EP1313810A2

EP1313810A2 - The use of a multiple dye system to enhance the spectral properties of dyed microparticles in an immunoassay

Info

Publication number: EP1313810A2
Application number: EP01966239A
Authority: EP
Inventors: Victor A. Manneh
Original assignee: SenDx Medical Inc
Current assignee: SenDx Medical Inc
Priority date: 2000-08-24
Filing date: 2001-08-22
Publication date: 2003-05-28
Also published as: CA2423112A1; WO2002016506A3; WO2002016506A2; AU2001286771A1

Abstract

This invention relates to infrared dyes and their use in microparticles. Specifically, combinations of at least two dyes that absorb in the infrared region of light are used to provide a broad absorption spectrum. The infrared dye composition is incorporated in or on microparticles. The microparticles may subsequently be used in determining the presence of a ligand of interest or physical property of a sample.

Description

The Use of a Multiple Dye System to Enhance the Spectral Properties of Dyed Microparticles in an Immunoassay

Field The present invention relates generally to the use of multiple dye system for preparing particles with enhanced spectral properties. These particles will be used in whole blood agglutination assays. Background

Publications and other reference materials referred to herein are incorporated herein by this reference.

For the most part diagnostic assays of physiological fluids or biological samples for one or more analytes have required clinical laboratory determinations although there has been an increasing focus on being able to carry out assay determinations in the doctor's office and in the home. Numerous systems have been developed in efforts to try to address the various problems associated with analyses carried out in the clinical laboratory.

In many instances blood is a source of a sample to diagnose a patient's health or to monitor the efficacy of drugs that have been administered to the patient. Blood as a source for the determination of these parameters has many deficiencies when used directly or even when diluted with buffer. These deficiencies include: rapid coagulation, the presence of a large number of light absorbing and fluorescent substances, variations in composition, susceptibility to changes in relation to reagents used in assays, and variations In the presence or absence of oxygen. These properties complicate the use of blood as a sample for diagnostic purposes. Various techniques have been employed to avoid these problems, e.g., high dilution, addition of anticoagulants, separation of blood into plasma and its cellular components, and the like. During such manipulations great care must be taken to avoid lysis of red blood cells to avoid the release of hemoglobin, which can interfere with diagnostic assays. Despite the problems associated with the use of blood as the sample medium, in many instances, blood is the only source that provides the information of interest. Therefore, identifying ways of using whole blood, while diminishing the interference from its constituents is highly desirable. There is, therefore, substantial interest in devising new approaches for using and manipulating blood for diagnostic purposes.

The use of microparticles or beads in assays would benefit from the ability to monitor agglutination at or near the isobestic point of hemoglobin, i.e., about 800 nm. However, the incorporation of dyes into the microparticles may encounter many problems. For example, the dyes may require harsh organic solvents to be brought into solution. These same solvents, if added to the microparticles, would melt them. Thus, there is a need for a method of incorporating or loading dyes into microparticles that leaves the microparticles intact and will not affect the surface characteristics of the microparticles.

Summary

The present invention is directed to a composition of matter useful in blood agglutination assays.

In one aspect of the invention there is a dye composition comprising at least two dyes. Preferably, the dyes absorb light in the infrared. The dyes each possess a distinct maximum absorption spectrum in the range of from about 750 nm to about 830 nm. The distinct maximum absorption spectrum for each dye is different from the other dyes in the composition. In a preferred embodiment, the dyes have a difference in their respective maximum absorption spectra of no greater than 70 nm. In a second aspect of the invention there are provided microparticles comprising an infrared dye mixture with a flat maximum absorption spectrum ranging from about 750 nm to about 830 nm.

In a further aspect of the mvention there is provided a method of preparing microparticles incorporating at least two dyes. Preferably, the dyes absorb light in the infrared. In one embodiment, the dyes are dissolved in an appropriate solvent resulting in a dye solution. The dye solution is added to a solution of undyed particles for a time sufficient to allow the dyes to be incorporated into the microparticles. The microparticles are subsequently purified by appropriate means. In a second embodiment, the microparticles are dyed in a sequential manner. The microparticles are dyed with a first infrared dye and purified. Subsequently, the microparticles that have incorporated the first infrared dye are dyed with a second infrared dye and purified.

In another aspect of the invention there is provided an improved agglutination assay of the type in which a sample is mixed with an aggregating system comprising microparticles to which are bound a binding component and any additional reagents necessary for the aggregation of said particles. The improvement in the agglutination assay resides in the utilization of microparticles incorporating at least two infrared dyes with a flat maximum absorption spectrum.

Description of the Figures Figure 1 is a graph of the absorption spectrum of the individual dyes (i.e., SI and Cl) and the mixture of dyes (SI plus Cl).

Figure 2 is the chemical structure of a preferred infrared absorbing dye, Cl . The chemical name for the Cl dye is Copper(II)-tetra-tert-butyl- tetrakis(dimethylamino)-29H-31H-phthalocyanine when the central metal ion is copper.

Figure 3 is the chemical structure of a preferred infrared absorbing dye, SI. The chemical name for the SI dye is Vanadyl-tetra-tert-butyl- tetrakis(dimethylamino)-29H-3 lH-phthalocyanine when the central ion is vanadate.

Figure 4 is the general chemical structure for substituted phthalocyanines. R¹, R , R³ and R⁴ are independently selected from the group consisting of H, CH₃, and short chain alkyl (C₂- C₅). Detailed Description The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications referred to herein are expressly incorporated by reference.

Definitions

As used herein, the following terms or abbreviations, whether used in the singular or plural, will have the meanings indicated: The term "Binding Component" is a molecule, agent, substance or compound, and the like, involved in particle aggregation.

