Abstract:
The present invention concerns an array-based analytical system and method having an enhanced sensitivity which allows for simple and rapid analysis of relative unmodified samples which comprises an analytical system of the type having a plurality of different first members of a specific binding pair affixed in an array thereupon, a mixture including at least one second member of a specific binding pair capable of binding to one of the first members so as to form a specific binding pair which is affixed to the support member, and a reporter system that produces a detectable signal indicative of the presence of the specific binding pair on the support member and wherein the reporter system includes an amplified reporter system that is independent of layering.

Description:
RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. application Ser. No. 10/123,713 filed Apr. 16, 2002, which claims priority of U.S. application Ser. No. 09/430,429 filed Oct. 29, 1999. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to analytical systems wherein arrays of at least one material such as oligonucleotides, DNA and/or RNA and/or fragments thereof, peptides, protein fragments, polypeptides, polysaccharides, cell fragments, cells and tissues is disposed on a support member and is contacted with a mixture which may or may not include a material which includes binding pair members which bind to at least one of the materials comprising the array. Most specifically, the invention relates to analytical systems of this type which further include an amplified reporter system that are not dependent on layering. 
     BACKGROUND OF THE INVENTION 
     Many analytical techniques and systems are based upon the ability of various materials to form a specific binding pair. As used herein, a specific binding pair is a system wherein the two components share an affinity for each other so as to cause one of the components contained in a mixture of materials to bind to the other upon contact. Either or both components of a specific binding pair may be organic or inorganic. Some examples of specific binding pairs are antibodies and antigens, nucleic materials such as DNA, RNA and fragments thereof, free nucleotides, metallic moieties and nucleic acids or proteins, metal ions-organic ligands, biotin and avidin, folic acid-folate binding protein, polysaccharides-polysaccharide binding protein, sulfhydryls and sulfhydryl reactive groups such as maleimides and haloacetyl derivatives, amines and amine reactive groups such as succinimidyl esters and isothiocyanates, etc. 
     Typical assays based upon the formation of specific binding pairs include a reporter system which provides a detectable signal indicative of the formation of a specific binding pair. For example, one of the members of the pair can be provided with a label which can comprise a fluorescent material, a radioactive material, any other signaling moiety, or a material which is further reactive with another species to form a colored complex or some other such detectable reaction product. The reporter system in these types of assays is commonly referred to as a layered-type system wherein successive layers of reagents such as labeled antibodies or nucleic acid probes are applied one after another in successive manipulation to generate a detectable signal. 
     Recently, a number of technologies have been developed which enable the production of very large arrays comprised of one or more differing materials such as oligonucleotides, DNA and/or RNA and/or fragments thereof, peptides, protein fragments, cell fragments, cells and tissues disposed upon a support body. The various members comprising the array are each capable of forming a unique, specific binding pair with their appropriate counterpart, and such arrays have great utility for rapidly screening mixtures for the presence or absence of a large number of materials. Techniques for the fabrication of such arrays will be found, for example, in U.S. Pat. Nos. 5,744,305; 5,489,678; 5,445,934; 5,405,783; 5,329,028; 5,143,854 and 4,419,444, the disclosures of which are incorporated herein by reference. The formation of specific binding pairs is detected in such arrays by utilizing conventional reporter technology, of the type described hereinabove. 
     There is often a need to increase the sensitivity of such assays. For example, in many instances, species will be present in the mixture at very low concentrations; hence, the detectable signal produced thereby will be very weak. Target amplification techniques, such as polymerase chain reaction (PCR) amplification may be applied to a sample containing nucleic materials so as to increase the concentration of these materials. However, PCR reaction, can be time consuming and difficult to implement. Therefore, it will be appreciated that there is a need for an array-based analytical system and method having an enhanced sensitivity which does not require such complex sample preparation or manipulation. The enhanced sensitivity of an assay of this type would allow for rapid and simple analysis of relatively unmodified biological fluids, preparations and the like. As will be described in detail hereinbelow, the present invention incorporates an amplification system into an array-based analysis. The system of the present invention may be utilized for the analysis of materials such as oligonucleotides, DNA and/or RNA and/or fragments thereof, peptides, protein fragments, polysaccharides, cell fragments, cells and tissues. 
