WO1998020351A1 - Assays using reference microparticles - Google Patents
Assays using reference microparticles Download PDFInfo
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- WO1998020351A1 WO1998020351A1 PCT/US1997/020045 US9720045W WO9820351A1 WO 1998020351 A1 WO1998020351 A1 WO 1998020351A1 US 9720045 W US9720045 W US 9720045W WO 9820351 A1 WO9820351 A1 WO 9820351A1
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- microparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/805—Optical property
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/806—Electrical property or magnetic property
Definitions
- the present invention relates to the field of diagnostics, and more particularly to the use of assays to detect the presence of an analyte of interest in a given fluid.
- the agglutination reaction has long been used in qualitative and quantitative assays for a wide variety of bacteria, cell-surface antigens, serum proteins and other analytes of clinical interest. Agglutination usually results from the reaction between antibodies and antigens of interest to produce aggregates which can be detected and/or measured in various ways.
- particles such as polystyrene microparticles (commonly known as "latex") are coated with a material which binds an antigen of interest, and the coated particles are mixed with a sample, such as blood or other bodily fluid. Depending upon the particular antigen, the interaction may cause the particles to agglutinate with one another, or it may retard agglutination.
- the number of agglutinated particles present after the particles are reacted with the bodily fluid will indicate the amount of the antigen present.
- the same types of reaction have been utilized for the detection of specific antibodies by the agglutination reaction caused by the addition of the corresponding antigen which binds the antibody.
- a number of particle-based immunoassays have been developed to exploit the specificity of antigen- antibody reactions, while avoiding the problems associated with conventional diagnostic methodology such as radiochemical labeling. For example, turbidimetric and nephelometric methods monitor light scattered from many particles in bulk solution. The former technique measures light transmission through a suspension of particle aggregates, whereas the latter technique directly measures scattered light in specific directions. These methods offer the advantage of not requiring a separation step; however, they have proven satisfactory only for the analysis of single analytes in a sample.
- agglutination assays have been developed using optical flow particle analyzers that sense aggregate formation or the degree of non- agglutination, by the measurement of forward scattered light using particles having different sizes. The presence and/or amount of an analyte in a sample has been determined by measuring the extent of agglutination or the number of non-reacted or non- agglutinated particles.
- 4,279,617, to Masson et al. involves the steps of mixing a liquid sample with a first particulate reagent which binds the antigen or antibody of interest to form a complex, and then adding to the mixture a second, different particulate reagent which binds to the complex to form an agglutinate, but which does not bind with the free first particulate reagent.
- the presence or the amount of the antigen or antibody is determined by assaying the non-agglutinated first or second particulate reagent.
- the '617 patent teaches that it is merely necessary to count the number of first reagent particles remaining free in the reaction mixture, and then comparing the result with the number of such particles added in the first step.
- Masson et al. coined the acronym PACIA (particle counting immunoassay) to describe agglutination assays that involve counting the number of free or non-reacted particles as an indication of the presence of an analyte in a sample.
- Masson discloses the use of an AutoCounter to measure forward light scatter from only non-agglutinated particles within a certain size range, to the exclusion of all other particles.
- the PACIA method however, has limited use, particularly due to inaccuracies caused by non-specific aggregation reactions and rheumatoid factor interference. See, Newman, et al., Ann. Clin. Biochem. .29:22-42 (1992), and Limet, et al., J. Immunol. Meth. 21:25-32 (1979).
- U.S. Patent No. 4,184,849 to Cambiaso et al. also teaches the use of optical counting system such as an AutoCounter in an agglutination assay, wherein the presence of antibodies or antigens in a liquid is detected by mixing the sample with two different particulate reagents which mutually agglutinate, but which agglutination is inhibited by the particular antibody or antigen being assayed.
- the amount of antigen or antibody present in the sample may be measured by determining the extent of agglutination or non-agglutination, the latter by simply counting the non-agglutinated particles or by using an identifying label.
- U.S. Patent No. 4,191,739 to Uzgiris, et al. is directed to an agglutination assay wherein the presence of a protein in a sample is determined by detecting multiplets of particles having a size formed by aggregation of only those particles having a first predetermined size with particles having a second predetermined size, using resistive pulse analysis.
