Patent Publication Number: US-2017370938-A9

Title: Prostate antigen standards and uses thereof

Description:
RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62/139,365, filed Mar. 27, 2015, and entitled “PROSTATE ANTIGEN STANDARDS AND USES THEREOF,” which is incorporated herein by reference. 
    
    
     BACKGROUND OF INVENTION 
     Elevated blood levels of total prostate-specific antigen (PSA) are associated with prostate-related disorders, including prostate cancer. There is considerable evidence that measuring levels of isoforms of PSA separately, along with measurement of human kallikrein 2 (hK2), leads to improved predictions relating to the presence of prostate cancer in a subject. Generally, assays to measure elevated levels of prostate antigens, such as PSA isoforms and hK2, utilize antigen standard curves to quantify prostate antigen levels. 
     SUMMARY OF INVENTION 
     Aspects of the disclosure relate to a recognition that currently used standards for prostate assays require the use of large quantities of prostate antigens, which is not cost-effective. Furthermore, the wide range of concentrations encompassed by standards in currently used assays may lead to imprecise quantification of antigen concentration because the standard ranges are not properly aligned with the quantities of antigen present in typical clinical sample. Accordingly, it has been recognized that there is a need for new standards that are cost-effective and provide increased accuracy of antigen level quantification. 
     Many assays to quantify prostate antigen levels rely on comparison of detected antigen signal to a set of values obtained from a set of standard antigen concentrations. However, currently used assays are not cost-effective because they require large amounts of prostate antigen standards. For example, some prostate antigen tests rely on standards having a maximum concentration of up to 350 ng/mL of iP SA or up to 22 ng/mL of hK2 per assay. Furthermore, the large prostate antigen range encompassed by currently used antigen standards may lead to inaccurate quantification of prostate antigen levels in a sample and increase the risk of an incorrect diagnosis. The instant disclosure solves these problems by providing cost-effective compositions and methods for improved quantification of prostate antigen levels. 
     Accordingly, in one aspect, the disclosure provides a method for quantifying levels of a prostate antigen. In some embodiments, the method involves performing an immunoassay to detect presence of a prostate antigen in a sample; and quantifying levels of the prostate antigen detected in the sample in relation to a minimal set of informative prostate antigen standards detected using the same immunoassay. In some embodiments, a minimal set of informative prostate antigen standards comprises at least two titers containing predetermined quantities of prostate antigens, in which the largest titer of the set is between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects and ii) 75-fold higher than that blood prostate antigen concentration. In some embodiments, a quantity of the smallest non-zero titer of the set is below the limit of quantification of the immunoassay. In some embodiments, a target population of subjects consists of men, the median age of which is in a range of 60 to 70 years. In some embodiments, a target population of subjects consists of men, the lower quartile age of which is in a range of 55 to 65 years. In some embodiments, a target population of subjects consists of men, the upper quartile age of which is in a range of 65 to 75 years. In some embodiments, a prostate antigen is selected from the group consisting of: total prostate specific antigen (tPSA), free prostate specific antigen (fPSA), intact prostate specific antigen (iPSA), and human kallikrein 2 (hK2). In some embodiments, a prostate antigen is iPSA or hK2. In some embodiments, a prostate antigen is intact prostate specific antigen (iPSA). In some embodiments, the smallest non-zero titer of the set is 0.025 ng/mL. In some embodiments, the largest titer of the set is 15 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of the following titers of prostate antigen standard: 0 ng/mL, 0.025 ng/mL, 0.089 ng/mL, 0.322 ng/mL, 1.157 ng/mL, 4.167 ng/mL, and 15 ng/mL. In some embodiments, the prostate antigen is hK2. In some embodiments, the smallest non-zero titer of the set is 0.002 ng/mL. In some embodiments, the largest titer of the set is 8 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of the following titers of prostate antigen standard: 0 ng/mL, 0.002 ng/mL, 0.011 ng/mL, 0.055 ng/mL, 0.290 ng/mL, 1.524 ng/mL, and 8 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of seven titers of prostate antigen standard. In some embodiments, each intermediate titer of prostate antigen standard is equidistant from the titer above and below it on a logarithmic scale. In some embodiments, the immunoassay is selected from the group consisting of DELFIA®, Enzyme Linked Immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, western blot assay, and immunoprecipitation assay (IPA). In some embodiments, the immunoassay is a DELFIA®. In some embodiments, the immunoassay is an ELISA. In some embodiments, a sample is obtained from a human subject. In some embodiments, a sample is a prostate tissue biopsy. In some embodiments, a sample is a blood or blood plasma sample. 
     In some aspects, the disclosure provides a prostate antigen detection kit for quantifying levels of a prostate antigen, the kit comprising a minimal set of informative prostate antigen standards for quantifying levels of a prostate antigen in a sample using an immunoassay, in which each prostate antigen standard is present in a container as a solution. In some embodiments, a minimal set of informative prostate antigen standards comprises at least two titers containing predetermined quantities of prostate antigens, in which the largest titer of the set is between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects and ii) 75-fold higher than that blood prostate antigen concentration. In some embodiments, the quantity of the smallest non-zero titer of the set is below the limit of quantification of the immunoassay. In some embodiments, a prostate antigen is selected from the group consisting of: total prostate specific antigen (tPSA), free prostate specific antigen (fPSA), intact prostate specific antigen (iPSA), and human kallikrein 2 (hK2). In some embodiments, the prostate antigen is iPSA or hK2. In some embodiments, a prostate antigen is intact prostate specific antigen (iPSA). In some embodiments, the smallest non-zero titer of the set is 0.025 ng/mL. In some embodiments, the largest titer of the set is 15 ng/mL. In some embodiments, the minimal set of informative prostate antigen standards consists of the following titers of prostate antigen standard: 0 ng/mL, 0.025 ng/mL, 0.089 ng/mL, 0.322 ng/mL, 1.157 ng/mL, 4.167 ng/mL, and 15 ng/mL. In some embodiments, a prostate antigen is hK2. In some embodiments, the smallest non-zero titer of the set is 0.002 ng/mL. In some embodiments, the largest titer of the set is 8 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of the following titers of prostate antigen standard: 0 ng/mL, 0.002 ng/mL, 0.011 ng/mL, 0.055 ng/mL, 0.290 ng/mL, 1.524 ng/mL, and 8 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of seven titers of prostate antigen standard. In some embodiments, each intermediate titer of prostate antigen standard is equidistant from the titer above and below it on a logarithmic scale. 
     In some aspects, the disclosure relates to a method for preparing prostate antigen standards for quantifying levels of a prostate antigen in a sample, the method comprising: (i) obtaining an antigen associated with prostate cancer; (ii) preparing a stock solution of the antigen in a suitable buffer, in which the stock solution is at a titer between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects and ii) 75-fold higher than that blood prostate antigen concentration; and (iii) preparing a minimal set of prostate antigen standards by serial dilution of the stock solution, such that each intermediate titer of prostate antigen standard is equidistant from the titer above and below it on a logarithmic scale. In some embodiments, a target population of subjects consists of men, the median age of which is in a range of 60 to 70 years. In some embodiments, a target population of subjects consists of men, the lower quartile age of which is in a range of 55 to 65 years. In some embodiments, a target population of subjects consists of men, the upper quartile age of which is in a range of 65 to 75 years. In some embodiments, the prostate antigen is selected from the group consisting of: total prostate specific antigen (tPSA), free prostate specific antigen (fPSA), intact prostate specific antigen (iPSA), and human kallikrein 2 (hK2). In some embodiments, the prostate antigen is iPSA or hK2. In some embodiments, the prostate antigen is intact prostate specific antigen (iPSA). In some embodiments, the smallest non-zero titer of the set is 0.025 ng/mL. In some embodiments, the largest titer of the set is 15 ng/mL. In some embodiments, the minimal set of informative prostate antigen standards consists of the following titers of prostate antigen standard: 0 ng/mL, 0.025 ng/mL, 0.089 ng/mL, 0.322 ng/mL, 1.157 ng/mL, 4.167 ng/mL, and 15 ng/mL. In some embodiments, the prostate antigen is hK2. In some embodiments, the smallest non-zero titer of the set is 0.002 ng/mL. In some embodiments, the largest titer of the set is 8 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of the following titers of prostate antigen standard: 0 ng/mL, 0.002 ng/mL, 0.011 ng/mL, 0.055 ng/mL, 0.290 ng/mL, 1.524 ng/mL, and 8 ng/mL. In some embodiments, a minimal set of informative prostate antigen standards consists of seven titers of prostate antigen standard. In some embodiments, each intermediate titer of prostate antigen standard is equidistant from the titer above and below it on a logarithmic scale. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a non-limiting box and whisker plot of hK2 levels (ng/mL) of a population of subjects (N=1012). 
         FIG. 2  shows a non-limiting box and whisker plot of iPSA levels (ng/mL) of a population of subjects (N=1012). 
     
