WO2013180657A1 - Dosages chromatographiques rapides par cohydratation contrainte basés sur des ligands d'affinité à sensibilité élevée - Google Patents

Dosages chromatographiques rapides par cohydratation contrainte basés sur des ligands d'affinité à sensibilité élevée Download PDF

Info

Publication number
WO2013180657A1
WO2013180657A1 PCT/SG2013/000220 SG2013000220W WO2013180657A1 WO 2013180657 A1 WO2013180657 A1 WO 2013180657A1 SG 2013000220 W SG2013000220 W SG 2013000220W WO 2013180657 A1 WO2013180657 A1 WO 2013180657A1
Authority
WO
WIPO (PCT)
Prior art keywords
analyte
affinity ligand
particles
agent
hydrated
Prior art date
Application number
PCT/SG2013/000220
Other languages
English (en)
Inventor
Peter Gagnon
Original Assignee
Agency For Science, Technology And Research
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 Agency For Science, Technology And Research filed Critical Agency For Science, Technology And Research
Publication of WO2013180657A1 publication Critical patent/WO2013180657A1/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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials

Definitions

  • Embodiments disclosed herein relate to methods for enhancing the performance of affinity-ligand based assays such as immunoassays.
  • Affinity ligand based assays such as antibody-based immunoassays can suffer certain limitations in speed and sensitivity of detection when faced with certain analytes such as very large proteins, virus particles, bacteria, and other microbes due, at least in part, to their size. Large analytes can make it difficult to achieve high capture efficiency, especially when the capture method is via an antibody passively immobilized on the walls of a 96-well plate. Immunoassays for large analytes can also be impaired by the low diffusivity in aqueous solutions.
  • ligand assays Another limitation of ligand assays is the frequent requirement for the formation of a chemical bond between the solid phase and the analyte to be measured, or a chemical bond between the analyte and a capture ligand immobilized on a solid phase.
  • a purification method called steric exclusion captures large proteins and virus particles on a hydrophilic solid phase in the presence of nonionic organic polymer solutions, such as polyethylene glycol (Lee et al, J. Chromatography A, 1221 (2012) 162-170). Elution of the captured protein or virus may be achieved by reducing the concentration of the nonionic organic polymer. This technique has also been described as constrained cohydration chromatography.
  • the invention provides in certain aspects, methods, devices and kits for the performance of immunoassays and other assays for detection of analytes such as cells, viruses, large proteins and other molecules or substances where a sample containing an analyte or suspected of containing an analyte is combined with one or more detectably labeled affinity ligands.
  • the affinity ligands have specific binding affinity for the analyte of interest, so as to form complexes between at least some of the analyte and at least some of the affinity ligand.
  • the sample is contacted with a hydrated surface of an undissolved material in the presence of a constraining agent in an amount sufficient to cause at least a fraction of the analyte-affinity ligand complexes to be retained at the hydrated surface of the undissolved material.
  • the undissolved material having the analyte-affinity ligand complexes retained at its hydrated surface is separated from the un-complexed affinity ligand and other materials in the sample.
  • the analyte-affinity ligand complex is typically released from the undissolved material by reducing the amount of the constraining agent.
  • the presence or absence of the analyte is detected qualitatively or quantitatively through the detection of the label on the affinity ligand by an appropriate monitoring device.
  • methods and associated compositions and kits are provided for practice of the invention in the context of an immunoassay or a process using multi-well plates wherein the undissolved surface comprises a monolith bearing a hydrated surface.
  • methods and associated compositions and kits are provided for practice of the invention in the context of an immunoassay or a process wherein the monolith is plumbed to a chromatograph, which is in turn plumbed to an auto-sampler which may be programmed to introduce samples for analysis to the column over a series of runs.
  • various elements of such a system may be duplicated and multiplexed to increase analytical throughput.
  • methods and associated compositions and kits are provided for practice of the invention in the context of an immunoassay or a process using multi-well plates wherein the undissolved surface comprises magnetic nanoparticles bearing hydrated surfaces in wells of the multi-well plate.
  • methods and associated compositions and kits are provided for practice of the invention in the context of an immunoassay or a process wherein the undissolved surface comprises a hydrated monolith, or multiple monoliths which may be multiplexed to support parallel processing, including in conjunction with robotically controlled multi-well plate systems.
  • the invention in certain embodiments exploits the ability of hydrated (hydrophilic) surfaces to retain analytes of interest at their hydrated surfaces in the presence of sufficient concentrations of constraining agents.
  • the invention provides the advantage that detectably labeled affinity ligands which are not bound to an analyte will not be retained at the hydrated surface of the undissolved material (such as particles, chromatographic monolith, etc) and therefore the labeled affinity ligand which is complexed with the analyte can be rapidly separated from labeled affinity ligand which is not complexed with the analyte of interest.
  • the invention permits the concentration of the analyte so as to enhance the sensitivity of the detection of the analyte.
  • the invention has the effect of lowering non-specific background interference, thereby improving accuracy and reproducibility, as well as leading to further increases in sensitivity.
  • Samples amenable to use of certain aspects of the invention can be from any source suspected of containing the analyte of interest including, but not limited to, environmental samples, clinical samples, pharmaceutical or biologic manufacturing samples and others.
  • Analytes to which the invention may be applied advantageously particularly include large proteins such as IgM, Factor VIII and Factor VIII-vWF complexes, viruses and virus-like particles, bacteria, cellular organelles, and cells.
  • Figures and examples disclosed herein include experimental data documenting the fundamental features of the invention through the retention behavior of various analytes under conditions that may be manipulated to develop the detailed specifications of a particular assay.
  • Figure 1 shows the relationship between the percentage of an IgM-analyte retained by constrained co-hydration at the surface of a hydrated monolith and pH for different percentages of a constraining agent, PEG-6000 (8%, 9%, 10%, 11%, and 12%) as described in Example 1.
  • Figure 2 shows the relationship between the percentage of IgM-analyte retained by constrained co-hydration at the surface of a hydrated monolith and concentration of NaCl for different percentages of a constraining agent, PEG-6000 (8%, 9%, 10%) as described in Example 2.
  • Figure 3 shows the relationship between the percentage of IgM-analyte retained by constrained co-hydration at the surface of a hydrated monolith and percentages of a constraining agent, PEG of varying molecular weights (6000, 4000, and 2000) as described in Example 3.
  • Figure 4 shows the relationship between the percentage of a target species retained by constrained co-hydration at the surface of a hydrated monolith and percentages of a constraining agent PEG-6000 for three analytes (Phage Ml 3, IgM, and IgG) as described in Example 4.
  • Figure 5 shows the performance of constrained co-hydration convective chromatography of a virus as described in Example 10 including the influence of the constraining agent PEG-6000 upon the timing of the elution of contaminants and the target virus, as described in Example 5.
  • Figure 6 shows a diagram of the overall process, beginning with a virus-containing solution of unknown content at upper left, followed by addition of a fluorescently labeled monoclonal antibody, leading to the binding of that ligand to the virus.
  • the three panels, beginning at lower left, show the stages of the process on a hydrated monolith.
  • the chromatogram at far right illustrates a hypothetical fluorescence monitor profile over the course of conducting the assay.
  • Figure 7 shows a time-course plot for a virus elution, in accordance with embodiments disclosed herein.
