EP1880207A2 - Methodes d'immunodosage de particules competitif par microfluoroscopie - Google Patents

Methodes d'immunodosage de particules competitif par microfluoroscopie

Info

Publication number
EP1880207A2
EP1880207A2 EP06759493A EP06759493A EP1880207A2 EP 1880207 A2 EP1880207 A2 EP 1880207A2 EP 06759493 A EP06759493 A EP 06759493A EP 06759493 A EP06759493 A EP 06759493A EP 1880207 A2 EP1880207 A2 EP 1880207A2
Authority
EP
European Patent Office
Prior art keywords
sample
microparticles
analyte
mixture
well
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06759493A
Other languages
German (de)
English (en)
Other versions
EP1880207A4 (fr
Inventor
Julius Tyczkowski
Dipak Mahato
Alan Chalker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Embrex LLC
Original Assignee
Embrex LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Embrex LLC filed Critical Embrex LLC
Publication of EP1880207A2 publication Critical patent/EP1880207A2/fr
Publication of EP1880207A4 publication Critical patent/EP1880207A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K45/00—Other aviculture appliances, e.g. devices for determining whether a bird is about to lay
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K45/00—Other aviculture appliances, e.g. devices for determining whether a bird is about to lay
    • A01K45/007—Injecting or otherwise treating hatching eggs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • 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
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes

Definitions

  • the present invention relates generally to methods of measuring analytes in a sample, more particularly, to methods of measuring analytes using a competitive particle immunoassay.
  • Conventional methods of sexing birds include feather sexing, vent sexing, and DNA or blood sexing. About three-thousand (3,000) chicks can be feather-sexed per hour at a cost of about 0.7 to 2.5 cents per chick. About fifteen hundred (1 ,500) chicks can be vent-sexed per hour at a cost of about 3.6 to 4.8 cents per chick. DNA or blood sexing is performed by analyzing a small sample of blood collected from a bird.
  • the present invention provides a method of detecting the presence of an analyte in a sample, comprising: mixing together a sample, a plurality of microparticles having competitor molecules bound thereto, and a fluorescently labeled binding protein that specifically binds an analyte; placing the mixture in a receptacle for a period of time sufficient to allow the microparticles to settle; and determining the presence of the analyte in the sample via fluorescence microscopy by detecting the number of settled microparticles that are fluorescently labeled in one or more fields and comparing the number of fluorescently labeled microparticles with a predetermined value, wherein a number below the predetermined value indicates that the analyte is present above a threshold level in the sample.
  • the invention also provides a method of determining the gender of an avian embryo in an egg, comprising: mixing together a sample of allantoic fluid from an avian egg with a plurality of microparticles having competitor molecules bound thereto, and a fluorescently labeled binding protein that specifically binds to an estrogenic steroid compound; placing the mixture in a receptacle for a period of time sufficient to allow the microparticles to settle; and determining the presence of the estrogenic steroid compound in the sample by detecting fluorescently labeled settled microparticles via fluorescence microscopy, wherein the presence of the estrogenic steroid compound in the sample above a threshold amount indicates that the avian embryo is female.
  • the invention provides a method of detecting the presence of an analyte in a sample, comprising: mixing together a liquid sample, a plurality of buoyant microparticles having competitor molecules bound thereto, and a fluorescently labeled binding protein that specifically binds an analyte; placing the liquid mixture in a receptacle for a period of time sufficient to allow the buoyant microparticles to float near the surface of the liquid mixture; and determining the presence of the analyte in the sample via fluorescence microscopy by detecting the number of microparticles that are fluorescently labeled and comparison of the number of fluorescently labeled microparticles with a predetermined value, wherein a number below the predetermined value indicates that the analyte is present above a threshold amount in the liquid sample.
  • Figure 1 is a flow chart that illustrates methods of determining the presence of an analyte in a sample according to embodiments the present invention.
  • Figure 2 is a top view of an exemplary sample tray that can be used to carry out embodiments of the present invention.
  • Figure 3 is a side view of an exemplary sample tray illustrating variability in the elevation of the well bottoms.