- the method purports to enhance the accuracy of the assay by accounting for the initial distribution of non-specifically formed multiplets.
- U.S. Patent No. 4,851,329 to Cohen, et al. is directed to an agglutination assay wherein the cluster size distribution of the agglutinated particles is determined by optical pulse particle size analysis.
- a standard quantitative relationship may be established by measuring the ratio of a number of particle dimers to the number of particle monomers, or any other measure of cluster size distribution, in a given serial dilution of known concentration of analyte. These assay methods are limited to single analyte analysis.
- One aspect of the present invention is directed to an assay for determining the presence of an analyte in a sample, which involves the following steps:
- the assay method also includes the further step (d) of comparing the test value to a control value for a non-reactive sample which does not contain the analyte, in which case the presence of the analyte in the sample is then determined by calculating a change value from the test value and the control value.
- the test value is the ratio of the number of non-reacted test microparticles to the number of reference microparticles found in step (c) after the reaction.
- the control value found in step (d) is the ratio of the number of non-reacted test microparticles to the number of reference microparticles obtained using the same procedures with the non-reactive sample.
- the control value may be referred to as the "control ratio", and the test.
- the change value may be referred to as the "test ratio".
- the change value may be calculated by obtaining the difference between the control ratio and the test ratio, and dividing that difference by the control ratio. The result may be expressed as a percentage. In a more preferred embodiment, the change value is calculated by dividing the control ratio by the test ratio, and this result may also be expressed as a percentage.
- test and control values represent loss of non-agglutinated particles, rather than formation of particular agglutinates in a particular size range, inaccuracies caused by formation of larger or smaller agglutinates are substantially eliminated.
- test value and control value are computed as a relationship between the number of non- agglutinated test microparticles and the number of inert reference particles, such as the test ratio and control ratio discussed above, errors due to inaccuracies in dilution of the reaction mixture and inaccuracies in particle counting are also substantially eliminated.
- the method further provides increased resolution of each type of test particle, thus allowing for multiple simultaneous tests. It also reduces inaccuracies inherent in counting large aggregates of particles.
- the reference microparticles further enhance the accuracy and precision of the assays of the present invention because they serve as an overall on-board diagnostic for both the instrumentation and the assay method per se.
- the quality of the response generated by the reference microparticles such as histogram width or rate of microparticles detected, may be indicative of a systemic disturbance involving fluid (e.g., clogging), electrical or optical features of the system.
- Aggregation of reference microparticles, e.g., dimerization, trimerization, etc., above a predetermined arbitrary value may be indicative of non- specific binding of the test microparticles.
- the reference microparticles further enhance dilution accuracy as a function of the rate at which they are counted. They can identify or otherwise account for phenomena such as system drift.
- Another aspect of the present invention is directed to an assay for determining the presence of at least two analytes in a sample, which involves the following steps:
- This aspect of the present invention preferably includes the further step (d) of comparing the first test value to a first control value for a non-reactive sample (which does not contain the first analyte) to determine the presence of the first analyte, and comparing said second test value to a second control value .
- kits for use in an assay for determining the presence of an analyte in a sample contains the following elements:
- test microparticles having disposed thereon a binding molecule which binds the analyte
- test and the reference microparticles are present in predetermined amounts or ratios in each reaction container in said kit.
- kits for use in assays for determining the presence of multiple analytes in a sample which contain the following elements: (a) first test microparticles having disposed thereon a first binding molecule which binds the first analyte;
- the microparticles are provided in dried form, and suitably packaged to facilitate mixing with a sample.
- the predetermined amounts of the each of the dried microparticles may be separately disposed in wells of a multi-well, sealed container, the addition of a fluid sample to which causes the reconstitution of the dried microparticles and the formation of a reaction mixture, which allows a specific binding reaction between the analyte(s) and the respective binding molecule(s) to occur.
- the dried microparticles may also be provided in admixture in the form of a composition which may be reconstituted upon contact with a sample fluid.
- the assay methods of the present invention may be advantageously practiced to determine the presence of the variety of analytes, such as antibodies specific to Rubella, herpes simplex virus (HSV) , syphilis, cytomegalovirus, and toxoplasma.
- analytes such as antibodies specific to Rubella, herpes simplex virus (HSV) , syphilis, cytomegalovirus, and toxoplasma.