"Microparticle" as used herein refers to a particle composed of any convenient composition, such as, for example, bioglass, organic polymers, e.g., polyacrylonitrile, polystyrene, polycarbonate, polymethacrylate, combinations thereof, or the like, or other material than can be made to absorb in the infrared by the incorporation of infrared absorbing dyes. Also, there will be many regions in the visual region in which the particle composition will be substantially transparent, as distinguished from carbon or colloid microparticles that do not transmit light over the visual and infrared region. However, when the light used for the determination is in the range of about 800±10 nm, then the microparticles may absorb over a broad wavelength range and may be opaque, such as colloidal carbon or metal particles, carbon dyed latex particles, Ti0₂ containing particles, and the like. The particles generally have a diameter smaller than about 25 μm, more preferably smaller than about 10 μm. The particles may preferably have a diameter size in the range of about 1-10 μm, more preferably from about 2-8 μm. For the most part, the particle composition without the dye will not absorb significantly in the infrared region of interest, usually absorbing less than about 25% of the total light absorbed in that region compared to a particle that has incorporated the infrared absorbing dyes. The terms microparticle, particle and bead are used interchangeably herein. "Infrared" means electromagnetic radiation at wavelengths longer than the red end of visible light and shorter than microwaves. Visible light has wavelengths of about 400 - 700 nm. Thus, light with a wavelength longer than about 700 nm and shorter than about 1000 nm is generally recognized as infrared. "Infrared absorbing dye" means a dye that will absorb light in the infrared.

The dyes will absorb light in the range of about 750 nm to about 830 nm, particularly in the range of about 780nm to about 820 nm. Dyes include, but are not limited to, bacteriochlorin, bacteriochlorophytin, meropolymethine dyes, benzoannulenes, vinylogous porphorins, polymethine dyes, cyanines and mercyanines and the like. Specific dyes of interest include dyes of the class of phthalocyanines, napthalocyanines, metaled napthalocyanine dyes, and modified natural bacterochlorines. Specific examples of dyes include Copper(II)-tetra-tert- butyl-tetrakis(dimethylamino)-29H-31H-phthalocyanine and a second dye is Vanadyl-tetra-tert-butyl-tetrakis(dimethylamino)-29H-31 H-phthalocyanine as shown in Figures 2 and 3. Although shown with copper or vanadate as the metal ion in Figures 2 and 3, respectively, other metal ions may be used. Preferred dyes may have various metal ions, such as, for example, iron, magnesium, zinc, nickel, cobalt, and copper, in the center held in by four coordination bonds. The dyes may be incorporated directly into the microparticle itself, through polymerization or passive adsorption. Alternatively, the dyes may be linked to the bead in combination with a binding component, such that the dyes do not leach from the surface.

"Linking Component" means a molecule capable of being attached to the particle via covalent bonding on one site and a second site available to attach or link another molecule. The molecule that is linked via the linking component to the particle may be a protein, ligand of interest or a dye. More than one class of molecules may be linked to the particle via the linking component. For example, a protein and a dye, two dyes and the like may be linked to the particle.

"Multiple dye microparticles" means a microparticle into which have been incorporated at least two absorbing dyes, preferably infrared. The incorporated dyes will have been chosen such that the combination of dyes utilized will have a flat absorption spectrum in the region of interest. The terms microparticle, bead and particle are used interchangeably herein.

In order to perform whole blood immunoassays without on-line separation of red blood cells it is important to use a detection system in the near infrared (IR) region close to 805 nm. At this wavelength referred to as the isobestic point the absorption of oxyhemoglobin and reduced hemoglobin is identical. Therefore, at the isobestic point the oxygenation state of the blood does not contribute to the imprecision in the assay. In accordance with the subject invention, the character of a sample is determined by combining the sample with particles which absorb in the infrared, where the rate and/or extent of aggregation of the particles is modulated by the character of the sample. Normally, the character of the sample will be associated with the presence and amount of a component of interest. In other situations the character may be associated with the activity of the sample in relation to its effect on an event, e.g., clotting. Other reagents may also be present, depending upon the nature of the component and the protocol of the assay. After sufficient time for any aggregation to occur, the assay mixture is illuminated with infrared light and the change in absorption determined. The value obtained may be compared to a standard for a quantitative determination of the amount of component in the sample.

The method is flexible and can be used to assess several parameters, including the presence of a component in the sample, the character of the sample, or even the combined effect of several components in the sample on the penultimate agglutimetric reaction. However, for the purposes of the description, the description will refer to the component of interest and its functional activity, rather than the character of the sample.

Any sample can be used. The method is particularly advantageous for those samples that contain entities which might interfere with spectrophotometric determination at wavelengths other than infrared. The sample may be any physiological fluid, environmental fluid, processing fluid, effluent or influx. The subject methodology finds immediate application with physiological fluids, more particularly blood or plasma. By using the subject methodology, less care in preparing the plasma is required, since released hemoglobin and other metal or nonmetal porphyrins will have reduced interference in the methodology.

Particular samples may include, as indicated, blood, plasma, cerebrospinal fluid, saliva, urine, and the like, more particularly, those samples which have an interfering substance which absorbs or emits light in the range of about 300nm to about 700nm Therefore, the subject methodology finds use, particularly with whole blood, by employing infrared light, where the signal from the sample is not significantly affected by the variations in absorption resulting from changes in oxygenation of the sample.