     SUMMARY OF THE INVENTION 
     The present invention provides a method for enhancing the sensitivity of array-based analytical assay systems which comprises a support member having at least one different first member of a specific binding pair affixed in an array thereupon, a mixture which may include at least one second member of a specific binding pair capable of binding to one of the first members so as to form a specific binding pair which is affixed to the support member, and a reporter system that produces a detectable signal indicative of the presence or absence of the specific binding pair on the support member wherein the reporter system comprises an amplified reporter system that is independent of layering. A commercial reagent package capable of performing the sequence of analysis process steps is also disclosed. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. 
       Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: 
         FIG. 1  is a schematic illustration of a catalyzed reporter deposition system in accordance with the present invention; 
         FIG. 2  is a schematic illustration of a further stage in the catalyzed reporter deposition system in accordance with the present invention; 
         FIG. 3  is a further schematic illustration of a stage of the catalyzed reporter deposition system in accordance with the present invention; 
         FIG. 4  is a further schematic illustration of a stage of the catalyzed reporter deposition system in accordance with the present invention; 
         FIG. 5  is a further schematic illustration of a stage of the catalyzed reporter deposition system in accordance with the present invention; 
         FIGS. 6A-C  are reproductions illustrating a comparison of direct and amplified array analysis wherein (A) illustrates the results for direct analysis using 100 μg total RNA, (B) illustrates the results obtained for direct analysis using 4 μg total RNA, and (C) illustrates the results for amplified analysis using 4 μg RNA; 
         FIG. 7  is a prior art detection slide of mouse monoclonal antibody (top) and streptavidin; 
         FIG. 8  is an amplified detection slide, of mouse monoclonal antibody (top) and streptavidin; and 
         FIG. 9  depicts a unit of four adjacent circular spots arranged on the support member according to the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In accord with the present invention, an array-based binding assay incorporates an amplified reporter system utilized for the analysis of various materials including oligonucleotides, DNA and/or RNA and/or fragments thereof, peptides, protein fragments, polysaccharides, cell fragments, cells and/or tissues. As used herein, an amplified reporter system means a system, in which the formation of one specific binding pair will give rise to a multitude of reporter species. This is in contrast to a nonamplified system such as a system wherein a fluorescently tagged antibody reacts with an appropriate antigen to form a specific binding pair which can include fluorescent tags or labels thereupon without generating any signal amplification from the formation of the specific binding pair. 
     One particularly preferred group of amplified reporter systems comprises enzymatically amplified reporter systems with catalyzed reporter deposition (CARD) being one particularly preferred amplification system. CARD amplification is a novel method of signal amplification which is disclosed in U.S. Pat. Nos. 5,731,158; 5,583,001 and 5,196,306, the disclosures of which are incorporated herein by reference. The method uses an analyte dependent enzyme activation system (ADEAS) to catalyze the deposition of reporter or hapten groups (labels) onto the solid phase of an assay support. These enzymatically deposited labels are detected directly or indirectly, which results in signal amplification and improved detection limits. 