- HSV herpes simplex virus
- syphilis syphilis
- cytomegalovirus cytomegalovirus
- toxoplasma toxoplasma.
- samples may be assayed, including blood and other bodily fluids such as serum, plasma, saliva, cerebrospinal fluid (CSF) , urine and eluates of solids.
- Fig. 1 is cross-sectional view of a multi- well container in which a preferred embodiment of the present invention may be carried out. Best Mode of Carrying Out Invention
- sample it is meant to include biological fluids such as blood, plasma, serum, saliva, CSF, and urine, and any other type of fluid, cell or material which is suspected of containing an analyte of interest.
- cells it is meant any sample obtained from a bodily organ or other tissue, e.g. a tumor, from which a cellular suspension can be prepared.
- analyte any substance for which there exists a specific binding member (i.e., a binding molecule which binds the analyte) , or for which a specific binding member can be prepared, and which will bind the specific binding member in an assay.
- Representative analytes include antigens, haptens, antibodies, and combinations thereof, including proteins, peptides, amino acids, hormones, steroids, vitamins, drugs, nucleic acids and metabolites thereof.
- Antibodies, antigens, or nucleic acids of interest that may be detected in accordance with the present invention include any antibody, antigen, or nucleic acid which is capable of agglutinating upon contact or reaction with a binding molecule, and particularly antibodies and antigens whose presence in a biological sample is indicative of an inflammatory condition or a disease state.
- Exemplary antibodies include anti- rubella antibody, anti-HSV I antibody, anti-syphilis antibody, anti-cytomegalovirus antibody, anti- toxoplasma antibody, anti-fungal antibodies, anti- parasite antibodies, and anti-HIV antibodies.
- Exemplary antigens of interest include proteins, e.g.
- hapten a low-molecular weight, physiologically active substance, which alone is incapable of producing an antibody response in a mammal, but when bound with a substance which in itself is antigenic, is capable of producing an antibody, as well as reacting with the antibody.
- Typical haptens include drugs such as androgens, estrogens, progestogens, corticoids, thyroid hormones, physiologically active amines, medicines, and the like, and their metabolic by-products.
- binding molecule which binds the analyte it is meant a member of a specific binding pair whereby one of the molecules of the pair, via chemical or physical means, specifically binds to the second molecule, and exhibits negligible cross-reactivity with other substances.
- Typical binding molecules include antigens, antigen fragments, receptors, nucleic acids, polyclonal or monoclonal antibodies, and fragments or complexes thereof. Such binding molecules specific for a given analyte may be obtained from commercial sources or may be provided in accordance with standard procedures.
- test microparticles having disposed thereon a binding molecule which binds the analyte
- reference microparticles microparticles which are substantially inert to, i.e., non-reactable with, the analyte(s), and the binding molecule(s)
- the test microparticles may be prepared from any natural or synthetic material capable of having a binding molecule, e.g., an antigen or antibody, disposed thereon.
- Such materials include glass, acrylamide or methacrylate, nylon, microscopic oxide powders, latex polymeric materials and their magnetic derivatives, e.g., polymers of olefinically unsaturated monomers such as polystyrene, acrylonitrile and polybutadiene and derivatives and copolymers thereof (see, e.g., Bangs, L.B., Uniform Latex Particles. Seragen, Indianapolis, 1984 and U.S. 4,305,925), dextrans, cellulose and derivatives thereof, as well as natural particulate materials such as red blood cells, pollens, liposomes, and bacteria.
- the reference particles may be prepared from the same materials as the test particles, or from different materials.
- the reference particles should be substantially inert to (i.e., non-reactable with) the binding materials disposed on the test particles, as well as the analyte (s) .
- the reference particles may be rendered inert in accordance with known procedures such as chemical treatment. Preferred materials for the reference particles are listed above.
- the chemical composition and/or size of the test and reference microparticles are selected so that they are resolvable at the sensitivity level of the detector from each other in the chosen manner in which the non-reacted (i.e., non-agglutinated) test microparticles and the reference microparticles are counted.
- any other characteristic or measurable property capable of generating a uniform detector response will provide an appropriate and reliable basis upon which to select microparticles for use in the disclosed assays.
- each of the test microparticles and the reference microparticles is of a uniform size.