The sample may be subject to pre-treatment, depending upon the nature of the sample. Generally, the sample may be used without significant sample manipulation preparation. However, preparation may include dilution, concentration, extraction, chromatography, electrophoresis, and the like. Desirably, there will be minimum sample preparation. In accordance with the subject invention, whole blood may be used, which is diluted less than about 10- fold, usually diluted less than about 5-fold, preferably less than about 1-fold, and, more preferably less than about 0.5-fold. The blood will be modified to prevent coagulation, by using various anti-coagulants. Anti-coagulants include citrate, heparin, thrombin inhibitors, and the like. Conveniently, citrate is employed in a small volume in relation to the volume of the whole blood sample, generally less than about 25% v/v, usually less than about 10% v/v, and may be less than about 1% v/v.

Illustrative compounds include drugs of abuse, such as tetrahydrocannabinol, morphine, heroin, ***e, and methamphetamine, barbiturates, tranquilizers and antidepressants, e.g. librium, diazepams, and tricyclics, diphenylhydantoin, immunosuppressants, e.g. cyclosporine and FK506, cardiovascular drugs, e.g. digitonin, nitroglycerin, etc., clotting inhibitors, e.g. Warfarin, heparin, low molecular weight heparin, aggregation activators, e.g iso- TRAP, analgesics, anaesthetics, antihypertensive reagents, e.g. renin inhibitors, lipid A, toxins, Ilb-IIIa antagonists including compounds such as RGD and KGD- based peptidomimetics, one subset of these compounds includes Searle compound 54701, Searle compound 57101, ReoPro (Centacor), Integrilin (Cor), Roche Ro440-3888, Hoechst S 1197, Merck L-738,167, TAK 029 (Tap Holdings), Boehringer Ingelheim BIBU 52ZW.

The compounds may be macromolecular compounds, which will have a molecular weight of at least about 5kD, more usually at least lOkD and generally less than about 1 million kD, more usually less than about 600,000 kD. These compounds may include various natural or synthetic polymers, such as polypeptides, nucleic acids, polysaccharides, lignins, polylipids, combinations, such as mucopolysaccharides, glycoproteins, sulfonated polysaccharides, lipopolysaccharides, and the like. Illustrative macromolecular compounds include insulin, blood factors, e.g.

Factor V, VI, VII, VIIIc, VIII vw, IX, X, X, XI and XII, soluble histocompatibility antigens, e.g. sHLA, β-amyloid, HIV gpl20 and p41, CD3, CD28, B7, glutamic acid dehydrogenase, tissue plasminogen activator, colony stimulating factors: G, M, and GM, porphyrins, complement proteins, bacterial and fungal proteins, protista proteins, viral proteins).

Finally, the component of interest may be a combination of one or more different categories of compounds, such as viruses, organelles, such as mitochondria, prokaryotes and eukaryotes, such as bacteria, fungi, protista, chlamydia, mammalian cells, such as platelets, cancer cells, e.g. leukemia and lymphoma, and the like. Viruses of interest include HIV, HTLV, papilloma virus, herpes virus, hepatitis viruses, adenoviruses, rhinoviruses, and the like.

The particles which are employed will generally be smaller than about 50 μ, more usually smaller than about 25 μ, usually being at least about 0.1 μ, preferably from about 1-10 μ, more preferably from about 2-8 μ. The composition of the particle may be any convenient composition, such as bioglas, organic polymers, e.g. polyacrylonitrile, polystyrene, polycarbonate, polymethacrylate, combinations thereof, or the like, or other material which absorbs in the infrared or can be made to do so with infrared absorbing dyes. For the most part, the particle composition without the dye will not absorb significantly in the infrared region of interest, usually absorbing less than about 25% of the total light absorbed in that region compare to the particle doped with the infrared absorbing dye. Also, there will be many regions in the visual region in which the particle composition will be substantially transparent, as distinguished from carbon or colloid particles which do not transmit light over the visual and infrared region. However, when the light used for the determination 2 in the range of about 800 ± 10 nm, then the particles may absorb over a broad wavelength range and may be opaque, such as colloidal carbon or metal particles, carbon dyed latex particles, Ti0₂ containing particles, and the like. Usually, at least 50 weight %, preferably at least about 75 weight %, will be of a size or diameter within the range indicated. Use of the wavelength range 800 ± 10 nm is of particular interest. In whole blood or even in diluted blood, to accurately measure a ligand of interest whether by transmission, scattering or fluorescence, the measurement should be under conditions where changes in the character of the blood sample do not influence the result. Desirably, the measurement wavelength is chosen such that there is no effect from the absorption characteristics of the rod cell hemoglobin. The absorption characteristics of hemoglobin depend upon whether it is reduced hemoglobin (Hb) or oxyhemoglobin (Hb0₂). Only at a few specific wavelengths, referred to as isobestic points, are the absorption characteristics of Hb and Hb0₂ the same. If a measurement is made in whole blood other than at an isobestic point, the reproducibility of the measurement requires that the ration of Hb-Hb0₂ be the same for each measurement. Otherwise, the change in background absorption of the measurement wavelength will cause an artificial change in the level of the received signal. The optimal isobestic point from an instrumentation standpoint is at 805 nm, since this is the isobestic point with the lowest absorption. The only measurement requirement for particles that are to be used in a whole blood assay is that they have either strong absorbance or fluorescence at 800 ± 10 nm. The manner in which this is achieved, using dyed particles or opaque particles, does not affect the operability of the subject invention.

The particles may be modified in a variety of ways. The particles may be chemically activated by having functional groups present on the surface of the particles, or be coated with a compound, e.g. protein, which may serve to substantially irreversibly (under the conditions of the processing and assay) bind to the dye. The coating compound may be the binding component, which will be involved in the aggregation of the particles, or other compound, usually being a protein. Alternatively, depending on the nature of the particles, the particles may not have chemically active groups, but rather provide binding by adsorption. In addition, infrared absorbing dyes which are stable under the conditions of formation of the particles, e.g. extrusion, may be mixed with the polymer prior to particle formation and the particle formed with the dye distributed throughout the particle.