    
    
     DETAILED DESCRIPTION OF DISCLOSURE 
     Aspects of the disclosure provide standards for quantifying the level of a prostate antigen in a sample. The standards described in the disclosure represent an improvement over currently used standards because they are cost effective (e.g., require a lower amount of prostate antigen) and provide sets of values that more closely aligned with clinically-relevant ranges of prostate antigens. The disclosure relates, in part, to a recognition that calibrating the titer range to a minimal set of informative prostate antigen standards significantly reduces cost while simultaneously providing increased accuracy of prostate level quantification. Thus, in some aspects, the disclosure provides improved methods for quantifying levels of a prostate antigen. The methods typically involve performing an immunoassay to detect presence of a prostate antigen in a sample and quantifying levels of the prostate antigen detected in the sample in relation to a minimal set of informative prostate antigen standards detected using the same immunoassay. 
     As used herein, a “minimal set of informative prostate antigen standards” is a set of prostate antigen standards for a particular immunoassay that includes at least two titers containing predetermined quantities of prostate antigens, in which the quantity of the smallest titer of the set is below the limit of quantification of the immunoassay and in which the quantity of the largest titer of the set is between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects (e.g., men suspected of having prostate cancer or of being in need for a biopsy) and ii) 100-fold higher than that blood prostate antigen concentration. In some embodiments, the largest titer of the set is between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects and ii) 75-fold higher than that blood prostate antigen concentration. In some embodiments, the largest titer of the set is between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects and ii) 50-fold higher than that blood prostate antigen concentration. In some embodiments, the largest titer of the set is between i) the blood prostate antigen concentration of the upper quartile of a target population of subjects and ii) 25-fold higher than that blood prostate antigen concentration. 
     In some embodiments, the quantity of the smallest non-zero titer of the set is below the limit of quantification of the immunoassay. As used herein, the term “limit of quantification” refers to the smallest amount of material detectable and quantifiable by using the immunoassay. In some embodiments, the limit of quantification is the smallest amount of material detectable and quantifiable with a coefficient of variation of less than 25%, less than 15%, less than 5%, or less than 1% by using the immunoassay. 
     In some embodiments, the quantity of the smallest non-zero titer of the set is below the limit of detection of the immunoassay. As used herein, the term “limitation of detection” refers to the smallest amount of material detectable but not necessarily quantifiable by using the immunoassay. 
     Prostate Antigen Standards 
     In some aspects, the disclosure provides prostate antigen standards for quantifying the level of a prostate antigen in a sample. As used herein, a “prostate antigen standard” is a substantially homogenous preparation of a prostate antigen suitable for establishing a reference against which an unknown quantity of the prostate antigen can be measured. In some embodiments, a prostate antigen standard comprises a preparation of prostate antigen that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% pure. However, in some embodiments, the standard comprises a homogenous preparation of prostate antigen in a medium (e.g., as a solution) similar to that of the sample to be tested (e.g., a blood, serum, urine or plasma sample). For example, the prostate antigen standard may be spiked into a blood, serum, urine or plasma sample that is otherwise free of the prostate antigen. In this way, the background of the prostate antigen standard and the unknown sample are similar or the same, which can improve accuracy of detection and quantification. 
     In some embodiments, concentration curves are used to determine the concentration of a particular antigen in a sample. For example, a concentration curve may be produced by plotting relative fluorescence units (RFU) versus a plurality of a known concentrations of an antigen (e.g. a set of antigen concentrations). The RFU value obtained from a sample having an unknown antigen concentration may then be compared to the concentration curve to determine the antigen concentration of the sample. 
     In some embodiments, the standard material comprises a set of antigen titers. As used herein, the term “set of antigens” refers to a plurality of discrete antigen titers. In some embodiments, each antigen titer of a set is housed in a different container. In some embodiments, a set of antigens standards is produced from a master stock having an antigen titer higher than the maximum antigen concentration of the set. In some embodiments, a set of antigens standards is produced from a master stock by serially diluting the stock. In some embodiments, a set of antigens is produced from a master stock having an antigen concentration at the maximum antigen concentration of the set. In some embodiments, a set of antigens is between 1 and 20 antigen titers, 5 and 15 antigen titers, or 8 and 12 antigen titers. In some embodiments, a set of antigens is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antigen titers. 
     In some aspects, the disclosure relates to a finding that establishing a minimal set of prostate antigen standards significantly reduces cost while simultaneously providing increased accuracy of prostate antigen level quantification. As used herein, the term “informative” refers to a characteristic of an antigen or set of antigens to provide clinically-relevant quantification of an antigen in a sample having an unknown antigen amount or concentration. For example, a standard having a broad range of antigen concentrations may produce a set of values (e.g., a concentration curve) with clinically relevant antigen concentrations falling outside the useful range of the values. This can reduce the predictive power of the values provided and thus tends to be less informative. On the other hand, use of standards having an range of antigen concentrations consistent with likely antigen amounts or concentrations in a target population is informative because the resulting values (e.g., concentration curve) have a higher number of clinically relevant antigen concentrations falling within the useful range of the values (curve). 
     In some aspects, the disclosure relates to the distribution of prostate antigen concentrations in a set of informative prostate antigen concentrations. In some embodiments, each informative prostate antigen concentration of a set is equidistant from the informative prostate antigen concentration above and/or below it. As used herein, the term “equidistant” refers to the number of units between prostate antigen concentrations. For example, the concentrations 1 ng/mL, 2 ng/mL, 3 ng/mL, and 4 ng/mL are each equidistant from (i.e., 1 ng/mL) the concentration above and/or below on a linear scale. The units may be measured on any appropriate scale, such as a linear scale or a logarithmic scale (e.g., log 10 , natural log (LN), etc.). In some embodiments, each informative prostate antigen concentration of a set is equidistant from the informative prostate antigen concentration above and/or below it on a logarithmic scale. For example, an informative set of iPSA antigens may be 0 ng/mL, 0.025 ng/mL, 0.089 ng/mL, 0.322 ng/mL, 1.157 ng/mL, 4.167 ng/mL, and 15 ng/mL. As another example, an informative set of hK2 antigens may be 0 ng/mL, 0.002 ng/mL, 0.011 ng/mL, 0.055 ng/mL, 0.290 ng/mL, 1.524 ng/mL, and 8 ng/mL. Equidistant concentrations of an antigen may be prepared by any suitable method, such as serial-dilution, or dissolving a quantity of antigen in an appropriate buffer to obtain a solution having an informative antigen concentration. 
