EP1880207A2

EP1880207A2 - Competitive particle immunoassay methods utilizing fluorescence microscopy

Info

Publication number: EP1880207A2
Application number: EP06759493A
Authority: EP
Inventors: Julius Tyczkowski; Dipak Mahato; Alan Chalker
Original assignee: Embrex LLC
Current assignee: Embrex LLC
Priority date: 2005-05-11
Filing date: 2006-05-10
Publication date: 2008-01-23
Also published as: JP2008541090A; WO2006124456A3; CN101218506A; CA2607284A1; BRPI0608805A2; MX2007014096A; IL187051A0; EP1880207A4; AU2006247747A1; KR20080015844A; WO2006124456A2

Abstract

The present invention provides methods of measuring analytes in a sample by competitive imunoassay utilizing flurescence microscopy In particular embodiments, the invention provides a method of determining the gender of an avian embryo in ovo by determining the presence of an estrogenic steroid compound m a sample of embryonic fluid (e g , allantoic fluid or blood) from the avian egg (Fig 1).

Description

COMPETITIVE PARTICLE IMMUNOASSAY METHODS UTILIZING FLUORESCENCE MICROSCOPY

RELATED APPLICATION INFORMATION This application claims the benefit of United States Provisional Application Serial No. 60/679,717, filed May 11, 2005; the disclosure of which is incorporated herein in its entirety.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

Discrimination between poultry eggs on the basis of some observable quality is a well-known and long-used practice in the poultry industry. "Candling" is a common name for one such technique, a term which has its roots in the original practice of inspecting an egg using the light from a candle. As is known to those familiar with eggs, although egg shells appear opaque under most lighting conditions, they are in reality somewhat translucent, and when placed in front of a direct light, the contents of the egg can be observed.

In hatchery management, it may be desirable to separate birds based upon various characteristics, such as gender, diseases, genetic traits, etc. For example, it may be desirable to inoculate male birds with a particular vaccine and inoculate female birds with a different vaccine. Sex separation of birds at hatch may be important for other reasons as well. For example, turkeys are conventionally segregated by sex because of the difference in growth rate and nutritional requirements of male and female turkeys. In the layer or table egg industry, it is desirable to keep only females. In the broiler industry, it is desirable to segregate birds based on sex to gain feed efficiencies, improve processing uniformity, and reduce production costs.

Unfortunately, conventional methods of sexing birds may be expensive, labor intensive, time consuming, and typically require trained persons with specialized skills. 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.

It would be desirable to identify the sex of birds, as well as other characteristics of birds, prior to hatching. Pre-hatch sex identification could reduce costs significantly for various members of the poultry industry. Although conventional candling techniques can discriminate somewhat effectively between live and non-live eggs, these conventional candling techniques may not be able to reliably determine gender and other characteristics of unhatched birds.

SUMMARY OF THE INVENTION

As one aspect, 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.

As yet another aspect, 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.

These and other aspects of the invention are set forth in more detail in the description of the invention that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now is described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity. Broken lines illustrate optional features or operations unless specified otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprise," "comprises" and "comprising," and like terms, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y." As used herein, phrases such as "from about X to Y" mean "from about X to about Y."

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being "on", "attached" to, "connected" to, "coupled" with, "contacting", etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, "directly on", "directly attached" to, "directly connected" to, "directly coupled" with or "directly contacting" another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" to another feature may have portions that overlap or underlie the adjacent feature.

Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of "over" and "under". The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

It will be understood that, although the terms "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.

Referring now to 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. In the case of avian eggs, 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.

In other embodiments, 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. According to particular embodiments of the present invention, 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). For example, for chicken eggs, 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, and the fourth quarter of in ovo incubation is from about the fifteenth to twentieth day of incubation. In particular embodiments, the sample is from a chicken egg at about the eighteenth or nineteenth day of in ovo incubation. In other embodiments, 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. In addition, 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.

Mixing of the 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. However, 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). As used herein, 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 μ\.

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. In particular embodiments, 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).

Because the mixture is observed via fluorescence microscopy, as described below, one or more portions of the receptacle are generally optically transparent. For example, if the receptacle is a well, 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. In the situation where observation/imaging is done through the top 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. Thus, in particular embodiments, the methods of the invention do not use confocal microscopy.

As another advantage, according to the methods of the invention there is no need to separate the unbound labeled binding protein from the microparticles beyond the separation that is achieved by allowing the microparticles to settle as described above.

