MXPA06004884A - Method for adjusting the quantification range of individual analytes in a multiplexed assay - Google Patents

Abstract

The invention provides general methods for adjusting the inherent quantification range of a particular set of analytes in assays that employ capture reagents immobilized on solid supports. Specifically, the quantification range of a given analyte is adjusted to higher concentration regions by the addition of free capture reagent specific for that analyte, leaving the range of the remaining analytes the same and thereby permitting the simultaneous and accurate quantification of a plurality of analytes over a wide range of concentration values.

Description

Published: For two-letter codes and other abbreviations, refer to the "Guid- - without intemational search report and to be republished anee Notes on Codes and Abbreviations" appearing at the beginning - upon receipt of that report no regular issue of the PCT Gazette.

METHOD FOR ADJUSTING THE INTERVAL OF QUANTIFICATION OF INDIVIDUAL ANALYTS IN A MULTIPLEXED TEST FIELD OF THE INVENTION The invention is directed to multiplexed assays for analytes. Specifically, the invention is directed towards methods and reagents for simultaneously quantifying high and low abundance analytes that may be contained within a biological fluid. BACKGROUND OF THE INVENTION The ability to quantify multiple levels of analytes in biological fluids or extracts promises to revolutionize biological and medical research. In particular, measuring the levels of protein analytes in an organism, called the proteome, are key to understanding the current state of the organism and will change as the state changes; The diagnostic potential of such information is widely appreciated. Such proteomic measurements are the direct analogue of genomic measurements made with DNA microarrays with several important differences. Gene expression arrays typically quantify mRNA levels in a sample, and these levels do not always correlate well with protein levels. In addition, no information regarding the post-translation modification of proteins, can be Ref .: 172429 extracted from gene expression data, while capture reagents, such as nucleic acid ligands or antibodies, can be elaborated to discriminate between different protein modifications. Finally, the physiological range of protein levels in an organism vary over a wider range, at least 10 logs, of mRNA levels, which encompass approximately 4 to 5 logs. For example, cytokines typically appear at subfemtomolar concentrations while many complement proteins approach micromolar concentrations. The wide range of physiological levels of analyte has a challenging problem for multiplexed measurements of analytes within a simple experiment. To date, protein levels have been measured individually with assays designed for each analyte of interest. The low level analytes can be detected with signal amplification schemes and high abundance analytes can simply be diluted in order to bring the physiological levels to the optimal quantification range of the assay. Obviously, no general solution of this kind can exist for proteomic measurements, since it is necessary to measure low and high abundance proteins simultaneously. In principle, highly abundant analytes could be measured with capture reagents with affinities, quantified by the dissociation constant K¿, comparable to their physiological levels. This becomes problematic from a specificity point of view, since the weakest specific interactions compete with a variety of weak non-specific ones. Such non-specific interactions are primarily responsible for the background effects and therefore adjust the lower limit of detection. Also, when trials are multiplexed, protocols must be adjusted to accommodate those that function more poorly; weaker interactions are more likely to have short shutdown speeds compared to high affinity ones, and therefore can limit -the -effectivity -of the de-washed background, for example. Clearly, it is desirable to use high affinity, high specificity capture reagents-including, but not limited to, antibodies and nucleic acid ligands-in a microarray fit. With uniformly high affinity capture reagents, the lower limit of detection is generally comparable between analytes; it is the upper limit of quantification that is difficult to design for each analyte within a multiplexed assay. For assays that employ high affinity interactions, it is the complete concentration of the capture reagent that adjusts the upper limit of the quantification in a sample. The concentration of capture reagents Individuals in a microtiter plate, or on spheres, etc., is limited to nanomolar concentrations at best, and is more typically in the range of 10 to 100 picomolar for microarrays. The detection of low level analytes limits the dilution of the sample to approximately 10%; it becomes difficult to simultaneously measure the most highly abundant analytes with endogenous levels exceeding nM. The object of the present invention is to provide a general method for adjusting the inherent quantization range of a particular group of analytes to higher-concentration regions, leaving the interval of the remaining beads equal, and thereby allowing the simultaneous and precise quantification of a plurality of analytes. analytes over a wide range of concentration values. BRIEF DESCRIPTION OF THE INVENTION The invention includes a method for decreasing the amount of a first analyte in a biological fluid that is capable of binding to a first capture reagent immobilized on solid support, without decreasing the amount of a second analyte in the same fluid. biological, which is able to bind to a second capture reagent immobilized on solid support. The method involves contacting the biological fluid with a quantity of the first free capture reagent in solution. The adition of an amount of the first free capture reagent in solution specifically quantitatively titrates the amount of the first analyte captured in the assay, decreasing the saturation levels of the first analyte to quantifiable levels. In embodiments in which the constant dissociation, Kdl of the first analyte for the first capture reagent is greater than the concentration, Cs, of the first capture reagent immobilized on the solid support, the concentration of the first free capture reagent in solution is preferably greater than the dissociation constant, more preferably 10 times higher. In embodiments in which the dissociation constant, Jd / of the first analyte for the first capture reagent is less than the concentration, Cs, of the first capture reagent immobilized on the solid support, the concentration of the first capture reagent free in solution Is it preferably higher than Cs? more preferably 10 times greater. The methods can be applied to multiplexed assays in which thousands of analytes must be analyzed simultaneously in a biological fluid. For each abundant analyte, a quantity of the cognate capture reagent can be added to the biological fluid, in order to displace the concentrations of those abundant analytes at quantifiable levels, while maintaining the desired sensitivity for low abundance analytes. The invention also provides a method for determining the concentration of an analyte in a biological fluid. The method involves providing a solid support on which a first quantity of a capture reagent that is capable of binding to the analyte in the biological fluid is immobilized. The solid support is then contacted with a mixture comprising the biological fluid to be evaluated and a second quantity of the capture reagent. The amount of the analyte bound to the solid support is then measured. The concentration of the analyte in the biological fluid can be determined based on the measurement of the amount of the analyte that has bound to the solid support, the concentration of the second quantity of the capture reagent in the mixture, and the K¿ of the reagent of the analyte. capture. The invention also provides a method for decreasing the non-specific binding of an analyte in a biological fluid to a non-cognate capture reagent, immobilized on a solid support. The method involves contacting the biological fluid with the free capture reagent capable of specifically binding to the analyte. BRIEF DESCRIPTION OF THE FIGURES Figure 1 describes a standard curve shift using a multiplexed aptamer microarray.