  • the invention provides methods for detecting an analyte in a sample including the steps of (i) contacting the sample suspected of containing the analyte of interest with a plurality of affinity ligands labeled with one or more detectable or signal- generating moieties, where the affinity ligands have specific binding affinity for the analyte of interest, so as to form stable complexes between at least some of the analyte and at least some of the affinity ligand; (ii) contacting the sample with a hydrated surface of an undissolved material in the presence of a constraining agent in an amount sufficient to cause at least a fraction of the analyte-affinity ligand complexes to be retained at the hydrated surface of the undissolved material; (iii) separating the undissolved material with the analyte-affinity ligand complexes retained at its hydrated surface from the affinity ligand in the sample which is not complexed with the analyte; and
  • the method includes the additional step of dissociating the analyte-affinity ligand complexes from the hydrated surface of the undissolved material prior to the detecting step.
  • the dissociating step includes reducing the concentration of the constraining agent in contact with the analyte-affinity ligand complexes retained at the hydrated surface of the undissolved material to a degree to cause to the dissociation.
  • the dissociating step includes washing the undissolved material having the analyte-affinity ligand complexes retained at its hydrated surface with a solution lacking the constraining agent.
  • the dissociating step includes washing the undissolved material having the analyte-affinity ligand complexes retained at its hydrated surface with a solution having a declining concentration of the constraining agent.
  • the invention provides methods including the step of concentrating the analyte-affinity ligand complexes prior to the detecting step.
  • the concentrating step occurs prior to the detecting step.
  • the concentrating may occur by means of loading a large volume of dilute sample on the hydrated surface. The ability to load large volumes can be beneficial compared to conventional analytical methods which may not be able to accommodate large sample volumes due, at least in part, to volumetric limitations of the container.
  • the invention provides methods including the step of cleaning extraneous and potentially interfering substances from the sample prior to the detecting step.
  • washing steps may occur by one or more aspiration- refill steps.
  • the cleaning (or washing) embodiments provided herein may occur through continuous application of clean buffer in any volume sufficient to eliminate substances that may interfere with the most sensitive possible detection of the analyte of interest.
  • the invention provides methods of capturing analytes and concentrating them on a surface without the need to form a chemical bond between the analyte and a hydrated surface. In certain such embodiments, the invention provides methods of capturing and concentrating analytes on a hydrated surface without the need for an
  • intermediate capture ligand such as an antibody or other immobilized affinity ligand.
  • certain analytical systems may require the use of a non-specific affinity capture surface or immobilized specific capture affinity ligand to mediate the assay, Such surfaces and ligands may greatly restrict the range of analytes that may be analyzed by a particular assay method.
  • the present invention may accommodates any analyte above a size corresponding to a hydrodynamic diameter of about 10 nm or greater.
  • the affinity ligand bearing a detectable or signal-generating moiety may be a polyclonal or monoclonal antibody, or derivative form of an antibody such as an Fab fragment, F(ab')2 fragment, VHH domain, single chain antibody (SCFV), or other naturally occurring or synthetic ligand such as bacterial ligand, phage-display ligand, peptide ligand, an oligonucleotide, avidin, biotin, or other affinity construct of a type suitable for performing ligand assays.
  • an antibody such as an Fab fragment, F(ab')2 fragment, VHH domain, single chain antibody (SCFV), or other naturally occurring or synthetic ligand such as bacterial ligand, phage-display ligand, peptide ligand, an oligonucleotide, avidin, biotin, or other affinity construct of a type suitable for performing ligand assays.
  • the detectable or signal-generating label of the affinity ligand is a fluorophore, chemiluminophore, fluorescence quencher, chemiluminescence quencher, radionuclide, magnetic resonance label, an enzyme, or other entity covalently, reversible-covalently, or non-covalently attached to the affinity ligand.
  • the affinity ligand may be engineered in such a way as to include its own inherent signal generating capability.
  • the detecting step includes detection of an optical, electrical or change of state as signal.
  • the signal is an optical signal detected as an affinity ligand concentration dependent change in fluorescence,
  • the signal is an electrical signal detected as an affinity ligand concentration-dependent change in current, resistance, mass to charge ratio, or ion count.
  • the signal is a change in state signal detected as an affinity ligand concentration dependent change in size, solubility, mass, radionuclide decay, magnetic resonance, or resonance.
  • more than 50% of the analyte-affinity ligand complexes is retained at the hydrated surface of the undissolved material. In certain such embodiments, substantially all of the analyte-affinity ligand complexes is retained at the hydration surface of the undissolved material.
  • the analyte is a cell, virus, virus-like particle, cell fragment, cellular organelle, protein or other molecule.
  • the analyte is a bacterial cell or a microbial cell.
  • the surface of the undissolved material has one or more polar chemical moieties.
  • the polar chemical moiety is hydroxyl group.
  • the polar chemical moiety is an amine, imine, ureide, carbohydrate, amino acid, peptide, a cationic charged group or an anionic charged group.
  • the polar chemical moiety is a carbohydrate such as glucose, mannose, galactose, lactose, a monosaccharaide, a disaccharide and a polysaccharide.
  • the polar chemical moiety is a ureide such as urea, uric acid, allantoin, hydantoin.
  • the polar chemical moiety is histidine, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, or selenocysteine.
  • the hydrophilic (hydrated) surface of the undissolved material at which the analyte-labeled affinity ligand complex is captured may include naturally occurring or synthetic particles or monoliths.
  • the hydrophilic nature of the surface may be endowed by chemical groups with a high affinity for water.
  • the surface may naturally include such groups, or they may be added synthetically, for example by coating the surface of the undissolved material, or affixing certain types of chemical moieties.
  • the chemical groups may have an elevated hydrogen bonding affinity for water, including simple hydroxyl groups, or polyhydroxyl groups such as certain polysaccharides, or ureides such as urea, allantoin, or uric acid, among others.
  • the chemical groups may have an electrostatic affinity for water, including negatively charged examples such as carboxyl, phospho, or sulfo groups; or positively charged examples such as primary, secondary, tertiary, or quaternary amino groups, or combinations thereof.
  • the conditions under which the invention is practiced may be designed to eliminate interfering secondary influences of the hydrophilic groups, for example in the case of negatively or positively charged moieties, including sufficient salt in the operating buffers to suspend electrostatic interactions between the analyte, the affinity ligand or the signal-generating moiety to which the ligand is attached.
  • the constraining agent includes soluble substances that interact with proteins in such a way that they are said to be preferentially excluded from protein surfaces.
  • the constraining agent is ammonium sulfate, sodium citrate, potassium citrate, and potassium phosphate.
  • the constraining agent is an aqueous soluble uncharged linear or branched non-ionic organic polymer such as, for example, polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone, dextran, starch, or cellulose.
  • the constraining agent is polyethylene glycol.
  • the polyethylene glycol has an average polymer weight between 1000 and 10,000 Daltons, or an average polymer weight between 4,000 and 6,000 Daltons. In certain such embodiments, the polyethylene glycol is provided at a concentration between approximately 2% and 50% (w/v). In certain embodiments, the constraining agent is a zwitterionic compound or polymer.
  • the undissolved material is a convective chromatography material, such as a monolith, a membrane, or nonporous particles.
  • the convective chromatography material is nonporous silica particles.
  • the convective chromatography material is a monolith.