- test and reference particles should be of different sizes.
- the size of the carrier microparticles is in the range of from about O.l ⁇ to about 20 ⁇
- the size of the reference microparticles is in the range of from about O.l ⁇ to about 20 ⁇ .
- Assays which utilize optical counting techniques may also resolve or distinguish between test and reference microparticles based upon their respective refractive indices. It is well known that particles of different compositions will scatter light differently in different directions. See, M. Kerker, The Scattering of Light and Other Electromagnetic Radiation, Academic Press, N.Y. 1969, and U.S. Patent No. 5,369,037 to Hansen et al.
- the binding molecules may be disposed on the test microparticles in accordance with standard techniques such as physical (passive) absorption, facilitated (forced) absorption and covalent coupling. For example, the binding molecule may be covalently attached to the test microparticle surface by modification with chemical functional groups capable of protein attachment.
- U.S. Patent No. 4,181,636 teaches carboxylated latex polymers coupled to immunologically active materials through a water soluble activating agent.
- U.S. Patent No. 4,210,723 teaches shell-core latex polymer particles of 0.15-1.5 u diameter having free epoxy groups on the particle surfaces.
- U.S. Patent No. 4,264,766 is directed to latex polymers having active groups such as carboxyl and amino groups to which water soluble polyhydroxy compounds can be covalently attached, and which upon treatment with an activating agent, e.g., carbodiimide, may be covalently coupled to an immunologically active agent.
- an activating agent e.g., carbodiimide
- test microparticles are polystyrene particles
- the sulfate charge groups which stabilize the particles may be substituted by a variety of other functional groups at the particle surface, such as hydroxyl, carboxyl, amine, acrylamide, and polymeric carboxylate groups, which facilitate the disposition (e.g., coating or adsorption) of the binding molecules on the microparticle surface.
- other functional groups at the particle surface such as hydroxyl, carboxyl, amine, acrylamide, and polymeric carboxylate groups, which facilitate the disposition (e.g., coating or adsorption) of the binding molecules on the microparticle surface.
- binding molecule-coated test microparticles may also be obtained commercially.
- the test microparticles and reference microparticles desirably are packaged together, ready for use in a test.
- a preferred package is a cup as illustrated in Fig. 1.
- Cup 10 is defined by wall 12 having inner and outer surfaces 13 and 13 * , respectively.
- Cup 10 desirably is formed from an inert polymeric material such as polypropylene, polyethylene, or other suitable thermoplastic material.
- the bottom of inner surface 13 contains recesses or wells 14 and 16, in which dried test microparticles 14' and reference microparticles 16*, respectively, are contained.
- the cup is closed by removable or pierceable sealing element 19 , which may be a heat- sealed metal foil or other membrane.
- the microparticles can be placed into the wells after drying.
- the microparticles are dispensed into separate wells as slurries and then dried in situ in the wells.
- the wells maintain the microparticles separate from one another prior to drying.
- the microparticles can mix with one another, but such mixing will not evoke a reaction in the absence of a liquid medium.
- this embodiment of the present invention is not limited to the illustrated features. It is not necessary to separate the individual test particles and the referenced microparticles, e.g., to be disposed in separate, individual wells. It is even more preferred that the assay be practiced in a non- recessed or welled container.
- the microparticles may be provided in liquid form.
- a sample suspected of containing an analyte of interest is then mixed with predetermined amounts of the test microparticles having disposed thereon the binding molecule which binds the analyte, and the reference microparticles under suitable conditions to form a liquid reaction mixture and to allow a measurable reaction between the binding molecule and the analyte to occur.
- the microparticles may be added to the sample, or vice versa .
- the sample and microparticles can be mixed with a liquid diluent, typically an aqueous diluent.
- the reaction is conducted in the container illustrated in Fig. 1.
- seal 19 is pierced or separated from lip 18 of the cup, and the microparticles are reconstituted by the addition of the sample (optionally with aqueous diluent) to be tested, with appropriate diffusion or agitation such as stirring or shaking.
- the reconstituted microparticles are sonicated.
- test microparticles and the reference microparticles are counted and compared to each other to establish a test value.
- these microparticles are counted cytometrically by passing the suspension through an electronic or optical flow particle analyzer (FPA) which detects particle aggregation by monitoring the size of the individual particles or aggregates thereof as they flow individually through either an electronic or optical sensing zone, respectively.