A binding component is bound to the particle surface that provides for aggregation of the particles. The aggregation may be a result of the interaction of the binding component with the same or a different component on another particle or with an agent in the medium, which agent may be the compound of interest, a member of a specific binding pair, or a catalytic agent, e.g. an enzyme, which interacts, usually reacts, with the binding component to modify the binding component to cause aggregation. The specific binding pair will usually consist of the binding component and the component of interest, a reagent that competes with the component of interest for binding to the binding component, or a reagent that binds to the component of interest. These may be illustrated by: an antigen and antibodies to the antigen as the binding component; a dimer of the component of interest binding to Fab as the binding component; and fibrinogen and thrombin. The binding component bound to the surface will vary widely as to its nature, depending upon the component of interest and the protocol which is employed. The binding component may be a small molecule, as small molecules were described previously, or a higher molecular weight molecule, or even in some instances, combinations such as virus or cell fragments or intact viruses or cells. Any of the compounds previously discussed may serve as the binding component. In one group of assays employing specific binding pairs for aggregation, where one is interested in binding to naturally occurring or synthetic components of interests, specific receptors may be employed, such as naturally occurring receptors, e.g. enzymes, lectins, surface membrane proteins, etc., or antibodies, either antisera or monoclonal antibodies. In other assays, one may employ one member of a naturally occurring specific binding pair, such as fibrin (prepared in the assay medium from, fibrinogen), that can bind to various proteins. The use of fibrinogen in conjunction with the platelet protein GPIIb/IIIa will be discussed in greater detail subsequently. Various integrins may be used in conjunction with various adhesive proteins and vice versa. Antibodies may be assayed, where one could have the epitope which binds to the antibody bound to the particle. The epitope could be present as a small molecule, such as a synthetic organic molecule or an oligopeptide, or could be a polyepitopic molecule where one or more antibodies in the medium bind to the various epitopes of the antigen. Where the component of interest is monoepitopic, one may employ as a reagent a dimer or higher order of the monoepitopic compound, which reagent will serve to crosslink. Where nucleic acids are concerned, one may provide for oligonucleotides bound to the particles that bind to different sites on the strand of interest. Alternatively, one may prepare a strand that has repeats of the same sequence as a reagent that can compete with the nucleic acid component of interest, so as to cross-link the particles. Also, as indicated previously, one may use combinations of naturally occurring specific binding pairs, such as CD4 and gpl20, P-selectin and L-selectin and their correlative homing receptors, CD3 and MHC, integrin adhesion receptors and their adhesive ligands, growth receptors and growth factors, cytokines and their cell surface receptors. As already alluded to, other reagents may be present. Particularly, where a monoepitopic compound is the component of interest. With a monoepitopic compound, where specific binding pairs are involved for cross-linking, in order to get cross-linking, one will need at least a dimer of such component or a mimetic analog thereof. Usually the reagent will have not more than about 5 of the cross- linking epitopes present. With this polyepitopic reagent in the absence of the component of interest, there will be aggregation. Increasing amounts of the component of interest will reduce the amount and rate of aggregation. Alternatively, one may use multibinding receptors that will crossreact with the binding component and the component of interest. The component of interest will fill the binding sites of the receptors, preventing crosslinking, again reducing the amount and rate of aggregation. In this way, one can detect monoepitopic compounds.

One may assay compounds which activate or inhibit catalysts, whether naturally occurring or synthetic, particularly enzymes which can activate the binding component to cause aggregation, e.g. thrombin and fibrinogen, casein or fibronectin and transamidases, etc.

Other reagents which may be present include substances which may modify the component of interest, such as activating a particular cellular function, upregulating or downregulating expression of a particular surface membrane protein, competing with the component of interest for the binding component on the particle, blocking binding by a substance which competes with the component of interest for binding to the binding component present on the particle, e.g. alleles, isotypes, etc., in order to avoid false positives associated with the competitive substance, and the like. These additional reagents will be selected in accordance with the nature of the component of interest, the protocol of the assay, and the like.

In each case the amount of the other reagents will be determined empirically. If one is using a polyepitopic reagent for competition with a monoepitopic component of interest, the reagent will be selected to give the highest sensitivity over the dynamic range of interest. This may vary from less to greater than stoichiometric and may be readily determined. One varies the concentration of the reagent with the lowest anticipated concentration of the component of interest and the highest anticipated concentration of the component of interest. One may choose one or two intermediate points to determine the greatest sensitivity at these intermediate points. By graphing the results, one can determine the concentration of the reagent which will provide the most sensitive result over the dynamic range, a higher response being required at the lower part of the range than at the higher part of the range. In carrying out the subject method, the sample, which may have been subject to prior preparation, is combined with the necessary reagents with mild agitation. Various conventional procedures for preparation of the sample may be employed. Depending upon the nature of the sample, the sample may be protected from the atmosphere or be in contact with the atmosphere. Protection from the atmosphere may be achieved by employing sealed containers, where the containers are sealed with a septum, and the sample is introduced by means of a needle through the septum, with the receiving container being evacuated or containing an inert gas.

Conveniently, relatively large or small samples may be taken and only small aliquots used in the assays. Thus, the assay volume may be from about 5μl to 500μl usually from about 25μl to 250μl, and conveniently from about 25μl to 150μl.