Once the microparticles have settled, 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, according to embodiments of the present invention, is more qualitative than quantitative in nature. For example, in particular embodiments, 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.

Further, according to the present invention, it is not necessary to quantify unbound fluorescence (i.e., background) and/or to quantify bound fluorescence and/or to determine the ratio or the difference between these two values. Thus, in particular embodiments, 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.

As used herein, terms such as "quantify," "quantitative" and like terms have the generally understood meaning in the art, e.g., relating to the determination of an amount (e.g., weight and/or concentration) of an indicated substance.

In particular embodiments, 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. For example, in methods of practicing the invention to determine the gender of an avian embryo in an egg by detecting an estrogenic steroid compound(s) in an embryonic fluid (e.g., blood or allantoic fluid), 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.

In other representative embodiments, 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. To illustrate, 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.

For example, 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.

As another example, 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. In particular embodiments, 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. Alternatively, 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. For example, the cutoff value can be determined from a known negative and/or positive control sample. In one particular embodiment, 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. According to this embodiment of the invention, 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. For example, with respect to methods of determining the gender of an avian embryo in ovo by determining the presence of an estrogenic steroid compound(s), if 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. Thus, 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.

According to one representative determination method, 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." In particular embodiments, 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. Commonly used cluster analysis techniques are (1) 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; (2) 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). In particular embodiments, hierarchical clustering with Ward's minimum variance method (distance between two clusters is the sum of squared deviations from points to centroids; objective is to minimize the within-cluster sum of squares) 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). In particular embodiments, 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. Machine vision is well understood those skilled in the art of the present invention, and need not be described further herein.

To illustrate, when using the Cognex VisionPro system, a Blob find algorithm can be used that searches for blobs of pixels that are a certain size (in number of pixels). Alternatively, 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.

Other methods of imaging may be utilized including but not limited to Wavefront Coding techniques (CMD Optics, Inc., Boulder Colorado).

According to some embodiments of the present invention, 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. Conversely, microparticle fluorescence in the image below a cutoff value indicates a concentration of the analyte above a threshold level in the sample.

Alternatively in other embodiments, rather than imaging techniques, 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). Such methods include, but are not limited to, laser-based distance-determining systems (e.g., the Displacement Sensor CD4 Series from RAMCO Innovations USA).

According to some embodiments of the present invention, the microparticles are generally spherical beads having a diameter of between about three microns and about fifteen microns (3μ -15μ). According to a particular embodiment, the microparticles are generally spherical beads having a diameter of less than about six microns (6μ). 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.

According to some embodiments of the present invention, 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, ERσ 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. For example, in the case of an antibody and/or a receptor, 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.

Thus, according to 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.

Any suitable fluorescent dye(s) can be used to label the binding protein. According to embodiments of the present invention, 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.

As another advantage, 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. Thus, in embodiments of the invention 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.

Various 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 17β, estrone, estriol and conjugated derivatives thereof. Particular conjugated derivatives include but are not limited to glucuronide and sulfate derivatives of estradiol, estradiol 17β 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. coli, Newcastle disease virus, Marek's disease virus, infectious bronchitis virus, influenza (e.g., avian influenza), Bursal disease virus, and the like. Exemplary analytes from non-biological sources (i.e., not from a subject) 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).

Referring to Fig. 2, an exemplary 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.

As illustrated in Fig. 3, 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. To compensate for this, 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).

According to other embodiments of the present invention, 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.

In representative embodiments, 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). Embodiments of the present invention are particularly suitable for use in determining the gender of an embryo in an avian egg. Moreover, 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.

Referring to Fig. 4, a method of determining the gender of an avian embryo in ovo according to some embodiments of the present invention is illustrated. A sample of allantoic fluid is obtained (Block 110) and then mixed together with microparticles (e.g., 5μ 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). Once the microparticles have settled, 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.

In particular methods of determining the gender of an avian embryo in ovo according to the present invention, 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.

In representative embodiments of determining the gender of an avian embryo in an egg, a sample of allantoic fluid from the egg is mixed together with microparticles (e.g., 5μ polystyrene beads) having estradiol bound thereto and with a fluorescently labeled monoclonal antibody that specifically binds to estradiol. Alternatively, the method can use a fluorescently labeled estrogen receptor to detect the concentration of estrogenic steroid compounds in the allantoic fluid sample.

According to particular embodiments, 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. When the analyte is an estrogenic steroid compound(s), the method can further comprise treating the sample to release the underivatized compound. For example, enzymatic treatment (e.g., glucuronidase and/or sulfatase) can be used to release estradiol and estrone from estradiol and estrone glucuronide and sulfate derivatives, which are commonly found, for example, in allantoic fluid.