- FPA optical flow particle analyzer
- Electronic-type FPAs typically measure the electrical impedance of the reaction mixture by passing the reaction mixture through a narrow orifice and measuring the electrical impedance through the orifice. Where the liquid in the mixture is electrically conductive and the particles are electrically nonconductive, the electrical impedance across the orifice increases when a particle or particle agglomerate passes through the orifice. The degree of increase is directly related to the size of the microparticle or agglutinate, and signals representing particles of different sizes are differentiated from one another. As discussed below, optical FPAs distinguish particle sizes and the formation of microparticle agglutinates by measuring various characteristics of light scatter of the individual microparticles. Examples of optical FPAs are disclosed in U.S. Patent Nos.
- test microparticles and reference microparticles may be distinguished on the basis of size and/or refractive index, fluorescence or absorbance.
- a narrow stream of the reaction mixture is passed through a flow cell illuminated with a beam of light from an incident light source such as a laser.
- the flow stream configuration and light beam configuration are selected so that individual microparticles or individual microparticle agglutinates pass through the light beam one after the other, and so that only one microparticle or agglutinate is in the beam at any time.
- each microparticle or agglutinate Light which is scattered, reflected or emitted by each microparticle or agglutinate as it passes through the beam of incident light is detected by appropriate sensors to yield a signal for each microparticle or agglutinate.
- the characteristics of individual signals are different for the different types of microparticles and agglutinates.
- the various types of microparticles or agglutinates are counted by categorizing each signal as representative of a particular type of microparticle or agglutinate.
- the scattered light intensity produced by each microparticle or agglutinate is directly related to the size of the particle or agglomerate, and to the refractive index of the microparticle or agglutinate. Therefore, scattered light signals within one range of intensities represent single non-agglutinated test microparticles, whereas signals in another range represent the non-agglutinated reference microparticles. By counting the signals within each range, the number of non-agglutinated test microparticles and non-agglutinated reference microparticles in the reaction mixture may be counted.
- Signals in yet another range of intensities represent agglutinates of two test microparticles (i.e., dimers)
- signals in still other ranges represent agglutinates of three or more test microparticles (i.e., trimers and multimers)
- Signals in a further range of intensities represent agglutinates of two reference microparticles (i.e., dimers)
- signals in an even further range represent agglutinates of three reference microparticles (i.e., trimers, etc., collectively referred to as self-agglutinates.
- the number of test microparticles and reference microparticles may be determined by also counting those microparticles that are in the form of self- agglutinates such as dimers and trimers.
- the signal classification and counting procedure may be performed by determining a statistical distribution such as a histogram based on a particular characteristic (e.g., pulse height) of the light scatter generated from each type of microparticle.
- the histogram serves as a statistical summary of the numbers and types of microparticles detected by the FPA, by creating a frequency distribution of microparticles.
- a histogram is created by digitizing the peak height of the pulses generated from each illuminated microparticle, using an analog-to-digital converter, and transferring each such digitized value (e.g., expressed in volts) that lies above an arbitrarily established value (e.g., to eliminate false triggering due to noise) to a computer program.
- This step may be accomplished using a suitable collection scheme such as direct memory access involving at least one data collection cycle.
- the program plots the number of signals as a function of digitized value such as voltage.
- a window or range which encompasses the expected spread in pulse height values, is defined for each type of microparticle or agglutinate.
- the collection scheme may be modified to establish a predetermined number of counts for any single window.
- different microparticle populations are identified by establishing pre-defined windows or by automatic peak seeking and classification.
- FPA operation and of particle classification and counting techniques, are merely exemplary.
- Other FPA techniques rely on signals other than scattered light, such as light emitted by fluorescent particles or light reflected by the microparticles. Still others use combinations of characteristics to differentiate signals, such as the relationship between fluorescent light emitted by each particle and the light scattered by such particle. Any of the known techniques for counting and categorizing microparticles can be utilized.
- the sum of the counts (i.e., individual data points) in the windows defining the non-reacted test microparticles and the reference microparticles is then calculated.
- the accuracy of the assays may be further enhanced by counting any such agglutinates and multiplying by the number of individual reference microparticles in each type of agglutinate.