The sample is combined with the particles and any other reagents under conditions where the particles are rapidly dispersed throughout the sample. The particles and other reagents may be present as a dry composition or dispersed with a small amount of liquid. Usually the volume of the particles and reagents will be not more than about an equal volume to the sample, preferably less than about 50% of the sample volume, more preferably less than about 25% of the volume sample. A reading is taken at 0 time or some convenient interval to obtain a 0 value, which is the value in the absence of significant aggregation. Readings may then be taken from time to time. Automated instrumentation can be employed to mix the sample with the particles and any other reagents, heat the assay mixture to the desired temperature, carry out any necessary operations during the assay, monitor the assay mixture to take the first reading, for example, when the sample has reached the desired temperature, take additional readings, as appropriate, and then calculate the assay result for the sample, with any other descriptive information associated with the sample. The concentration of particles in the medium will be optimized in accordance with the nature of the component of interest, the dynamic range of the component of interest, the nature of the sample medium, and the like. The amount of the particles may be determined empirically. Generally, the aggregation media absorption coefficient should be at least twice the absorption coefficient of the sample, preferably at least three times, more preferably at least about four times, and may be ten times or more. In the absence of any substantial background in the infrared, there is no effective ratio.

The time for mixing may be varied widely, usually being at least about 1 sec. and not more than about 5 min., usually not more than about 2 min., and preferably for about 5 sec. to 1 min. The particular manner of agitation is not critical to this invention, so long as it provides for thorough mixing, without preventing the formation of aggregates. If desired, mild agitation may be maintained during the course of the assay, again insuring that there is homogenous distribution of the particles and any other particulate matter, while insuring that aggregation is not impeded.

The temperature for the assay may be varied widely, depending upon the nature of the component of interest. Conveniently, ambient temperatures may be employed, although elevated temperatures that can be controlled and maintained are preferred. Where nucleic acids are involved, the temperature may be elevated, so as to enhance the degree of stringency of hybridization. Thus, the temperature may vary from about 15-90°C, where with other than nucleic acids, the temperature will generally vary from about 25-40°C. Usually, with nucleic acids the temperature will generally be in the range from about 20-90°C, more usually in the range of about 30-85°C. With nucleic acids, stringencies may be achieved by using salts, organic solvents, and the like. However, with other than nucleic acids, normally the only addition will be a buffer, if at all, where the buffer will range from about 5-10 in pH, more usually from about 6-9, and at a concentration of from about 10-500 mM, more usually from about 25-250 mM. The time for the assay will vary depending upon the manner in which the measurement is taken. Where zero time is carefully controlled, one may take one or two measurements at different time intervals to determine the absolute infrared transmission at the time intervals or determine the rate of formation of the aggregation. Alternatively, one may take a plurality of measurements over the time course of the assay and analyze the slope beginning at a fixed time from the time of mixing. The data may be analyzed by any convenient means, particularly using an algorithm that can manipulate the data in relation to calibrators and/or controls. The total time of the readings from the zero time (time of mixing), may range from about 10 sec. to 5 min., more usually about 30 sec. to 5 min., and preferably about 30 sec. to 2 min.

Usually, the result will be compared to a calibrator, which may be performed concomitantly or have been performed previously or may be provided as a standard curve. The calibrators will vary depending upon the nature of the component of interest. Samples having known amounts of the component of interest may be prepared and performed in the assay and the results charted so as to be able to translate the measurement obtained with the sample to the standard. In some instances controls will be used, where the base value may vary depending on the source of the sample. The particular control will be associated with the sample and the component of interest. The subject invention finds particular application in conjunction with the determination of platelets and platelet function. Platelet adhesive function is an extreme test of the subject methodology in the sensitivity to various factors of platelets. First, platelets can be activated to varying degrees by the physical manipulation of blood and by the release of factors elicited when blood vessels are damaged on venipuncture. Second, is the effect of the time between drawing the blood and testing: for techniques requiring plasma this time is necessarily longer and therefore less desirable. In addition, the mechanical action needed to separate plasma from red cells can activate the platelets to varying degrees and also result in variable cell recovery. When measuring the effectiveness of inhibitors of platelet adhesive function, there is the issue of the relatively fast off- rate. The rapid off-rate of an inhibitor means that its effect will be underestimated if the sample is diluted prior to assay, and in some cases, even if the dilution occurs during the assay. Also, since platelet aggregation, fibrinogen binding and, in some cases, inhibitor binding are calcium dependent, the choice of anticoagulant may be important in accurately determining platelet function levels. Variability in absorption, metabolism, etc., of the anti-IIb/IIIa drugs may lead to large differences in pharmacokinetics. The subject methodology allows for a rapid determination of the effective level of inhibition of platelet adhesive function and/or the ability of platelets to aggregate. This information, permits accurate decision making on timing and frequency of dosing with anti-platelet drugs aimed at inhibiting clot formation.

Where platelet aggregation is to be measured, because of interest in the platelet status of an individual, which may be the natural status or the status resulting from administration of a drug, the sample will be in effect whole blood, which has been subjected to less than about 50%, preferably less than about 20% dilution.

The whole blood is drawn desirably in the substantial absence of air. Conveniently, a Vacutainer is employed for capturing and holding the blood sample. The Vacutainer desirably includes a small volume of a solution of sodium citrate generally in the range of about 3-5% sodium citrate having a volume in the range of about 0.05-0.5 ml. The blood sample should be obtained from an extremity free of peripheral venous infusions. Conveniently, the needle should be at least about 21 gauge. The first tube which is withdrawn is discarded, the second tube or subsequent tubes being used. Mild agitation, simply gently inverting the Vacutainer is employed to insure the mixture of the anticoagulant with the sample. The sample in each container may range from about 1-10 ml, more usually from about 1-8 ml, conveniently from about 1-5 ml. The sample should not be stored for an unduly long period, generally storage before the assay should not exceed 1 hour.

