ASSAY METHODS

Abstract: New methods for performing sandwich assays have been discovered. The assays utilize dual capture probes and eliminate noise resulting from nonspecific binding of detector probe to capture probe.

Description: ASSAY METHODS Field Of The Invention The invention relates to methods a d kits for detecting target molecules in samples. In particular, the target molecules can be nucleic acid fragments from clinical samples. Embodiments of the invention provide methods for rapid and sensitive detection of target nucleic acids. Background The utility of detecting target nucleic acid sequences in clinical diagnostics is well established. Various methodologies have been developed for detecting particular target nucleic acid sequences as diagnostic of the presence of the target organism. These include hybridization assays which rely upon one or more tailored nucleic acid probes which are able to hybridize or bind to sequences of nucleic acid within the target nucleic acid. The hybridization assays developed to date remain limited due to the presence of artificial signal or noise generated during the assay and competitive with the signal generated by captured target nucleic acid. For example, sandwich hybridization assays have been developed which employ two separate nucleic acid probes able to hybridize to mutually exclusive nucleic acid sequences within the target nucleic acid. One of the probes, the detector probe, is constructed to carry a detectable label moiety to permit detection of the captured target. The second probe, the capture probe, is constructed to permit binding to a support as well as the target to facilitate sample purification and isolation of the captured target. Under hybridization or binding conditions, the two probes hybridize with the target nucleic acid to form a capture probe:target:detector probe ternary complex. Introduction of the support under binding conditions results in capture of the ternary complex by the support The detector probe can then be isolated and measured to determine the presence or quantity of target in the sample. The sensitivity of such sandwich hybridization assays is known to be limited, however, because the detector probe will bind to the assay support directly notwithstanding the absence of target in the sample. The binding of detector probe directly to support in the absence of target is referred to as nonspecific binding. Additionally, the sample to be assayed may also have other nucleic acid sequences of some similarity to the sequence selected as the target, known as pseudo targets. These pseudo targets, at high levels associated with detector probe, may also bind nonspecifϊcally to the assay support and, further, bind to the detector probe thereby binding the detector probe to the support in the absence of target and creating noise. In addition, pseudo target associated with detector probe can hybridize with a few mismatches to the capture probe and, thus, be captured on the assay support This binding of detector probe to support through a nucleic acid sequence similar to the target is referred to as nonspecific hybridization. Nonspecific binding and nonspecific hybridization generate detectable signal or noise which is cumulative with the signal generated by capture of the ternary complex and target The problem of noise due to nonspecific binding and nonspecific hybridization is often exacerbated by the presence of excess detector probe in the assay to facilitate rapid and complete formation of the target* detector probe hybridization. Conventional sandwich hybridization assays are able to detect to a limit of about 10 8 targets. However, diagnostic assays often seek to detect the presence of targets present in samples at concentrations much lower than the 10 8 target threshold presently permitted by conventional assays. Researchers have attempted to eliminate or, at least, lessen the noise from sandwich hybridization assays. One method, hereafter referred to as Reversible Target Capture or RTC, has been effective in reducing such background noise. Reversible Target Capture involves cycles of capture and release of the ternary complex from a succession of supports. Initially, following conventional sample preparation to expose the target nucleic acid for detection, capture and detector probes are added to the sample medium. In the presence of target and upon imposition of binding conditions the ternary complex is formed. Upon introduction of a suitable support, the ternary complex is captured by the support However, a certain amount of detector probe binds nonspecifically to the support The support and bound ternary complex are then removed from the sample medium, typically washed, and then introduced to a second sample medium. Releasing conditions are then imposed causing the ternary complex to be released from the support The original support is then removed from the second sample medium, thereby removing some of the nonspecifically bound detector probe from the assay. Thereafter, a second support is introduced to the second sample medium, binding conditions again imposed and the ternary target containing complex is again captured. Since some of the detector probe nonspecifically bound to die original support may also have been released into the second sample medium by imposition of releasing conditions and, thus, be available for nonspecific binding to the second support, additional cycles of release and capture may be necessary to achieve maximal sensitivity for the assay. Typically, three cycles of target capture are sufficient to reduce noise below detectable limits in a conventional sandwich hybridization assay format Reversible Target Capture and other methods aimed at reducing noise resulting from nonspecific binding of the detector probe are described in detail in European Patent Application No. 87309308.2 (Publication No. 0265244), Analytical Biochemistry, 181, 345-359 (1989), and Analytical Biochemistry, 181, 360-370 (1989). All are hereby incorporated by reference. Sandwich hybridization assays supplemented with Reversible Target Capture are able to extend the level of sensitivity to about 10 6 targets. Notwithstanding the success of these techniques, however, those in die art have continued to look for additional ways to enhance the sensitivity of the assay. These include the use of amplification schemes to further amplify the target or signal generated by the assay. For example, European Patent Application No. 88312135.2 (Publication No.0328829) describes improved assay methods which amplify the target polynucleotide before detecting it Amplification is accomplished enzymatically using polymerase enzymes. For example, target DNA and the enzyme core RNA polymerase can be used to form RNA complements to the target DNA. The RNA complements are then detected. Similarly, target RNA can be replicated by enzymes such as Qβ-replicase and reverse transcriptase before detection. These techniques are fully compatible with Reversible Target Capture and can be used to enhance assay sensitivity to approximately 10 6 targets. These techniques are described more fully in European Patent Application No. 88312135.2 which is incorporated herein by reference. Alternatively, assay sensitivity can be enhanced by amplifying the signal produced by the detector probe or, more particularly, the detector probe itself. This can be accomplished by adopting midivariant (MDV-1) RNAs for use as detector probes. Midivariant-like RNAs can serve as templates for Qβ-replicase and can be replicated thereby. Hence, detector probes can be constructed for amplification by Qβ-replicase to further enhance the assay. The use of Qβ-replicase amplification in nucleic acid sandwich hybridization assays to enhance assay sensitivity to approximate by 10 6 targets is described by Cahill et al., Clinical Chemistry, 37, 1482 (1991) which is incorporated herein by reference. Notwidistanding the advances made to date, the sensitivity of nucleic acid sandwich hybridization assays remains limited by background noise generated during the assay. Hence, it would be beneficial to increase assay sensitivity to a point where the sensitivity of the assay is limited by the ability of the assay to detect signal from the target only, and not by background noise produced during the assay. Accordingly, it is, an object of the present invention to provide a sandwich hybridization assay of enhanced sensitivity. It is another object of the present invention to provide a sandwich hybridization assay for which the sensitivity of the assay is limited by the signal generated by target and not by background noise generated during the assay. Summary Of The Invention It has now been discovered that significantly more