The curve to the left (circles) is the standard curve in buffer for the protein, angiogenin using aptamer 1069-1 without soluble aptamer, while the two curves displaced to the left were generated using 5 1 nM (squares) and 10 nM (triangles) of soluble 1069-1 in the test diluent. Figure 2 depicts a standard curve shift for angiogenin using a multiplexed aptamer microarray, and seven soluble aptamers at varying concentrations. The curve to the left (circles) is the standard curve in the shock absorber and the curve to the right (frames) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are displayed visually as . filled markers and seven serum measurements are shown as open markers on the two standard curves. Figure 3 depicts a standard curve shift for endostatin using a multiplexed aptamer microarray and seven soluble aptamers to various concentrations. The curve to the left (circles) in the standard curve in the buffer, and the curve to the right (frames) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are shown as filled markers and seven measurements in serum are visually displayed as markers open on the two standard curves. Figure 4 depicts a standard curve shift for IgE using a multiplexed aptamer microarray, and seven soluble aptamers at various concentrations. The curve to the left (circles) is the standard curve in the buffer, and the curve to the right (frames) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are shown as filled markers and seven serum measurements are shown as open markers on the two standard curves. Figure 5 depicts a standard curve shift for P-selectin using a multiplexed aptamer microarray, and seven soluble aptamers at various concentrations. The curve to the left (circles) is the standard curve in the buffer, and the curve to the right (frames) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are shown as filled markers and seven serum measurements are shown as open markers on the two standard curves. Figure 6 depicts a standard curve shift for TIMP-1 using a multiplexed aptamer microarray, and seven soluble aptamers at various concentrations. The curve to the left (circles) is the standard curve in the buffer, and the curve to the right (frames) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are shown as filled markers and seven serum measurements are shown as open markers on the two standard curves. Figure 7 depicts a standard curve shift for lactoferrin using a multiplexed aptamer microarray, and seven soluble aptamers at various concentrations. The curve to the left (circles) is the standard curve in the shock absorber, and the curve more to the _right .. (squares) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are shown as filled markers and seven serum measurements are shown as open markers on the two standard curves. Figure 8 depicts a standard curve shift for L-selectin using a multiplexed aptamer microarray, and seven soluble aptamers at various concentrations. The curve to the left (circles) is the standard curve in the damper, and the curve to the right (frames) is the standard curve with the soluble aptamer in the test diluent. The data points of the standard curve are shown as filled markers and seven measurements in serum are shown as open markers on the two standard curves. Figure 9 describes six serum sample peaks for IgE. The serum peaks (higher curves) are observed to converge towards the standard curve in the buffer (filled circles) without evidence of matrix effects. Figure 10 illustrates that the concentrations computed in Figure 9 are in excellent concordance with the peak values, even for the lowest peak levels within large endogenous levels. Figure 11 describes six peaks of serum sample for IgE_. The serum_peaks _ (higher curves) are observed to converge towards the standard curve in the buffer (filled circles) without evidence of matrix effects. Figure 12 illustrates that the concentrations computed in Figure 11 are in excellent concordance with the peak values, even for the lowest peak levels within large endogenous levels. Figure 13 depicts in the bar graph format the coefficients of variation for all aptamers in a microarray using seven individual soluble aptamers. The data is presented as bars where 50% of the measurements fall inside the boxes, the median is denoted by the white bar through the bar and the lines above and below the bar indicate the data interval. DETAILED DESCRIPTION OF THE INVENTION Definitions Various terms are used herein to refer to aspects of the present invention. To assist in the clarification of the description of the components of this invention, the following definitions are provided: The term "capture reagent" means a molecule-or-a multi-molecular complex that can be linked to an analyte. The capture agents are preferably linked to their analyte link partners in a substantially specific manner. The capture reagent can optionally be a recombinant or synthetic biomolecule of natural origin. Antibodies or antibody fragments and nucleic acid ligands (aptamers) are highly suitable as capture agents. Antigens can also serve as capture agents for protein analytes, since they are capable of binding to the antibodies. A receptor that binds to a protein ligand is yet another example of a possible capture reagent. It is understood that the agents of capture are not limited to agents that only interact with their partners of binding of analytes through non-covalent interactions. The capture agents may also optionally be covalently linked to the