  • Figure 4 is a flow chart that illustrates methods of determining the presence of an estrogenic steroid compound in an allantoic fluid sample from an avian egg according to embodiments of the present invention.
  • Figure 5 is a flow chart that illustrates methods of determining the presence of an analyte in a liquid sample using buoyant microparticles according to embodiments of the present invention.
  • phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y.
  • phrases such as “between about X and Y” mean “between about X and about Y.”
  • phrases such as “from about X to Y” mean “from about X to about Y.”
  • the device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
  • the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
  • first, second, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a “first” element, component, region, layer or section discussed below could also be termed a “second” element, component, region, layer or section without departing from the teachings of the present invention.
  • the sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.
  • Embodiments of the present invention provide rapid and inexpensive methods for the detection of analytes. Embodiments of the present invention are compatible with high-throughput automation involving minimal sample handling and manipulation.
  • FIG. 1 a method of detecting the presence of an analyte in a sample, according to some embodiments of the present invention is illustrated.
  • a sample of material is obtained (Block 10) and then mixed together with microparticles having competitor molecules bound thereto and with a fluorescently labeled binding protein that specifically binds an analyte (Block 20).
  • a sample may be obtained from virtually any source including organic and inorganic materials, and in particular embodiments may be a biological sample (e.g., from a subject or an avian egg).
  • exemplary biological samples encompass biological fluids and tissues, including but not limited to urine, feces, blood, plasma, serum, lymph, cerebrospinal fluid, milk, allantoic fluid, yolk, amniotic fluid, subgerminal fluid, tissue, tissue homogenate and mixtures thereof.
  • illustrative sample materials may be obtained from allantoic fluid, blood, amniotic fluid, tissue, tissue homogenate, and the like, or an extract of any of the foregoing.
  • the sample can be a soil sample, a water sample, a wastewater sample, and the like, or an extract of any of the foregoing.
  • Embodiments of the present invention are not limited to avian eggs at a particular day (e.g., day eleven) during the embryonic development period.
  • the sample is from an egg in the last half, the third quarter or the fourth quarter of in ovo incubation (i.e., of embryonic development).
  • the last half of incubation is from about the tenth to twentieth day of incubation
  • the third quarter of in ovo incubation is from about the tenth to fourteenth day of incubation
  • the fourth quarter of in ovo incubation is from about the fifteenth to twentieth day of incubation.
  • the sample is from a chicken egg at about the eighteenth or nineteenth day of in ovo incubation.
  • samples are taken from turkey eggs on about the fourteenth to twenty-seventh day of incubation, on about the fourteenth to twentieth day of incubation, on about the twenty-first to twenty- seventh day of incubation, or on about the twenty-fifth or twenty-sixth day of incubation.
  • methods according to embodiments of the present invention may be used with any type of avian egg, including, but not limited to, chicken, turkey, duck, geese, quail, pheasant, crane, parakeet, parrot eggs and the like.
  • sample, microparticles and binding protein may be performed in various steps. For example, the sample and binding protein may be mixed together first, and then the microparticles are added to the sample-binding protein mixture.
  • embodiments of the present invention are not limited to any particular order of mixing the components together.
  • the mixture may be stored for a period of time (Block 30), although this step is not necessary in all embodiments. According to some embodiments, the mixture may be stored for between about one minute and about forty-five minutes. According to other embodiments, the mixture may be stored for longer than sixty minutes (e.g., about two to six hours or about three to four hours).
  • the mixture is then placed in a receptacle for a period of time sufficient to allow the microparticles to settle out to the bottom of the mixture (Block 40).
  • the terms “settle,” “settling” and the like mean that the microparticles are substantially deposited on the bottom of the receptacle.
  • the time required for the microparticles to settle is a function of the microparticles and the sample, as would be understood by those skilled in the art. For example, a mixture with high viscosity may require a longer time period for the microparticles to settle than would a mixture having a lower viscosity.
  • the amount of mixture placed in a receptacle may be virtually any amount.
  • An exemplary amount may be between about 1 and 25, 50, 100 or 250 ⁇ .
  • the receptacle may be virtually any type of apparatus capable of retaining the mixture.