  • the monolith architecture may be supported by polymers such as a polyacrylate, polymethacrylate, styrenedivinylbenzene, or others; by minerals such as silica or mineral oxides; by crystalline or fused crystalline or ceramic materials such as apatites; by metallic compounds such as titania or alumina; or combinations of any of the foregoing or other materials, or combinations of any of the foregoing with secondary coatings to render the surface hydrophilic or enhance the hydrophilicity of the surface.
  • the monolith architecture may also incorporate nanoparticles to increase surface area.
  • the monolith has average channel sizes between about 1 micron and 200 micron, or between about 1 micron and 2 microns, or between about 10 micron and 20 microns, or between about 20 micron and 200 microns.
  • the monolith is coated with a polymer and /or is chemically modified to increase its degree of surface hydration.
  • the convective chromatography material surface is coated with a polymer and /or is chemically modified to increase its degree of surface hydration.
  • the hydrophilic (hydrated) surface of the undissolved material at which the analyte-labeled affinity ligand complex is captured may include naturally occurring or synthetic particles or monoliths.
  • the hydrophilic nature of the surface may be endowed by chemical groups with a high affinity for water.
  • the surface may naturally include such groups, or they may be added synthetically, for example by coating the surface of the undissolved material, or affixing certain types of chemical moieties.
  • the chemical groups may have an elevated hydrogen bonding affinity for water, including simple hydroxyl groups, or polyhydroxyl groups such as certain polysaccharides, or ureides such as urea, allantoin, or uric acid, among others.
  • the chemical groups may have an electrostatic affinity for water, including negatively charged examples such as carboxyl, phospho, or sulfo groups; or positively charged examples such as primary, secondary, tertiary, or quaternary amino groups, or combinations thereof.
  • the conditions under which the invention is practiced may be designed to eliminate interfering secondary influences of the hydrophilic groups.
  • sufficient salt may be included in the operating buffers to suspend electrostatic interactions between the analyte, the affinity ligand or the signal-generating moiety to which the ligand is attached.
  • organic solvents or surfactants may be included to suppress non-specific hydrophobic interactions.
  • the H, conductivity and salt concentration of the buffers delivered to the column are selected to discourage direct chemical interaction between the affinity ligand-analyte complexes or affinity ligand and such charged moieties.
  • the sample is combined with a buffer containing at least one constraining agent prior to delivery of the sample to the convective chromatography material and wherein such convective chromatography material is equilibrated with the constrained hydration agent prior to such delivery of the sample.
  • the methods of the invention include the additional steps of washing the hydrated convective chromatography material with a solution comprising the constraining agent in an amount sufficient to remove contaminants not retained at the hydrated surface of the undissolved material, and dissociating the target species from the convective chromatography material.
  • the step of dissociating the analyte comprises washing the convective chromatography material with a solution where the solution either (i) does not contain the constraining agent, (ii) contains the constraining agent in an amount insufficient to retain the analyte at the hydrated surface of the convective chromatography material, (iii) contains an agent which effects dissociation of the analyte from the hydrated surface of the convective chromatography material, (iv) a combination of (i) and (iii), or (iv) a combination of
  • the agent which effects dissociation of the target species from the hydrated surface of the convective chromatography material may be a surfactant, urea, a ureide, a chaotropic salt, sodium thiocyanate, potassium thiocyanate, sodium perchlorate, potassium perchlorate or a guanidine salt.
  • the undissolved material comprises particles.
  • the particles are nanoparticles or microparticles and such particles may be magnetic, paramagnetic or high-density.
  • the particles may be or include metal-core particles having a polymer coating, metal core particles having a cellulose coating, glass particles, polyacrylate, polymethacrylate, styrenedivinylbenzene, thiophilic magnetic particles, cellulose coated tungsten carbide particles, silica particles, agarose particles, cellulose particles, and composite particles.
  • the particle size may be between about 10 nm and about 500 ⁇ , or between about 100 nm and about 50 ⁇ , or between about 100 nm and about 4 ⁇ , or between about lOOnm and about 3 ⁇ , or between about lOOnm and about ⁇ , or between about 200nm and about 2 ⁇ , or between about 200nm and about 500nm, or between about 500 nm and about ⁇ , or between about 5 ⁇ and about 50 ⁇ .
  • the step of contacting the sample with the particles occurs prior to the step of treating the sample with the constraining agent.
  • the particles are provided in a plurality of wells in a multi- well plate.
  • the multi-well plate may be a 96 well plate and particles for use in practicing the invention may be in some or all of the wells of the plate.
  • the particles in the wells of the plate are magnetic or paramagnetic.
  • the wells are configures such that a magnetic field can be applied to attract the particles to the sides of the well so that optical detection can be performed along a vertical path through the bottom of the well.
  • an additional step of dissociating the affinity ligand-analyte complexes from the particles and then separating the affinity ligand-analyte complexes from the particles may be performed after the step of separating the particles with the affinity ligand- analyte complexes retained at the hydrated surfaces of the particles from the affinity ligand in the sample which is not complexed with the analyte.
  • the pH, conductivity and salt concentration of the buffers delivered to the column are selected to discourage direct interaction between the affinity ligand-analyte complexes and such charged moieties.
  • the step of dissociating the analyte comprises washing the particles with a solution where the solution either (i) does not contain the constraining agent, (ii) contains the constraining agent in an amount insufficient to retain the analyte at the hydrated surface of the particles, (iii) contains an agent which effects dissociation of the analyte from the hydrated surface of the particles, (iv) a combination of (i) and (iii), or (iv) a combination of (ii) and (iii).
  • the agent which effects dissociation of the analyte from the hydrated surface of the particles may be a surfactant, urea, a ureide, a chaotropic salt, sodium thiocyanate, potassium thiocyanate, sodium perchlorate, potassium perchlorate or a guanidine salt.
  • the constraining agent is added to the sample and the particles over a period of time under 5 seconds, under 10 seconds, under 30 seconds, under one minute, from about one minute to about five hours; between 2 minutes and 15min; between 2 minutes and 30 minutes; between 10 minutes and 30 minutes; between 2 minutes and 2 hours; or between about 2 minutes and 1 hour.
  • the constraining agent may be mixed with the sample as the sample is introduced to the hydrated surface. This mode of sample application may offer special benefits when the invention is practiced on a hydrated monolithic support, where it permits sample application to be achieved in seconds.
  • the particles are separated from the liquid component of the mixture by centrifugation, sedimentation, decantation, subjecting the mixture to a magnetic field, or by filtration.
  • the separated particles are washed with a solution comprising the constraining agent.
  • the step of dissociating is carried out in a single step of adding a dissociating buffer to the particles. In certain embodiments, the step of dissociating the target species is carried out incrementally through the reduction of the concentration of the constraining agent.
  • the method additionally provides for the detection of a second analyte such as for example the detection of two different cells or a cell and virus, or two different kinds of virus.
  • the invention provides for the detection of 3, 4, 5, or a plurality of analytes.
  • the sample is contacted with more than one distinct species of detectably labeled affinity ligand with each species of affinity ligand having binding specificity for one or more of the analytes and each distinct affinity ligand having a detectably different label.
  • each analyte of interest will have one affinity ligand which binds specifically to such analyte and not other analytes; in certain of such embodiments, each affinity ligand will have a detectably distinct label and the signal which permits detection of each such label will uniquely represent the presence of the corresponding analyte.