A small portion of the sample may now be transferred to a cuvette far measurement. Generally, the volume may range from about 25-500μl more usually from about 75-250μl . Conveniently the cuvette contains the particles which have been coated with fibrinogen. The platelets may be activated by the addition of various agents, which serve to activate the platelets. Illustrative agents include iso-TRAP (See U.S. Patent No. 5,455,228), TRAP, ADP, collagen, thrombin, ristocetin, or any combination thereof. Any convenient activator may be employed. Iso-TRAP is employed at a concentration in the range of about 1 to 5, preferably about 2 μmol/L. The activating agent may be incorporated with the bead reagent to which the blood sample is added. The beads and other reagents may be dry, so as to not dilute the sample, although in some instances a small amount of liquid may be present, desirably less than about 25% of the volume of the sample. The particles are conveniently polystyrene particles of a size in the range of about 2 to 8 microns, which have been coated with fibrinogen by passive adsorption or by covalent linkage in accordance with conventional ways. Generally, the weight of fibrinogen to the weight of particles will be in the range of about 1:1000 to 1:10. The amount of beads should provide a ratio between the agglutination media absorption coefficient and whole blood absorption coefficient of greater than about 4: 1 at 800 nm, generally not more than about 10:1 at 800 nm. The optimal absorption ratio may be achieved by configuring both the light-absorbing characteristics of the agglutination media and the concentration of the agglutination media in the assay sample.

The mixture of citrated whole blood, particles and activating agent is gently agitated to insure homogeneity and the mild agitation is continued so as to maintain homogeneity without impeding aggregation formation. The temperature for the medium will be maintained at a constant temperature. After a short time, generally under 30 sec, usually under about 10 sec, readings are begun by illuminating the sample with light at about 800 nm. The total time for the readings will generally be under about 5 min, usually under 3 min, where, when one is determining the rate of change to determine the change in slope with time, the number of data points per second may range from about 0.01 to 100, more usually from about 1 to 50. Thus one may take readings at constant intervals of from about 0.01 sec to about 1.5 sec, usually from about 0.02 sec to 1 sec. Otherwise, data points may be taken as convenient, there being at least one data point, more usually at least two data points, frequently not fewer than 1 per minute. The change in transmissibility with time is determined by any convenient technique, conveniently employing a conventional spectrophotometric detector for the infrared.

As a control, blood containing a coagulation inhibitor is treated with a reagent, conveniently an antibody or fragments thereof which completely neutralizes the inhibitor. It is found that the baseline far platelet activity can vary widely with time for a patient and between patients, so that by neutralizing the inhibitor one can get the baseline value for platelet activity for the particular sample. This may then be used for comparison with the results obtained with the sample to determine the platelet activity in the presence of the inhibitor. Illustrative compounds which find use as coagulation inhibitors include Searle compounds 5470 IB and 57101 A, which are potent Ilb/IIIa function blocking drugs. Antisera or monoclonal antibodies or binding fragments thereof can be used to block the action of the inhibitor and the resulting uninhibited sample used for the control. The control would be used in the same way as the sample and could be run concurrently so that the same conditions are employed for the control as are employed for the sample.

The amount of inhibitor neutralizing agent which is employed will provide for complete neutralization of the inhibitor and excesses may be used, usually not more than about five-fold excess of the maximum concentration of the inhibitor, as anticipated from the dose given to the patient, without significant dilution of the sample, usually less than about 50% dilution, usually less than about 25% dilution. Where antiserum or fragment thereof is used, high affinity titers should be used, desirably 50% maximum binding should be at a titer ranging from at least about 1:10,000 and maybe 1:100,000 or more, preferably at least about 1 :25,000. By employing this technique, one may establish a baseline rate, or any other Ilb/IIIa functional test baseline parameter.

As a calibrator, the subject particles without fibrinogen, serving as surrogate platelets, can be combined with thrombin in an appropriate buffered medium. This reagent may then be combined with the particles coated with fibrinogen in the same manner as the sample. If desired, the buffered medium may be augmented with-blood constituents, such as red blood cells, serum albumin, immunoglobulins, or other significant constituent of blood, which does not participate in the aggregation of the particles. A convenient buffer medium is a HEPES-sodium chloride buffer comprising from l-5mg/ml protein e.g. BSA. If one wishes, this technique as modified may also be used in evaluating the activity of various proteins in the blood, which are associated with thrombin activation and aggregation. These proteins include FV, FVIIIc, FIX, and other factors previously described. Thus, by adding a blood sample to a prepared mixture, which may be dry and require reconstitution or a concentrated solution, which contains the necessary blood factors for coagulation, except for the factor to be measured, and the fibrinogen coated particles, a change in the rate of aggregation will be related to the activity of the factor of interest in the sample. The result may then be related to calibrators having known amounts of the factor of interest. After the sample has been combined with the reagents, desirably it will be heated to a temperature above room temperature, but below interference with the assay, so as to insure that the temperature can be controlled without adversely affecting the assay result. Desirably, the temperature should be at least 25°, preferably in the range of 30-40°, more preferably about 37°C. While not essential, it is preferable that the sample be mildly agitated during the incubation and measurement of the aggregation. For agitation, metal beads may be moved up and down, magnetic beads oscillated at a slow rate or other means employed for mild agitation.

The sample volume can be quite small, usually being not less than about lOμl, more usually not less than about 25μl, and desirably not more than about lml, preferably not more than about 500μl, more preferably not more than about 250μl.