  • Exemplary receptacles include, but are not limited to, microscope slides, sample wells, and the like.
  • the receptacle is a well of a multi-well plate, e.g., a 4-well, 12-well, 24-well, 96-well, 384- well or 1536-well plate.
  • the method can be practiced to detect the presence of an analyte in one receptacle at a time or, alternatively, in multiple receptacles (e.g., within a multiwell plate).
  • one or more portions of the receptacle are generally optically transparent.
  • the bottom portion of the well is typically optically transparent so that the mixture can be observed and/or imaged through the bottom of the well.
  • the top of the well should be optically transparent.
  • One of the advantages of the present invention is that it can be practiced with conventional fluorescence microscopy, thereby avoiding the necessity for more expensive equipment associated with other techniques, such as confocal microscopy.
  • the methods of the invention do not use confocal microscopy.
  • the presence of an analyte in the sample is determined via fluorescence microscopy by detecting the fluorescently labeled binding protein bound to the settled microparticles (Block 50).
  • Analyte determination is more qualitative than quantitative in nature.
  • qualitative determinations of an estrogenic steroid compound(s) in a sample can be used to determine whether an avian embryo is male or female without the necessity of utilizing more expensive microscopy techniques in order to quantify the amount of the estrogenic steroid compound(s) in the sample.
  • the methods of the present invention do not quantify unbound fluorescence (Ae., background) and/or do not quantify bound fluorescence and/or do not determine the ratio or the difference between these two values.
  • the presence of the analyte is determined by comparison with a cutoff value. Fluorescence above the cutoff value indicates that the analyte is not present or is present at a low concentration and fluorescence below the cutoff value indicates that the analyte is present above a threshold value.
  • fluorescence above the cutoff value indicates a male embryo (i.e., low levels of the estrogenic steroid compound(s) in the embryonic fluid) and fluorescence below the cutoff value indicates a female embryo (i.e., relatively high levels of the estrogenic steroid compound(s) in the embryonic fluid).
  • the methods of the invention can be used to make other qualitative determinations, e.g., presence of contamination, a pathogen or a controlled substance above a threshold amount in a sample.
  • the invention is practiced to determine the presence of a pathogen in a sample by detecting antigen (e.g., surface antigen) or antibodies that specifically bind to the pathogen.
  • antigen e.g., surface antigen
  • the method can be practiced to detect antigen or antibodies in blood, serum or plasma or embryonic fluid from an egg (e.g., yolk, blood, amniotic fluid, allantoic fluid) so as to determine the presence of the pathogen and/or previous exposure to the pathogen by an individual subject, by a maternal parent such as a laying hen (i.e., in the case of maternal antibodies) and/or by a flock or herd of avian or animal subjects, respectively.
  • an egg e.g., yolk, blood, amniotic fluid, allantoic fluid
  • the presence of antibodies (Ae., maternal antibodies) against avian influenza can be detected in yolk from avian eggs or in blood, plasma or serum from post-hatch avian subjects to determine the presence of avian influenza and/or prior exposure to avian influenza by a subject, a laying hen (Ae., in the case of maternal antibodies) and/or the flock.
  • the presence of avian influenza surface antigen can be detected in water samples or in blood; plasma or serum from post-hatch avians or avians in ovo to determine the presence of avian influenza and/or prior exposure to avian influenza by a subject, a laying hen (Ae., in the case of maternal antibodies) and/or the flock.
  • the cutoff value can be determined by any means known in the art, and is optionally a predetermined value.
  • the cutoff value is predetermined in the sense that it is fixed, for example, based on previous determinations of the presence of known amounts of the analyte and/or previous assays.
  • the term "predetermined" value can also indicate that the method of arriving at the cutoff value is predetermined or fixed even if the particular value varies among assays for the same analyte or may even be determined for every assay run.
  • the cutoff value can be determined from a known negative and/or positive control sample.
  • the method of determining the presence of the analyte comprises counting or "scoring" the number of fluorescently labeled microparticles in one or more microscope fields by fluorescence microscopy.