  • an affinity ligand may bind specifically to more than one analyte, preferably a subset of all analytes; in certain such embodiments, the combined signals of the various representing detection of various subsets of the total set of analytes will permit the determination of the presence (including quantitation) or absence of each analyte.
  • two or more detectably labeled affinity ligand -analyte complexes with distinct labels are detected simultaneously.
  • the distinct labels are distinct fluorophores having different fluorescence spectra and the labels are detected by a multi- wavelength fluorescence detector.
  • the invention provides an apparatus or a kit adapted for the convenient practice of a method of the invention.
  • the invention provides a platform for rapid high-sensitivity affinity ligand-based assays of cells, viruses and other target species of biomolecular character.
  • Antimicrobial immunoassays especially antiviral and antibacterial immunoassays are provided.
  • the invention is practiced using chromatographic, particularly convective chromatographic apparatus, preferably with optical such as fluorescent detection of the labeled affinity ligand (e.g. fluorophore labeled antibody) from the eluate of the apparatus.
  • the invention provides advantages of greater speed, higher capacity and/or higher sensitivity than conventional methods of conducting immunoassays.
  • certain embodiments of the present invention may provide high surface areas, convective mass transport, and a non-bioaffinity mechanism for rapid high-capacity broad-spectrum capture of microbes. This combination of enhancements can in certain embodiments permit rapid quantification with high sensitivity.
  • the invention provides a labeled (fluorescent, enzyme, etc.) antibody (or other affinity ligand) which is combined with a sample that needs to be evaluated for the presence/quantity of a particular analyte or target species such as a virus, bacteria, or protein.
  • a labeled antibody or other affinity ligand
  • the sample is contacted with a hydrated surface in the presence of polyethylene glycol (PEG, or other excluded solute or constraining agent) such that the antibody-associated analyte is retained at the hydrated surface, while unreacted antibody and other sample components are washed away, such as through the action of washes or buffers passing through a chromatographic column or monolith.
  • PEG polyethylene glycol
  • the analyte can then be dissociated from the hydrated surface by reducing or eliminating the constraining agent.
  • the hydrated surface is a monolith
  • the antibody is labeled with a fluorophore
  • a downstream fluorescent monitor may be used to measure the amount of fluorescent signal.
  • the hydrated surface comprises hydrated magnetic nanoparticles in a magnetically enabled 96-well plate
  • the antibody is labeled with an enzyme
  • the analyte complexed with the labeled affinity ligand may be dissociated from the nanoparticles through reduction of the concentration of the constraining agent and the magnetic nanoparticles may be separated from the analyte complexed with the labeled affinity ligand by application of a magnetic field.
  • a sufficiently clear optical path may be provided for detection of a signal related to the presence of the labeled affinity ligand in the well.
  • the label is an enzyme
  • an appropriate substrate for the enzyme may added to the well and resulting signal read with a corresponding fluorescent, visible, chemometric, or luminometric monitor.
  • the plate version can accommodate multiple samples but will require
  • the chromatography format (fluorescent- antibody/monolith) of the invention can be used to monitor multiple microbe species simultaneously, for example by using several antibodies, each specific for a different analyte, each labeled with a different fluorophore, and all read upon elution by a multi-wavelength fluorescent detector.
  • the chromatography format in particular has the ability in certain embodiments to concentrate analyte from a large volume of dilute sample, which can offer the opportunity to amplify the signal in comparison with conventional immunoassays and thereby permit higher sensitivity.
  • the sensitivity of the methods will support use of samples taken from saliva, blood or other bodily fluids such as samples collected for clinical, diagnostic or research purposes.
  • the invention can also accommodate simultaneous reading of mixed virus/bacteria-containing samples such as may be of interest with applications with monitoring phage therapy or phage treatment of foods.
  • both the monolith and plate-based systems may be used with customized integrated equipment systems to promote optimal operation. Additionally, certain embodiments may benefit from the development of special lines of labeled antibodies adapted for use with the invention.
  • the plate format be practiced with kits including prefilled plates where wells are filled with the appropriate magnetic particles. Definitions
  • amino acid refers to a class of organic molecules of natural or synthetic origin that contain carbon, hydrogen, oxygen, and nitrogen in an arrangement including an amine group, a carboxylic acid, and a side chain.
  • organic nucleation centers favor species of low solubility. The effective concentration of such agents varies with the identity of the amino acid and the characteristics of the biological product being processed by the invention. By extension, small peptides may also be employed as organic nucleation centers.
  • Affinity ligand refers to a substance which has the ability to recognize a particular analyte or feature of analyte in such a way that it binds only to that analyte and not to others.
  • Examples may include a polyclonal or monoclonal antibody, or derivative form of an antibody such as an Fab fragment, F(ab')2 fragment, VHH domain, single chain antibody (SCFV), or other naturally occurring or synthetic ligand such as bacterial ligand, phage-display ligand, peptide ligand, an oligonucleotide, avidin, biotin, or other affinity construct of a type suitable for performing ligand assays.
  • Analyte refers to the substance to be detected and measured in a sample of unknown composition.
  • a given sample may contain multiple species of analytes.
  • Antibody refers to an immunoglobulin, composite, or fragmentary form thereof.
  • the term may include but is not limited to polyclonal or monoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.
  • Antibody may also include composite forms including but not limited to fusion proteins containing an immunoglobulin moiety.
  • Antibody may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc and other compositions, whether or not they retain antigen- binding function.
  • An antibody may serve as an affinity ligand as described above, or it may be the analyte to which a particular assay is directed, especially in the case of large antibodies such as IgM, and Fc-fusion proteins.
  • Carbohydrate refers to a class of organic molecules of natural or synthetic origin that contain carbon, hydrogen, and oxygen with the general formula Cm(H20)n.
  • Species constituting organic nucleation centers suitable for performing certain embodiments of the invention include but are not limited to cellulose, starch, and poorly soluble sugars.
  • Chromatography support refers to a fixed chromatography bed that may be used to conduct chromatography.
  • the chromatography support may comprise a monolith, a membrane, a packed column of non-porous particles, or particles added directly to a liquid containing an analyte to be quantitated.
  • Constraining agent sometimes a "Precipitating agent;” “Preferentially excluded solute” or “preferentially excluded agent,” “sterically excluded solute” or
  • sterically excluded agent refers to an agent defined by its preferential exclusion from surfaces of proteins or other biological target species or which otherwise promotes the retention of a target biological molecule or structure at a hydrated surface without requiring any modes of chemical attraction to effect such retention.
  • Such substances may include but are not limited ' to so-called kosmotropic salts, nonionic or zwitterionic organic polymers, and amino acids.
  • kosmotropic salts such as ammonium sulfate, sodium citrate, and potassium phosphate, among others.
  • Another group comprises non-ionic organic polymers such polyethylene glycol (PEG), polypropylene glycol (PPG), and polyvinylpyrrolidone, among others.
  • PEG has a structural formula HO-(CH2-CH2-0)n-H.
  • Examples include, but are not limited to compositions with an average polymer molecular weight ranging from less than 100 to more thanl 0,000 daltons.
  • Another group comprises amino acids such as glycine.
  • Exosomes refers to a type of organelle associated with stem cells.
  • Fractor VIII Fact eight is clotting protein.
  • Fibrinogen is clotting protein.
  • Highly hydrated surface or “hydrophilic surface” refers to a surface that interacts strongly with water, potentially through hydrogen bonding, electrostriction, or some combination of the two mechanisms. Such interactions may be mediated by chemical groups such as hydroxyls, negative charges, or positive charges, or uncharged polar groups.