analytes with which they bind. For example, the capture reagent may be a photocrosslinking nucleic acid ligand that is photo-reticular to its analyte binding partner after linkage and photoactivation. The term "cognate" is sometimes used to indicate that a particular analyte binds in a substantially specific manner to a particular capture reagent, eg, an analyte binds in a substantially specific manner to its capture reagent. -cognate, but can be linked in a non-specific manner to other non-cognate capture reagents (whose non-cognate capture reagents in turn are linked in a substantially specific manner to other analytes). As used herein, the term "analyte" refers to any compound that is to be detected in an assay via its link to a capture reagent. An analyte can be a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, transition state analog, cofactor, inhibitor, drug , coloring, nutrient, growth factor, cell, tissue, etc., without limitation. As used herein, the term "biological fluid" refers to a mixture of macromolecules obtained from an organism. This includes, but not limited to, blood plasma, urine, semen, saliva, lymphatic fluid, meningeal fluid, amniotic fluid, glandular fluid, and cerebrospinal fluid. It also includes experimentally separated fractions of all procedures. The term "biological fluid" also includes solutions or mixtures containing homogenized solid material, such as faeces, tissues, and samples of hyopathy. As used herein, "solid support" is defined as any surface to which molecules can be linked through links either covalent or non-covalent. This includes, but is not limited to, membranes, plastics, paramagnetic spheres, loaded paper, nylon, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold and silver. Any other material known in the art that is capable of having functional groups such as the amino, carboxyl, thiol or hydroxyl groups incorporated on its surface is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces, grooved surfaces, and cylindrical surfaces, for example, columns. The multiple capture reagents, each specific for a different analyte, can be linked to specific sites ("addresses") on the surface of a solid support in a dirigible format to form an array, also referred to as a "microarray" or as a "biochip" By way of non-limiting example only, an array can be formed with a flat solid support, the surface of which is linked to the capture reagents. By way of non-limiting example only, an array can also be formed by the coupling of the capture reagents to spheres, followed by La. placing the spheres in an array format on another solid support, such as a microtiter plate. As used herein, "nucleic acid ligand" is a nucleic acid of non-natural origin that has a desirable action on a target or target. Nucleic acid ligands are also referred to in this application as "aptamers". A desirable action includes, but is not limited to, the objective link, the catalytic change of the target, the reaction to the target in a way that modifies / alters the objective or functional activity of the target, the covalent link to the target as in a 'suicide inhibitor, facilitating the reaction between the target and another molecule. In the preferred embodiment, the This action is of specific binding affinity for a target molecule, said molecule being a target of a two-dimensional chemical structure different from a polynucleotide that binds to the nucleic acid ligand through a mechanism that predominantly depends on the mating of atson / Crick bases or the triple helix linkage, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule. Nucleic acid ligands include nucleic acids that are identified from a candidate nucleic acid mixture, said nucleic acid ligand is a ligand of a given objective, by the method comprising: a) contacting the candidate mixture with the objective, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture can be divided from a remnant of the candidate mixture; b) dividing the increased affinity nucleic acids from the rest of the candidate mixture and c) amplifying the increased affinity nucleic acids to produce a mixture enriched in nucleic acid ligands, whereby the nucleic acid ligands of the target molecule are identified . This process, called the SELEX process, is described in U.S. Patent Application Serial No. 07 / 536,428, filed June 11, 1990, entitled "Systematic Evolution of Ligands by Exponential Enrichment ", now abandoned, U.S. Patent No. 5,475,096 entitled" Nucleic Acid Ligands ", and U.S. Patent No. 5,270,163 (see also 091/19813) entitled" Nucleic Acid Ligands " each of which is specifically incorporated by reference herein A particularly important form of the SELEX process is described in U.S. Patent Application Serial No. 08 / 123,935, filed on September 17, 1993, and the U.S. Patent Application No. 08 / 443,959 filed May 18, 1995, both entitled "Photosensing of Nucleic Acid Ligands", and both now abandoned, and U.S. Patent No. 5,763,177, Patent of US Pat. United States No. 6,001,577, O95 / 08003, United States Patent No. 6,291,184, United States Patent No. 6,458,539 and United States Patent Application Serial No. 09 / 723,718, filed on November 28, 2000, each of which is entitled "Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and SELEX in Solution", and each of which describes a method based on the SELEX process for selecting nucleic acid ligands containing photoreactive groups capable of binding and / or photocrosslinking to and / or photoinactivating a target molecule.