     Various types of antigens are contemplated by the disclosure. Examples of antigens include but are not limited to peptides, proteins, lipoproteins, glycoproteins, and small molecules (e.g., haptens). Other non-limiting examples of antigens include nucleic acids, such as DNA, RNA and oligonucleotides. In some embodiments, the disclosure provides standards comprising prostate antigens. As used herein, the term “prostate antigen” refers to an antigen that is produced or expressed in the prostate of a subject. Examples of prostate antigens include but are not limited to total prostate specific antigen (tPSA), free prostate specific antigen (fPSA), intact prostate specific antigen (iPSA) and human kallikrein 2 (hK2). In some embodiments, the standard material comprises an informative set of iPSA concentrations. In some embodiments, the standard material comprises an informative set of hK2 concentrations. 
     As used herein, the term “subject” refers to a mammal having or suspected of having cancer, or being tested for cancer. Examples of subjects include humans, non-human primates (e.g., marmosets and monkeys), pigs, horses, cats, dogs, rats, and mice. In some embodiments, a subject is a human subject. In some embodiments, the subject is a human having or suspected of having prostate cancer. In some embodiments, the subject is a human being tested for prostate cancer. Human subjects may or may not display signs or symptoms of prostate cancer, including a need to urinate frequently, especially at night, difficulty starting urination or holding back urine, weak or interrupted flow of urine, painful or burning urination, difficulty in having an erection, painful ejaculation, and/or blood in urine or semen. 
     Containers for standard materials are also contemplated by the disclosure. Standard materials as described herein may be contained in any suitable vessel. Non-limiting examples of suitable vessels include vials (e.g., glass vials or plastic vials), test tubes, blister packs, bottles, pouches, and assay plates. In some embodiments, a set of antigen concentrations (e.g., a set of informative prostate antigen concentrations) may be housed in a plurality of containers. For example, a set of seven informative prostate antigen standards may be stored in seven sealed tubes, each tube housing a single informative prostate antigen standard. Generally, standard materials are in liquid phase while being contained by a container. However, the skilled artisan recognizes that a standard material, for example a protein antigen, may be contained in a powder form (for example, in a lyophilized format) and reconstituted into an informative antigen concentration with an appropriate amount of solvent (for example, water or a buffer solution). 
     In some embodiments, the disclosure relates to kits comprising standard materials. Kits may include a standard material, related equipment for performing an assay (e.g. assay plates, appropriate buffers, sample collection instruments and apparatus, etc.) and instructions for performing an assay using the standard material. In some embodiments, the disclosure provides a kit having a plurality of containers, each container housing a standard material. In some embodiments, each container comprises an informative prostate antigen concentration of a set of informative prostate antigen concentrations. In some embodiments, kits described herein further comprise instructions for quantifying levels of prostate antigen from a sample. 
     Methods of preparing prostate antigen standards are also contemplated by the disclosure. In some embodiments, the disclosure provides a method for preparing a prostate antigen standard for quantifying levels of a prostate antigen, the method comprising (i) obtaining an antigen associated with prostate cancer; (ii) preparing a stock solution of the antigen in a suitable buffer or solvent; and, (iii) preparing a set of informative concentrations from the stock solution, in which each informative concentration of the set has a concentration that is equidistant from the concentration above and/or below it on a log scale. In some embodiments, the antigen associated with prostate cancer is a prostate antigen. In some embodiments, the prostate antigen is selected from the group consisting of tPSA, fPSA, iPSA, and hK2. In some embodiments, the prostate antigen is iPSA or hK2. Non-limiting examples of suitable buffers and/or solvents include water, phosphate-buffered saline (PBS), and tris-buffered saline (TBS). A set of informative antigen concentrations from a stock solution may be prepared, for example, by serially diluting the stock solution to arrive at a plurality of informative antigen concentrations. 
     Assays for Quantification of Prostate Antigen Levels 
     In some aspects, the disclosure relates to methods for quantifying prostate antigen levels. The disclosure is based, in part, on the discovery that the prostate antigen standards described by this disclosure allow for more cost-effective and accurate quantification of prostate antigens than currently used prostate antigen standards. 
     Accordingly, in some aspects, the disclosure provides a method for quantifying levels of a prostate antigen, the method comprising, performing an immunoassay to detect presence of a prostate antigen in a sample and quantifying levels of the prostate antigen detected in the sample based on values derived from detection of a set of informative prostate antigen concentrations by the immunoassay. For example, in some embodiments, the informative set of antigen concentrations is an informative set of iPSA concentrations. In some embodiments, the informative set of antigen concentrations is an informative set of hK2 concentrations. Additional methods for detecting the presence of prostate antigens are disclosed, for example in U.S. Ser. No. 61/972,099, filed on Mar. 28, 2014, the content of which is incorporated herein by reference in its entirety. 
     Immunoassays 
     Levels of prostate specific antigens (e.g., tPSA, iPSA, fPSA, and hK2) can be assessed by any appropriate method. In some embodiments, antibodies or antigen-binding fragments are provided that are suited for use in immunoassays. Immunoassays utilizing such antibody or antigen-binding fragments may be competitive and non-competitive immunoassays, in either a direct or indirect format. Non-limiting examples of such immunoassays are dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA®), Enzyme Linked Immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay (immunometric assay), flow cytometry, western blot assay, immunoprecipitation assays, immunohistochemistry, immuno-microscopy, lateral flow immuno-chromatographic assays, and proteomics arrays. 
     In some embodiments, the immunoassay is DELFIA®. DELFIA® (dissociation-enhanced lanthanide fluorescence immunoassay) is a time-resolved fluorescence (TRF) intensity technology that is designed to detect the presence of a compound or biomolecule using lanthanide chelate labeled reagents. DELFIA® assays are flexible, compatible with a variety of plate readers, and, as a wash-based technology, are compatible with various sample types (e.g., blood, serum, plasma, cells, etc.). The technology is based on fluorescence of lanthanide chelates (Europium, Samarium, and Terbium). The fluorescence decay time of these lanthanide chelate labels is much longer than traditional fluorophores, allowing efficient use of temporal resolution for reduction of autofluorescent background. The large Stokes&#39; shift (difference between excitation and emission wavelengths) and the narrow emission peaks contribute to increasing signal-to-noise ratio. Sensitivity is further increased because of the dissociation-enhancement principle: the lanthanide chelate is dissociated and a new highly fluorescent chelate is formed into a protective micellar solution. Unlike traditional ELISA, DELFIA® does not involve the use of an enzyme (e.g., horse radish peroxidase, HRP) to generate a detectable signal. 