For convenience, kits can be provided comprising some or all of the reagents that find use in the subject invention. The kit will have the particles for use with the component of interest. In addition, neutralizing immunoglobulins may be provided for removing inhibitor in a sample to serve as a control. Calibrators may be provided providing particles with the appropriate binding component mixed with any other reagents associated with the assay and, if desired, a source of the component of interest, either in measured amounts or in bulk. For platelet aggregation, a combination of thrombin and uncoated particles may be supplied. Also, of convenience, would be Vacutainers comprising 0.1- lml of 1-5M anticoagulant, e.g. sodium citrate. Of particular interest for the kit is a container containing one or more of the appropriate reagents in order to reduce the manipulative steps for the assay. For example, a container, such as a cuvette, may be provided containing the particles and, as appropriate, other reagents for the assay.

The particles are loaded with a dye that absorbs in the infrared. Various dyes have been reported as useful in the absorption range. These dyes include bacteriochlorin, bacteriochlorophytin, meropolymethine dyes, benzoannulenes, vinylogous porphorins, polymethine dyes, cyanines and merocyanines, and the like. Specific dyes of interest are Copper(II)-tetra-tert-butyl- tetrakis(dimethylamino)-29H-3 lH-phthalocyanine and Vanadyl-tetra-tert-butyl- tetrakis(dimethylamino)-29H-3 lH-phthalocyanine. The particular dye which is selected is one of convenience, availability, stability, compatibility with the particle and the like. These dyes may be incorporated directly into the particle itself, through polymerization or passive adsorption. The dyes may be loaded individually (i.e., sequentially) or in combination (i.e., simultaneously). Alternatively, the dyes may be linked to the bead in combination with the linking component, such that they do not leach from the surface. Irrespective of the loading method used, the conditions are such that the particle surface is unaffected with respect to the ability to agglutinate under appropriate conditions.

The dyes will absorb light in the range of about 750 nm - 900 nm, particularly in the range of about 750 - 850 nm. For samples with high levels of red blood cells, the light will be at about 800 nm ± 10 nm, which is the isobestic point for oxyhemoglobin and reduced hemoglobin. The amount of dye employed with the particles will vary with the extinction coefficient of the dye in the light range of interest, the required sensitivity of the assay, the size of the particles, the mode of binding of the dye to the particles, compatibility of the dye with the particle matrix, and the like. Usually, loading will be in the range of about 1 to 20 weight percent, more usually 5 to 15 weight percent.

Dyes which find a particular use in the present invention are phthalocyanines. Phthalocyanines have a planar structure, with a cyclic 16- member ring system consisting of four bridged pyrole rings and four nitrogen atoms at the four meso positions. The outer four ethylene units are essentially pure double bonds, and they can be replaced by rings to give tetrabenzoporphorine. The 16 center ring system which contains 18p electrons is the basic chromophore of porphorine. This unique structure gives the phthalocyanines its unique spectral properties in the near infrared. Metal free phthalocyanines absorb at approximately 700 nm (e=162,000). The metal complexes shift the absorption to either shorter or longer wavelength, most metals shift the absorption to a much shorter wavelength, but some, such as lead absorb at much longer wavelength than the metal free phthalocyanines

The complexes formed between transition metals and phthalocyanines (metollophthalocyanines and Metallonaphthalocyanines) are chemically very stable to light and heat. They are formed by condensation of opthalodinitriles in the presence of an appropriate metal. Some of the metals used in the formation of the metalophthalocyanines besides the copper (Cu) and the Vanadium (V) are magnesium (Mg), zinc (Zn), and cobalt (Co). We successfully prepared carboxylated microparticles with a flat absorption maximum by incorporating multiple dyes that have distinct absorption maximum close to 805 nm. This resulted in a flat maximum absorption spectrum across a broad range wavelength from 780-820 nm. The current invention describes a multiple dye system to be utilized in the dye loading of particles ranging in size from 0.1 to 10 microns. The resultant particles will have the desired spectral properties while their conjugating ability remains intact.

Examples The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. Example 1 Preparation of Dye Solution Dyes are prepared to a final concentration of 10 mg/mL. In this example a dye solution of two dyes is prepared. For each x mL of carboxylated-modified polystyrene latex particles (CML) at 4% solids an amount of dye equal to 0.25x mL is required. Thus, for example, 10 mL CML requires 2.5 mL Combination Dye Solution with final concentration of 10.0 mg/mL.

To prepare a Combination Dye Solution, dissolve 7.5 mg SI dye in 0.4375 mL CH₂C1₂ and 0.8125 mL ethanol to yield a final concentration of 6.0 mg/mL. Dissolve 17.5 mg Cl dye in 0.4375 mL CH₂C1₂ and 0.8125 mL ethanol to yield a final concentration of 14.0 mg/mL. Sonicate both dye solutions for approximately 5 minutes to ensure complete dissolution is achieved. Combine the two dye solutions together into a single container (1:1 volume ratio of the 6.0 mg/mL SI dye and 14.0 mg/mL Cl dye). Mix well by vortexing. Protect from light until ready to use. Store in a glass container at all times.

To prepare a Single Dye Solution, dissolve either dye as initially described for the Combination Dye Solution above. The initial 1.25 mL dye solution is then diluted with 0.4375 mL CH₂C1₂ and 0.8125 ethanol to a final volume of 2.5 mL and a final concentration of 3 mg/mL (SI dye) or 7 mg/mL (Cl dye).