The resulting nucleic acid ligands are referred to interchangeably as "photocrosslinking nucleic acid ligands" and "photoaptamers". Automated methods and apparatus for the generation of nucleic acid ligands, including photocrosslinking nucleic acid ligands, are provided in U.S. Patent Application Serial No. 09 / 993,294, filed on November 21, 2001, U.S. Patent Application Serial No. 09 / 815,171, filed March 22, 2001, U.S. Patent Application Serial No. 09 / 616,284, filed July 14, 2000, Application for U.S. Patent Serial No. 09 / 356,233, filed July 16, 1999, and U.S. Patent No. 6,569,620, each of which is entitled "Method and Apparatus for the Automated Generation of Acid Ligands. Nucleic ". Photocrosslinking of nucleic acid ligands, produced by the fotoSELEX process, have particular utility as capture reagents, in multiplexed diagnostic assays or medical prognostic tests. In such an embodiment, the photorecycling nucleic acid ligands of the targets involved in the disease are linked to a flat solid support in an array format, and the solid support is then placed in contact with a biological fluid to be analyzed for the presence or absence of the objectives. Photocrosslinking nucleic acid ligands are photoactivated, and the solid support is washed under aggressive, very demanding conditions (preferably under conditions that denature nucleic acids and / or proteins) in order to remove all non-specifically bound molecules. The bound target is not removed because it is covalently crosslinked to the nucleic acid ligand via the photoreactive group. Protein targets bound by photocrosslinking nucleic acids can then be detected using a reagent or reagents that label proteins and not nucleic acids with a detectable portion. Such or such reagents are referred to as Universal Protein Stains ("UPS") and are described in PCT / US03 / 04142, filed on February 10, 2003, entitled "Methods for the Multiplexed Evaluation of Ligands of Nucleic Acid of Photocrosslinking " The ability to photo-reticular, followed by demanding washing, allows diagnostic tests and unparalleled sensitivity and specificity forecasting to be performed. Arrays (also commonly referred to as "biochips" or "microarrays") of nucleic acid ligands, including photocrosslink nucleic acid ligands and aptamers, and methods for their manufacture and use, are described in U.S. Patent No. 6,242,246, U.S. Patent Application Serial No. 09 / 211,680, filed December 14, 1998, now abandoned, document 099/31275, U.S. Patent No. 6,544,776, U.S. Patent No. 6,503,715 and U.S. Patent No. 6,458,543, each of which is entitled "Diagnostic Biochip of Nucleic Acid Ligand." These patents and patent applications are collectively referred to as "biochip applications", and are each specifically incorporated herein by reference, in their -totality.- - - - Note that throughout this application, various publications and patent applications are mentioned; each one is incorporated by reference to the same degree as if each one specific and individually incorporated by reference.