     Antigens or antibodies or antigen-binding fragments can be immobilized, e.g., by binding to solid supports (e.g., carriers, membrane, columns, proteomics array, etc.). Examples of solid support materials include glass, polystyrene, polyvinyl chloride, polyvinylidene difluoride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, such as nitrocellulose, polyacrylamides, agaroses, and magnetite. The nature of the support can be either fixed or suspended in a solution (e.g., beads). 
     In some embodiments, labeled antibodies or antigen binding fragments may be used as tracers to detect antigen bound antibody complexes. Examples of the types of labels which can be used to generate tracers include enzymes, radioisotopes, colloidal metals, fluorescent compounds, magnetic, chemiluminescent compounds, and bioluminescent compounds. Radiolabeled antibodies are prepared in known ways by coupling a radioactive isotope such as  153 Eu,  3 H,  32 P,  35 S,  59 Fe, or  125 1, which can then be detected by gamma counter, scintillation counter, or by autoradiography. As discussed herein, antibodies and antigen-binding fragments may alternatively be labeled with enzymes such as yeast alcohol dehydrogenase, horseradish peroxidase, alkaline phosphatase, and the like, then developed and detected spectrophotometrically or visually. Suitable fluorescent labels include fluorescein isothiocyanate, fluorescamine, rhodamine, and the like. Suitable chemiluminescent labels include luminol, imidazole, oxalate ester, luciferin, and others. 
     An immunoassay may comprise contacting the sample, e.g., a plasma sample, containing an antigen with an antibody, or antigen-binding fragment (e.g., F(ab), F(ab) 2 ), under conditions enabling the formation of binding complexes between antibody or antigen-binding fragment and antigen. In some embodiments, a plasma sample is contacted with an antibody or antigen-binding fragment under conditions suitable for binding of the antibody or antigen-binding fragment to a target antigen, if the antigen is present in the sample. This may be performed in a suitable reaction chamber, such as a tube, plate well, membrane bath, cell culture dish, microscope slide, and other chamber. In some embodiments, an antibody or antigen-binding fragment is immobilized on a solid support. An antibody or antigen binding fragments that binds to an antigen in a sample may be referred to as a capture antibody. In some embodiments, the capture antibody comprises a tag (e.g., a biotin label) that facilitates its immobilization to a solid support by an interaction involving the tag (e.g., a biotin-streptavidin interaction in which the streptavidin is immobilized to a solid support). In some embodiments, the solid support is the surface of a reaction chamber. In some embodiments, the solid support is of a polymeric membrane (e.g., nitrocellulose strip, Polyvinylidene Difluoride (PVDF) membrane, etc.). In other embodiments, the solid support is a biological structure (e.g., bacterial cell surface). Other exemplary solid supports are disclosed herein and will be apparent to one of ordinary skill in the art. 
     In some embodiments, the antibody and antigen-binding fragment is immobilized on the solid support prior to contacting with the antigen. In other embodiments, immobilization of the antibody and antigen-binding fragment is performed after formation of binding complexes. In still other embodiments, antigen is immobilized on a solid support prior to formation of binding complexes. In some embodiments, a tracer may be added to the reaction chamber to detect immobilized binding complexes. In some embodiments, the tracer comprises a detectably labeled secondary antibody directed against the antigen. In some embodiments, the tracer comprises a detectably labeled secondary antibody directed against the capture antibody. In some embodiments, the primary antibody or antigen-binding fragment is itself detectably labeled. 
     In one embodiment, immunoassay methods disclosed herein comprise immobilizing antibodies or antigen-binding fragments to a solid support; applying a sample (e.g., a plasma sample) to the solid support under conditions that permit binding of antigen to the antibodies or antigen-binding fragment, if present in the sample; removing the excess sample from the solid support; applying a tracer (e.g., detectably labeled antibodies or antigen-binding fragments) under conditions that permit binding of the tracer to the antigen-bound immobilized antibodies or antigen-binding fragments; washing the solid support and assaying for the presence tracer. 
     In some embodiments, the antibody and antigen-binding fragment is immobilized on the solid support after contacting with the antigen in a reaction chamber. In some embodiments, the antibody and antigen-binding fragment is immobilized on the solid support prior to contacting with the antigen in a reaction chamber. In either case, a tracer may be added to the reaction chamber to detect immobilized binding complexes. In some embodiments, a tracer comprises a detectably labeled secondary antibody directed against the antigen. In some embodiments, the tracer comprises a detectably labeled secondary antibody directed against the primary antibody or antigen-binding fragment. As disclosed herein, the detectable label may be, for example, a radioisotope, a fluorophore, a luminescent molecule, an enzyme, a biotin-moiety, an epitope tag, or a dye molecule. Suitable detectable labels are described herein. 
     In some embodiments, it has been found that performing certain immunoassays in low pH buffer leads to more sensitive antigen detection. Accordingly, in some embodiments, a tracer antibody is contacted with a capture antibody in a buffer having a pH in a range of 6.5 to less than 7.75 such that the tracer binds to the capture-antibody- antigen complex. In some embodiments, the buffer pH is about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6. 
     It should be appreciated that in any of the assays disclosed herein capture antibodies may be swapped with tracer antibodies. 
     In some embodiments, an immunoassay that measures the level of fPSA involves contacting fPSA present in the plasma blood sample with a capture antibody specific for fPSA under conditions in which the first capture antibody binds to fPSA, thereby producing a capture-antibody-fPSA complex; and detecting the capture-antibody-fPSA complex using a tracer. The capture antibody may be a H117 antibody. In some embodiments, the tracer comprises a 5A10 antibody or fragment thereof (e.g., a F(ab) fragment). In some embodiments, the tracer comprises an antibody or fragment thereof (e.g., a F(ab) fragment) that has at least 80%, at least 85%, at least 90%, or at least 99% amino acid sequence identity to a 5A10 antibody. In some embodiments, the tracer comprises an antibody or fragment thereof (e.g., a F(ab) fragment) that has at least 90%, at least 91%, at least 92% at least 93% at least 94% at least 95%, at least 96% at least 97% at least 98%, or at least 99% amino acid sequence identity to a 5A10 antibody. 
     The heavy and light chain sequences of 5A10 antibody, which may be incorporated into fragments, are shown below: 
     