  • chemical groups such as hydroxyls, negative charges, or positive charges, or uncharged polar groups.
  • the presence of hydratable chemical groups may be a basic feature of the native composition of a given material, or it may result from or be enhanced secondarily by chemical modification to immobilize such groups on the surface, including but not limited to carbohydrates and ureides. So-called hydrophobic surfaces are generally considered not to be highly hydrated, but surfaces that include strongly hydratable groups in combination with hydrophobic residues may nevertheless be sufficiently hydrated to practice the invention.
  • Kosmotropic salts may include any of a variety of salts including but not limited to ammonium sulfate, sodium sulfate, potassium phosphate, sodium citrate, potassium citrate, and sodium chloride.
  • the effective concentration of such salts varies with the identity of the salt and the characteristics of the biological product being processed by the invention.
  • Non-ionic organic polymer refers to a naturally occurring or synthetic hydrocarbon composed of linked repeating organic subunits that lack charged groups. It may be linear, dominantly linear with some branching, or dominantly branched. Examples suitable to practice the invention include but are not limited to dextran, starch, cellulose, polyethylene glycol (PEG), polypropylene glycol, and polyvinylpyrrolidone (PVP). PEG has a structural formula HO-(CH2-CH2-0)n-H. Examples include, but are not limited to compositions with an average polymer molecular weight ranging from less than 100 to more than 10,000 daltons.
  • the average molecular weight of commercial PEG preparations is typically indicated by a hyphenated suffix.
  • PEG-6000 refers to a preparation with an average molecular weight of about 6,000 daltons.
  • the effective concentration of such agents varies with the identity of the polymer and the characteristics of the biological product being processed by the invention.
  • Organelle refers to component of a cell, usually understood to be a functional component, metaphorically equivalent to the organs of higher organisms. Organelles are understood to include such structures as mitochondria, ribosomes, and exosomes, among others.
  • Organic solvent refers to naturally occurring or synthetic organic compounds existing in a liquid state. Examples suitable to practice the invention include but are not limited to ethylene glycol, propylene glycol, dimethyl sulfoxide, ethanol, and phenoxyethanol.
  • Particle refers to an undissolved material that may be used to practice the invention. Particles may range in size from less than 100 nm to more than 100 microns. They may be porous or non-porous. They may be of inorganic origin, such as silica; or they may be or organic origin. Organic particles may comprise naturally occurring materials such a starch, allantoin, cellulose, agarose, or dextran, or their synthetic analogues; or purely synthetic polymers such as polymethacrylates, polyacrylates, styrenedivinylbenzene, or many others. Particles may be of uniform structure throughout, or they may be compound, consisting of an inner core of one material such as a metal alloy. They may have an applied surface that is highly hydrated or permits the attachment of chemical groups to produce a highly hydrated surface.
  • Preferential exclusion describes an interaction whereby certain dissolved substances are deficient in the immediate area surrounding a hydrophilic (hydrated) surface, in comparison to the concentration of the same substances in the bulk solution.
  • Preferentially excluded solutes particularly include so-called precipitating salts, non-ionic polymers, and amino acids.
  • Preferential hydration refers to an area immediately adjacent to the surface of a protein or a hydrated solid material, in which the concentration of water is greater than the concentration of water in the bulk solvent as the result of a preferentially excluded substance (constraining agent) being displaced from that area.
  • the thickness of the preferential hydration zone is generally equivalent to the radius of the preferentially excluded substance (constraining agent). It is to be understood preferential hydration water is distinct from hydration water associated with the surface of a protein by hydrogen bonding or electrostriction.
  • Protein refers to any of a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed principally of one or more chains of amino acids linked by peptide bounds.
  • the protein may be of natural or recombinant origin. Proteins may be modified with non-amino acid moieties such as through glycosylation, pegylation, or conjugation with other chemical moieties. Examples of proteins include but are not limited to antibodies, clotting factors, enzymes, and peptide hormones.
  • Stem cells refer to a class of cells known for their ability to differentiate into any type of tissue.
  • Ureide refers to a cyclic or acyclic organic molecule of natural or synthetic origin that comprises one or more urea moieties or derivatives thereof.
  • the invention provides ureides such as urea, uric acid, hydantoin, allantoin (CAS number 97-59-6; alcloxa, aldioxa, hemocane, ureidohydantoin, 5-ureidohydantoin, glyoxylureide, glyoxylic acid diureide, 2,5-dioxo-4-imidazolidinyl urea), purines, and derivatives thereof.
  • urea such as urea, uric acid, hydantoin, allantoin (CAS number 97-59-6; alcloxa, aldioxa, hemocane, ureidohydantoin, 5-ureidohydantoin, glyoxylureide, glyoxylic acid diure
  • the invention provides organic molecules of the formula R-CO-NH-CO-NH2 or R- CO-NH-CO-NH-CO-R' or R' R"NH-CO-NR"'R"" where the relevant "R-groups” may be H or any organic moiety.
  • Virus refers to an ultramicroscopic (roughly 20 to 300 nm in diameter), metabolically inert, infectious agent that replicates only within the cells of living hosts, mainly bacteria, plants, and animals: composed of an RNA or DNA core, a protein coat, and, in more complex types, a surrounding envelope.
  • Examples include but are not limited to a dsDNA virus, a ssDNA virus, a dsRNA virus, a (+)ssRNA virus, a (-)ssRNA virus, a ssRNA RT virus and a dsDNA-RT virus; an adenovirus, a herpes virus, a poxvirus, a parvovirus, a reovirus, a picornavirus, a togavirus, an orthomyxovirus, a rhabdovirus, a retrovirus, a hepadanvirus, a papillomavirus, a Human Immunodeficiency Virus (HIV), an influenza virus, dengue virus, Japanese encephalitis virus, West Nile virus, and bacteriophages.
  • HIV Human Immunodeficiency Virus
  • virus is understood to include virus particles for use as vectors for gene therapy, for use as vaccines, and as replacements for antibiotics. It is also understood to include so-called pseudovirions, which may be described as virus particles that have been recombinantly modified to conserve their ability to generate protective immunity while eliminating their ability to cause infection. Viruses are in most cases understood to be analytes in the context of the present invention.
  • Von Willebrands Factor is a clotting protein. It binds to Factor VIII, forming so- called anti-hemophiliac factor, with a molecular weight of about 2 million Daltons.
  • Preferentially excluded agents suitable for practicing the invention particularly include nonionic organic polymers such as polyethylene glycol (PEG), generally of a molecular weight ranging from 600 to 6000 Daltons, but also lower and higher molecular weights.
  • PEG polyethylene glycol
  • Other nonionic organic polymers may be used effectively, such as but not limited to polypropylene glycol, dextran, and others.
  • Other preferentially excluded agents suitable for practicing the invention may include kosmotropic salts from the group comprising ammonium sulfate, sodium sulfate, sodium or potassium citrate, potassium phosphate, and others; and amino acids such as glycine. Experimental data indicate that different preferentially excluded agents confer different selectivities.
  • nonionic organic polymers Another valuable benefit of nonionic organic polymers is their chemical gentleness. High concentrations of PEG have a longstanding reputation for stabilizing proteins, they are well tolerated by living cells, and many FDA-approved human-injectable therapeutic formulations employ PEG as an inactive ingredient.
  • Chromatography supports suitable for practicing the invention particularly include media with surfaces that are highly hydrated.
  • Many polymer-based media unfunctionalized for adsorption chromatography have surfaces that are densely populated by hydroxyl groups that interact strongly with water, leaving the surface highly hydrated.
  • Surfaces that have been functionalized with highly hydrated materials are also ideal, potentially including immobilized sugars, starch, other carbohydrates, and ureides.
  • Positively and negatively charged groups also tend to be highly hydrated, and can be used to practice the invention at high salt conditions where charge interactions between biological sample components and the support are effectively suspended.