sensitive sandwich hybridization assays obtain when certain modifications are made to the Reversible Target Capture assay method. The sensitivity of the new assays is limited by me signal generated as a result of detected target and not by background noise. Introduction of the new assay methods into a hybridization assay utilizing a signal amplification scheme such as using Qβ-replicase to replicate an RNA-like template within d e detector probe permits the sensitivity of the assay to be enhanced by a factor of 10 3 . Thus, sensitivity can be increased by up to three orders of magnitude. Previous efforts aimed at reducing background noise in sandwich hybridization assays have been directed to reducing the noise resulting from the nonspecific binding of labeled detector probe to the supports used in the assay. Although techniques such as Reversible Target Capture are effective in reducing such noise, residual noise remained in the assay. It has now been discovered that this residual noise can be reduced significantly to the point where it is eliminated entirely. It has been discovered that this residual noise can be eliminated by using two or more separate capture probes and modifications of the RTC format It is believed the new assay formats eliminate noise due to nonspecific binding of the detector probe to the capture probe. Prior Reversible Target Capture methods utilized a single capture probe and preserved the capture probe:target:detector probe ternary complex throughout die assay. Accordingly, detector probe nonspecifically bound to die capture probe is maintained throughout the target release and recapture cycles of RTC and is present at the end of die assay to generate noise in competition with the signal resulting from the captured target This noise which remains in the assay during target release and recapture is termed cyclable noise. The methods described herein are effective at removing cyclable noise as well as the noise resulting from nonspecific binding of detector probe to support In effect the methods can produce a sandwich hybridization assay entirely free of background noise. The invention comprises two classes of Dual Capture assays. The Class I method comprises d e steps of contacting the sample with a first capture probe and detector probe which in the presence of target bind to d e target to form a first capture probe:target:detector probe ternary complex; separating die target.detector probe segment of the ternary complex from the first capture probe; contacting the sample witii a second capture probe which binds to the target to form a second capture probe:target:detector probe ternary complex; minimizing the amount of detector probe nonspecifically bound to the second capture probe; and monitoring the second ternary complex for the presence or absence of detector probe. The Class π method comprises the steps of contacting the sample with a first probe and a second capture probe which in die presence of target bind to the target to form a target complex; minimizing the amount of second capture probe present in the sample; contacting the sample with detector probe and fresh first capture probe which in the presence of target binds to the target forming a quaternary complex comprising a second target complex of the second capture probe.target-detector probe and bound first capture probe; minimizing the amount of detector probe bound to die second capture probe of die second target complex; and monitoring the second target complex for the presence or absence of detector probe. More particularly, a preferred embodiment of die Class I method of the invention comprises the steps of contacting the sample medium to be assayed for target with a first capture probe and detector probe which, in the presence of target bind to the target to form a first ternary complex of first capture probe.target-detector probe; contacting the sample with a first support wherein die first support binds to the first capture probe thereby binding d e first ternary complex to the support; substantially separating the first support and first ternary complex from die sample medium; introducing die first support and first ternary complex to a second medium and releasing die target.detector probe segment of die first ternary complex from die first support and capture probe; substantially separating the first support and capture probe from the second medium; introducing a second support and second capture probe to the second medium to form a second ternary complex of second capture probe: targetdetector probe bound to d e second support; substantially separating the second support and second ternary complex from d e second medium; introducing d e second support and second ternary complex to a third medium and releasing the second ternary complex from the second support; substantially separating the second support from the tiiird medium; and detecting die presence of target in die tiiird medium. Additional cycles of target capture and release can be performed as necessary to complete the reduction of noise due to nonspecific binding of detector probe to support A preferred embodiment of die Class II method comprises the steps of contacting the sample to be assayed with a first capture probe and a second capture probe wherein die first and second capture probes bind to the target to form a target complex; contacting the sample with a second support which binds to the second capture probe thereby binding d e target complex to the second support; substantially separating die second support and bound target complex from the sample; introducing die second support and bound target complex into a second medium and releasing the target complex from the second support; substantially separating die second support from die second medium; contacting the second medium widi a first support which binds to die first capture probe thereby binding die target complex; substantially separating the first support and bound target complex from the second medium; introducing die first support and bound target complex into a third medium and releasing die second capture probe: target segment of die target complex from the first support and first capture probe; substantially separating the first support and first capture probe from the tiiird medium; contacting the third medium widi detector probe and fresh first capture probe which in die presence of target bind to the target to form a quaternary complex comprising a second target complex of the second capture probe:target:detector probe and bound first capture probe; contacting die tiiird medium widi fresh first support which binds with die first capture probe to bind die quaternary complex; substantially separating the first support and bound quaternary complex from the third medium; introducing die first support and bound quaternary complex into a fourth medium and releasing d e second target complex from the first support and first capture probe; substantially separating the first support and bound first capture probe from the fourth medium; contacting the fourth medium wid second support which binds to die second capture probe; substantially separating die second support and bound second target complex from the fourth medium; introducing die second support and bound second target complex into a fifth medium and releasing the second target complex from the second support ; substantially separating the second support from die fifth medium; and monitoring the second target complex for the presence or absence of detector probe. Brief Description of the Figures Figure 1 presents side by side for comparison the results from a matrix of experiments demonstrating signal and noise production for nucleic acid hybridization assays for targets from Chlamydia trachomatis using the Dual Capture Class I mediodology of die invention and standard Reversible Target Capture methodology. Figure 2 presents side by side for comparison the results from a matrix of experiments demonstrating signal and noise production for nucleic acid hybridization assays for targets from Chlamydia trachomatis using separate embodiments of die Dual Capture Class I methodology of die invention. Figure 3 presents side by side for comparison the results from a matrix of experiments demonstrating signal and noise production for nucleic acid hybridization assays for targets from Chlamydia trachomatis using die Dual Capture Class II mediodology of the invention and a comparable Reversible Target Capture mediodology. Detailed Pescriptic-n Of Th? Invention It has now been discovered tiiat the sensitivity of sandwich assays can be enhanced considerably using assay methods which are modifications of conventional sandwich assays using die Reversible Target Capture