  • a microparticle is counted or scored as positive for fluorescence if the level of fluorescence is above the detection setting of the microscope and/or is above the level of detection set by an image processing system that is used to evaluate the images captured by the microscope.
  • it is not necessary to quantify the amount of fluorescence (i.e., labeled binding protein) bound to each microparticle and/or to quantify the amount of unbound background fluorescence.
  • All fluorescent microparticles are scored as positive for fluorescence regardless of the amount of labeled binding protein bound thereto or the amount of labeled protein that remains unbound.
  • a number of fluorescent microparticles above a cutoff value indicates that the analyte is not present or is present at a low concentration (i.e., is not present above a threshold amount).
  • a number of fluorescent microparticles below a cutoff value indicates that the analyte is present in the sample above a threshold amount.
  • the number of fluorescent microparticles is above the cutoff value, it indicates a low level of estrogenic steroid compound(s) below a threshold amount is present and that the embryo is a male. If the number of fluorescent microparticles is below the cutoff value, it indicates a relatively high concentration of the estrogenic steroid compound(s) above a threshold amount is present and that the embryo is a female.
  • the methods of the invention can be used in assays that provide yes/no types of output (for example, to determine male/female or presence/absence of an analyte above a threshold) without the need to quantify the amount of bound fluorescence and/or without the need to quantify the amount of unbound fluorescence.
  • the method can be practiced so that a number of fluorescent microparticles falling on or near the cutoff value can be scored as indicating that the analyte is present above a threshold amount or not, depending on the end result desired. For example, in the case of gender sorting avian eggs, values falling on or very close to the cutoff value can be scored as males if it is more advantageous to erroneously classify some female birds as males rather than vice versa.
  • a distribution of the fluorescence scores for a plurality of samples is determined and the cutoff value can be set at any point within the distribution, for example, by using statistical methods known in the art such as "cluster analysis.”
  • the cutoff is set at the point where approximately 50% of the samples are below and approximately 50% of the samples are above the cutoff.
  • This embodiment is particularly suited to determinations in which there is a biphasic distribution of analyte.
  • the distribution of sample values can be determined by any method known in the art, for example, it can be based on pooling the results of a number of assay runs or can be determined internally, i.e., based on each assay run.
  • Cluster analysis refers to a variety of multivariate techniques whose purpose is to put objects into groups suggested by the data, such that objects within a cluster are similar and objects in different clusters are dissimilar. Cluster analysis does not assume that the number or membership of groups is known beforehand, although the number of groups may be specified or examples of group membership provided. Cluster analysis can be used to group either variables (typically employing Pearson correlation) or cases (typically employing the squared Euclidean distance [sum of squared distances]). Clusters may be either overlapping, disjointed, hierarchical or fuzzy. Cluster analysis may be used to analyze interval data, count data or binary data, and if different variables are used data can be standardized prior to analysis.
  • K-means clustering an iterative process in which at each step cases are grouped into the cluster with the closest center, and the cluster centers are recalculated, continuing on until no further changes occur in centers or a maximum number of iterations is reached;
  • hierarchical clustering an agglomerative process which begins with combining the closest pair of objects into a cluster and at each subsequent step, joining pairs of objects, pairs of clusters or an object in a cluster until all data are clustered together into a dendrogram (tree).
  • hierarchical clustering with Ward's minimum variance method is used to select the cutoff value.
  • the scoring of the fluorescent microparticles can be done by eye or, alternatively, with digital imaging software, which is well-known in the art (e.g., National Instruments Image Builder, NIH Image, Cognex VisionPro).
  • machine vision is used to image the fluorescent microparticles.
  • Machine vision enables automated visual inspection, and a machine vision system conventionally includes the following components: a vision processor (either host- based or embedded), a video monitor to display images, vision software to process and analyze images, a user interface, a camera, and lighting.
  • a vision processor either host- based or embedded
  • a Blob find algorithm can be used that searches for blobs of pixels that are a certain size (in number of pixels).
  • template matching algorithms can be employed. For example, a template of a single bead can be entered and the algorithm can search an image for regions that match the template.
  • Wavefront Coding techniques CMD Optics, Inc., Boulder Colorado.