     Operation of one catalyzed reporter deposition system is shown schematically in  FIGS. 1-5 .  FIG. 1  depicts a support member  12  having an array of first members of a specific binding pair supported thereupon. The support member  12  is planar and adjacent array spots of first members are intended to be flooded with a single, binding pair member-containing solution. In contrast, prior art arrays have traditionally relied on the formation of separate analyte solution columnar volumes overlying each array spot to prevent signal interference between adjacent spots. Surprisingly, the present invention shows no appreciable signal interference in spite of the absent analyte isolation, a high degree of amplication, and small array spot sizes. As shown in  FIG. 1 , two members of the array  14  and  16  are depicted. As discussed above, these members can comprise materials such as oligonucleotides, DNA and/or RNA and/or fragments thereof, peptides, protein fragments, polysaccharides, cell fragments, cells, tissues, organometallic species illustratively including manganese or magnesium containing organometallics, and organic ligands capable of binding metal ions, and each is capable of binding to a specific material so as to form a specific binding pair. Organic ligands capable of chelating metal ion typically have a molecular weight of less than 500 Daltons and contain Lewis base moieties. In a typical assay, the support member  12  may be polymeric or glass and may be in the form or shape of any solid or porous support, and it will include a number of receptor sites  18  thereupon. These receptor sites  18  function to bind an activated, labeled conjugate, as will be described hereinbelow. The receptor sites  18  may comprise chemically active sites, such as phenolic sites normally present on the support member  12 , or they may comprise a material separately added to the support, such as a proteinaceous material, a phenolic based material, or any other such compound capable of interacting with the activated conjugate, as will be described hereinbelow, or the support surface itself may be chemically reactive. 
       FIG. 2  depicts a further stage in the use of the analytical system. As shown therein, the array is contacted with a mixture which may include one or more second members or analytes of a specific binding pair, capable of binding to at least one of the immobilized first members on the support  12 . As specifically shown in  FIG. 2 , the mixture includes two different second members  20 ,  22 . As illustrated, the second member  20  has bound to the immobilized first member  14  to form a specific binding pair  24 . The other second member  22  is not capable of binding to either of the first members  14 ,  16 , and does not form a specific binding pair, and in a subsequent step is washed away or otherwise removed from the region of the support member  12 . 
     Referring now to  FIG. 3 , there is shown a further step in the method. As shown therein, an enzyme  26  is coupled to the specific binding pair  24 . While the Figures imply that the enzyme  26  is joined to the specific binding pair  24  after the specific binding pair is formed, the methodology of the present invention does not require this sequence of events. In some instances, the enzyme  26  may be coupled to the second member  20  prior to the formation of the binding pair, while in other instances, the enzyme  26  may be coupled after formation of the specific binding pair. Coupling can be accomplished by specific or nonspecific binding reactions. In some particular instances, the enzyme itself will be the second member of the specific binding pair, in which case, formation of the specific binding pair will inherently incorporate the enzyme. In any instance, the net result of the foregoing is that an enzyme  26  will be immobilized upon the support member  12  only at those locations in the matrix at which a specific binding pair is formed. The enzyme, in one specific embodiment, comprises horseradish peroxidase (HRP), although other enzymes may be utilized in other embodiments of the invention. Surprisingly, the free-radical nature of the reaction does not spatially spread a given array spot to proximal array spots. 
     Optionally, the enzyme  26  is coupled to the second member  20  directly without an intermediate bridging unit such as another antibody. Unlike usage application in the instance of ELISA (Enzyme-Linked ImmunoSorbent Assay) as evidenced in Bobrow et al. (Catalyzed reporter deposition, a novel method of signal amplification, Journal of Immunological Methods, 137:103-112, 1991), the present invention does not necessarily require a secondary antibody to form the specific binding pair  24 , and therefore the secondary antibody is optionally excluded in the formation of the specific binding pair  24 . It is appreciated that the recognition and detection of the first member  14  is only analyte-specific at the formation step of the binding pair  24 . The attachment of the enzyme  26  to a specific binding pair such as pair  24  is universal for the detection of other different analytes such as the first member  16 . 
     In one particular instance, the complex formed by the enzyme  26  and the specific binding pair includes only a one antibody. 