technique described, for example, in European Patent Application No. 873093082. The methods disclosed here substantially eliminate the background noise that would otherwise accrue during die assay and, thus, limit the sensitivity of die assay. In doing so, die methods disclosed here extend die sensitivity of the assays by up to three orders of magnitude and result in assays with sensitivity limited by true signal response rather than noise. The mediods of die invention are compatible widi sandwich assays generally. They are particularly advantageous in sandwich hybridization assays for nucleic acid targets. The methods are also amenable to kits, automation and instrumentation. Thus, the mediods disclosed herein permit rapid, convenient and sensitive sandwich assays for conventional assay targets. The assay methods disclosed herein are advantageous because they address a heretofore unappreciated noise component of the sandwich assay. Previously, assay mediods had been developed to reduce noise resulting from nonspecific binding of detector probes to supports used in sandwich assays and to materials in the sample which are somewhat similar to the actual target In nucleic acid hybridization assays, tiiese can be nucleic acid sequences which bear some similarity to the targets. The assay mediods of die invention are generally termed Dual Capture Class I and Class π assays. In contrast to conventional sandwich assays which utilize a single capture probe throughout the assay, the Dual Capture assays use multiple capture probes in die assay. Moreover, although it is necessary that die capture probes be able to bind to both the supports and targets used in die assay, it is also a feature of die invention that the capture probes are constructed and selected for use in die assay such that some bind more strongly to the assay support than the target and so can be separated from die target while remaining bound to the support, while others bind more strongly to the target than the support and so can be separated from die support while remaining bound to die target For example, in the Dual Capture Class I assay, the capture probes may bind to the same or different regions of the target but are also constructed such that the first capture probe is more readily separated from die target tiian die support to which it binds while die second capture probe is more readily separated from the support than the target Initially, the first capture probe and detector probe are contacted widi die sample and allowed to bind widi die target to form a first ternary complex. Next, a first support which binds to die first capture probe is contacted widi die sample. The target and detector probe are then captured dirough the first capture probe to die support The first support and bound, first ternary complex are substantially separated from the sample medium and introduced to a second medium. Thereafter, the target and detector probe segment of die ternary complex is released and separated from die first capture probe and support which are dien removed from the second medium. Presumably, removal of the support and first capture probe removes detector probe nonspecifically bound to the support and to die capture probe, thereby removing background noise from the assay. Next, the target and detector probe can be recaptured by first introducing die second capture probe to form a second ternary complex and dien a second support to die second sample medium. Thereafter, the second support and second ternary capture probe: target.detector probe complex can be transferred to a third medium for processing. Then die second support can be separated from die second ternary complex and separated from die tiiird medium. The ternary complex is men monitored for the presence or absence of detector probe. When target is present in die original sample, detector probe will be detected. Presumably, separation of the second support from the third medium removes detector probe nonspecifically bound to the second support The capture and release using first and second capture probes can be performed in either order. Moreover, additional cycles of capture and release using either capture probe or still another capture probe can be performed to further reduce background noise as needed. As illustrated in Example 1, one cycle using the first capture probe and two cycles using the second capture probe eliminate almost all of the background noise. Similarly, the Dual Capture Class II method also uses separate capture probes to remove background noise. In this method, die separate capture probes bind to separate regions of the target The separate capture probes are again constructed such that one will bind more strongly to a support than the target while die otiier will bind more strongly to the target than support. Moreover, the capture probes and supports are selected so that there is minimal cross reactivity between them. The first capture probe and first support are selected so as to have minimal affinity for the second support and second capture probe, respectively. In the Class II method, both capture probes are bound to die target to begin the assay. One of the capture probes will be used for target capture and release cycling in completion of the assay. Accordingly, steps are taken to minimize the amount of any excess of this capture probe from the assay, i.e., any capture probe not specifically bound to die target before introduction of die detector probe into the assay. For example, die first and second capture probes are combined widi die target and bind thereto to form a target complex. Excess first capture probe not bound to die target can be removed by capturing the target complex with a second support specific for the second capture probe but which has no affinity for the first capture probe. The second support and captured target complex can be separated from die sample medium and transferred to a second medium for further processing. The second capture probe is constructed to bind more strongly to the target than the second support Thus, the target complex can then be separated from the second support. The second suport can be separated from die second medium. Thereafter, the target complex can be captured widi a first support specific for die first capture probe. This first capture probe is constructed to bind more strongly to die first support than to die target Thus, the first support and captured target complex can be separated from die second medium and introduced into a third medium for further processing. The target and second capture probe can dien be released from the first support and first capture probe. The first support and first capture probe can then be separated from the third medium presumably removing excess second capture probe nonspecifically bound to die first support which may cause noise in die assay. Next die target and second capture probe can be recaptured first by a fresh supply of first capture probe in the presence of detector probe and d en by a first support The first support and captured quaternary complex comprising a second target complex of the second capture probe:target:detector probe and bound first capture probe can be separated from the third medium and introduced to a fourth sample processing medium. Next, the second target complex can be released from the first support and first capture probe. These can be separated from the sample medium tiiereby removing excess detector probe nonspecifically bound to die first support and first capture probe and reducing background noise from the assay. Next one or more cycles of capture and release of the second target complex can be performed widi die corresponding second support to further reduce background noise. Additionally, one or more cycles of capture and release using the first capture probe and first support can also be performed. The additional capture and release cycles may be performed in any order. Finally, the second target complex is monitored for die presence or absence of detector probe indicating die presence or absence target in die original sample. Those skilled in the art will appreciate diat binding of probes to supports and target is facilitated by imposition of suitable binding conditions. Similarly, release of targets and probes from supports can be facilitated by imposition of releasing conditions. Binding and releasing conditions are well known in die art and include modulation of sample temperature and salt concentrations in sample solutions. Those skilled in die art will also appreciate that wash steps can be interposed between capture and release steps in both Dual Capture assay formats to further reduce background noise. Those skilled in the art will also