       
         
           
               
            
               
                 5A10 Heavy chain 
               
               
                 (SEQ ID NO: 1) 
               
               
                 EVQLVESGPGILQPSQTLSLTCSFSGFSLSTTGMGVSWIRQPSGKGLEWL 
               
               
                   
               
               
                 AHLYWDEDKRYNPSLKSRLTISEDSSRNQVFLKITSVGPADSATYYCARK 
               
               
                   
               
               
                 GYYGYFDYWGQGTALTVSS 
               
               
                   
               
               
                 5A10 Light chain 
               
               
                 (SEQ ID NO: 2) 
               
               
                 DIVMTQSQKFMSTSVGDRVSVTCKASQNVNTDVAWYQQKPGQSPKALIFS 
               
               
                   
               
               
                 TSYRSSGVPDRFTGSGSGTDFTLTITNVQSEDLAEYFCQQYSNYPLTFGA 
               
               
                   
               
               
                 GTKVDLN 
               
            
           
         
       
     
     In some embodiments, an immunoassay that measures the level of iPSA involves contacting iPSA present in the plasma blood sample with a capture antibody specific for free PSA, which includes iPSA and nicked PSA, under conditions in which the second capture antibody binds at least to iPSA, thereby producing a capture-antibody-iPSA complex and detecting the capture-antibody-iPSA complex using a second tracer. In some embodiments, the tracer comprises a 4D4 antibody. In some embodiments, the capture antibody is a 5A10 antibody or fragment thereof (e.g., a F(ab) fragment). 
     In some embodiments, an immunoassay that measures the level of tPSA involves contacting tPSA present in the plasma blood sample with a capture antibody specific for tPSA under conditions in which the third capture antibody binds to tPSA, thereby producing a capture-antibody-tPSA complex; and detecting the capture-antibody-tPSA complex using a third tracer. In some embodiments, the tracer comprises a HSO antibody. In some embodiments, the capture antibody is a H117 antibody. 
     In some embodiments, an immunoassay that measures the level of hK2 involves contacting PSA in the plasma blood sample with blocking antibodies specific for PSA; contacting hK2 present in the plasma blood sample with a fourth capture antibody specific for hK2 under conditions in which the fourth capture antibody binds to hK2, thereby producing a capture-antibody-hK2 complex; and detecting the capture-antibody-hK2 complex using a fourth tracer. In some embodiments, the tracer comprises a 7G1 antibody. In some embodiments, the capture antibody is a 6H10 F(ab) 2 . In some embodiments, the blocking antibodies comprise a 5H7 antibody, a 5H6 antibody, and a 2E9 antibody. 
     Table 1 below lists antibodies and antigen-binding fragments that may be used in the methods disclosed herein and their corresponding epitopes. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Antibodies and Epitopes/Sources of Antibodies 
               
            
           
           
               
               
               
            
               
                 Antibody Name 
                 Epitope 
                 Reference or Source 
               
               
                   
               
               
                 F(ab) 2  6H10 
                   
                 Becker et al. 2000. Sensitive 
               
               
                   
                   
                 and Specific Immunodetec- 
               
               
                   
                   
                 tion of Human Glandular 
               
               
                   
                   
                 Kallikrein 2 in Serum. Clin 
               
               
                   
                   
                 Chem. 46(2), 198-206. 
               
               
                 2E9 
                 amino acids 79-93 and/or  
                 Lilja et al. 1991. Prostate- 
               
               
                   
                 80-91 of PSA protein  
                 Specific Antigen in Serum 
               
               
                   
                 (SEQ ID NO: 3) 
                 Occurs Predominantly in 
               
               
                   
                   
                 Complex with alpha-1- 
               
               
                   
                   
                 Antichymotrypsin. Clin 
               
               
                   
                   
                 Chem. 37(9), 1618-1625. 
               
               
                   
                   
                 Piironen, et al. Determina- 
               
               
                   
                   
                 tion and analysis of  
               
               
                   
                   
                 antigenic epitopes 
               
               
                   
                   
                 of prostate specific 
               
               
                   
                   
                 antigen (PSA) and human 
               
               
                   
                   
                 glandular kallikrein 2 (hK2) 
               
               
                   
                   
                 using synthetic peptides and 
               
               
                   
                   
                 computer modeling. Protein 
               
               
                   
                   
                 Science (1998), 7: 259-269 
               
               
                 5F7 
                   
                 Nurmikko et al. 2000. 
               
               
                   
                   
                 Production and Character- 
               
               
                   
                   
                 ization of Novel Anti- 
               
               
                   
                   
                 Prostate-specific Antigen  
               
               
                   
                   
                 (PSA) Monoclonal  
               
               
                   
                   
                 Antibodies That Do Not  
               
               
                   
                   
                 Detect Internally Cleaved  
               
               
                   
                   
                 Lys145-Lys146 
               
               
                   
                   
                 Inactive PSA. Clin Chem. 
               
               
                   
                   
                 46(10): 1610-1618. 
               
               
                 5H6 
                 amino acids 225-237 of PSA 
                 Nurmikko et al. 2000.  
               
               
                   
                 protein (SEQ ID NO: 3) 
                 Supra 
               
               
                 7G1 
                   
                 Nurmikko et al. 2000.  
               
               
                   
                   
                 Supra 
               
               
                 Fab 5A10 
                 amino acids 75-89, 80-94 
                 Eriksson et al. 2000. Dual- 
               
               
                   
                 and/or 82-39 of PSA protein  
                 label time-resolved 
               
               
                   
                 (SEQ ID NO: 3) 
                 immunofluorometric assay  
               
               
                   
                   
                 of free and total Prostate- 
               
               
                   
                   
                 specific Antigen Based on 
               
               
                   
                   
                 Recombinant Fab Frag- 
               
               
                   
                   
                 ments. Clin Chem 46(5),  
               
               
                   
                   
                 658-666. Piironen et al.  
               
               
                   
                   
                 Supra 
               
               
                 4D4 
                 amino acids 130-144 of PSA 
                 U.S. Pat. No. 7,872,104 
               
               
                   
                 protein (SEQ ID NO: 3) 
                   
               
               
                 H117 
                   
                 U.S. Pat. No. 5,672,480 
               
               
                 H50 
                   
                 U.S. Pat. No. 5,672,480 
               
               
                 5A10 
                 amino acids 75-89, 80-94 
                 U.S. Pat. No. 5,939,533, 
               
               
                   
                 and/or 82-39 of PSA protein  
                 European Collection of 
               
               
                   
                 (SEQ ID NO: 3) 
                 Animal Cell Cultures 
               
               
                   
                   
                 Accession number  
               
               
                   
                   
                 93091201. 
               
               
                   
                   
                 Piironen et al. Supra 
               
               
                   
               
            
           
         
       
     