     Referring now to  FIG. 4 , there is shown a further stage in the operation of the analytical system of the present invention wherein the support member  12  having the specific binding pair  24  and associated enzyme  26  immobilized thereupon, is contacted with a labeled conjugate  28 . The labeled conjugate  28  includes a substrate (S) for the enzyme  26 , and a label (L). The substrate is a material which is activatable by the enzyme so as to cause it to bind to the receptor sites  18  on the support member  12 . The receptor sites  18  may be reactive components of the support member  12  or may be added to the support member  12 . The label can be any detectable label, such as a fluorescently detectable label, a hapten (e.g. biotin), a radioactive label, or a chemically reactive, color forming label, through redox, chemiluminescence or other mechanism, or any other signaling moiety. As will be seen from  FIG. 4 , the enzyme  26  creates an activated conjugate  28 N, and as seen in  FIG. 5 , this activated conjugate  28 N binds to the receptor sites  18  in the region of the specific binding pair  24 . The unactivated conjugate  28  is not capable of binding to the receptor sites  18 ; hence, the label is displayed only proximate the specific binding pair  24 . As noted from  FIG. 5 , the formation of one specific binding pair  24  catalyzes the deposition of a number of labeled conjugates, thereby providing an amplified reporter system. 
     The support member  12  of an inventive analytical system is pre-treated with receptor deposition. Receptors, preferably suspended or dissolved in liquid form, are deposited onto the support member in pre-determined sites thereupon. Receptors so deposited are grouped into spots in various forms such as a circular spot, a polygon spot, a square spot, or a rectangular spot, preferably a circular spot or a rectangular spot. Within each spot there contains one or more molecules of a first member such as the first member  14  or the first member  16 . The spots are arranged on the support member  12  so as to provide a functional distance between any two adjacent spots. The functional distance, Fd, is the shortest distance between the boundaries of any two adjacent spots whereby signal bleeding between the two adjacent spots are prevented or minimized. The functional distance, Fd, varies with the shape of spots arranged on the support member  12 . 
       FIG. 9  depicts a unit of four adjacent circular spots which are of equal functional size Fs. For a circular spot, the functional size Fs is the diameter of the spot. The functional width defines the smallest amount of a circular area on the support member  12  which is capable of delivering an optimal dynamic range of signal. The functional width varies with the density of the receptors  18  and often the occurrence of the second member  20 . For the circular spot, Fs is a value in the range of 5 to 100 micrometers, preferably in the range of 20 to 400 micrometers, and more preferably in the range of 40 to 150 micrometers. The functional distance Fd has a value in the range of 10 to 200 percent of that of Fs and preferably in the range of 50 to 150 percent of that of Fs. 
     A total number of the spots upon which the receptors are deposited varies with the type of the support member  12 . In particular instances where the support member  12  is a conventional glass slide (1×3 in) or a nitrocellulose membrane strip of equivalent size, the total number is within the range of 4 to 400, preferably within the range of 8 to 200, and more preferably within the range of 16 to 100. 
     An inventive analytical system enables an end user to customize and group particular analytes to be detected. A total number of the analytes that are detectable varies with the type of the support member  12 . Since conventional microarray applications require a much higher number of analytes, often in the number of 3000 and up and a small group of analytes the versatility is of these microarrays is limited. This limitation is overcome by the present invention in which the identities and the total number of the analytes are determined by the end user. The duplicates of each analyte is aligned as a row or a column on the support member  12  and different rows or columns are grouped so as to be corresponding to a particular experimental variation to a sample wherein the analyte is contained. Due to the versatility of the inventive analytical system, the end user is able to construct different systems corresponding to respective metabolic research needs. An exemplary list of groups of analytes includes those detecting glycolysis, citric acid cycle, pentose phosphate pathway, glycogen biosynthesis, galactose pathway, beta-oxidation pathway, cholesterol biosynthesis, urea cycle, amino acid metabolism, oxidative phosphorylation, cell cycle, signaling pathway, and blood coagulation pathway. Analytes involved in one of more of above biomedical pathways are included on a support member  12 . Optionally an internal control with duplication is included in each system so that data generated thereto is comparable to one another. 
     A commercial reagent package according to the present invention contains a support derivatized as detailed herein, along with peroxidase, enzyme, coupling agent, and labeling agent reagents. Instructions are supplied to perform the inventive amplified detection as detailed herein. 