appreciate that capture probes are often added separately from the corresponding supports for the convenience of the assay to facilitate complex formation. However, capnire probes and supports can be added togedier without otherwise disrupting die performance of the assay. The Dual Capture methods of die invention are compatible with conventional sandwich assay procedures. The methods are compatible with a wide array of supports such as filters, dipsticks, and dispersible particles. The methods are compatible with conventional sandwich assay targets such as antigens and nucleic acid sequences, and dieir corresponding antibody and nucleic acid binding partners as probes. The methods are compatible widi conventional labeling moieties and detection schemes. The Dual Capture methods are particularly well-suited for nucleic acid hybridization assays. The methods apply equally well to assays for DNA and RNA targets. Nucleic acid probes and supports are readily constructed widi the binding characteristics required for Dual Capture. For example, target specific nucleic acid sequences can be derivatized with one member of a ligand binding pair such as biotin. When used in conjunction widi supports derivatized widi die other member of the ligand binding pair, such as streptavidin for biotin, such probes will bind to both target and supports such that die binding is stronger to the supports than target Thus conditions can be imposed whereby the probe will separate from the target but not the particles. Similarly, such probes and supports can be made by derivatizing target specific nucleic acid sequences widi deazadeoxyguanosine residues and d e corresponding supports, with deoxycytidine residues. Those skilled in the art will appreciate that other probe-support combinations can be devised widi die requisite properties. Probes which bind more strongly to target than supports are well known and routinely used in conventional sandwich hybridization assays. For example, target specific nucleic acid sequences can be derivatized with homopolymeric tails of specific nucleotide residues such as deoxyadenylate residues dA. When used in conjunction with supports derivatized with deoxytiiymidine residues dT, such dA-tailed probes will bind to both target and dT derivatized supports such that die binding is stronger to die targets tiian the supports. Thus conditions can be imposed whereby die probe will separate from the support but not die target Nucleic acid hybridization assays are also compatible widi supports which are dispersible witiiin the sample media. Such supports are particularly advantageous because they enable rapid hybridization between capture probe and die support and, hence, a more rapid assay procedure. Examples of such supports are polystyrene beads and magnetic particles. Those skilled in die art will appreciate, however.that it may be difficult to capture and separate all of such supports from a sample medium. Nevertheless, tiiose skilled in the art will be able to separate substantially all of such supports without compromising the assay. The methods of die invention are amenable to use widi kits. Such kits include supplies of first and second capture probes specific for the Class I or Class π method to be used. The kits also include a supply of detector probe. All probes are specific for the target to be detected. Additionally, die kits can include supplies of appropriate supports and reagents to facilitate binding, releasing and washing during the assay. EXAMPLES Materials Paramagnetic particles derivatized widi oligo dT and dCi4 were prepared for use in the Examples as follows. Sub-micron ferric oxide particles (Advanced Magnetics, Inc., Cambridge, MA) were derivatized widi dTi4 or dCi4 oligomers using the method described by Moπissey, Anal. Biochem., 181:345-349 (1989). The beads were blocked for 4 hrs. at 65 * C in Bead Blocking Buffer [lOOmM Tris 4.9% BSA (Bovine Serum Albumin, Sigma Fraction 5, 0.5% Sarkosyl (Sigma), lOmM EDTA, 0.05% Bronopol (Indlex, Philadelphia, PA), 0.01% antifoam [Dow-Coming FG-10], ph 7.8. The final suspension of particles was 0.25% (W/V) solids widi a binding capacity, of 300 pmol (deoxyadenylate) dAso per mg. Just before use, particles were separated from the buffer and made up to 0.045% in fresh Bead Blocking Buffer and Bead Blocking Buffer plus 4M guanidine hydrochloride (GuHCl). Paramagnetic particles derivatized widi streptavidin were obtained from Promega (Madison, WI). Purified 16S rRNA from Chlamydia trachomatis was used as the target in all examples. The 16S rRNA was purified using conventional techniques. Various nucleic acid sequences are incorporated into the probes used in d e Examples. These are Sequence Ref. No. Sequence (5' - 3') 3018 (Sequence ID No. 1) CCΠTAA(X3TΓACKX GATGCCCAAA 781 (Sequence ID No. 2) OπAA∞πACTOTCA GO^ 1660 (Sequence ID No. 3) CTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTC- AGTTCCAGTGT C29 (Sequence ID No.4) GGGGACCCCCCCGGAAGGGGGGGACGAG- GUGCGGGCACCUCGUACGGGAGUUCGAC- CGUGACGCGUCGUCGAGUUGAGGCCUUU- ACCCCACCAACUAGCUGAUAUCAC- AUACUGAAUCUUUAGTCACGGGCU- AGCGCUUUCGCGCUCUCCCAGGUGACGC- CUCGUGAAGAGGCGUGACCUUCGUGCGU- UUCGGUAACGCACGAGAACCGCCACGCU- GCUUCGCAGCGUGGCCCCUUCGCGCAGC- CCGCUGCGCGAGGUGACCCCCCGAAGGG- GGGUUCCC The nucleic acid sequences 3018, 781, and 1660 were made using conventional beta- cyanoediyl phosphoramidite chemistry and conventional nucleic acid syndiesizer equipment such as die 380-B Synthesizer from Applied Biosystems, Foster City, CA. The sequences were modified for use as capture and detector probes as follows. The sequence 3018 (Sequence ID No. 1) was biotinylated to form probe B-3018 by first modifying die 5' end of sequence 3018 (sequence ID No. 1) to contain a primary amine. This was accomplished by addition of an amino-propyl-modified cystidine phosphoramidite. Deprotection of the phosphates and nucleotide bases was accomplished by standard mediods. The crude oligonucleotide mixtures were purified by reverse phase HPLC. The amino- modified oligonucleotide was labeled widi biotin using the Fluo Reporter Biotin Labeling Kit, F-2610 from Molecular Probes, Inc. The sequences 781 (Sequence ID No. 2) and 1660 (Sequence ID No. 3) were tailed widi about 150 deoxyadenylate residues at die 3' end of each sequence to form probes dA- 781 and dA-1660. This was accomplished by incubation widi terminal deoxynucleotidyl transferase (Life Sciences, Inc.) following the method of Nelson et al, Methods in Enzymology, 68: 41-47, 1979. The sequence 3018 (Sequence ID No. 1) was also tailed wid about 150 deazadeoxyguanosine residues (C7dG) at d e 3' end of the sequence to form probe C7dG-3018. This was accomplished using the metiiod described by Nelson et al. The sequence C29 (Sequence ID No. 4) was used as the detector probe in all examples without further modification. The RNA sequence C29 (Sequence ID No. 4) was made from a DNA sequence complementary to C29 (Sequence ID No. 4) prepared by conventional methods. The complementary sequence was cloned into a transcriptase vector. C29 was transcribed from the cloned DNA using a Ty RNA polymerase kit from Promega. Examples 1-3 demonstrate matrix experiments illustrating various embodiments, features and advantages of die invention. For example, Example 1 provides a side by side comparison of the Class I Dual Capture Methodology of die invention widi standard Reversible Target Capture Methodology. The example demonstrates die detection of targets using these assay methodologies and also die various components of noise generated during these assays for purposes of comparison. Example 1 demonstrates die noise generated during die assays attributable to nonspecific binding of detector probe to support and detector probe to capture probe. Example 1. This example compares signal and noise from the Dual Capture Class I methodology of the invention with those from standard Reversible Target Capture methodology in die detection of Chlamydia trachomatis. The Dual Capture assays used B- 3018 and dA-781 capture probes. The RTC assays used only die dA-781 capture probe. The assays were performed as follows. Five solutions were prepared: Solution 1 was made up to comprise 700 microliters of 1.4 * 10 6 purified target molecules, 70ng B-3018 capture probe, 1.4 * 10 12 molecules of C29 detector probe in 4M guanidine hydrochloride (GuHCl), 125mM Tris pH 7.8 and 16mM EDTA. Solution 2 was made up to comprise 700 microliters of 1.4 * 10 6 purified target molecules, 70ng dA-781 capture probe, 1.4 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. Solution 3 was made up to comprise 700 microliters of 1.4 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. Solution 4 was made up to comprise 1600 microliters of 160ng B-3018 capture probe, 3.2 * 1012 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. Solution 5 was made up to contain 1600 microliters of 160ng dA-781 capture probe, 3.2 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. An 8(A-H) x 12(1-12) 96 well rack for 1.5mi polypropylene tubes (Micronics, Flow Laboratories, McLean, VA) was set up. Rfty microliter aliquats of solutions 1-5 were added to die tubes as follows: 50μl (10 s target molecules plus probes) Solution 1 was added to tubes A-B 1-6. 