     In some aspects, the disclosure relates to the use of immunoassays for quantifying the level of an antigen in a sample. As used herein, the term “quantification” refers to determination of antigen amount or concentration in a sample based upon a prostate antigen standard having a set of informative concentrations of the antigen. For example, the level of iPSA may be quantified by contacting a set of informative iPSA concentrations with a detectable antibody; producing a set of values (e.g., a concentration curve) based upon detectable output of each antibody-bound informative iPSA concentration; contacting a sample with unknown iPSA concentration with the detectable antibody; measuring the detectable output of the antibody-bound iPSA of the sample; and, quantifying the iPSA level of the sample based upon a comparison with the set of values produced by detection of the informative concentrations. 
     In some embodiments, a set of values are produced by obtaining the detectable output of antibodies. In some embodiments, detectable outputs are obtained by fluorescence readers, for example fluorescent microscopes, microplate readers and/or UV spectrophotometers. In some embodiments, fluorescence readers are connected to a computer. In some embodiments, the computer comprises software (for example, SoftMax Pro™ or Gen5 Data Analysis) for the calculation of concentration curves from detectable outputs. 
     Aspects of the disclosure may be implemented using a computer. For example, the amount of a prostate antigen in a sample may be determined in relation to a prostate antigen standard by obtaining immunoassay measurements of the prostate antigen in the sample and comparing those, through the use of a computer, to a prostate antigen amounts in minimal set of prostate antigen standards. For example, computer systems described by the disclosure may be used to detect the output of antibody-antigen binding or to create a standard curve using the output values obtained from a set of informative antigen concentrations. The computer system may include one or more processors and one or more computer-readable non-transitory storage media (e.g., memory and one or more non-volatile storage media). The processor(s) may control writing data to and reading data from the memory and the non-volatile storage device in any suitable manner, as the aspects of the present disclosure described herein are not limited in this respect. 
     To perform any of the functionality described herein, e.g., to compute prostate levels based on a minimal set of informative prostate antigens, the processor(s) may execute one or more instructions, such as program modules, stored in one or more computer-readable storage media (e.g., the memory), which may serve as non-transitory computer-readable storage media storing instructions for execution by the processor. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Embodiments may also be implemented in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote computer storage media including memory storage devices. Data inputs and program commands may be received by the computer through a input interface. The input interface may comprise a keyboard, touchscreen, USB port, CD drive, DVD drive, or other input interface. 
     It should be appreciated that various embodiments may be formed with one or more of the above-described features. The above aspects and features may be employed in any suitable combination as the present disclosure is not limited in this respect. It should also be appreciated that the drawings illustrate various components and features which may be incorporated into various embodiments. For simplification, some of the drawings may illustrate more than one optional feature or component. However, the disclosure is not limited to the specific embodiments disclosed in the drawings. It should be recognized that the disclosure encompasses embodiments which may include only a portion of the components illustrated in any one drawing figure, and/or may also encompass embodiments combining components illustrated in multiple different drawing figures. 
     EXAMPLES 
     Example 1 
     Sequences for PSA and Human Kallikrein 2 
       
     
       
         
           
               
            
               
                 PSA protein 
               
               
                 (SEQ ID NO: 3) 
               
               
                 IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVIL 
               
               
                   
               
               
                 LGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLL 
               
               
                   
               
               
                 RLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCV 
               
               
                   
               
               
                 DLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQ 
               
               
                   
               
               
                 GITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP 
               
               
                   
               
               
                 hK2 protein 
               
               
                 (SEQ ID NO: 4) 
               
               
                 IVGGWECEKHSQPWQVAVYSHGWAHCGGVLVHPQWVLTAAHCLKKNSQVW 
               
               
                   
               
               
                 LGRHNLFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSHDLMLL 
               
               
                   
               
               
                 RLSEPAKITDVVKVLGLPTQEPALGTTCYASGWGSIEPEEFLRPRSLQCV 
               
               
                   
               
               
                 SLHLLSNDMCARAYSEKVTEFMLCAGLWTGGKDTCGGDSGGPLVCNGVLQ 
               
               
                   
               
               
                 GITSWGPEPCALPEKPAVYTKVVHYRKWIKDTIAANP 
               
            
           
         
       
     
     Example 2 
     Improved Standard Materials for Detecting Prostate Antigens 
     Previous iPSA standards had a maximum concentration ranging from 166 to 350 ng/mL. This example describes the reformulation of iPSA standards by a serial dilution of the maximum concentration of 15 ng/mL, as shown in Table 2. The Informative Set shown below provides several benefits over the prior standard sets. First, the reduction of the maximum iPSA concentration lowers cost approximately 11- to 23-fold because less antigen is required. Second, the reduced range of the assay allows more points (e.g., values) of the calibration curve to fall within the clinically-relevant range of iPSA concentrations for diagnosis of prostate cancer. Finally, the use of seven serially-diluted concentrations (e.g., equidistant concentrations) allow specifications to be placed on the raw signal values detected from the set, which improves quality control from lot-to-lot of the standards and run-to-run of the assay. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Comparison of iPSA Standard Sets (ng/mL) 
               
            
           
           
               
               
               
               
            
               
                 Prior Standard  
                 Prior Standard  
                 Prior Standard  
                 Informative 
               
               
                 Set 1 
                 Set 2 
                 Set 3 
                 Standard Set 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 0 
                 0 
                 0 
                 0 
               
               
                 0.002 
                 0.016 
                 0.023 
                 0.025 
               
               
                 0.095 
                 0.033 
                 0.084 
                 0.089 
               
               
                 0.330 
                 0.22 
                 0.209 
                 0.322 
               
               
                 3.70 
                 2.2 
                 2.06 
                 1.157 
               
               
                 37.0 
                 23.3 
                 19.8 
                 4.167 
               
               
                 350  
                 237 
                 166 
                 15 
               
               
                   
               
            
           
         
       
     
     Human kallikrein 2 (hK2) standards were reformulated in a similar manner. Previously available hK2 standards have a maximum concentration of 13.1 ng/mL to 22.7 ng/mL, as shown in Table 3. The Informative Set shown below provides several benefits over the prior standard sets. First, the reduction of the maximum hK2 concentration lowers cost approximately 1.6- to 2.8-fold because less antigen is required. Second, the reduced range of the assay allows more points (e.g., values) of the calibration curve to fall within the clinically-relevant range of hK2 concentrations for diagnosis of prostate cancer. Finally, the use of seven serially-diluted concentrations (e.g., equidistant concentrations) allow specifications to be placed on the raw signal values detected from the set, which improves quality control from lot-to-lot of the standards and run-to-run of the assay. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Comparison of hK2 Standard Sets (ng/mL) 
               
            
           
           
               
               
               
            
               
                 Prior Standard 
                 Prior Standard 
                 Informative 
               
               
                 Set 1 
                 Set 2 
                 Standard Set 
               
               
                   
               
            
           
           
               
               
               
            
               
                 0 
                 0 
                 0 
               
               
                 0.001 
                 0.002 
                 0.002 
               
               
                 0.011 
                 0.023 
                 0.011 
               
               
                 0.047 
                 0.055 
                 0.055 
               
               
                 0.12 
                 0.211 
                 0.290 
               
               
                 1.3 
                 2.01 
                 1.524 
               
               
                 13.1 
                 22.7 
                 8.0 
               
               
                   
               
            
           
         
       
     
