12 . The system of  claim 1 , where at least one type of said capture particle comprises a moiety chosen from the group consisting of carbonyls, amines, thiols, imines, and vinyls. 
     
     
         13 . The system of  claim 1  wherein said capture particles have a magnetic core or coating. 
     
     
         14 . The system of  claim 1  where said capture particle identification parameter of at least one of said types of analyte-specific capture particles is selected from the group consisting of size, electromagnetic emission profile, and intensity of electromagnetic emission profile. 
     
     
         15 . The system of  claim 1  where at least one type of said capture particle is immobilised on said detection surface by a tethering molecule, a physical force, or incorporation of said capture particle within a matrix. 
     
     
         16 . The system of  claim 15  where said immobilisation is accomplished using covalent attachment, ionic interaction, hydrogen bonding, or Van der Waals interaction. 
     
     
         17 . The system of  claim 15  where said immobilisation is accomplished using electrostatic forces and/or positive or negative pressure. 
     
     
         18 . The system of  claim 15  where said matrix is selected from the group consisting of liquid-derived solid matrices, resins, glues, adhesives, and gels. 
     
     
         19 . The system of  claim 1  where said capture particle is a biological or synthetic molecule for which the target analyte shows affinity at a concentration of 10 millimolar or less. 
     
     
         20 . The system of  claim 19 , wherein said capture particle comprises a capture molecule selected from the group consisting of an antibody, antibody fragment, Fab region, receptor or receptor fragment, lectin, substrate of the target analyte, vitamin, an inorganic molecule, and derivatives thereof. 
     
     
         21 . The system of  claim 1  where said detector molecule is a biological or synthetic molecule for which the target analyte has affinity at a concentration of 10 millimolar or less. 
     
     
         22 . The system of  claim 21 , where said detector molecule is selected from the group consisting of an antibody, antibody fragment, Fab region, receptor, receptor fragment, lectin, substrate of the target analyte, vitamin, an inorganic molecule, and derivatives thereof, said capture molecule linked to a chromophore, or fluorophore or luminescent moiety. 
     
     
         23 . The system of  claim 1  where said first type of detector molecule with a first type of chromophore and/or fluorophore and/or luminescent moiety is used in the same assay as one or more different detector molecules, each with different chromophore and/or fluorophore and/or luminescent moiety. 
     
     
         24 . The system of  claim 1  where said analyte is a soluble inorganic, biological or synthetic molecule. 
     
     
         25 . The system of  claim 1 , wherein said computer has instructions stored therein to perform one of more of the following steps:
 (1) a Capture Particle Distinguishing Step, comprising:
 (i) moving said multi-well plate holder, 
 (ii) focusing said lens, 
 (iii) capturing an image of a capture particle using at least a first filter, 
 (iv) identifying and classifying said capture particle; 
   (2) a Recording Step, comprising:
 (i) re-imaging said capture particle using a second filter different from said first filter, thereby recording of an image of said fluorescent or luminescent complex; 
 (ii) measuring the intensity of fluorescence or luminescence characteristic of said detector molecule; 
   (3) an Analysis Step, comprising:
 (i) using of said measurement described in said step (2) to determine the presence and/or quantity of at least one analyte. 
   
     
     
         26 . The system of  claim 1  where said computer has
 (a) instructions stored therein to image an additional field of view within a well of a multi-well plate, 
 (b) instructions to determine a number of capture particles of each subset to be imaged to achieve a degree of precision of 85% prior imaging of other wells, 
 (c) instructions to move said well plate holder and re-focusing said lens followed by repeating said Capture Particle Distinguishing Step and said Recording Step on an additional field of view. 
 
     
     
         27 . The system of  claim 1 , where said computer has instructions stored therein to perform the steps:
 (a) predict fluorescence or luminescence values under conditions where said detector molecule fluorescence or luminescence exceeds the limit of detection (‘white-out’), comprising:   (b) repeating said Recording Step at a lower sensitivity of fluorescence or luminescence detection; and   (c) calculating the predicted fluorescence or luminescence that would have been obtained at the higher sensitivity of fluorescence or luminescence detection at which ‘white-out’ was observed; wherein said predicted value then being used in said Analysis Step.   
     