50μl (10 5 target molecules plus probes) Solution 2 was added to tubes A-B 7-12. 50μl Solution 3 was added to tubes C 1-12. 50μl Solution 4 was added to tubes D-H 1-6. 50μl Solution 5 was added to tubes D-H 7-12. The content of the rack can be represented schematically as follows. Rack setup: <- Dual Capture > <- Standard RTC > 1 2 3 4 5 6 7 8 9 10 1 1 12 A <— 105 target + B-3018 + C29—> <~10*5 target + dA-781 + C29--> B <— 105 tar et + B-3018 + C29— > <— 105 target + dA-781 + C29— > C < C29 > <- C29 > D < B-3018 + C29 > <- dA-781 + C29 > E < B-3018 + C29 > <- dA-781 + C29 > F < B-3018 + C29 > < dA-781 + C29 > G < B-3018 + C29 > < dA-781 + C29 > H < B-3018 + C29 > <- dA-781 + C29 > All tubes were incubated for 30 min. at 37 * C to allow the Chlamydia target to hybridize to capture probe and detector probe. Following incubation, a 350μl suspension of 0.023% streptavidin derivatized particles in Bead Blocking Buffer [4% BSA (Bovine Serum Albumin, Sigma Fraction 5), 0.5% Sarkosyl, lOOmM Tris, 20mM EDTA, 0.01% antifoam], pH 8.0 was added to each of tubes A-H 1-6. A lOOμl suspension of 0.045% dTi4 derivatized particles in Bead Blocking Buffer containing 4M GuHCl was added to each of tubes A-H 7-12. After mixing on a platform vortex, die tubes were incubated at 37 * C for 5 min. to capture he ternary capture probe: target detector probe complexes. The particles and captured ternary complexes were collected at die sides of die tubes using a magnetic separations device as described in U.S. Patent No. 4,988,618 to Li et al., which is incorporated herein by reference. The supernatants were aspirated away. Next, die particles were washed to remove detector probe unbound to die target. Two hundred microliters of Wash Buffer [300mM Nad, 0.5% Sarkosyl, lOOmM Tris, 20mM EDTA, 0.5% BSA, 0.1% antifoam], pH 8.1 was added to each tube. The tubes were mixed and incubated at 37 * C for 2 min. The particles and captured ternary complexes were collected at die sides of d e tubes as described above. The supernatants were aspirated away. The particles were washed twice more in he same manner. Next, 160μl of Guanidine Thiocyanate (GuSCN) Buffer [2.5M GuSCN, lOOmM Tris, lOmM EDTA, 0.5% Sarkosyl, and 0.5% BSA], pH 7.8 was added to each tube. After mixing, the tubes were incubated at 37 * C for 5 min. This resulted in release of the targetdetector probe segment of the ternary complex in tubes A-H 1-6, and release of die capture probe: targetdetector probe ternary complex in tubes A-H 7-12. The B-3018 capture probe remains bound to die streptavidin particles in tubes A-H 1-6. After 5 min. die particles were collected at die sides of die tubes using a magnetic separation device as described above. The supernatants were transferred to clean tubes. Next, 40μl of 500ng/ml dA-781 capture probe in lOmM Tris, lmM EDTA was added to each supernatant of tubes A-H 1-6. Forty microliters of lOmM Tris and lmM EDTA was added to each supernatant of tubes A-H 7-12. The tubes were incubated for 30 min. at 37'C to allow dA-781 capture probe to hybridize to die target detector probe segment in tubes A-H 1-6. Three hundred microliters of 0.05% dTπ particles in Bead Blocking Buffer was added to all tubes. The ternary complexes were captured on die dT particles. The particles were collected at die sides of d e tubes as described above and die supernatants were aspirated away. The particles were dien washed widi Wash Buffer as described above to remove detector probe unbound to die target The supernatant was aspirated away. Next, die ternary complexes were released from the particles by addition lOOμl Release Buffer [25mM NaCl, lOOmM Tris, 20mM EDTA, 0.2% Sarkasyl, 0.05% Bronopol and 0.05% BSA], pH 8.1 as described above. The supernatants widi released ternary complexes were transferred to clean tubes. Next, 200μl of a suspension of 0.045% oligo dTi4 particles in Buffer A [4% BSA, 4M GuHCl, lOOmM Tris, 20mM EDTA, 0.5% Sarkosyl, and 0.1% antifoam], pH 7.8 was added to each of d e released supernatants. The ternary complexes were captured onto die particles as described above. The particles were collected at die sides of die tubes as described above and die supernatants were aspirated away. The particles were dien washed twice in pre-Amp Wash Buffer [300mM KCl, 50mM Tris, lmM EDTA], pH8.0. The supernatants were aspirated away. Next 150μl of Pre- Amp Release Buffer [50mM Tris, lmM EDTA, 0.5% NP-40] was added to each tube. After mixing, the tubes were incubated at 37 * C. This resulted in release of the ternary complexes. The particles were separated from the supernatants as described before. The presence of target was determined by replicating the MDV-like C29 detector probes with Qβ-replicase in the presence of the intercalating dye propidium iodide and measuring the time required to produce a positive response. More particularly, lOOμl of the supernatant from each tube was added to a lOOμl. mixture of 220mM Tris pH 7.8, 0mM MgCl2, 600μM each of the nucleotide triphosphates ATP, CTP, GTP and UTP (Pharmacia), 50ng propidium iodide and lμg of purified Qβ-replicase (specific activity of 2,000 units/mg). The Qβ-replicase was obtained as described in U.S. Patent No. 5,141,857 which is incorporated herein by reference. The tubes were capped and kept on ice until ready for measurement The 96 tubes were processed simultaneously. Each tube was maintained at 37 * C± 0.25 * C. Each tube was excited at 510nm and read at 610nm every 40 sec. for a period of 45 min. The output fluorescence was analyzed numerically for response time. The response time was taken as the intersection of die fluorescent response with die baseline (no response). The intersection was determined using third order polynomials to fit the fluorescent response. The baseline was averaged. The response time represents a quantifiable determination of the presence of target as it is inversely proportional to the log of the number of template molecules present in die sample. Cahil et al., Clin. Chem. 37: 1482- 1485, 1991. Figure 1 shows the cumulative results for each method. The Figure shows the responses generated by assays employing die Dual Capture format of the invention and the standard RTC format In addition, Figure 1 shows the time for responses generated by die assays in the presence of target (true signal) and in die absence of target (noise). Ten of the 12 tubes for the Dual Capture method including target (A-B 1-6) were measured. All 10 produced a positive response. These are indicated by spots in the column above T. Eleven of die 12 tubes for the standard RTC mediod including target (A-B 7-12) were measured. All 11 produced a positive response. Twenty-three of the 30 tubes for the Dual Capture method including capture probes and detector probe but omitting target (D-H 1-6) were measured. Only 1 of the 23 produced a positive response. This is indicated by die spot in die column above CN. The response is attributable to the cyclable noise resulting from nonspecific binding of die C29 detector probe to die capnire probes. Twenty-eight of die 30 tubes for die standard RTC mediod including capture probe and detector probe but omitting target (D-H 7-12) were measured. All 28 produced a positive response attributable to cyclable noise. All 6 of the 6 tubes for each assay method including C29 detector probe but excluding target and capture probe (Dual Capture: C 1-6; RTC: C 7-12) were measured. Responses here would be attributable to nonspecific binding and/or hybridization of detector probe to the supports used in the assays. However, none of die tubes for either mediod produced a positive response. This is indicated by die absence of spots in die column above NSH. The results depicted in Figure 1 demonstrate the superiority of the Dual Capture Class I methodology of die invention over standard RTC mediodology. As Figure 1 demonstrates, both methodologies are able to detect die signal generated in die presence of target Similarly, both methods are able to eliminate the background signal (noise) generated by nonspecific binding of detector probe to assay supports. However, the Dual Capnire methodology is clearly superior in eliminating or substantially reducing die background signal resulting from cyclable noise. Figure 1 demonstrates that such cyclable