  • Surfaces with some hydrophobic character may be suitable if they possess an overall polar character sufficient to dominate the surface, which will exclude most surfaces intended for conducting hydrophobic interaction chromatography.
  • Dissociation of biological products bound to a support may be achieved by adding an agent that interferes with the ability of the preferentially excluded agent to maintain binding of the target product.
  • agents include preferentially surfactants, urea, neutral salts, polysaccharides, amino acids, and chaotropic salts such as sodium or potassium thiocyanate, sodium or potassium perchlorate and guanidine; or such agents can be applied in conjunction with reducing the concentration of the preferentially excluded agent, though in most cases the simple method of reducing the preferentially excluded agent will be preferred.
  • selectivity is dominantly a function of the size of the product of interest, with the strength of retention increasing in proportion to the size of the product. This is consistent with the fact that protein hydration is proportional to size. A practical consequence is that the concentration of preferentially excluded (constraining) agents required to achieve binding is inversely proportional to product size. Good virus binding can be achieved at PEG- 6000 concentrations as low as about 2%, while IgM antibodies may employ about 9% to about 12%, and IgG antibodies may employ about 15% or more.
  • the equilibration buffer may include a buffering compound to confer adequate pH control.
  • Such compounds include but are not limited to acetate, phosphate, citrate, MES,
  • the pH of the equilibration buffer may range from about pH 4.0 to about pH 9.0.
  • the equilibration buffer may contain MES at a concentration of about 50 mM at a pH of 5.8, sodium chloride at a concentration of 600 mM, and PEG-6000 at a concentration of 6%.
  • the sample may be equilibrated to conditions compatible with the column equilibration buffer as part of the sample loading process.
  • the sample may be pumped onto the column through one line, a concentrated solution of the preferentially excluded solute pumped onto the column simultaneously through another line, and the contents of the two lines may be mixed immediately before the column to minimize the pre-column time interval during which the product is exposed to the preferentially excluded agent, as a means of limiting the degree of precipitation that may occur before the sample flows onto the column.
  • the concentration of the preferentially excluded agent being applied through one line is 125% of the target concentration of the mix, and the mixing ratio of preferentially excluded agent to sample is 80% agent, 20% sample.
  • the concentration of the preferentially excluded agent being applied through one line is 200% of the target concentration of the mix, and the mixing ratio of preferentially excluded agent to sample is 50% agent, 50% sample.
  • these conditions provide broadly effective starting points, with the main necessity being to determine the amount of preferentially excluded agent required in the final mix to achieve the desired product binding characteristics.
  • the relative concentration of the preferentially excluded agent and the proportioning factor may be varied to identify the conditions that support that highest capacity in combination with the lowest mixed sample volume.
  • the preferentially excluded agent is PEG-8000, or PEG-
  • the formulation of the equilibration buffer may be substantially different from the formulation of the equilibrated sample.
  • a 1.2 M sodium chloride might be added to the sample, while the equilibration buffer contains only 200 mM sodium chloride with the objective of maintaining a net sample salt concentration of 400 mM sodium chloride (in-line dilution proportion 80:20), then washing immediately thereafter with 200 mM sodium chloride.
  • Such a tactic might be used when the higher salt concentration was due to higher capacity binding but the lower salt concentration was more effective for washing away a particular subset of contaminants.
  • the two buffers might also differ with respect to pH or the presence, absence, or amounts of other agents. A similar effect could be accomplished by optionally applying a second wash buffer.
  • the column is washed following sample loading, by a wash buffer, usually of the same composition as the equilibration buffer, to remove unbound contaminants from the chromatography support.
  • a wash buffer usually of the same composition as the equilibration buffer
  • the composition of the wash buffer may differ from the composition of the equilibration buffer.
  • more than one wash buffer may be applied, each with a different
  • composition each of which may also be distinct from the formulation of the equilibration buffer.
  • the product is then dissociated from the chromatography support solely by reducing the concentration of the preferentially excluded (constraining) agent, for example by creating a gradient from Hepes PEG to just Hepes buffer.
  • the reduction of the preferentially excluded (constraining) agent is accompanied by an increase of one or more elution enhancing substances by including such substances in the gradient end-point buffer.
  • elution enhancing substances might include, urea, arginine, or a surfactant, among others.
  • Preferentially excluded agents suitable for practicing certain embodiments of the invention particularly include nonionic organic polymers such as polyethylene glycol (PEG), generally of a molecular weight ranging from about 2,000 to about 6,000 Daltons but also lower and higher molecular weights, such as from about 600 to about 10,000 Daltons.
  • PEG polyethylene glycol
  • concentrations of PEG have a long-standing reputation for stabilizing proteins, they are well tolerated by living cells, and many FDA-approved human-injectable therapeutic formulations employ PEG as an inactive ingredient. Experimental data indicate that they similarly protect viruses.
  • nonionic organic polymers may be used effectively, such as but not limited to
  • preferentially excluded agents suitable for practicing the invention may include kosmotropic salts from the group comprising ammonium sulfate, sodium sulfate, sodium or potassium citrate, potassium phosphate, and others; and amino acids such as glycine.
  • Particles suitable for practicing certain embodiments of the invention particularly include particles with surfaces that are highly hydrated. Many polymer-based media
  • unfunctionalized for adsorption chromatography have surfaces that are densely populated by hydroxyl groups that interact strongly with water, leaving the surface highly hydrated.
  • Surfaces that have been functionalized with highly hydrated materials are also ideal, potentially including immobilized sugars, starch, other carbohydrates, and ureides.
  • Positively and negatively charged groups also tend to be highly hydrated, and can be used to practice the invention at high salt conditions where charge interactions between biological sample components and the support are effectively suspended.
  • Surfaces with some hydrophobic character may be suitable if they possess an overall polar character sufficient to dominate the surface, which will exclude most surfaces intended for conducting hydrophobic interaction chromatography.
  • Naturally occurring particles may also be useful in certain embodiments, such as particles comprising starch or ureides.
  • synthetic particles having magnetic or paramagnetic properties may be used to practice the invention.
  • Porous particles offer the potential for various proteins, including both the contaminants and the product of interest, to diffuse into the pores, and then gradually leak out at various stages of the process, compromising purity or product recovery, or both.
  • Porous particles it may generally be beneficial, in some embodiments, to employ particles having a porosity that is sufficiently small to prohibit diffusive entry of the analyte or the labeled affinity reagent used to make the analyte detectable.
  • Non-porous particles may be useful to avoid the potential issue of diffusive entry of analyte into the particle altogether.
  • particles may range in size from less than 100 run to more than 200 ⁇ . This covers the range from nanoparticles to microparticles, both of which have been shown to be effective.
  • Particles may be composed of any material, including polymers, minerals, or metals, any of which may be of compound structure with a surface composition different from the interior of the particles. This may specifically include iron-core particles designed to be collected by magnetic separators.
  • the quantity of particles should be of an amount sufficient to accommodate binding of all of the analyte. This can be determined easily by