     
         28 . The system of  claim 1  where said computer has instructions stored therein to recognise and exclude said capture particles and/or said fluorescent or luminescent complexes that are in close enough proximity to interfere with each other's said detector molecule fluorescence or luminescence, and/or said capture particle' s fluorescence or luminescence. 
     
     
         29 . The system of  claim 1  where the computer has instructions stored therein to recognise debris, being objects that are not said capture particles, but have been imaged in said Capture Particle Distinguishing Step and Recording Step and said instructions include steps for exclusion of debris from incorporation into said Analysis Step. 
     
     
         30 . A method for detecting and quantifying an analyte, comprising:
 providing a system of  claim 1 ;   providing a sample containing the analyte to be measured;   incubating said sample with said capture particle before, during or after incubation of said first capture particle with a solution containing a known concentration of said first analyte, said capture particle having a fluorophore, luminescent moiety or chromophore conjugated thereto;   permitting said labelled analyte to compete with the unlabelled analyte for binding to said capture particle;   immobilising said capture particle on said detection surface in a well of a multiwell plate;   adding a first type of detector molecule to said well thereby producing a complex of said capture particle, analyte and detector molecule immobilised on said surface;   using an image-based detector to determine the amount of fluorophore, luminescent moiety or chromophore present on said complex; and   calculating the amount of said analyte in said sample.   
     
     
         31 . The method of  claim 30  where said capture particle is immobilised on said surface by electrostatic force. 
     
     
         32 . The method of  claim 30  where said analyte is an indicator of a disease, and can be used to screen for said disease, diagnose a disease, ascertain severity of any disease, or measure a patient's response to a treatment. 
     
     
         33 . The method of  claim 32 , wherein said disease is a human disease. 
     
     
         34 . The method of  claim 32  where said disease is a non-human animal or plant disease. 
     
     
         35 . The method of  claim 30  where measurement of said analyte is conducted on a sample derived from an in vitro or in vivo experiment. 
     
     
         36 . The method of  claim 30 , where said method is used to screen for the presence of an analyte in an industrial setting. 
     
     
         37 . The method of  claim 36 , where said industrial setting is selected from the group consisting of the following industries: private and public health, veterinary, cosmetic, agriculture, food production, water, pharmaceutical, diagnostic, biological laboratory, horticultural, fishery, marine crop, government agency, forensic, security, toxicological, environmental, biotechnology, institute of higher education, a college, a university, contract research organisation, central laboratory testing organisation, brewing, wine, spirit, bio-fuel, textile, chemical, paper, preservation, healthcare device, medical equipment, biomaterial, and prosthetic. 
     
     
         38 . The method of  claim 30 , where said analyte is an enzyme and said capture particle has a substrate attached thereto and said enzyme catalyzes an addition reaction to add an additional moiety to or a removal reaction that removes an already existing moiety from said substrate, said detection molecule having a chromophore, fluorophore, or luminescent moiety conjugated thereto binds to said additional moiety or to said existing moiety, further comprising;
 (a) incubating said capture particle and substrate with said enzyme and said additional moiety, thereby producing a complex of said capture particle, substrate and said additional moiety; or   (b) incubating said capture particle with said pre-existing moiety with said enzyme; and   (c) incubating said complex obtained in step (a) or step (b) with said detection moiety, and   (d) determining the presence and/or activity of said enzyme by detecting the fluorescence or luminescence on said detection moiety.   
     
     
         39 . The method of  claim 30 , where said analyte is an enzyme and said capture particle having a substrate having a fluorescent tag attached thereto and said fluorescent tag has a fluorescent-conjugated detector molecule comprising a flurophore, chromophore or luminescent moiety, thereby forming a labeled complex, further comprising;
 (a) incubating said labeled complex with a solution containing said enzyme; and   (b) determining the presence and/or activity of said enzyme by detecting and quantifying the loss of intensity of said fluorescence.   
     