- test value is a ratio of the number of non-reacted test microparticles to the number of reference microparticles.
- other test values based on these numbers may be used such as the difference between the number of unagglutinated test microparticles and the number of reference microparticles.
- the test value is compared to a control value for a non-reactive sample (which does not contain the analyte) , which is determined in accordance with the same procedure described above, and a change value is calculated from the test value and the control value.
- the change value is calculated as a decrease in test microparticles (relative to the original, predetermined population) , expressed as a percentage.
- a change ratio is calculated by subtracting the test ratio from the control ratio, dividing the difference by the control ratio, and multiplying by 100.
- the change ratio is calculated by dividing the control ratio by the test ratio, such that the change ratio increases with samples containing greater amounts of analyte. The change value may be compared to threshold limits generated in accordance with standard procedures, e.g., testing known positive and negative samples.
- an assay is considered positive and the presence of the analyte in the fluid sample is confirmed if the change value is above an upper threshold limit. Conversely, a change value that is below the lower threshold limit is indicative of a negative result.
- the assays of the present invention may also be used to detect a plurality of analytes in a given sample. To conduct an assay in accordance with this embodiment, it is necessary to provide test microparticles specific for each analyte to be determined, and which are resolvable as to each other and the reference microparticles in the given counting method.
- test microparticles having disposed thereon a first binding molecule which binds the first analyte
- second test microparticles having disposed thereon a second binding molecule which binds the second analyte, as well as the reference microparticles.
- Test values for each of the different non-reacted test microparticles are calculated based on the same number of reference microparticles.
- the reactants for a multi-analyte assay may be processed to a dry powder or tablet, individually, or together in admixture to form a composition.
- the reactants may be packaged in a kit which may include a container suitable for reconstituting the microparticles upon mixing with the sample and optional substances such as liquid diluents, and the like, and conducting the binding molecule-analyte reaction.
- a kit which may include a container suitable for reconstituting the microparticles upon mixing with the sample and optional substances such as liquid diluents, and the like, and conducting the binding molecule-analyte reaction.
- the peak height of scattered light produced by a microparticle or agglutinate is used as the criterion for classifying the microparticle or agglutinate as belonging to a particular type, such as an unreacted test microparticle, reference microparticle, agglutinate of test microparticles or the like.
- Light scatter signals generated by each type of particle passing through the FPA may be measured at low angles in the forward direction (i.e., from about 3 to about 7 degrees from the axis of the incident beam of light) , from about 85 to about 95 degrees (i.e., substantially side-scatter signals), or from about 95 to about 180 degrees (i.e., back scatter signals) .
- the assays out of present invention may be carried out wherein the non-reacted test microparticles and the reference microparticles are counted by measuring an electronic property of the reaction mixture such as resistive- pulse (impedance) , capacitance or resistance.
- the resistive-pulse technique consists of suspending the particles in an electrolytic solvent so that each particle which enters the pore displaces a portion of solvent resulting in a change in the pore's electrical conductivity. Since the change in conductivity is proportional to the volume of the particle, the size distribution of the sample is accumulated by sending thousands of particles through the pore. Because the measurement and subsequent signal processing is rapid, statistically meaningful distributions can be obtained in a short time, often in minutes.
- agglutinates of test microparticles it is not necessary to count agglutinates of test microparticles; information concerning the extent of agglutination is obtained from the count of non-agglutinated test microparticles remaining after reaction, taken in comparison with the number of reference microparticles.
- the count can be used as a cross-check on the assay.
- the ratio of non- agglutinated test microparticles to reference microparticles in the reacted mixture is substantially lower than the corresponding ratio in an unreacted control mixture, indicating substantial agglutination of the test microparticles, the count of agglutinated microparticles should be high. If it is not, the computer can issue an error signal.
- the three types of antigen were disrupted with detergent and sonication and diluted with carbonate/bicarbonate solution at pH 9.6 as follows: Toxoplasma antigen at 50 ⁇ g per L; rubella at 10 ⁇ g per mL; and CMV at 50 ⁇ g per mL. Washed polystyrene particles were suspended at a 2% W/V concentration in the same buffer and equal parts of particles and antigens were mixed, i.e., 1.6 ⁇ particles and Toxoplasma solution, 1.7 ⁇ particles and rubella solution and 1.9 ⁇ particles and CMV solution. The mixtures were rocked at room temperature, i.e., 2 hours for Toxoplasma and rubella and 1 hour for CMV.