Referring to Fig. 5, a method of detecting the presence of an analyte in a sample, according to some embodiments of the present invention is illustrated. 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). Once the microparticles have floated near the surface, 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). In this case, 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.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. 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.

2. The method of Claim 1 , wherein the receptacle comprises a microscope slide.

3. The method of Claim 1 , wherein the receptacle comprises a well, wherein a bottom portion of the well is optically transparent, and wherein the microparticles settle to the well bottom.

4. The method of Claim 1 , further comprising storing the mixture for a period of time prior to placing the mixture in a receptacle.

5. The method of Claim 4, wherein storing comprises storing the mixture for between about one minute and about forty-five minutes (1 min. - 45 min.).

6. The method of Claim 4, wherein storing comprises storing the mixture for more than about sixty minutes (60 min.).

7. The method of Claim 1 , wherein the sample is a biological sample.

8. The method of Claim 7, wherein the biological sample is from an avian egg.

9. The method of Claim 7, further comprising obtaining the biological sample prior to the mixing step.

10. The method of Claim 7, wherein the biological sample comprises material selected from the group consisting of allantoic fluid, yolk, urine, feces, blood, plasma, serum, amniotic fluid, subgerminal fluid, lymph, cerebrospinal fluid, milk, tissue, tissue homogenate, and a combination thereof.

11. The method of Claim 1 , wherein the microparticles comprise generally spherical beads having a diameter of less than about six microns (6μ).

12. The method of Claim 1 , wherein the microparticles comprise generally spherical beads having a diameter of between about three microns and about fifteen microns (3μ -15μ).

13. The method of Claim 1 , wherein the microparticles comprise material selected from the group consisting of polystyrene, melamine, nylon, polymethyl methacrylate (PMMA), silica, gold, and iron oxides.

14. The method of Claim 1 , wherein the binding protein is an antibody.

15. The method of Claim 14, wherein the antibody is a monoclonal antibody.

16. The method of Claim 14, wherein the antibody is a polyclonal antibody.

17. The method of Claim 1 , wherein the binding protein is a receptor.

18. The method of Claim 1 , wherein the fluorescently labeled binding protein is labeled with a fluorescent dye selected from the group consisting of a phycobiliprotein, a rhodamine dye or derivative thereof, a fluorescein dye or derivative thereof, an Alexa Fluor® dye, a BODIPY® dye, a cyanine dye or derivative thereof, and Texas Red.

19. The method of Claim 1 , wherein the analyte is selected from the group consisting of a protein, a peptide, a peptide growth factor, a cytokine, a steroid hormone, a protein hormone, an antibody, a pathogen, an environmental contaminant, an explosive, and an illegal substance.

20. The method of Claim 1 , wherein the analyte is an estrogenic steroid compound selected from the group consisting of estradiol, estrone, a conjugated derivative of estradiol, a conjugated derivative of estrone, and a combination thereof.

21. The method of Claim 1 , wherein the mixing step comprises first mixing the sample and binding protein together, and subsequently adding the microparticles to the sample-binding protein mixture.

22. The method of Claim 1 , wherein the step of determining the presence of the analyte in the sample via fluorescence microscopy comprises obtaining one or more images of the mixture via fluorescence microscopy imaging.

23. The method of Claim 3, wherein determining the presence of the analyte in the sample via fluorescence microscopy comprises: determining a median elevation of the bottom of the well; and obtaining at least two images of the mixture in the well at focal planes having respective elevations that are above and below the median elevation.

24. The method of Claim 3, wherein determining the presence of the analyte in the sample via fluorescence microscopy comprises: determining a median elevation of the bottom of the well; obtaining at least two images of the mixture in the well at focal planes having respective elevations that are above and below the median elevation; and obtaining an image of the mixture in the well at a focal plane at the bottom of the well.

25. The method of Claim 3, wherein determining the presence of the analyte in the sample via fluorescence microscopy comprises obtaining an image of the mixture in the well at a focal plane at the bottom of the well.

26. 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.

27. The method of claim 26, wherein a number of settled microparticles that are fluorescently labeled is determined and compared with a predetermined value, wherein a number below the predetermined value indicates that the estrogenic steroid compound is present above a threshold amount in the sample.

28. The method of Claim 26, wherein the binding protein is an antibody.

29. The method of Claim 28, wherein the binding protein is a receptor for an estrogenic steroid compound.