Adjustment of the Quantification Interval of Individual Analytes in a Multiplexed Test The optimal performance of an analytical assay occurs in the center of the limits of quantification, hereinafter referred to as "LOQ". LOQs are the highest and lowest concentration of the analyte that can be measured in a sample with acceptable accuracy and precision.

The highest concentration should preferably not exceed a loss in accuracy of 10% due to the deviation of linearity near saturation. The LOQ should preferably be commensurate with the physiological levels of interest. The concentration of the analyte at which the assay saturation occurs is due to a combination of characteristics, most importantly the concentration of the capture reagent and its affinity for the analyte. The concentrations of the capture reagent for the microarrays that measure the protein analytes are typically very low due to the micrometric scale of the characteristics-comprised of-the-capture-reagents.

Typical microarray capture reagent densities are ~ 104 molecules μm2 with characteristic areas of ~ 104 μm2. The replication characteristics, ie 4 or 5, in a sample of 100 μL, therefore, produce a total concentration of the capture molecules, Ct of: r - ^ characteristics x 1 molecules / μm2 \ O μm1 characteristics ^ j Q-J I ^ 6 x 3 O23 molecules / mol x l O "4 L Surfaces comprised of hydrogel layers, adding a third dimension to the flat surface, can increase this concentration tenfold. For high affinity capture reagents, such nucleic acid ligands and antibodies, with KdS that 1 nM, is this relatively low concentration of the capture reagent that establishes the upper limit of the quantification. Near saturation, the concentration of the capture reagent is much less than the Ka and analyte concentration [A], so that the fraction of the capture reagents occupied by the analyte bound at equilibrium is given by: ? : C] = [A] C, K ^ [A For a nM or better Kd, the upper LOQ will be approximately 5 x K < _ or 5 x Ct, whichever is greater. Clearly, for protein analytes that exceed nM concentrations (after appropriate dilution), the capture reagents will be saturated and accurate quantification is not possible. The present invention provides a method for increasing Ct for certain capture reagents whereby its standard curve - and hence the saturation point - is moved to higher concentration levels for the specific analyte for which Ct has been increased. The method involves adding the free capture reagent to the solution containing the analyte to be measured. For example, the free capture reagent can be added to the diluent that is used to dilute a biological fluid suspected of containing the analytes to be measured, before the application of the biological titer, to the surface of a microarray (whose microarray comprises the same capture reagent bonded thereto). The addition of free capture reagents in solution quantitatively titrates the amount of the analyte captured in the assay, decreasing the saturation levels of the analyte to quantified levels. The magnitude of the standard curve offset (and hence the displacement of the saturation point) depends on the amount of the immobilized capture reagent (assumed to be the same here for all capture reagents); the affinity of the capture-analyte reagent, and the amount of reactive-free capture in the solution containing the analyte. The strict linearity of the assay will be maintained at analyte concentrations that are well below the concentration of the capture reagent. Mathematically, this is easily observed starting with. the equilibrium binding equation for the capture of the analyte from the solution, A + C? A: C, where [C] and [A] are the concentration of the non-complexed capture reagent and the analyte and [A: C] is the concentration of the analyte bound to the capture reagent. The equations of balance between mass for this system are: A, = [A] H • l Oa), C, = [C] + [A • (3b) where At and Ct are the total, bound and unbound concentrations of the analyte and the capture reagent in the system. Ct is comprised of the immobilized surface and the free soluble capture reagent, whose total concentrations are denoted Cs and Cf, ie Ct = Cs + Cf. The linearity condition of the test, At «Ct, makes it possible to equal [C]« C, since only a small number of capture reagents will bind to the analyte under these conditions. Using the mass balance (equation 3), the condition of linearity and the equilibrium constant (equation 2) produces the following expression for the concentration of capture reagent, immobilized on the surface and free, bound to the analyte A C [A C.}. = • '• (4) 1 J Kd + C, Provided the complex affinity on the surface is the same as in solution, the concentration of the capture reagent immobilized with the bound analyte is simply the ratio of the surface capture molecules to the total capture molecules so many times the concentration of the complex formed, shown in equation 4, A? (5) Equation 5 can be easily rearranged to give the fraction of the capture reagent immobilized on the surface, bound to the analyte, Mi = - (6) c,?, + C, + cr The total concentration of analyte in a sample that gives rise to a particular fraction of the bound capture reagents, »_ [A: CJ] fB = -cT > depends linearly on Kd, Cs and Cf and is given by At (Cf) - f? K + s + Cf) (7) The annotation At (Cf) emphasizes the fact that, for a fixed Kd and Cs, in order to obtain a particular fB in the presence of the free capture reagent, the amount of the total analyte must be increased by an amount directly proportional to the concentration of the free capture reagent. Since Kd and Cs are essential characteristics of the capture reagent and the microarray, the two limits of the free equation are of interest, for example, where Kd > Cs and Cs > Kd. When Kd, > Cs, equation 7 is reduced to At (Cf) = fB (Kd + Cf) and the proportion of the total analyte required to give the same response in the assay to that required without the free capture reagent is simply TO,. { Cf) ^ Kd + Cf A, ®) K, Free capture reagent concentrations that are less than or equal to Kd result in little perceptible displacement in the test response. The free capture reagent present in the assay at a concentration of 10 x Kd results in a displacement about 10 times in the standard curve. Similarly, for capture reagents with Kd < Cs, the proportion of the analyte required to give an equivalent response to the assay without the free capture reagent is A ,. { Cj) Cx + Cf Free capture reagent concentrations that are less than or equal to Cs result in little change or perceptible displacement in the assay response. Free capture molecules at a concentration of 10 x Cs result in a displacement of approximately 10 times in the standard curve. The determination of the concentration of the free capture reagent required to displace the concentration range of a given analyte, such that it coincides approximately with the center of the LOQ, constitutes mere routine experimentation for a person skilled in the art, guided by the Equations 8 and 9.