noise observed widi die RTC mediod is competitive widi die signal generated by die 10 s targets. Thus, the sensitivity of the assay is limited by the cyclable noise to about 10 5 targets. In contrast the cyclable noise produced in the Dual Capture assay is almost eliminated entirely and is clearly distinguishable from the signal produced by die 10 s targets. Figure 1 demonstrates that the Dual Capture assay is much more sensitive than die standard RTC mediod. EXAMPLE 2 This example compares signal and cyclable noise for separate embodiments of die Dual Capture Class I method in die detection of Chlamydia trachomatis. One set of assays was performed using B-3018 and dA-781 capture probes as in Example 1. The second set of assays was performed using C7 dG-3018 and dA-781 capture probes. Both sets used die C29 detector probes. The assays were performed as follows. Five solutions were prepared: Solution 1 was made up to comprise 1400μl of 1.4 * 10 6 purified targets molecules, 140ng B-3018 capture probe, 2.8 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Solution 2 was made up to comprise 1400μl of 1.4 * 10 6 purified target molecules, 140ng C7dG-3018 capture probe, 2.8 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. Solution 3 was made up to comprise 1400μl of 2.8 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. Solution 4 was made up to comprise 3200μl of 320ng B-3018 capnire probe, 6.4 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125mM Tris pH 7.8, 16mM EDTA. Solution 5 was made up to comprise 3200μl of 320ng C7dG-3018 capture probe 6.4 * 10 12 molecules of C29 detector probe in 4M GuHCl, 125MM Tris pH 7.8, 16mM EDTA. An 8 (A-H) x 12 (1-12) 96 well rack for 1.5ml polypropylene mbes (Micronics, Flow Laboratories) was set up. One hundred microliter aliquats of solutions 1-5 were added to die tubes as follows: lOOμl (10 5 target molecules plus probes) Solution 1 was added to tubes A and B 1-6. lOOμl (10 5 target molecules plus probes) Solution 2 was added to mbes A and B 7-12. lOOμl Solution 3 was added to tubes Cl-12. lOOμl Solution 4 was added to mbes D-H 1-6. lOOμl Solution 5 was added to mbes D-H 7-12. Thus, die tubes A-H 1-6 demonstrate facets of the Dual Capture Method using B- 3018 capnire probe; mbes A-H 7-12 demonsttate facets of the mediod using C7dG-3018 capture probe. All tabes were then incubated for 30 min. at 37 * C to allow the Chlamydia target to hybridize to capture probe and detector probe. Following incubation a 300μl suspension of 0.027% streptavidin derivatized particles in Bead Blocking Buffer [4% BSA (Bovine Serum Albumin, Sigma Fraction 5), 0.5% Sarkosyl, lOOmM Tris, 20mM EDTA, 0.01% anti¬ foam], pH 8.0 was added to each of tubes A-H 1-6. A 200μl suspension of 0.045% d(C)i4- derivatized particles in Bead Blocking Buffer containing 4M GuHCl was added to each of tabes A-H 7- 12. After mixing, die tubes were incubated at 37 * C for 5 min. to capture die ternary capture probe: target * detector probe hybrids. The particles were then collected at die sides of d e tabes using a magnetic separations device as described in U.S. Patent No. 4,988,618. The supernatants were aspirated away. Next die particles in all tubes were washed to remove detector probe unbound to die target. Two hundred microliters of Low Salt Wash/Release Buffer [25mM NaCl, 0.2% Sarkosyl, 25mM Tris 5mM EDTA, 0.05% BSA, 0.05% Bronopol], pH 8.1 was added to each mbe. The tubes were then mixed on a platform vortex for 30 sec. and incubated at 37 * C for 2 min. The particles and captured hybrids were collected at die sides of die tubes using a magnetic separation device as described before. The supernatants were aspirated away. The particles were washed twice more in die same manner. Next lOOμl of Guanidine Thiocyanate (GuSCN) Buffer [2.5M GuSCN, lOOmM Tris, lOmM EDTA, 0.5% Sarkosyl and 0.5% BSA], pH 7.8 was added to each tube. After mixing, die tabes were incubated for 5 min at 37 * C. This resulted in release of the target detector probe segment of die ternary complex since the B-3018 and C7dG-30185 capture probes dissociate from the target but not from the particles. After 5 min die particles were collected at the sides of die tabes using a magnetic separations device as described above. The supernatants were transferred to clean tubes. Next, 50μl of S On ml dA-781 capture probe in lOmM EDTA was added to each supernatant The tabes were incubated for 30 min. at 37 * C to allow die dA-781 capture probe to hybridize to die target detector probe. Two hundred microliters of 0.05% 0 14 particles in Bead Blocking Buffer was added to all tabes. The ternary complexes were captured on die dTi4 particles. The particles were then collected at he sides of die tubes as described above and die supernatants were aspirated away. The particles were dien washed with High Salt Wash Buffer [300mM NaCl, 0.5% Sarkosyl, lOOmM Tris, 20mM EDTA, 0.5% BSA, 0.1% antifoam], pH 8.1 to remove detector probe unbound to die target Next die ternary complexes were released into lOOμl/tube of Low Salt Wash/Release Buffer as described above. The released supernatants were transferred to clean tabes. To each tabe was dien added 200μl of a suspension of dTi4 particles in Buffer A [4% BSA, 4M GuHCl, lOOmM Tris, 20mM EDTA, 0.5% Sarkosyl, and 0.1% antifoam], pH 7.8. The ternary complexes were captured onto die particles as described above. The particles were collected at the sides of die tubes as described above and die supernatants were aspirated away. The particles were then washed twice in die High Salt Wash Buffer. The ternary complexes were then released into lOOμl of the Low Salt Wash/Release Buffer. The released supernatants were transferred to clean tabes. Next, 200μl of a suspension of dTu particles in Buffer A was added to each supernatant and die ternary complexes were captured. The particles were collected at die sides of die tabes as before and die supernatants were aspirated away. The particles were then washed twice in Pre-Amp Buffer [300μM KC1, 50μlM Tris, lmM EDTA], pH 8.0 as described before. Finally, die ternary complexes were released into 150μl Pre-Amp Release Buffer [50mM Tris, lmM EDTA and 0.5% NP-40] as described before. The particles were separated from the supernatants as described before. The presence of target was determined as in Example 1. Figure 2 shows he cumulative results for each mediod wid respect to responses generated in the presence of target (column T) and by cyclable noise (column CN). All 12 of die 12 tabes for each assay mediod including target capture and detector probe (tabes A-B 1-6 using B-3018 capnire probe and A-B 7-12 using C7dG-3018 capnire probe) were measured. All tubes provided a positive response All of die tubes including capture and detector probes but omitting target (D-H 1-6 for B-3018 capture probe, D-H 7-12 for C7dG-3018 capture probe) were measured. Only 7 of the 30 tubes using B-3018 capnire probe produced a positive response. Only 3 of the 30 tubes using C7dG-3018 capture probe produced a positive response. These responses are attributable to cyclable noise. The results in Figure 2 demonsttate diat die assays are comparable in ability to distinguish signal resulting from target relative to signal attributable to cyclable noise. Example 3 This example compares signal and noise from the Dual Capture Class π mediodology of die invention widi tiiose from a comparable RTC method. The Dual Capture Class II assays used B-3018 and dA-1660 capture probes. These probes hybridize to exclusive sequences of the target Chlamydia trachomatis 16S rRNA. The nucleic acid sequence 1660 (Sequence ID No. 3) is a universal sequence to 16S eubacterial rRNA. The RTC assays used only dA-1660 capture probes. The assays were performed as follows. Nine solutions were prepared as follows: Solution 1 was made up to comprise 1 lOOμl of 1.1 * 10 7 purified target molecules, llOng B-3018 capture probe and llOng dA-1660 capture probe in 4M GuHCl, 125mM Tris pH 7.8, lόmM EDTA. Solution 2 was made up to comprise 1 lOOμl of 1.1 * 10 7 purified target molecules and llOng dA-1660 capture probe in 4M GuHCl, 125mM Tris, pH 7.8, 16mM EDTA. Solution 3 was made up to comprise 1 lOOμl of 1.1 * 10 6 purified target molecules, 110 ng B-3018 capture probe and llOng dA-1660 capture probe in 4M GuHCl 125mM Tris pH 7.8, lόmMEDTA. Solution 4 was made up to comprise llOOμl of 1.1 * 10 6 purified target molecules and 1 lOng dA-1660 capture probe 4M GuHQ, 125mM Tris pH 7.8, 16mM EDTA. Solution 5 was made up to comprise 1600μl of 160ng B-3018 capture probe and 160ng dA- 1660 capture probe in 4M GuHQ, 125mM Tris pH 7.8, 16mM EDTA. Solution 6 was made up to comprise 1600μl of 160ng dA-1660 capture probe in 4M GuHQ, 125mM Tris pH 7.8, 16mM EDTA. Solution 