     Example 3 
     Use of iPSA and hK2 Standards in the 4Kscore Assay 
     This example describes the use of the iPSA and hK2 standards described above in the 4Kscore test. The 4Kscore Test is a calibrated multivariate individualized risk score derived from an algorithm that includes the measurement of four kallikrein proteins associated with the prostate in a blood sample- Total Prostate Specific Antigen (tPSA), Free PSA (fPSA), Intact PSA (iPSA) and human Kallikrein 2 (hK2)-plus the following clinical information: the existence (or not) of a prior negative biopsy, patient&#39;s age, and observation (or not) of nodules upon digital rectal examination (DRE) status. The algorithm calculates the risk probability for finding high grade cancer (Gleason Score 7) if a prostate biopsy were to be performed. The test is intended to be used as an aid in the clinical decision on whether to proceed with prostate biopsy. 
     The concentrations of tPSA and fPSA are determined with commercial assays approved for use in human diagnostics by the FDA (Roche™ Cobas® instrument and Elecsys® assays). The concentrations of iPSA and hK2 are determined using a test developed to run on the PerkinElmer® AutoDELFIA® instrument. 
     Venous blood sample is collected in a K 2 EDTA tube. The tube size must be large enough to allow the collection of approximately 7.5 mL of blood (at least 5 mL of blood). To ensure good mixing with the EDTA, the tube of blood must be inverted eight times shortly after sample collection. These quantities of blood ensure that enough plasma can be obtained. The single determination of the four kallikrein markers requires approximately 1 mL of plasma. Two milliliters (2 mL) of plasma allow the assay to be re-run, if needed. 
     The presence of iPSA and hK2 are detected by a sandwich (noncompetitive) immunoassay. The iPSA assay employs two distinct mouse monoclonal antibody products. The capture probe is a biotinylated, recombinant His 6 -Cys-tagged Fab fragment of the monoclonal antibody 5A10 with specificity for fPSA (of which iPSA is a component). The tracer is a Europium-labelled monoclonal antibody 4D4, with specificity to iPSA and complexed PSA (PSA-ACT). In combination, the reagents are specific for iPSA. The hK2 assay employs five distinct mouse monoclonal antibody products. The capture probe is a biotinylated, F(ab)2 fragment of 6H10 monoclonal antibody with specificity for hK2 and tPSA. The tracer is a Europium-labelled monoclonal antibody 7G1, with specificity to hK2 and tPSA. The assay requires the use of blocker antibodies for tPSA. The sample is diluted with a cocktail of three tPSA monoclonal antibodies (clones 2E9, 5F7 and 5H6), and the tracer formulation also contains 5H6. In combination, the reagents are specific for hK2. 
     The assays are calibrated as follows. The iPSA assay is calibrated to the DELFIA® ProStatus™ PSA Free/Total Kit, which is in turn calibrated to the WHO 96/670 (tPSA) and 96/668 (fPSA). The ProStatus assay allows the quantitative determination of tPSA and fPSA, of which iPSA is a subset molecule. Assignment of iPSA calibration solutions is derived from the average recovery obtained for the tPSA and fPSA assays. The hK2 assay is calibrated to the DELFIA® ProStatus™ PSA Free/Total Kit, which is in turn calibrated to the WHO 96/670 (tPSA) and 96/668 (fPSA). The ProStatus assay allows the quantitative determination of tPSA and fPSA, and the tPSA component has an equimolar cross-reactivity with hK2. Assignment of hK2 calibration solutions is derived from the recovery obtained for the tPSA assay. 
     The signals obtained from the detection of iPSA and hK2 in the immunoassays are then compared to the signals obtained from the detection of a set of standards (e.g. the calibrator solutions) using the same iPSA and hK2 antibodies. The iPSA and hK2 assays each utilize 7 standards (e.g., calibrator solutions). Measurement of standards (e.g., calibrator solutions) is used to determine the reporting range of the assay. The reporting range is defined by the limit of quantification (LoQ) at the low end and the highest calibrator at the high end. For the iPSA and hK2 assays, the LoQ was determined using CLSI EP-17A. The reporting range of the assay is shown in Table 4 below. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 4Kscore Reporting Range for iPSA and hK2 
               
            
           
           
               
               
               
            
               
                   
                 iPSA 
                 hK2 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Assay range 
                 0.014-15 ng/mL 
                 0.022-8 ng/mL 
               
               
                   
               
            
           
         
       
     
     The expected values for iPSA and hK2 were obtained by analyzing data from the U.S. clinical trial for the 4Kscore Test, which included 1012 participants. The participants were divided into three groups based on the pathological examination of the tissues after the patient proceeded to biopsy. Data are shown below in Tables 5 and 6, and as box and whisker histograms in  FIG. 1  and  FIG. 2 . Results indicate that 99.9% of hK2 values (1011/1012) and 99.6% of iPSA values (1008/1012) fall with the reporting range of the assays. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Values of iPSA and hK2 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 iPSA (ng/mL) 
                 hK2 (ng/mL) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 Inter- 
                   
                 Inter- 
               
               
                   
                   
                   
                 quartile 
                   
                 quartile 
               
               
                   
                 N 
                 Median 
                 Range 
                 Median 
                 Range 
               
               
                   
               
               
                 Biopsy negative 
                 542 
                 0.416 
                 (0.268,  
                 0.069 
                 (0.042,  
               
               
                   
                   
                   
                 0.636) 
                   
                 0.107) 
               
               
                 Low-grade disease 
                 239 
                 0.469 
                 (0.311,  
                 0.081 
                 (0.055,  
               
               
                 (Gleason Score 6) 
                   
                   
                 0.654) 
                   
                 0.120) 
               
               
                 High grade disease  
                 231 
                 0.511 
                 (0.360,  
                 0.107 
                 (0.063,  
               
               
                 (Gleason &gt; Score 7) 
                   
                   
                 0.783) 
                   
                 0.176) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 iPSA and hk2 Percentile Data (All Subjects, N = 1012) 
               
            
           
           
               
               
               
            
               
                 percentile 
                 hK2 (ng/mL) 
                 iPSA (ng/mL) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 0.010 
                 0.068 
               
               
                 5 
                 0.021 
                 0.135 
               
               
                 10 
                 0.029 
                 0.189 
               
               
                 20 
                 0.045 
                 0.263 
               
               
                 25 
                 0.051 
                 0.301 
               
               
                 30 
                 0.055 
                 0.324 
               
               
                 40 
                 0.065 
                 0.384 
               
               
                 50 
                 0.078 
                 0.451 
               
               
                 60 
                 0.094 
                 0.520 
               
               
                 70 
                 0.112 
                 0.614 
               
               
                 75 
                 0.121 
                 0.670 
               
               
                 80 
                 0.136 
                 0.759 
               
               
                 90 
                 0.183 
                 0.984 
               
               
                 95 
                 0.249 
                 1.345 
               
               
                 99  
                 0.659 
                 3.315 
               
               
                   
               
            
           
         
       
     
     Assay performance for iPSA and hK2 was evaluated using CLSI EP5-A2 and CLSI EP6-A. Results are shown below in Table 7. Note that within each assay (e.g., iPSA or hK2) coefficient of variance (CV) values remain low (&lt;15%) across the reporting range. Additionally, the R 2  value for each assay is 1. Together, these data indicate high accuracy and precision across the reporting range of the assay. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Assay performance 
               
            
           
           
               
               
               
            
               
                   
                 iPSA 
                 hK2 
               
               
                   
               
               
                 Precision 
                 0.01-0.10 ng/mL CV ≦  
                 0.01-0.10 ng/mL CV ≦  
               
               
                   
                 15% 
                 10% 
               
               
                   
                 0.11-1.0 ng/mL CV ≦ 8% 
                 0.11-1.0 ng/mL CV ≦ 8% 
               
               
                   
                 1.1-15 ng/mL CV ≦ 5% 
                 1.1-8 ng/mL CV ≦ 10% 
               
               
                 Linearity 
                 R 2  = 1, slope 1.05 
                 R 2  = 1.00, slope = 1.02 
               
               
                 Accuracy by spike  
                 101.5% 
                 93.3% 
               
               
                 and recovery 
                   
                   
               