The presence of the free capture reagent for a given analyte does not affect the quantification of any other analyte in the assay. Therefore, it is possible to quantitatively displace the concentration of all highly abundant analytes in a multiplexed assay towards the center of the LOQ while retaining the desired sensitivity for low abundance analytes that could undoubtedly suffer from dilution of the sample. In this way, the method can be easily applied to multiplexed assays in which thousands of highly abundant analytes - as well as analytes of lower abundance - can be simultaneously measured. Optimal tuning of the multiplexed assays according to the methods provided herein, also allows the accurate measurement of up and down regulated analytes, which appear under certain non-standard conditions, including, but not limited to, disease states and reactions to medical treatment. In some embodiments, in addition to altering the quantification range of the individual analytes, in the multiplexed microarray assay, the free capture reagent can be used to reduce the non-specific binding of a cognate analyte to the non-cognate capture reagents. Since the specific interaction of a Analyte with its cognate capture reagent in solution will be greater than its affinity for non-cognate capture reagents on the array, the effective concentration of the free analyte is dramatically reduced, leading to less non-specific binding by this particular analyte. Therefore, in some embodiments, the free capture reagents are added to the diluent for the biological fluid, in order to bind and keep in solution those analytes that may be problematic for non-specific interactions with other non-coded capture reagents, about the arrangement. The methods provided herein may be used with any capture reagent. Suitable capture reagents include, but are not limited to, antibodies (including fragments thereof), antigens, receptors, proteins, peptides, nucleic acid ligands (including photocrosslinking nucleic acid ligands), and nucleic acid fusions -protein (as described in U.S. Patent No. 6,537,749, incorporated by reference herein in its entirety). In addition, the methods provided herein are not limited to use with microarrays, but can be used in any multiplexed assay in which capture reagents are associated with solid supports. For example, the methods provided in the present can be used with spherical-based flow cytometric assays, as described in U.S. Patent No. 6,449,562, incorporated by reference herein, in its entirety. The present invention provides the equipment comprising a microarray of capture reagents for multiplexed retention of a plurality of analytes found in a biological fluid. At least one of the capture reagents on the microarray is bound to an analyte that is present in the biological fluid at a concentration that is greater than the LOQ higher than that particular capture reagent. The kit also comprises a container that includes the free capture reagent corresponding to at least one capture reagent on the microarray that binds to the abundant analyte. The equipment may also comprise one or more buffers or diluents containers, or may be mixed with the biological fluid, together with the free capture reagent, before beginning the multiplexed assay. In its simplest form, the equipment can include the free capture reagent apart from the diluent to which the biological fluid is added.