Example 2 Particles with two dyes incorporated Carboxylated-modified polystyrene latex particles (CML) of various sizes ranging from 0.1-10 micrometers can be effectively dye loaded with a dye combination that is soluble in a solution mixture comprising of dichloromethanate and ethanol at a temperature range of 50-55°C. Have x amount of the CML (in mL) at 4.0%) solids in an appropriate size 3-neck round bottom flask equipped with a magnetic stir bar. Attach a condenser to the middle neck and cap the other two off with a stopper. Begin stirring the CML solution to create a vortex. Add 0.5x of ethanol and 0.002x of 2-(2-ethoxyethoxy)ethyl acetate (both in mL) to the CML solution and stir. The dye solution is introduced into the vigorously stirring CML latex solution at a controlled rate of 0.125x mL/hr for a maximum period of

2 hours for sufficiently large batches (> 10 mL @ 4% solids). The Combination Dye Solution must be placed in a glass container at all times, i.e., a glass syringe, during the entire addition process to prevent evaporation from occurring.

The resulting dyed latex particles are subsequently subjected to purification utilizing methods of solvent evaporation, filtration with glass fiber, and centrifugation. After the addition process is complete, dilute the dyed CML solution with 0.5x mL 0.1N NaOH. Filter the resultant solution through glass wool into an appropriate size single neck round bottom flask. Wash the glass wool with 0.1N NaOH (a few milliliters is sufficient). Attach the flask to a rotavapor and remove approximately ^γA of the total volume in the dyed CML solution. Transfer the solution to appropriate size vial(s). Centrifuge the solution at 2,000 rpm for approximately 10 minutes. Remove the supernatant and resuspend the pellet with a volume of 0. IN NaOH equal to the removed supernatant. Repeat the centrifugation and resuspension steps until the sample is clean - a homogeneous pellet and a completely clear supernatant are obtained. The final purified product should be free of major aggregation without the use of any surfactants, covalently conjugatable, and contain a sufficient level of absorbance intensity.

Example 3 Sequential loading of IR absorbing dyes Carboxylated-modified polystyrene latex particles (CML) of various sizes ranging from 0.1-10 micrometers can be effectively dye loaded with a dye combination that is soluble in a solution mixture comprising of dichloromethante and ethanol at a temperature range of 50-55°C. Each dye is prepared essentially as described above in Example 1. The dye to be loaded first is prepared such the amount of dye loaded is greater than the final concentration of the respective dye in the particles (as a finished product). The first dye is loaded at a higher concentration to compensate for leaching of the first dye during the loading of the second dye. The first dye solution is introduced into the vigorously stirring CML latex solution at as controlled rate for a maximum period of 2 hours for sufficiently large batches (> 10 mL @ 4% solids) yielding a dyed latex particle solution. The resulting dyed latex particles are subsequently subjected to purification utilizing methods of solvent evaporation, filtration with glass fiber, and centrifugation as described in Example 2. The second dye solution is introduced into the vigorously stirring dyed latex particles at as controlled rate for a maximum period of 2 hours for sufficiently large batches (> 10 mL @ 4% solids) yielding a multiple dyed particle solution. The resulting multiple dyed latex particles are subsequently subjected to purification utilizing methods of solvent evaporation, filtration with glass fiber, and centrifugation as previously described. The final purified product should be free of major aggregation without the use of any surfactants, covalently conjugatable, and contain a sufficient level of absorbance intensity.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. An infrared dye composition comprising at least two infrared dyes wherein the dyes each have a distinct maximum absorption spectrum in a range of from about 750 nm to about 830 nm that differs from the other, and the composition has a flat maximum absorption spectrum said range.

2. The infrared dye composition of Claim 1 comprising two dyes wherein one dye is Copper(II)-tetra-tert-butyl-tetrakis(dimethylamino)-29H-31H- phthalocyanine and a second dye is Vanadyl-tetra-tert-butyl- tetrakis(dimethylamino)-29H-3 lH-phthalocyanine.

3. Microparticles comprising an infrared dye mixture with a flat maximum absorption spectrum ranging from about 750 nm to about 830 nm.

4. The microparticles of Claim 3 wherein the maximum absorption spectrum is from about 780 nm to about 820 nm.

5. The microparticles of Claim 3 wherein the maximum absorption spectrum peak is about 805.

6. The microparticles of Claim 3 wherein the particles have a diameter of from about 0.1 micron to about 10 microns.

7. A method of preparing multiple dye microparticles, said method comprising:

(a) dissolving in an appropriate medium a first infrared dye to yield a first dye solution;

(b) combining the first dye solution with microparticles under conditions that allow for the first dye to be incorporated into the microparticles to yield a first dyed microparticles;

(c) purifying the first dyed microparticles;

(d) dissolving in an appropriate medium a second infrared dye to yield a second dye solution;

(e) combining the first dyed microparticles with the second dye solution under conditions that allow for the second dye to be incorporated into the first dyed microparticles to yield a multiple dye microparticles; and (f) purifying the multiple dye microparticles.

8. A method of preparing multiple dye microparticles, said method comprising:

(a) preparing a dye mixture comprising at least two infrared dyes wherein each dye has a distinct maximum absorption spectrum ranging from about 750 nm to about 830 nm different from the other dye;

(b) combining the dye mixture with microparticles under conditions that allow for the dyes to be incorporated into the microparticles; and (c) purifying the multiple dye microparticles prior to use.

9. The method of Claim 8 wherein step b further comprises conditions that allow for the dyes to be incorporated into the microparticles without effecting the surface of the particles.

10. An improved agglutination assay of the type in which a sample is mixed with an aggregating system comprising' microparticles to which are bound a binding component and any additional reagents necessary for the aggregation of said particles, wherein the improvement comprises:

(a) dying a first quantity of microparticles with a first infrared dye having a maximum absorption spectrum ranging from about 750 nm to about 830 nm;

(b) dying a second quantity of microparticles with a second infrared dye having a maximum absorption spectrum that is different from the first infrared dye and ranging from about 750 nm to about 830 nm;

(c) mixing the first and second quantities of dyed microparticles prior to use in the agglutination assay;

(d) utilizing said dyed particles in said assay.