     The methodology of the present invention may be implemented in accord with various array-based analyses of the type shown in the prior art including both layered and non-layered assays and incorporated hereinabove by reference. Specific chemistries for the catalysts, supports, substrates, labels and members of the specific binding pair will depend upon the exact nature and purpose of the assays, which, in view of the teaching presented herein and in the patents referred to herein, will be readily apparent to one of skill in the art. 
     EXAMPLES 
     Comparison of Direct and Amplified Array Analysis 
     Example 1 
     Nucleic Acid Detection 
     For direct detection, cyanine  5  labeled cDNA was prepared from 100 μg and 4 μg Jurkat total RNA using the MICROMAX Direct Reagent Kit (NEN Life Science Products, Boston, Mass.). The cyanine 5 labeled cDNA was hybridized to Practice Slides (MICROMAX Human cDNA Microarray System I, NEN Life Science Products, Boston, Mass.) according to MICROMAX Human cDNA System I-Direct (NEN Life Science Products, Boston, Mass.) kit directions. 
     For amplified analysis, biotin labeled cDNA was prepared from 4 μg Jurkat total RNA using the MICROMAX Human cDNA Microarray System I kit reagents and protocols. Hybridization to Practice Slides and amplified detection using streptavidin-HRP and cyanine 5 tyramide were according to the MICROMAX Human cDNA Microarray System I kit directions. 
     Slides were scanned on a GSI Lumonics ScanArray 5000 (Watertown, Mass.) scanner. 
     The results for direct analysis using 100 μg total RNA are shown in  FIG. 6A .  FIG. 6B  shows the results obtained for direct analysis using 4 μg total RNA. The results for amplified analysis using 4 μg total RNA is shown in  FIG. 6C . The loss of signal going from 100 μg to 4 μg of total RNA for direct analysis indicates that there is insufficient material available for adequate analysis. A greater amount of cells or tissue mass is required for the direct method. The signal for the inventive amplified analysis using 4 μg of total RNA is greater than that using 100 μg for direct analysis, allowing for much greater flexibility in analyzing small amounts of tissues or cells. 
     Example 2 
     Protein Detection 
     Aminosilane slides (Erie Scientific, Portsmouth, N.H.) were spotted with a mouse monoclonal antibody and streptavidin. For spotting, serial two-fold dilutions were made of each protein starting at 100 ug/ml in phosphate buffered saline (PBS). Slides were washed in PBS containing 0.1% Tween 20 (Sigma, St. Louis, Mo.) and blocked for 30 minutes with PBS, 0.1% Tween 20, 0.5% Bovine Serum Albumin (BSA, Sigma). The slides were then incubated for 30 minutes with Goat anti-Mouse Biotin (Sigma) diluted 1/100 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. 
     For conventional detection, slides were incubated for 30 minutes with Streptavidin-Cy3 (Jackson ImmunoResearch, West Grove, Pa.) diluted 1:100 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. The slides were rinsed in water and air dried. 
     For inventive amplified detection, slides were incubated for 30 minutes with Streptavidin-HRP (PerkinElmer Life Sciences, Boston, Mass.) diluted 1/100 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. The slides were then incubated for ten minutes with Biotinyl-Tyramide diluted 1/500 in Amplification Diluent (ELAST Kit, PerkinElmer Life Sciences) containing 2.5 M NaCl, and washed three times with PBS, 0.1% Tween 20. Slides were incubated for 30 minutes with Streptavidin-Cy3 diluted 1:200 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. The slides were rinsed in water and air dried. 
     Slides were scanned with a Packard (Downers Grove, Ill.) ScanArray 5000 scanner. 