- the l.l ⁇ polystyrene particles were washed and resuspended in 0.1% SDS and then rocked for 1.5 hours. The particles were then washed three times in carbonate/bicarbonate and then three times in 0.1 M glycine pH 7.0, 1% BSA, 5% sucrose and 0.1% sodium azide, and then resuspended in the same buffer at 1% W/V. A mixture of the four types of coated particles was then dried in individual cups. Each of the coated particle types was first sonicated briefly to disperse any aggregates and then a mixture of the four particle types was made.
- the following aliquots of the 1% W/V coated particle suspensions are mixed: IRP 3.5 ⁇ L; Toxoplasma 15 ⁇ L; Rubella 10 ⁇ L; and CMV 20 ⁇ L. This mixture was centrifuged to concentrate the mixture to 20 ⁇ L per cup to be filled. The mixture was then sonicated briefly to disperse any aggregates. Twenty ⁇ l of the mix were then added to each of 1500 cups.
- the cups containing the particle mixtures were then dried overnight in a 37°C vacuum oven. A metal stir bar was then added to each cup before the cup was sealed. The sealed cups were stored at room temperature until used.
- the required number of cups was placed in the CopalisTM One Immunoassay instrument (maximum number of cups is 24 per run) .
- the instrument pierced the cup seal, and added 180 ⁇ L of reaction buffer (0.5 M potassium bromide, 0.1 M glycine, pH 9.0, 1% BSA, 0.2% sodium azide, 1.5% polyethylene glycol, MW 6000-8000) .
- the instrument briefly sonicated eacj cup to disperse any aggregates and then added a sample to be tested to each cup, 20 ⁇ L of either human serum or control material. Controls consisted of samples with known presence or absence of antibodies specific for Toxoplasma gondii , rubella and
- the present invention has utility primarily as a diagnostic tool to identify infection or disease. Thus, it may be advantageously used by routineers in the relevant arts such as medical technicians to determine the presence of analytes in biological fluids.
- the present invention also accommodates fluid samples from other sources such as the environment.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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JP10521688A JP2000503404A (en) | 1996-11-04 | 1997-11-03 | Assays using reference microparticles |
AU51656/98A AU724443B2 (en) | 1996-11-04 | 1997-11-03 | Assays using reference microparticles |
EP97946500A EP0890102A4 (en) | 1996-11-04 | 1997-11-03 | Assays using reference microparticles |
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US08/743,164 US5858648A (en) | 1996-11-04 | 1996-11-04 | Assays using reference microparticles |
US08/743,164 | 1996-11-04 |
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WO1998020351A1 true WO1998020351A1 (en) | 1998-05-14 |
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PCT/US1997/020045 WO1998020351A1 (en) | 1996-11-04 | 1997-11-03 | Assays using reference microparticles |
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US (1) | US5858648A (en) |
EP (1) | EP0890102A4 (en) |
JP (1) | JP2000503404A (en) |
AU (1) | AU724443B2 (en) |
CA (1) | CA2240293A1 (en) |
WO (1) | WO1998020351A1 (en) |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001096868A2 (en) * | 2000-06-12 | 2001-12-20 | Sysmex Corporation | Immunoassay and immunoassay apparatus |
WO2001096868A3 (en) * | 2000-06-12 | 2003-01-30 | Sysmex Corp | Immunoassay and immunoassay apparatus |
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CN105738623A (en) * | 2014-12-26 | 2016-07-06 | 株式会社东芝 | Method and device for detecting sample |
US9804116B2 (en) | 2014-12-26 | 2017-10-31 | Kabushiki Kaisha Toshiba | Method and device for detecting sample |
Also Published As
Publication number | Publication date |
---|---|
AU724443B2 (en) | 2000-09-21 |
EP0890102A1 (en) | 1999-01-13 |
JP2000503404A (en) | 2000-03-21 |
US5858648A (en) | 1999-01-12 |
AU5165698A (en) | 1998-05-29 |
EP0890102A4 (en) | 2000-06-28 |
CA2240293A1 (en) | 1998-05-14 |
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