  • determining the presence of an analyte in a sample via fluorescence microscopy comprises obtaining one or more images of the mixture via fluorescence microscopy imaging.
  • a portion of a receptacle containing the mixture is generally optically transparent to permit imaging. For example, if the sample is placed in a well, the bottom of the well is typically optically transparent.
  • An image of the mixture in the well is obtained at a focal plane at the bottom of the well.
  • the level of fluorescence in an image can be determined in an automated fashion using image analysis software as described above.
  • Microparticle fluorescence in the image above a cutoff value indicates a low concentration of the analyte below a threshold level in the sample.
  • microparticle fluorescence in the image below a cutoff value indicates a concentration of the analyte above a threshold level in the sample.
  • determining the presence of an analyte in a sample by fluorescence microscopy comprises the use of any technique that can determine the distance between the settled microparticles and the objective lens of the microscope and/or change that distance to the focal distance of the microscope system (i.e., an autofocus system).
  • any technique that can determine the distance between the settled microparticles and the objective lens of the microscope and/or change that distance to the focal distance of the microscope system (i.e., an autofocus system).
  • Such methods include, but are not limited to, laser-based distance-determining systems (e.g., the Displacement Sensor CD4 Series from RAMCO Innovations USA).
  • the microparticles are generally spherical beads having a diameter of between about three microns and about fifteen microns (3 ⁇ -15 ⁇ ). According to a particular embodiment, the microparticles are generally spherical beads having a diameter of less than about six microns (6 ⁇ ).
  • Exemplary microparticle materials include, but are not limited to, polystyrene, melamine, nylon, polymethyl methacrylate (PMMA), silica, gold, iron oxides and combinations thereof.
  • the binding protein can be any protein or peptide that specifically binds to the analyte.
  • Suitable examples include, but are not limited to, antibodies, receptors, ligands, substrates, antigens, transport proteins, cytochrome P450, binding proteins such as insulin-like growth factor binding proteins, and any other specific binding partner of the analyte as known in the art.
  • the selection of a suitable binding protein is generally dependent on the nature of the analyte of interest and is within the purview of those skilled in the art.
  • an antibody may be a monoclonal or polyclonal antibody including antibody fragments.
  • the antibody or antibody fragment is not limited to any particular form and can be a bispecific, humanized, chimerized antibody or antibody fragment and can further be a Fab fragment, single chain antibody, and the like.
  • Illustrative receptors include protein hormone receptors, growth factor receptors, cytokine receptors, steroid hormone receptors (e.g., estrogenic steroid compound receptors), antibody receptors, and the like.
  • Binding proteins that specifically bind estrogenic steroid compounds include but are not limited to antibodies, receptors for estrogenic steroid compounds and aromatase.
  • Receptors for estrogenic steroid compounds include estrogen receptors (for example, ER ⁇ and/or ER/?).
  • the competitor molecule can be any molecule that competes for binding of the analyte to the fluorescently labeled binding protein.
  • Competition assays are well-known in the art and are generally based on the competition between an analyte in a sample and a known molecule (the competitor molecule(s) for binding to a binding protein.
  • the competitor molecule(s) and analyte need not be the same, although in particular embodiments they are, as long as the competitor molecule binds to the binding protein and thereby reduces the binding of the analyte to the binding protein and vice versa.
  • the analyte and competitor molecules can be different as long as they both bind specifically to the antibody or receptor (although not necessarily with the same affinity or avidity) and inhibit the binding of the other to the antibody or receptor.
  • Methods of affixing or binding molecules to microparticles are well-known in the art.
  • the present invention there is competition between the competitor molecules bound to the microparticles and the analyte in the sample for binding to the fluorescently labeled binding protein. If the absence of any analyte, the labeled binding protein will bind to the competitor molecules bound to the microparticles and will be detected as fluorescence associated with the microparticles. As the concentration of analyte in the sample increases, binding of the fluorescently labeled binding protein to the microparticles will decrease due to competition for binding to the labeled binding protein. Thus, the amount of fluorescence associated with the microparticles is generally inversely proportional to the amount of analyte in the sample.