               
                 High Dose Effect  
                 ≧100 ng/mL 
                 ≧90.2 ng/mL 
               
               
                 (Absence thereof) 
               
               
                   
               
            
           
         
       
     
     The clinical performance of the 4Kscore test was evaluated. A total of 1,012 patients were enrolled in a blinded, prospective clinical study at 26 urology centers in the United States. In order to obtain a cohort representative of current biopsy selection practice, enrollment was open to all men scheduled for a prostate biopsy, regardless of age, PSA, DRE, or prior biopsy. Each participant underwent a transrectal ultrasound guided prostate biopsy of at least 10 cores, and histopathologic examination was conducted according to the established practice at each center. Patients with a treatment history known to influence PSA levels were excluded. A blinded blood sample was collected prior to the biopsy and sent to OPKO Lab in Nashville, Tenn., for the testing of the four kallikrein markers (tPSA, fPSA, iPSA and hK2). The results of the four kallikrein markers, histopathology, age, DRE, and prior biopsy status data were un-blinded and analyzed by independent biostatisticians, and are shown in Table 8. Note again that median and interquartile levels of iPSA and hK2 were within the reporting range of the assay and have a highly significant p-value. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Patient Data Table 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Negative  
                 Low Grade  
                 High Grade  
                   
               
               
                   
                 Biopsy  
                 PCa  
                 PCa  
                   
               
               
                   
                 (N = 542;  
                 (N = 239;  
                 (N = 231;  
                   
               
               
                   
                 54%) 
                 24%) 
                 23%) 
                 p-value 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Age at Blood  
                 63 (57, 68) 
                 64 (59, 69) 
                 66 (61, 72) 
                 &lt;0.0001 
               
               
                 Draw, years 
                   
                   
                   
                   
               
               
                 &lt;50 
                 30 (5.5%) 
                 6 (2.5%) 
                 3 (1.3%) 
                   
               
               
                 50-75 
                 481 (89%) 
                 220 (92%) 
                 197 (85%) 
                   
               
               
                 &gt;75 
                 31 (5.7%) 
                 13 (5.4%) 
                 31 (13%) 
                   
               
               
                 Race 
                   
                   
                   
                 0.014 
               
               
                 African Ameri-  
                 44 (8.1%) 
                 14 (5.9%) 
                 27 (12%) 
                   
               
               
                 can 
                   
                   
                   
                   
               
               
                 Caucasian 
                 458 (85%) 
                 218 (91%) 
                 193 (84%) 
                   
               
               
                 Hispanic 
                 28 (5.2%) 
                 4 (1.7%) 
                 4 (1.7%) 
                   
               
               
                 Other 
                 10 (1.8%) 
                 3 (1.3%) 
                 4 (1.7%) 
                   
               
               
                 Unknown 
                 2 (0.4%) 
                 0 (0%) 
                 3 (1.3%) 
                   
               
               
                 Abnormal DRE 
                 127 (23%) 
                 50 (21%) 
                 70 (30%) 
                 0.045 
               
               
                 Prior Prostate 
                 139 (26%) 
                 38 (16%) 
                 22 (10%) 
                 &lt;0.0001 
               
               
                 Biopsy 
                   
                   
                   
                   
               
               
                 Total PSA,  
                 4.38 (2.88,  
                 4.62 (3.60,  
                 6.07 (4.37,  
                 &lt;0.0001 
               
               
                 ng/ml 
                 6.25) 
                 6.12) 
                 9.66)  
                   
               
               
                 &lt;4 
                 232 (43%) 
                 79 (33%) 
                 37 (16%) 
                 &lt;0.0001 
               
               
                 4-10 
                 274 (51%) 
                 146 (61%) 
                 140 (61%) 
                   
               
               
                 10-25 
                 36 (6.6%) 
                 11 (4.6%) 
                 39 (17%) 
                   
               
               
                 &gt;25 
                 0 (0%) 
                 3 (1.3%) 
                 15 (6.5%) 
                   
               
               
                 Free PSA,  
                 0.77 (0.51,  
                 0.80 (0.54,  
                 0.81 (0.61,  
                 0.038 
               
               
                 ng/ml 
                 1.20) 
                 1.15) 
                 1.27)  
                   
               
               
                 Free-to-Total  
                 21 (15, 26) 
                 17 (13, 25) 
                 13 (10, 19) 
                 &lt;0.0001 
               
               
                 PSA Ratio 
                   
                   
                   
                   
               
               
                 Intact PSA,  
                 416 (268,  
                 469 (311,  
                 511 (360,  
                 &lt;0.0001 
               
               
                 pg/ml 
                 636) 
                 654) 
                 783) 
                   
               
               
                 hK2, pg/ml 
                 69 (42, 107) 
                 81 (55, 120)  
                 107 (63, 176) 
                 &lt;0.0001 
               
               
                 4Kscore 
                 7 (3, 15) 
                 14 (6, 25) 
                 34 (17, 66) 
                 &lt;0.0001 
               
               
                 &lt;5% 
                 206 (38%) 
                 44 (18%) 
                 12 (5.2%) 
                 &lt;0.0001 
               
               
                 5%-10% 
                 130 (24%) 
                 57 (24%) 
                 13 (5.6%) 
                   
               
               
                 10%-15% 
                 77 (14%) 
                 29 (12%) 
                 23 (10%) 
                   
               
               
                 15%-20% 
                 46 (8.5%) 
                 27 (11%) 
                 22 (10%) 
                   
               
               
                 &gt;20% 
                 83 (15%) 
                 82 (34%) 
                 161 (70%) 
                   
               
               
                 Clinical T Stage 
                   
                   
                   
                   
               
               
                 T1A/B 
                   
                 2 (0.8%) 
                 1 (0.4%) 
                   
               
               
                 T1C 
                   
                 177 (74%)  
                 135 (58%) 
                   
               
               
                 T2A 
                   
                 40 (17%) 
                 36 (16%) 
                   
               
               
                 T2B 
                   
                 14 (5.9%)  
                 23 (10%) 
                   
               
               
                 T2C 
                   
                 6 (2.5%) 
                 31 (13%) 
                   
               
               
                 T3A 
                   
                 0 (0%) 
                 3 (1.3%) 
                   
               
               
                 T4 
                   
                 0 (0%) 
                 1 (0.4%) 
                   
               
               
                 TX 
                   
                 0 (0%) 
                 1 (0.4%) 
                   
               
               
                 Biopsy Gleason 
                   
                   
                   
                   
               
               
                 Grade 
                   
                   
                   
                   
               
               
                 6 
                   
                 239 (100%) 
                 0 (0%) 
                   
               
               
                 3 + 4 
                   
                 0 (0%) 
                 108 (47%) 
                   
               
               
                 4 + 3 
                   
                 0 (0%) 
                 59 (26%) 
                   
               
               
                 8 
                   
                 0 (0%) 
                 35 (15%) 
                   
               
               
                 9 
                   
                 0 (0%) 
                 26 (11%) 
                   
               
               
                 10 
                   
                 0 (0%) 
                 3 (1.3%) 
               
               
                   
               
            
           
         
       
     
     While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention. 
     The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” 
     The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. 
     As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (e.g. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of” “only one of,” or “exactly one of” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. 
     As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. 
     In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, e.g., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. 
     Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. 
     It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.