Examples The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. In particular it should be understood that the use of the nucleic acid ligands as the capture reagent and the proteins as the analytes in the following examples do not limit the nature of the capture reagent or the analyte which can be used with the general methods of the invention. invention.

Example 1: Displacement of the standard curve of a single analyte in a multiplexed assay The effect of introducing a free capture reagent into the solution containing the analytes to be measured can be illustrated by examining the standard curves in the buffer with or without the free capture molecules. A microarray on a hydrogel surface measuring 25 protein analytes and 33 different aptamers (some analytes are measured with multiple aptamers) were synthesized according to the methods provided in the biochip applications. Twenty-five proteins were serially diluted in buffer, without and with the free aptamer for angiogenin (1069-1 having Kd of 20 pM) and applied to the separated microarrays to simultaneously generate twenty-five standard curves. Without the free angiogenin aptamer, the upper limit of quantification for angiogenin is - 1 nM, a logarithm above the estimated Ct without the free aptamer in solution. The addition of 1 to 10 nM of free 1069-1 to the diluent displaces the standard curve for angiogenin by approximately 0.75 and 1.5 logs at higher concentrations. See figure 1. Only the standard angiogenin curve was displaced; none of the other 24 analytes were affected by the addition of 1069-1 to the diluent. In addition to generating standard curves, angiogenin levels were measured in two serum samples at a dilution of 20% with 0.1 nM and 10 nM of 1069-1 free. The Relative Fluorescence Units (RFU) values subtracted from. antecedent, for The two samples are summarized in Table 1. The effect of the free aptamer added to the diluent is reduced to signal on the aptamer 1069-1 on the array as the concentration of the free aptamer increases.

Table 1: Angiogenin measured in 20% serum for 0.1 nM and 10 nM free aptamer This is parallel to the displacement observed in the standard curve at higher concentrations. The same sample concentration will signal lower in an assay with the free aptamer present, since a proportion of the analyte is distributed between the aptamers on the microarray and free in solution.

Example 2: Simultaneous displacement of the standard curves of a plurality of analytes in a multiplexed assay Using the same microarray of 25 proteins in Example 1, seven individual aptamers were added to the diluent, for the incubation of the sample. . See table 2.

Table 2: Seven aptamers added to the sample incubation diluent together with the affinity for its target analyte and the K_ Along with the generation of the standard curve, seven serum samples were run with or without the free aptamer. The standard curves for these protein analytes, as well as the serum sample responses, are shown in Figures 2-6. The magnitude of the displacement of the standard curve depends on the amount of mobilized aptamers (assumed to be the same here for all aptamers), the affinity of the aptamer-analyte pair, and the amount of the free aptamer in the diluent. The smallest displacement is observed for lactoferrin which is less than one tenth of a log, while endostatin and XgE were moved by more than 2.5 logs of their initial buffer response. The free aptamer that is close to the K results in a less perceptible displacement. For aptamers with K < _ < C, a 10-fold concentration of the free aptamer above K results in a 10-fold shift in standard curves, see results for endostatin (Figure 3) and TIMP-1 (Figure 6). For aptamers with Kd < C, a 10-fold concentration of the free aptamer above the Ct results in a 10-fold shift in the standard curve, see results for P-selectin (Figure 5). Due to the uncertainty in the measured values of Kd, made in solution, as well as the uncertainty of the surface effects on the link affinities, the The results in table 2 are in reasonable agreement with the theory. No standard curve for the other 18 analytes measured with the microarray was affected by the presence of the free aptamer in the protein incubation diluent. Also, the desired effect of decreasing the serum sample responses was observed. All measurements for the analytes with the free capture molecules are smaller compared to those run in the buffer alone, see figures 2-6. A direct quantitative comparison of the measurements in serum with or without the free aptamer should be carried out with care_.and -._ that_.J. Measurement without, -, is usually outside the LOQ. However, most of the calculations are in agreement within a factor of 2-4. The values determined with the free aptamer present during the incubation are presumably more reliable, since these are well within the LOQ (which is the purpose of adding the free aptamer).