  • the fluorescent dye may include a phycobiliprotein (e.g., r-phycoerythrin, b-phycoerythrin, allophycocyanin and/or a phycobilisome), a rhodamine dye or derivative thereof, a fluorescein dye or derivatives thereof, an Alexa Fluor® dye, a BODI PY® dye, a cyanine dye or derivatives thereof (e.g., Cy-5, Cy-5.5 and Cy7), Texas Red, and any combination of the foregoing.
  • Methods of fluorescently labeling a protein or peptide for example, by binding the fluorescent dye molecules to the binding protein, are well known art and can be readily carried out by those skilled in the art.
  • the present invention does not require quantitation of unbound and/or bound fluorescence and/or determination of the difference between bound versus unbound fluorescently labeled binding protein.
  • it is not necessary to separate free label during the production of the fluorescently labeled binding protein, thereby avoiding this expensive and labor-intensive procedure, and permitting the use of less- expensive reagents.
  • analytes in a sample can be detected using methods according to embodiments of the present invention.
  • exemplary analytes in biological samples include, but are not limited to, proteins, peptides, cytokines, peptide growth factors, steroid hormones, protein hormones, pathogens (e.g., by detecting surface antigen and/or toxins), antibodies, and the like.
  • Steroid hormones include estrogenic steroid hormones including but not limited to estradiol, estradiol 17 ⁇ , estrone, estriol and conjugated derivatives thereof.
  • Particular conjugated derivatives include but are not limited to glucuronide and sulfate derivatives of estradiol, estradiol 17 ⁇ and estrone including estradiol-3-- glucuronide, estradiol-17-glucuronide and/or estrone-3-glucuronide.
  • Pathogens encompass bacteria, protozoa, yeast, fungal and viral pathogens, including but not limited to Giardia, Salmonella, Clostridia, Eimeria, E.
  • analytes from non-biological sources include, but are not limited to, environmental contaminants (e.g, fecal matter, pathogens, chemicals, protein hormones, steroid hormones including estrogenic steroid compounds, growth factors, etc.), explosives (e.g., TNT), and controlled substances (e.g., narcotics such as opiates, THC, and amphetamines or performance-enhancing substances such as steroids including androgens and growth hormone).
  • environmental contaminants e.g, fecal matter, pathogens, chemicals, protein hormones, steroid hormones including estrogenic steroid compounds, growth factors, etc.
  • explosives e.g., TNT
  • controlled substances e.g., narcotics such as opiates, THC, and amphetamines or performance-enhancing substances such as steroids including androgens and growth hormone.
  • sample tray 70 containing a plurality of sample wells 72 formed therein in various arrays is illustrated.
  • Each sample well 72 is configured to receive a sample mixture as described above.
  • Sample trays having various configurations and arrays of sample wells may be utilized in accordance with embodiments of the present invention. Sample trays may be formed from various materials and via various techniques. The present invention is not limited to use of the illustrated sample tray 70.
  • the elevations of the bottom of wells 72 in a sample tray 70 may vary.
  • the bottoms of the illustrated wells 72 have different elevations (E1 , E2, E3) relative to each other.
  • E1 , E2, E3 elevations relative to each other.
  • a median elevation of the bottom of each well is determined, and then at least two images of the mixture in a well are obtained at focal planes having respective elevations that are above and below the median elevation.
  • the images are then analyzed for microparticle fluorescence as described above.
  • This aspect of the invention is particularly useful with less-expensive sample trays in which may be more variability in the elevation of the well bottom (e.g., thermoform multiwell plates).
  • a median elevation of the bottom of each well is determined. At least two images of the mixture in the well are obtained at focal planes having respective elevations that are above and below the median elevation, and an image of the mixture in the well is obtained at a focal plane at the bottom of each well. The images are then analyzed for microparticle fluorescence as described above.
  • the invention can be practiced to measure any analyte of interest in a sample from an avian egg (e.g., pathogens, antibodies, hormones, growth factors, proteins, peptides, and the like).
  • analyte of interest in a sample from an avian egg
  • Embodiments of the present invention are particularly suitable for use in determining the gender of an embryo in an avian egg.