     
         40 . The method of  claim 30 , where said detection molecule is a specific ligand for an analyte molecule in a solution, further comprising:
 (a) incubating wells containing said detection molecule with solutions containing different known concentrations of said analyte; and   (b) calculating the dissociation constant, association constant or affinity of said ligand-specific molecule for said ligand.   
     
     
         41 . The method of  claim 30 , where said detection molecule is an analyte and said analyte is a ligand for said analyte, further comprising:
 (a) incubating wells containing said detection molecule with solutions containing different know concentrations of said ligand; and   (b) calculating the dissociation constant, association constant, or affinity of said ligand for said analyte.   
     
     
         42 . The method of either of  claim 40  or  41 , where the analyte is selected from the group consisting of small molecule therapeutic agents, peptide therapeutic agents, cell-derived receptors, DNA binding molecules, and antibodies. 
     
     
         43 . A kit, comprising:
 (1) a multi-well plate;   (2) a subset of capture particles with or without capture molecules pre-conjugated to said capture particles;   (3) a substance used to immobilise said capture molecules,   (4) a subset of detector molecules with or without pre-conjugation to a fluorophore, luminescent moiety, or chromophore;   (5) a fluorophore, luminescent moiety, or chromophore with a moiety facilitating conjugation or binding to said detector molecule;   (6) reagents for use in the preparation of said multi-well plate, said capture particles, said analytes, said samples, said detector molecules, or said fluorophore, luminescent moiety, or chromophore, for performing the assay;   (7) a covering for the top of said multi-well plate.   (8) instructions for use describing methods for preparation of said multi-well plates, said capture particles, said analytes, said samples, said detector molecules said fluorophore and/or luminescent moiety and/or chromophore, methods of said assay conduct or for reducing data derived from use of said kit.   
     
     
         44 . A system for image-based analysis of a plurality of analytes, comprising:
 a. a detection surface having a plurality of types of capture particles thereon, each of said types of capture particles having a uniquely identifiable spectral feature, and each type of said capture particles having at least one analyte-specific capture molecule attached thereto;   b. a plurality of types of analyte-specific detector molecules, each of said plurality of types corresponding to one of said types of analyte-specific capture molecules;   c. a light source;   d. an imaging sensor; and   e. a computer having a program stored in a computer memory device having instructions thereon to determine the type and amount of each of said plurality of analytes.   
     
     
         45 . The system of  claim 41 , said spectral feature being a size, luminescence, colour, electromagnetic emission profile, electromagnetic emission intensity or combinations thereof. 
     
     
         46 . The system of  claim 44  or  claim 45 , said analyte-specific detector molecule being selected from the group consisting of antibodies, antibody fragments, cell-derived receptors, and lectins. 
     
     
         47 . A method for image-based analysis of a plurality of analytes, comprising:
 a. providing a detection surface having a plurality of types of capture particles thereon, each of said types of capture particles having a uniquely identifiable spectral feature, and each type of said capture particles having at least one analyte-specific capture molecule attached thereto;   b. applying a sample containing two or more analytes to said detection surface thereby forming a plurality of analyte-specific capture molecule-analyte complexes (“ASCMAC”);   c. providing a plurality of types of analyte-specific detection molecules, each type of which comprises a luminescent moiety different from luminescent moieties of other types of detection molecules.   d. applying to said ASCMAC, a said plurality of analyte-specific detector molecules with conjugated luminescence moieties attached thereto thereby forming a plurality of ACSMAC-analyte-specific detector molecule complexes (“ASCMASDC”); and   e. capturing an image of said ASCMASDC.   
     
     
         48 . The method of  claim 47 , said spectral feature being a size, luminescence, colour, electromagnetic emission profile, electromagnetic emission intensity or combinations thereof. 
     
     
         49 . A system for image-based detection of analytes as described herein. 
     
     
         50 . A method for detecting and/or quantifying the amount of an analyte in a sample as described herein.