Example 3: Analysis of possible matrix effects There is a possibility that the presence of the free aptamers in Example 1 and 2 would introduce matrix effects that could have resulted in a deviation of the sample. For example, different serum samples may have different amounts of materials that they bind to the free aptamers, reducing their effect in a sample-dependent manner. To address such problems, a series of serum measurements with peak samples was performed. If there were large matrix effects, it could be expected that different serum samples would give rise to different magnitudes of displacement in the peak samples. This behavior was not observed. The peak curves all tended to converge on the standard shock curve at sufficiently high peak levels over the endogenous ones. This is illustrated in Figure 9 and Figure 10. The same concentration of protein used to generate the standard-curves was used only here. reached a maximum in six different serum samples. The data for all 25 peak analytes were simultaneously generated in the presence of the seven free aptamers in the diluent. Low peak protein levels are masked by large endogenous protein levels, for IgE and TIMP-1. As the peak concentration increased, the six samples tended to converge on the standard curves generated in the buffer. By subtracting the concentration of endogenous protein, computed from the peak sample without protein, a remarkable recovery of peak proteins is allowed. The computed values all fall close to the peak values. No evidence of individual sample deviation was present in these experiments and good analytical behavior was observed. None of the curves or calculated recovery values were different for the analytes with the free aptamers compared to those without them.

Example 4: Reproducibility of the multiplexed assays with the free capture reagent Six serum samples were measured seven times at a 20% dilution. The coefficient of measurement of the variation (CV: standard deviation divided by the mean concentration computed from seven replicates) was determined for each active analyte. These data are shown in Figure 11 as histogram statistics and show very reasonable CVs for all analytes in the multiplexed array with the exception of a noisy VEGF aptamer, 467-65. There is no appreciable difference in the CVs for measurements of analytes with the soluble aptamer versus those without it. Seventeen aptamers give median CVs of 10% or less, the remaining ones are between 10-20%. The average median CV is 9.8%, a very acceptable level of variation for multiple measurements in complex media. It is noted that in relation to this date, the best known method for carrying out the aforementioned invention is that which is clear from the present description of the invention.

Claims (12)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for decreasing the amount of a first analyte in a biological fluid that is capable of binding to a first capture reagent immobilized on a solid support, without decreasing the amount of a second analyte in the biological fluid that is capable of binding to a Second capture reagent immobilized on the solid support, is characterized in that it comprises contacting the biological fluid with the first free capture reagent in solution

2. The method according to claim 1, characterized in that the first capture reagent is a antibody.

3. The method according to claim 1, characterized in that the first capture reagent is a nucleic acid ligand.

4. The method according to claim 1, characterized in that the first analyte is a protein.

5. The method according to claim 1, characterized in that the dissociation constant, Ka, of the first analyte for the first capture reagent is greater than the concentration Cs, of the first capture reagent immobilized on the solid support, and wherein the concentration of the first free capture reagent in solution is greater than the dissociation constant.

The method according to claim 5, characterized in that the concentration of the first free capture reagent in solution is approximately ten times greater than the dissociation constant. 0 7.

The method according to the claim 1, characterized in that the dissociation constant, K of the First, the analyte for the first capture reagent is less than the concentration Cs of the first capture reagent immobilized on the solid support and where the concentration of the first reagent free capture in solution is greater than Cs.

The method according to claim 7, characterized in that the concentration of the first free capture reagent in solution is approximately ten times or greater than Cs.

9. A method for increasing the saturation point for an analyte of an immobilized capture reagent on a solid support, characterized in that it comprises contacting a solid support with the free capture reagent 5 in solution.

10. A method for determining the concentration of an analyte in a biological fluid, characterized in that it comprises: a) providing a first quantity of a capture reagent capable of binding to the analyte wherein the first quantity of the capture reagent is immobilized on a solid support; b) contacting the solid support with a mixture comprising the biological fluid and a second quantity of the capture reagent; c) measuring the amount of the analyte bound to the first quantity of the capture reagent; and d) calculating the concentration of the analyte in the biological fluid based on the measurement made in step c), the concentration of the second quantity of the capture reagent in the mixture of step b), and the Kd of the capture reagent.

11. A method for decreasing the non-specific binding of an analyte in a biological fluid to a non-cognate capture reagent, immobilized on a solid support, characterized in that it comprises contacting the biological fluid with a capture reagent capable of specifically binding to the analyte, wherein the capture reagent capable of specifically binding to the analyte is free in solution in the biological fluid.

12. A method for increasing the effective concentration of a capture reagent immobilized on a solid support, characterized in that it comprises contacting a solid support with the free capture reagent in solution.