     The results for conventional direct analysis for a quadruplet dilution series of mouse monoclonal antibody (top) and streptavidin (bottom) visualized with anti-mouse IgG biotin and streptavidin Cy3 are shown in  FIG. 7 . The results of the inventive amplified detection for the same quadruplet dilution series as per  FIG. 7  are shown in  FIG. 8 . Inventive detection is visualized with anti-mouse IgG biotin, streptavidin-HRP, biotinyl-tyramide and streptavidin Cy3. The inventive amplified detection is approximately 64 to 128 times more sensitive and approximately 32 times more sensitive for immobilized mouse monoclonal antibody and streptavidin, respectively, as compared to conventional direct detection. 
     Example 3 
     Cell Lysate Detection 
     Aminosilane slides (Erie Scientific, Portsmouth, N.H.) are spotted with an array of antibodies targeted to yeast proteins. Each antibody is spotted at a predetermined optimal concentration in phosphate buffered saline (PBS). Slides are washed in PBS containing 0.1% Tween 20 (Sigma, St. Louis, Mo.) and blocked for 30 minutes with PBS, 0.1% Tween 20, 0.5% Bovine Serum Albumin (BSA, Sigma). 
     Yeast cell lysate is prepared by incubating the cells in a sodium acetate buffer, pH 5.0, containing 15% CHAPS (Sigma), 8 M Urea (Sigma), 5 mM EDTA (Sigma) and 4 mM TCEP (Pierce, Rockford, Ill.). The pH is adjusted by adding 10 μl of 1 M BisTris-HCl buffer pH 6.5 per 100 μl lysate. To label the proteins with biotin, 15 μl of 0.1 M N-(biotinoyl)-N′-(iodoacetyl) ethylenediamine (Molecular Probes, Eugene, Oreg.) in dimethylsulfoxide (Aldrich, Milwaukee, Wis.) is added per 100 μl lysate and incubated for 60 min. at ambient temperature. Reagents are removed by dialysis against PBS. 
     Alternately, yeast cell lysate is prepared by suspending the cells in sodium acetate buffer, pH 5.0. The cells are disrupted using glass beads and a Mini-BeadBeater (BioSpec Products, Bartlesville, Okla.) according to the manufacturer&#39;s directions. Cell debris is removed by centrifugation for 15 min. at 12000×g. The extracted proteins are reduced with 4 mM TCEP, and the pH is adjusted by adding 10 μl of 1 M BisTris-HCl buffer pH 6.5 per 100 μl lysate. To label the proteins with biotin, 15 μl of 0.1 M N-(biotinoyl)-N′-(iodoacetyl) ethylenediamine (Molecular Probes, Eugene, Oreg.) in dimethylsulfoxide (Aldrich, Milwaukee, Wis.) is added per 100 μl lysate and incubated for 60 min., and the lysate is dialyzed against PBS. 
     The slides are incubated for 60 minutes with the labeled cell lysate and washed three times with PBS, 0.1% Tween 20. 
     For standard detection, slides are incubated for thirty minutes with Streptavidin-Cy3 (Jackson ImmunoResearch, West Grove, Pa.) diluted 1:100 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. The slides are rinsed in water and air dried. 
     For amplified detection, slides are incubated for 60 minutes with Streptavidin-HRP (PerkinElmer Life Sciences, Boston, Mass.) diluted 1/100 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. The slides are then incubated for ten minutes with Biotinyl-Tyramide diluted 1/500 in Amplification Diluent (ELAST Kit, PerkinElmer Life Sciences) containing 2.5 M NaCl, and washed three times with PBS, 0.1% Tween 20. Slides are incubated for thirty minutes with Streptavidin-Cy3 diluted 1:200 in PBS, 0.1% Tween 20, 0.5% BSA and washed three times with PBS, 0.1% Tween 20. The slides are rinsed in water and air dried. 
     Slides are scanned with a Packard (Downers Grove, Ill.) ScanArray 5000 scanner. Results were comparable to those detailed with respect to Example 2. 
     The foregoing drawings, discussion and description are illustrative of the general principles of the present invention, and some specific embodiments thereof, but are not meant to be limitations upon the practice of the present invention, since numerous modifications and variations will be readily apparent to one of skill in the art. It is the following claims, including all equivalents, which define the scope of the invention.