  • embodiments of the present invention may mitigate problems associated with current gender-identifying technologies including, but not limited to, reagent costs, disposal costs, instrument costs, instrument complexity and assay speed.
  • a sample of allantoic fluid is obtained (Block 110) and then mixed together with microparticles (e.g., 5 ⁇ polystyrene beads) having competitor molecules (e.g., an estrogenic steroid compound such as estradiol) bound thereto and with a fluorescently labeled (e.g., phycoerythrin labeled) binding protein (e.g., a monoclonal antibody) that specifically binds to an estrogenic steroid compound(s) (Block 120).
  • the mixture may be stored for a period of time (Block 130), as described above.
  • the mixture is then placed in a receptacle (e.g., a sample tray well as described above) for a period of time sufficient to allow the microparticles to settle out to the bottom of the mixture (Block 140).
  • a receptacle e.g., a sample tray well as described above
  • the presence of estrogenic steroid compound(s) in the sample is determined via fluorescence microscopy by detecting fluorescently labeled binding protein bound to the settled microparticles (Block 150). If there is a high level of estrogenic steroid compound(s) in the sample (i.e., a female egg), the fluorescently labeled binding protein will exhibit low binding to the beads. If there is a low level of estrogenic steroid compound(s) in the sample (i.e., a male egg), the fluorescently labeled antibody will exhibit high binding to the beads.
  • a number of settled microparticles that are fluorescently labeled is determined as described above and compared with a cutoff value, also as described above.
  • a number of fluorescently labeled particles below the cutoff value indicates that the estrogenic steroid compound is present above a threshold amount in the sample and that the embryo is a female.
  • a sample of allantoic fluid from the egg is mixed together with microparticles (e.g., 5 ⁇ polystyrene beads) having estradiol bound thereto and with a fluorescently labeled monoclonal antibody that specifically binds to estradiol.
  • microparticles e.g., 5 ⁇ polystyrene beads
  • a fluorescently labeled monoclonal antibody that specifically binds to estradiol.
  • the method can use a fluorescently labeled estrogen receptor to detect the concentration of estrogenic steroid compounds in the allantoic fluid sample.
  • the methods of the invention are quite sensitive and can detect as little as about 500, 250, 150, 100, 50 pg/ml or less of an estrogenic steroid compound(s) in a sample.
  • the method can further comprise treating the sample to release the underivatized compound.
  • enzymatic treatment e.g., glucuronidase and/or sulfatase
  • estradiol and estrone from estradiol and estrone glucuronide and sulfate derivatives, which are commonly found, for example, in allantoic fluid.
  • a liquid sample of material is obtained (Block 210) and then mixed together with buoyant microparticles having competitor molecules bound thereto and with a fluorescently labeled binding protein that specifically binds to an analyte (Block 220).
  • the mixture may be stored for a period of time (Block 230), as described above.
  • the mixture is then placed in a receptacle (e.g., a sample tray well as described above) for a period of time sufficient to allow the buoyant microparticles to float near the surface of the liquid mixture (Block 240).
  • the presence of an analyte in the sample is determined via fluorescence microscopy by detecting fluorescently labeled binding protein bound to the microparticles (Block 250).
  • fluorescence microscopy is conducted via the open end of a receptacle (e.g., a sample well).
  • Embodiments of the present invention may require no sample handling after the addition of reagents. Assay results may be read directly from a sample receptacle. Another advantage is that in particular embodiments the instrumentation can be relatively low maintenance equipment: a conventional microscopy system with halogen or LED illumination.

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Abstract

La présente invention concerne des méthodes de mesure d'analytes dans un échantillon par immunodosage compétitif par microfluoroscopie. Dans des modes de réalisation particuliers, l'invention concerne une méthode permettant de déterminer le genre d'un embryon aviaire in ovo par détermination de la présence d'un composé stéroïde oestrogénique dans un échantillon de fluide embryonnaire (par exemple un fluide allantoïque ou du sang) de l'ovocyte.