  • Typical experimental amounts may range from about 2% to about 20% particles, but the method can be practiced with lower and higher amounts without reducing its effectiveness.
  • a so-called moderately greater amount might consist of 10% more, or 50% more, or twice the minimum amount, depending on the level of variation in sample composition.
  • excess amounts of particles may interact non-specifically with the analyte, thereby preventing full dissociation of the analyte-affinity ligand complex. This may reduce the sensitivity of some assays.
  • the preferentially excluded (constraining) agent can be added as a liquid concentrate to increase the efficiency with which it is dispersed throughout the liquid. Addition may be performed manually or automated conveniently through the use of a pump.
  • the precipitating (constraining) agent may alternatively be added as a powder. This offers the advantage of reducing the final process volume, but increases the time period over which addition must occur since time must be allowed to permit the precipitating (constraining) agent to dissolve.
  • addition of precipitating (constraining) agents as liquid concentrates may be performed over 30 minutes or more, while addition of dry powder may be performed over 60 minutes or more. Subsequent experimentation will reveal the specific limits for any given application.
  • the invention provides a method for capturing and concentrating large biological structures (such as cells, organelles, and viruses) that are believed to work, without being bound by any specific theory, by trapping them at a non-reactive hydrophilic surface, exclusively through constrained sharing of water between their respective preferential hydration shells. Entrapment is induced and stabilized by constraining agents such as polyethylene glycol (PEG) that preferentially hydrate hydrophilic surfaces. Selectivity correlates with molecular size. Since binding is related to the size of the analyte, it is understood that additional mass contributed by the affinity ligand will generally have the effect of enhancing binding of the analyte-ligand complex. Retention increases with PEG size and concentration. Binding is enhanced near the isoelectric point (pi) of the analyte. Salt weakens retention by reducing PEG size. Virus binding capacity on monoliths is about 1 trillion particles per mL.
  • Example 1 A series of experiments was performed in which the binding characteristics of an analyte consisting of a purified IgM monoclonal antibody with an isoelectric point of about 5.5 were evaluated at different pH values and different concentrations of PEG- 6000. All these experiments were conducted with a 334 OH monolith at a flow rate of 4 mL/min. Results are shown in Fig. 1. 100% binding of the IgM was achieved in 10% PEG at pH 5.0. Fig. 1 shows that pH of 9.5 may have been adequate to achieve 100% binding.
  • Example 2 A series of experiments was performed in which the binding characteristics of an analyte consisting of an IgM were evaluated as a function of NaCl concentration. All experiments were conducted with PEG-6000 on a 334 ⁇ , OH monolith at a flow rate of 4 mL/min. Results are shown in Fig. 2. Binding efficiency was reduced dramatically with increasing salt concentration, requiring an increase in PEG-6000
  • Example 3 The effect of PEG size. A series of experiments in which binding of an analyte consisting of IgM was evaluated as a function of PEG size. All experiments were conducted on a 334 iL OH monolith at a flow rate of 4 mL/min. Results are shown in Figs. 3, showing that larger PEGs are able to achieve binding at lower concentrations.
  • Example 4 The effect of PEG concentration.
  • a series of experiments were conducted with analytes consisting of virus (mass 16.7 MDa), IgM (mass 960 kDa), IgG (mass 160 kDa), and bovine serum albumin (BSA, mass 67 kDa) at various PEG concentrations to evaluate the relative contribution of biomolecule size. All experiments were conducted with PEG-6000 on a 334 ⁇ OH monolith. Results are shown in Fig. 4. The species with the largest mass bound at the lowest PEG concentration, with smaller species requiring higher PEG concentrations to achieve equivalent binding. BSA barely began to bind at 15% PEG-6000. These results highlight the size selectivity of the technique. Parallel experiments conducted using insoluble starch or allantoin as a substrate in place of an OH monolith have shown that the same relationship persists regardless of which highly hydrated (hydrophilic) substrate is employed.
  • Example 5 Dynamic binding capacity of virus. Dynamic binding capacity of bacteriophage Ml 3 was determined by feeding phage at a constant concentration at 6% PEG, until phage began to flow through the 334 ⁇ OH monolith column as indicated by an increase in UV absorbance. This occurred at the point at which 9.9 x 10el2 virus particles had been loaded. This example illustrates that binding capacity is unlikely to be exceeded with the application of small samples such as typically used for analysis.
  • Example 6 Binding efficiency and recovery of virus. Analysis by infectivity and chromatography indicate that constrained co-hydration chromatography of bacteriophage Ml 3 as described above achieved more than 90% binding efficiency from crude unpurified feed stream, and that essentially all of that was recovered in the eluate. This example illustrates that the method achieves a high degree of accuracy by virtue of efficient capture and dissociation.
  • Example 7 Purification efficiency of virus. Analysis by ELISA for E. coli host proteins indicated that purification by constrained co-hydration chromatography removed 99.8% of the protein contaminants from bacteriophage Ml 3 in this single purification step. Subsequent purification by a follow-on anion exchange chromatography step reduced host cell protein by a combined total of 99.99%. AccuBlue assays showed that constrained cohydration chromatography removed more than 90% of DNA, and the subsequent anion exchange step reduced it by another 10 fold. This example illustrates the ability of the invention to eliminated substances that might interfere with sensitivity or accuracy.
  • Example 8 Virus purification. And 1 mL OH-monolith was equilibrated to 6% PEG- 6000, 50 mM Hepes, 600 niM NaCl, pH 7.0. This was done by equilibrating with a 50:50 mixture of 12% PEG-6000, 50 mM Hepes, 600 mM NaCl, pH 7.0 to 50 mM Hepes, 600 mM NaCl, pH 7.0. 100 mL of bacteriophage Ml 3 was loaded by in-line dilution with the 12% PEG buffer, yielding 200 mL of virus sample in 6% PEG.
  • Example 9 Monolith-based CCC isolation of virus from a complex mixture.
  • Polymethacrylate monoliths with a hydroxylated (OH) surface and average 2 ⁇ channels were used to evaluate the ability of constrained co-hydration to achieve selective retention of virus.
  • Mass transfer in monoliths occurs by convection, which is unaffected by the diffusivity limitations that encumber columns packed with porous particles [Hahn, R. et al., A., Sep. Sci. TechnoL, 37 1545-1565 (2002).; Strancar, A. et al, Adv. Biochem. Eng. Biotechnol. 76 49-85 (2002); Jungbauer, A., J. Chromatogr. A, 1065 3-12 (2005)].
  • Bacteriophage Ml 3 occurs as a weakly flexible rod with a length of 916 nm, a diameter of 7.2 nm, and a molecular weight of about 16.7 MDa24.
  • Filtered E.coli supernatant was loaded through one pump and diluted in-line with 12% PEG-6000 delivered through another pump at the same flow rate.
  • Pre-monolith residence time of the virus in the 6% PEG mix was calculated to be about 1 second, insufficient for significant precipitation to occur prior to virus contact with the monolith surface. Transit time through the monolith for unretained substances was about 5 seconds. More than 90% of the virus from a 2.5 L sample was retained by a 0.34 mL monolith, highlighting the rapid binding kinetics and high concentration factor.
  • the monolith was subsequently washed with 6% PEG, 600 mM NaCl, 50 mM Hepes, pH 7.0, then eluted with a linear gradient to 50 mM Hepes, 600 mM NaCl, pH 7.0.
  • the elution profile is reminiscent of affinity chromatography, with nearly all contaminants flowing through the column and the virus recovered in a 2.5 mL fraction, as indicated in Figure 6.
  • Dynamic binding capacity determined with purified phage was 9.9 x 10 12 cfu/mL of monolith.
  • Host cell ELISA documented 99.8% reduction of E.coli proteins. AccuBlue documented 92% reduction of
  • Example 10 Virus was immobilized using the monolith-based format of the invention while simultaneously establishing that antibody reagents up to 1 million Daltons in size are not retained at the hydrated surface of the monolith under the conditions at which virus is retained. Unreacted labeled antibodies were not expected to be retained at the hydrated surface of the monolith as antibodies even when labeled are typically substantially smaller than
  • Example 11 Amplified detection of virus on a hydrated monolithic support. 10 microliters of fluoresceinated anti-bacteriophage Ml 3 coat protein (Santa Cruz Biotechnology,
  • Buffer A was 50 mM Hepes, 500 mM NaCl, 12% PEG-6000, pH 7.0. Buffer