7 was made up to comprise 1 lOOμl of 4mM GuHQ, 125mM Tris pH 7.8, 16mM EDTA. Solution 8 was made up to comprise 2500μl of 750ng B-3018 capnire probe and 3.8 * 10 12 molecules of C29 detector probe in lOmM Tris lmM EDTA, pH 8.0. Solution 9 was made up to comprise 2500μl of 750ng dA-781 capture probe and 3.8 * 10 12 molecules of C29 detector probe in lOmM Tris, lmM EDTA, pH 7.8. Eight (A-H) x 10 (1-10) wells of an 8 x 1296 well rack for 1.5ml polypropylene tubes was set up. One hundred microliter aliquats of solutions 1-7 were added to the tubes as follows: lOOμl (10 6 target molecules plus capnire probes) Solution 1 was added to tabes A-B 1-5. lOOμl (10 6 target molecules plus capnire probes) Solution 2 was added to tabes A-B 6-10. lOOμl (10 5 target molecules plus capture probes) Solution 3 was added to tabes C-D 1-5. lOOμl (10 s target molecules plus capnire probes) Solution 4 was added to tabes C-D 6-20 lOOμl Solution 5 was added to tabes E-G 1-5. lOOμl Solution 6 was added to tabes E-G 6-10. lOOμl Solution 7 was added to tabes H 1-10. All tabes were incubated for 30 min. at 37 * C to allow the Chlamydia target to hybridize to die capture probes. Following incubation, a 200μl suspension of 0.045% d(T)i4 derivatized particles in Bead Blocking Buffer pH 7.8 containing 4M GuHQ was added to each tube. After mixing on a platform vortex, die tabes were incubated at 37 * C for 5 min. to capture the capture probe: target hybrids. The particles were collected at the sides of the tabes using a magnetic separations device as described previously. The supernatants were aspirated away. Next, die particles were washed to remove capture probe unbound to die target Two hundred microliters of High Salt Wash Buffer [300mM NaCl, 0.5%Sarkosyl, lOOmM Tris, 20mM EDTA, 0.5% BSA, 0.1% antifoam], pH 8.1 was added to each tube. The tabes were mixed on a platform vortex and incubated at 37 * C for 2 min. The particles and captured hybrids were collected at die sides of die tabes using a magnetic separations device as described in U.S. No.4,988,618. The supernatants were aspirated away. The particles and hybrids were washed again in die same High Salt Wash Buffer. Next, lOOμl of Low Salt Wash Release Buffer [25mM NaCl, 0.2% Sarkosyl, 25mM Tris, 5mM EDTA, 0.5% BSA, 0.05% Bronopol], pH 8.1 was added to all tabes. After mixing, die tabes were incubated for 5 min. at 37 * C to release the target capture probe hybrids from the particles. The particles were collected at the sides of die tabes. The supernatants were transferred to new tubes. Next lOOμl of a suspension of 0.04% streptavidin derivatized paramagnetic particles in Bead Blocking Buffer also containing 1M GuHCl was added to each tube. After mixing, the tubes were incubated at 37 * C for 5 min. to recapture die target captare probe hybrids. The particles were dien collected at die sides of the tabes and die supernatant was aspirated away. The particles and captured hybrids were then washed once each widi die High Salt Wash Buffer and Low Salt Wash Buffer to remove capnire probe unbound to die target Next lOOμl of Guanidine Thiocyante (GuSCN) Buffer [2.5M GuSCN, lOOmM Tris, lOmM EDTA, 0.5% Sarkosyl, and 0.5% BSA], pH 7.8 was added to all tabes. After mixing, the tubes were incubated at 37 * C. for 5 min. to release the target dA-1660 capture probe hybrids. Under tiiese conditions die B-3018 capture probe used in die Dual Capture Class π assays remain bound to die streptavidin particles. The particles were then collected at die sides of die tubes as described before. The supernatants were transferred to clean tubes. Next 50μl of Solution 8 (containing B-3018 captare probe and detector probe) was added to tubes A-H 1-5. Similarly, 50μl of solution 9 (containing dA-781 capture probe and detector probe) was added to tabes A-H 6-10. The tubes were incubated for 30 min. at 37 * C to allow ternary capture probe: target: detector probe complexes to form. Next 150μl of a suspension of 0.04% streptavidin derivatized particles in Bead Blocking Buffer was added to each of tabes A-H 1-5. Similarly, 300μl of a suspension of 0.05% d(T)i4 derivatized particles in Bead Blocking Buffer was added to each of tubes A-H 6-10. The tubes were incubated at 37 * C for 5 min. to allow capture of the ternary complexes to the particles. The particles and captured ternary complexes were collected at die sides of die tubes as described before. The supernatants were then aspirated away. The particles were then washed widi High Salt Wash Buffer as described to remove detector probe unbound to die target Next lOOμl of 2.5M GuSCN Buffer was added to all tabes. After mixing, the tabes were incubated at 37'C for 5 min. to release ternary complexes. More particularly, in tabes A-H 1-5 die ternary dA-1660 capture probe: target * detector probe complex is released into the Buffer while die B-3018 capnire probe remains bound to die particles. In tabes A-H 6- 10, the complex includes the two capture probes, target and detector probe. The particles were collected at die sides of die tabes and die supernatants were transferred to clean tabes. Next 200μl of a suspension of 0.045% dTi4 particles in Bead Blocking Buffer was added to each supernatant The complexes were captured onto die particles as described previously. The particles were collected at die sides of he tabes and die supernatants were aspirated away. The particles were washed twice in die High Salt Wash Buffer. The ternary complexes were then released in lOOμl of Low Salt Wash/Release Buffer. The supernatants ternary were transferred to clean tubes. Next 200μl of a suspension of dTu particles in Buffer A was added to each supernatant The complexes were recaptured onto the particles as described above. The particles were separated from die supernatant and washed twice in Pre-Amp Wash Buffer [300mM KQ, 50mM Tris, lmM EDTA], pH 8.0. The ternary captare probe: target: detector probe complexes were released into 150μl Pre-Amp Release Buffer [50mM Tris, lmM EDTA and 0.5% NP-40 as described previously. The particles were separated from the supernatants as described before. The presence of target was determined as in Examples 1 and 2. Figure 3 shows the cumulative results for each method widi respect to responses generated in the presence of c target for 10 6 target molecules (A-B 1-10) and 10 s target molecules (C-D 1-10) and by cyclable noise (E-G 1-10) in the columns T 6 , T s and CN, respectively. All 10 of the 10 tubes for each Dual Capture Class π assay with target (10 6 and 10 s targets), capture and detector probes were measured. All tabes provided a positive response. All 10 tabes for die RTC assay method widi 10 s targets, capture and detector probes were measured. All 10 provided a positive response. Nine of the 10 tubes for die RTC mediod widi 10 s targets, capture and detector probes were measured. All 9 provided a positive response. Fourteen of the 15 tabes including capnire and detector probes but excluding target were measured for each assay method. Only 1 of die 14 mbes using the Dual Capture Qass II method provided a positive response . In contrast all 14 of the 14 tabes using die comparable RTC mediod provided a positive response. More importantly, this signal attributable to cyclable noise is comparable to and indiscriminate from the true signal produced by 10 s targets. Accordingly, die sensitivity of die RTC mediod illustrated here is limited by cyclable noise. The sensitivity of die Dual Capture Qass II method, however, is not so limited and easily exceeds 10 s targets. Sequence Listing (1)GENERAL INFORMATION: (i)APPLICANT: Shah, Jyotsna King, Walter Liu, Jing Smith, James Popoff, Sonya Serpe, Eugene (ii)TITLE OF INVENTION: ASSAY METHODS (iii)NUMBER OF SEQUENCES: 4 (iv)CORRESPONDENCE ADDRESS: (A)ADDRESSEE: Amoco Corporation, c/o Patents and Licensing (B)STREET: 200 East Randolph Drive (C)CITY: Chicago (D)STATE: Illinois (E)COUNTRY: USA (F)ZIP: 60601 (v)CCMPUTER READABLE FORM: (A)MEDIUM TYPE: 3.5 inch/800K bytes-Macintosh formatted diskette; Double-sided, Double Density (B)COMPUTER: Macintosh LC (C)OPERATING SYSTEM: Macintosh 7.0.1 (D)SOFTWARE: MacWrite II (MacWrite II and Text-only Files) (vi)CURRENT APPLICATION DATA: (A)APPLICATION NUMBER: (N3E AVAILABLE AS OF YET) (B)FILING DATE: October 9, 1992 (C)CLASSIFICATION: (NONE AVAILABLE AS OF YET) (vii)PRIOR APPLICATIO .DATA: (A)APELICATICN N*BER: N/A (B)FILING DATE: N/A (viii)ATTORNEY/AGENT INFORMATION: (A)NAME:Galloway, Nbrval B. (B) REGISTRATION NUMBER: 33, 595 (C) REFERENCE DOCKET NUMBER: 32455 (÷b TEI-EXrfcMlOTCATICN INFORMATION: (A) TELEPHONE: (312) 856-7180 (B) TELEFAX: (312) 856-4972 (C) TE EX: (2)INFORMATION FOR SEQ ID NO:l: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH: 26 NUCLEOTIDES (B)TYPE: NUCLEIC AC3D