EP06759493A 2005-05-11 2006-05-10 Methodes d'immunodosage de particules competitif par microfluoroscopie Withdrawn EP1880207A4 (fr)

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PCT/US2006/018085 WO2006124456A2 (fr) 2005-05-11 2006-05-10 Methodes d'immunodosage de particules competitif par microfluoroscopie

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AU (1) AU2006247747A1 (fr)
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JP5564491B2 (ja) * 2008-04-29 2014-07-30 サイケメディクス コーポレイション 固相多分析物アッセイ法
JP5419012B2 (ja) * 2010-01-12 2014-02-19 独立行政法人理化学研究所 プローブが固定された基体の製造方法及び製造装置
EP2742352B1 (fr) * 2011-08-12 2018-02-28 Siemens Healthcare Diagnostics Inc. Détection de stéroïdes sexuels
AU2013297168B2 (en) 2012-07-30 2019-05-30 In Ovo Holding B.V. Gender, viability and/or developmental stage determination of avian embryos in ovo
US10032270B2 (en) 2013-03-15 2018-07-24 Richard H. Turner System and methods for the in vitro detection of particles and soluble chemical entities in body fluids
JP2017524899A (ja) * 2014-05-22 2017-08-31 ゾエティス・サービシーズ・エルエルシー 性別および他の特徴に従って選択的に卵を処理するための方法と装置
WO2018216022A1 (fr) 2017-05-25 2018-11-29 Eggxyt Ltd Procédés de détermination du sexe d'embryons aviaires dans les œufs non éclos et moyens correspondants
DE102015226490A1 (de) * 2015-12-22 2017-06-22 Universität Leipzig Verfahren zur in ovo Geschlechterbestimmung von Küken
HUE055153T2 (hu) 2016-05-24 2021-11-29 In Ovo B V Módszer és rendszer a roncsolásmentes in ovo meghatározásához a szárnyas nemnek
WO2020075104A1 (fr) * 2018-10-10 2020-04-16 Bord Bar Gholamreza Mirage d'embryon de poulet par le biais d'une injection de fluorescence et de phosphorescence à l'oiseau mâle
NL2023176B1 (en) 2019-05-22 2020-12-01 In Ovo B V Composition comprising eggs and use of such composition
EP4425180A1 (fr) * 2023-07-28 2024-09-04 Mabtech Ab Essai immunologique utilisant des microsphères magnétiques

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JPH0829422A (ja) * 1994-07-12 1996-02-02 Aisin Seiki Co Ltd 蛍光集合体を用いた検出方法
ID21888A (id) * 1996-10-04 1999-08-05 Embrex Inc Metode pemisahan "in ovo" burung
US6365339B1 (en) * 1998-09-04 2002-04-02 Bechtel Bwxt Idaho, Llc Gender determination of avian embryo
DE10054382A1 (de) * 2000-10-27 2002-05-08 Attomol Gmbh Molekulare Diagno Verfahren und Testkit zum Nachweis von Analyten in einer Probe
US7041439B2 (en) * 2001-04-17 2006-05-09 Embrex, Inc. Methods and apparatus for selectively processing eggs having identified characteristics
US7083941B2 (en) * 2001-04-23 2006-08-01 Lifesensors, Inc. Compositions and methods for gender sorting
JP2004028682A (ja) * 2002-06-24 2004-01-29 Juki Corp 目的物質の測定方法
JP2004150997A (ja) * 2002-10-31 2004-05-27 Kyogo Ito サイトカインの測定方法
JP4257513B2 (ja) * 2003-09-12 2009-04-22 学校法人早稲田大学 バイオセンシング方法

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CA2607284A1 (fr) 2006-11-23
AU2006247747A1 (en) 2006-11-23
JP2008541090A (ja) 2008-11-20
EP1880207A4 (fr) 2010-09-08
CN101218506A (zh) 2008-07-09
IL187051A0 (en) 2008-02-09
WO2006124456A2 (fr) 2006-11-23
WO2006124456A3 (fr) 2007-08-09
BRPI0608805A2 (pt) 2010-11-30
KR20080015844A (ko) 2008-02-20
MX2007014096A (es) 2008-02-07

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