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (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 concerne des procédés et des matières servant à la détection par dosage immunologique rapide d'analytes cibles à sensibilité élevée. Selon l'invention, une immobilisation transitoire par cohydratation contrainte d'analytes cibles est accomplie par contact avec des anticorps marqués spécifiques de l'analyte cible pertinent. Des anticorps liés à l'analyte sont retenus pendant que des anticorps non liés et d'autres matière sont évacués par lavage; et après libération de l'anticorps lié à l'analyte cible, la quantité de l'analyte est déterminée par détection quantitative du marqueur de l'anticorps
PCT/SG2013/000220 2012-05-31 2013-05-30 Dosages chromatographiques rapides par cohydratation contrainte basés sur des ligands d'affinité à sensibilité élevée WO2013180657A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261653774P 2012-05-31 2012-05-31
US61/653,774 2012-05-31

Publications (1)

Publication Number Publication Date
WO2013180657A1 true WO2013180657A1 (fr) 2013-12-05

Family

ID=49673728

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SG2013/000220 WO2013180657A1 (fr) 2012-05-31 2013-05-30 Dosages chromatographiques rapides par cohydratation contrainte basés sur des ligands d'affinité à sensibilité élevée

Country Status (1)

Country Link
WO (1) WO2013180657A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023196262A1 (fr) * 2022-04-03 2023-10-12 Biosensing Instrument Inc. Procédés d'analyse rapide de concentration d'analyte pour de multiples échantillons
EP3371302B1 (fr) * 2015-11-05 2024-01-03 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de purification de compositions de virus et de telles compositions obtenues

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090247735A1 (en) * 2007-01-09 2009-10-01 Bio-Rad Laboratories, Inc. Enhanced capacity and purification of protein by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers
WO2012169970A1 (fr) * 2011-06-08 2012-12-13 Agency For Science, Technology And Research Purification de produits biologiques par chromatographie avec cohydratation forcée

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090247735A1 (en) * 2007-01-09 2009-10-01 Bio-Rad Laboratories, Inc. Enhanced capacity and purification of protein by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers
WO2012169970A1 (fr) * 2011-06-08 2012-12-13 Agency For Science, Technology And Research Purification de produits biologiques par chromatographie avec cohydratation forcée

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "MaxDiscoveryTM Respiratory Syncytial Virus ELISA Kit Manual", BIOO SCIENTIFIC, 2010, pages 1 - 12, Retrieved from the Internet <URL:http://www.biooscientific.com/ProductsServices/LifeScienceProducts/ReporterAssays/MaxDiscovery%E2%84%A2RespiratorySyncytialVirus(RSV)ELISAKit> [retrieved on 20130718] *
ZHOU, J. ET AL.: "A scalable process for purification of recombinant adeno-associated virus serotype 9", MOLECULAR THERAPY, vol. 18, no. SUPPL., 19 May 2010 (2010-05-19), WASHINGTON, DC, UNITED STATES, pages S28 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3371302B1 (fr) * 2015-11-05 2024-01-03 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé de purification de compositions de virus et de telles compositions obtenues
WO2023196262A1 (fr) * 2022-04-03 2023-10-12 Biosensing Instrument Inc. Procédés d'analyse rapide de concentration d'analyte pour de multiples échantillons

Similar Documents

Publication Publication Date Title
Liangsupree et al. Modern isolation and separation techniques for extracellular vesicles
Delaunay-Bertoncini et al. Immunoaffinity solid-phase extraction for pharmaceutical and biomedical trace-analysis—coupling with HPLC and CE—perspectives
US20170218011A1 (en) Purification of biological products by constrained cohydration chromatography
EP3228709B1 (fr) Transporteur lié à la protéine tim, procédés d&#39;obtention, d&#39;élimination et de détection de vésicules membranaires extracellulaires et de virus à l&#39;aide dudit transporteur et kit comprenant ledit transporteur
US9695216B2 (en) Materials and methods for removing endotoxins from protein preparations
EP2616809B1 (fr) Isolation et déglycosylation des glycoprotéines
WO2016069764A1 (fr) Procédés pour la préparation rapide de glycosylamines étiquetées et pour l&#39;analyse de biomolécules glycosylées les produisant
Stevenson et al. Selective extraction of proteins and other macromolecules from biological samples using molecular imprinted polymers
WO2013180655A1 (fr) Liaison sélective de cibles biologiques à des uréides à phase solide
Du et al. L-histidine functionalized multi-walled carbon nanotubes for on-line affinity separation and purification of immunoglobulin G in serum
Ott et al. Preparation of epoxy‐based macroporous monolithic columns for the fast and efficient immunofiltration of Staphylococcus aureus
Pichon Aptamer-based and immunosorbents
WO2013180657A1 (fr) Dosages chromatographiques rapides par cohydratation contrainte basés sur des ligands d&#39;affinité à sensibilité élevée
US10669307B2 (en) Device, use of said device, and method for separating substances with an improved utilization of the capacity of chromatographic media
Multia Development of fast, reliable and automated isolation and fractionation methods for nanosized subpopulations of human biomacromolecules
CN112114074A (zh) 乳铁蛋白适配体亲和柱及其制备方法与应用
Konečná et al. Optimalization of deoxyribonucleic acid extraction using various types of magnetic particles
WO2010002455A1 (fr) Membranes asymétriques poreuses
Alasel et al. Development of a borreliosis assay: Mannan coated polyethylene sinter bodies as a new platform technology
Huang Rapid Isolation and Quantification of Exosome and Lentivirus by Hydrophobic Interaction Chromatography on a Polyester Capillary-Channeled Polymer Fiber Stationary Phase
EP0246103A2 (fr) Séparations par bioaffinité et par échange d&#39;ions utilisant des supports d&#39;échange liquides
EP3832307A1 (fr) Support en phase solide et kit
Pichon 1Department of Analytical, Bioanalytical Sciences and Miniaturization, UMR CBI, ESPCI Paris, PSL Research University, Paris, France; 2Sorbonne University, Paris, France
DOROBANŢU et al. IMMUNOAFFINITY CHROMATOGRAPHY BASED ON ANTIGEN-TYPE NANOIMMUNOSORBENTS USED IN SEPARATION AND CHARACTERIZATION OF ANTIDICAMBA ANTIBODIES
Lu et al. A novel purity analysis method of bovine milk-derived exosomes by two-dimensional ultra-performance liquid chromatography

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: 13796654

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13796654

Country of ref document: EP

Kind code of ref document: A1