SEQUENCE (C)STRANDEDNESS: SINGLE STRANDED (D)TOPOLOGY: LINEAR (ii)MXECULE TYPE: DNA PROBE (A)DESCRIPTION: CHLAMYDIA TRACHOMATIS SPECIFIC PROBE FOR 16S RIBOSCMAL RNA, FOR FIRST CAPTURE, (ix)FEATURE: (A)NAME/KEY: GTS OLIGO NUMBER 3018 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTTTTAACGT TACTCGGATG CCCAAA 26 (2)INFORMATION FOR SEQ ID ND:2: (i)SEQUENT CHARACTERISTICS: (A)LENGTH: 36 NUCLEOTIDES (B)TYPE: NUCLEIC ACID SEQUENCE (C)STRANDEDNESS: SINGLE STRANDED (D)TOPOLOGY: LINEAR (ii)MXECULE TYPE: DNA PROBE (A)DESCRIPTION: CHLAMYDIA TRACHOMATIS SPECIFIC PROBE FOR 16S RIBOSCMAL RNA, FOR SECOND CAPTURE, (ix)FEATURE: (A)NAME/KEY: GTS OLIGO NUMBER 781 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTTTAAOGTT ACTOQGAIQC CCAAATATOG CCACAT 36 (2)INFORMATION FOR SEQ ID N0:3: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH: 45 NUCLEOTIDES (B)TYPE: NUCLEIC ACID SEQUENCE (C)STRANDEDNESS: SINGLE STRANDED (D)TOPOLOGY: LINEAR (ii)MOLECULE TYPE: DNA PROBE (A)DESCRIPTION: BACTERIAL PROBE FOR THE MAJORITY OF EUBACTERIAL 16S RIBOSCMAL RNA, FOR CLASS II ASSAY. (ix)FEATURE: (A) AME/KEY: GTS OLIGO NUMBER 1660 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CIGCTGCCTC CCGTAGGAGT TTGGGCCGTG TCTCAGTTCC AGTGT 45 (2)INFORMATION FOR SEQ ID N0:4: (i)SEQUENCE CHARACTERISTICS: (A)LENGTH: 280 NUCLEOTIDES (B)TYPE: NUCLEIC ACID SEQUENCE (C)STRANDEDNESS: SINGLE STRANDED (D)TOPOLOGY: LINEAR (ii)MOLECULE TYPE: RNA PROBE (A)DESCRIPTION: CHLAMYDIA 16S RIBOSCMAL RNA DETECTOR PROBE, REPLICABLE BY Q-BETA REPLICASE, IN VITRO, (ix)FEATURE: (A)NAME/KEY: GTS OLIGO NUMBER 1126, C29 RNA MXECULE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGGGACCCCC CCGGAAGGGG GGGACGAGGU GCQGGCACCU CGUACGGGAG UUCGACCGUG ACGCGUCGUC GAGUUGAGGC CUUUACCCCA CCAACUAGCU GAUAUCACAU ACUGAAUCUU UAGUCACGGG CUAGCGCUUU CGCGCUCUOC CAGGUGACGC OJOGUGAAGA GGCGUGACCU UCGUGCGUUU CQGUAACGCA CGAGAACCGC CACQCU3CUU CGCAGCGU3G CCCCUUCGCG CAGCOCGCUG CGCGAGGUGA OCOCCCGAAG GGGGGUUCCC 280

Claims: Accordingly, diat which is claimed is: 1. A mediod for assaying a sample for target comprising the steps of: a. contacting the sample widi a first capture probe and detector probe which in the presence of target bind to the target to form a first capture probe:target detector probe ternary complex; b . separating the targetdetector probe segment of the ternary complex from the first capture probe; c. contacting die sample with a second captare probe which in the presence of target binds to die target to form a second capnire probe:targetdetectσr probe ternary complex; d. minimizing the amount of detector probe nonspecifically bound to die second capture probe; and e. monitoring the second ternary complex for die presence or absence of detector probe. 2. The method of claim 1 wherein the targetdetector probe segment of the ternary complex is separated die first capture probe and die amount of detector probe non¬ specifically bound to die die second capnire probe is minimized by steps comprising: a. contacting the sample with a first support which binds to die first capture probe; b. substantially separating the first support and bound first ternary complex from the sample; c. introducing die first support and bound first ternary complex to a second medium and releasing the targetdetector probe segment of the first ternary complex from the first support and first capture probe; d. substantially separating the first support and first capture probe from the second medium; e. introducing a second capture probe to the second medium which in the presence of target binds to die target to form a second capture probe: targetdetector probe ternary complex; f . contacting die second medium widi a second support which binds to die second capture probe; g. substantially separating die second support and bound second ternary complex from die second medium; h . introducing die second support and bound second ternary complex to a third medium and releasing the second ternary complex from die second support; and i. substantially separating the second support from the third medium. 3. The mediod of Claim 1 wherein die target is a polynucleotide. 4. The method of claim 1 wherein one or more dispersible supports are used. 5. The method of claim 2 further comprising one or more cycles of the steps comprising: a. contacting die third medium widi a third support wherein the third support binds to die second captare probe; b . substantially separating the diird support and bound second ternary complex from the tiiird medium; c. introducing the tiiird support and bound second ternary complex into a fourth medium and releasing die second ternary complex from the third support; and d . substantially separating the tiiird support from the fourth medium. The method of claim 5 wherein the target is a polynucleotide. 7. The method of claim 5 wherein the supports are dispersible in the sample media. 8. The method of claim 2 further comprising two cycles of target capture and release. 9. A method for assaying a sample for target comprising the steps of: a. contacting the sample with a first capture probe and a second captare probe which in the presence of target bind to die target to form a target complex; b. minimizing the amount of second capture probe present in the sample; c. contacting the sample with detector probe and fresh first capture probe which in die presence of target bind to die target forming a quaternary complex comprising a second target complex of die second capture probe:target:detector probe and bound first capture probe; d. minimizing the amount of detector probe bound to die second captare probe of the second target complex; and e. monitoring die second target complex for the presence or absence of detector probe. 10. The mediod of claim 9 wherein the amount of second captare probe present in the sample is minimized by steps comprising: a. contacting the sample with a second support which binds to die second capture probe; b. substantially separating the second support and bound target complex from die sample; c. introducing the second support and bound target complex into a second medium and releasing the target complex from the second support; d. substantially separating the second support from the second medium; e. contacting the second medium containing the target complex with a first support which binds to die first capture probe; f . substantially separating the first support and bound target complex from the second medium; g. introducing he first support and bound target complex into a third medium and releasing the second capture probe:target segment of the target complex from the first support and first capture probe; and h. substantially separating the first support and first capture probe from he third medium. 11. The mediod of claim 10 wherein the amount of detector probe bound to die second captare probe of the second target complex is minimized by steps comprising: a. contacting the third medium with fresh first support which binds widi die first capture probe; b. substantially separating the first support and bound quaternary complex from the tiiird medium; c. introducing die first support and bound quaternary complex into a fourth medium and releasing the second target complex from the first support and bound first capture probe; and d. substantially separating the first support and bound first captare probe from the fourth medium. 12. The method of claim 11 further comprising one or more cycles of the steps comprising: a. contacting the fourth medium widi second support which binds to d e second capture probe; b . substantially separating the second support and bound second target complex from the fourth medium; c. introducing die second support and bound second target complex into a fifth medium and releasing the second target complex from the second support; and d . substantially separating the second support from die fifth medium. 13. The mediod of claim 11 further comprising one or more additional cycles of target capture and release. 14. The method of claim 11 wherein the supports are dispersible in the sample media. 15. The mediod of claim 9 wherein the target is a polynucleotide. 16. A kit for performing the mediod of claim 1 comprising a supply of first capture probes, a supply of separate second capnire probes and a supply of detector probe. 17. A kit for performing the method of claim 9 comprising a supply of first capture probes, a supply of second captare probes and a supply of detector probes.

Inventors: KING WALTER, LIU JING, POPOFF SONYA, SERPE EUGENE, SHAH JYOTSNA, SMITH JAMES, KING, WALTER, LIU, JING, POPOFF, SONYA, SERPE, EUGENE, SHAH, JYOTSNA, SMITH, JAMES
Applicants: AMOCO CORP, AMOCO CORPORATION
Application year: 1993
Main classification: C12Q1
IPC codes: C07H21, C12N15, C12Q1