WO2014095092A1

WO2014095092A1 - Double-stranded dna binding to a solid support and related methods and kits

Info

Publication number: WO2014095092A1
Application number: PCT/EP2013/055476
Authority: WO
Inventors: Pau Bruguera; Marta CAMPS; Roser LLEVADOT
Original assignee: Instrumentation Laboratory, Inc.
Priority date: 2012-12-21
Filing date: 2013-03-15
Publication date: 2014-06-26

Abstract

The invention provides double-stranded DNA (dsDNA) stably attached to a solid support and to methods of making and methods of using the dsDNA attached to a solid support. The invention is generally in the field of assays for detection of antibodies against dsDNA which is of importance in autoimmune disease diagnostics.

Description

DOUBLE-STRANDED DNA BINDING TO A SOLID SUPPORT AND RELATED

METHODS AND KITS

Background of the Invention

Many molecular applications utilize synthetic oligonucleotides attached to solid supports. It is well known how to attach single stranded oligonucleotides or single stranded DNA (ssDNA) to a solid support either by a covalent bond or through biotin streptavidin or avidin binding. For the immobilization, the single stranded oligonucleotides or ssDNA must be modified with a functional group in order to attach to a reactive group on a solid surface. The most commonly used modifications for solid support attachment are amine, thiol and acrydite modifications for covalent binding and biotin for streptavidin/avidin binding.

The attachment of a modified single stranded oligonucleotides or modified ssDNA to a solid support is a well know technique. The only way described so far to attach double stranded DNA to a solid support is by hybridization of a complementary ssDNA to a single stranded DNA or single stranded oligonucleotide already bound to the particle to form a double stranded complex.

Hybridization is the process of establishing a non-covalent, sequence-specific interaction between two complementary strands of nucleic acids into a single complex. The complex is easily dissociated by denaturation, which is the process by which double- stranded deoxyribonucleic acid unwinds and separates into single strands through the breaking of hydrogen bonding between the bases. Denaturation can be induced by heat and also by chemicals. Therefore, double stranded DNA attached to a solid support via such non-covalent hybridization is susceptible to becoming single stranded DNA easily due to a temperature increase or to the buffer resuspension conditions.

It is therefore an object of the disclosed invention to provide a solid support with dsDNA stably attached thereto.

It is another object of the disclosed invention to provide a method of making dsDNA stably attached to a solid support.

It is another object of the disclosed invention to provide a method of detecting autoantibodies to dsDNA. Summary of the Invention

According to various embodiments described herein, the present invention describes dsDNA stably attached to a solid support, the attached DNA being partially single- stranded and partially double-stranded and the double-stranded DNA portion being circular. In one or more aspects, the DNA is attached to the solid support through a covalent bond. In further aspects, the DNA is attached to the solid support through biotin-streptavidin or biotin-avidin binding. In one or more aspects, the solid support is beads, particles, paramagnetic particles, microtiter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or avidin or modified to enable formation of a covalent bond with DNA or protein.

According to various embodiments described herein, the present invention describes a method of making dsDNA stably attached to a solid support by attaching a ssDNA or single stranded oligonucleotide to a solid support, hybridizing a circularizable oligonucleotide to the ssDNA or single stranded oligonucleotide, ligating the circularizable oligonucleotide, and extending the ssDNA to form a circular dsDNA portion. The circularizable oligonucleotide comprises a 3^L target probe portion and a 5' target probe portion complementary to regions in the ssDNA or single stranded oligonucleotide. In order to attach or immobilize a ssDNA or single stranded oligonucleotide to a solid support, the ssDNA or single stranded oligonucleotides must be modified with a functional group in order to have attachment to a reactive group on a solid surface. In one or more aspects the attaching of the ssDNA or single stranded oligonucleotide to the solid support is via covalent bond formation. In further aspects, the attaching of the ssDNA or single stranded oligonucleotide to the solid support is via biotin-streptavidin or biotin-avidin binding.

Single stranded oligonucleotides or ssDNA can be attached to flat two-dimensional surfaces, such as glass slides, as well as to three-dimensional surfaces such as micro- beads and micro-spheres. After attaching the single stranded oligonucleotides or ssDNA to the solid support the circularizable oligonucleotide comprising a 3' target probe portion and a 5' target probe portion complementary to regions in the ssDNA or single stranded oligonucleotide is added to or contacted with the solid support with immobilized single stranded oligonucleotide or ssDNA in the presence of appropriate conditions to allow hybridization of the circularizable oligonucleotide to the immobilized ssDNA or single stranded oligonucleotide. After allowing for hybridization, ligase is added to or contacted with the solid support with immobilized ssDNA or single stranded oligonucleotide and hybridized circularizable oligonucleotide. The ligase catalyzes the formation of a phosphodiester bond between the 5 '-phosphate and 3 '-hydroxy 1 groups of the circularizable oligonucleotide to form circular DNA. The ligation reaction results in circularly closed molecules that are catenated to the single stranded oligonucleotide or ssDNA which is bound to the solid support. After forming circular DNA hybridized to the immobilized single stranded oligonucleotide or ssDNA, a DNA polymerase that is not capable of causing extension- dependent strand displacement is added to or contacted with the solid support with immobilized ssDNA or single stranded oligonucleotide and hybridized circular DNA to extend the 3'-hydroxyl end of the single stranded oligonucleotide or ssDNA to form a circular dsDNA portion. The polymerization step after the ligation of the circularizable oligonucleotide forms a double stranded DNA structure immobilized on a solid surface, the resulting structure being very stable and resistant to denaturation due to the formation of catenated structure. In one or more aspects, the solid support is microspheres beads, particles, paramagnetic particles, microtiter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or avidin or modified to enable formation of a covalent bond with DNA or protein. In further aspects, the extending of the ssDNA or single stranded oligonucleotide to form a circular dsDNA portion is via a DNA polymerase that is not capable of causing extension-dependent strand displacement.

According to various embodiments described herein, the present invention describes a method of detecting autoantibodies against dsDNA by providing a solid support with dsDNA stably attached thereto , the attached DNA being partially single-stranded and partially double-stranded and the double-stranded DNA portion being circular, contacting a biological sample from a patient with the solid support with dsDNA stably attached and detecting binding of autoantibodies to the dsDNA. In one or more aspects, the DNA is attached to the solid support through a covalent bond. In further aspects, the DNA is attached to the solid support through biotin-streptavidin or biotin-avidin binding. In one or more aspects, the solid support is micro-spheres, beads, particles, paramagnetic particles, microtiter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or avidin or modified to enable formation of a covalent bond with DNA or protein. In one or more aspects, the biological sample comprises a body fluid or tissue sample. The sample may be, for example, blood, blood products, urine, saliva, cerebrospinal fluid or lymph. In one or more aspects, the detecting of binding of autoantibodies to dsDNA is via luminescence, colorimetry or fluorescence. According to various embodiments described herein, the invention provides for a kit for detecting the presence autoantibodies to dsD A in a sample material. In one embodiment, the kit comprises an circularizable oligonucleotide and a single stranded oligonucleotide or ssDNA. The circularizable oligonucleotide comprises a 3' target probe portion and a 5' target probe portion complementary to regions in the single stranded oligonucleotide or ssDNA. In one or more aspects, the single stranded oligonucleotide or ssDNA is modified with a reactive functional group at the 5'- terminus. In one or more aspects, the single stranded oligonucleotide or ssDNA is modified with biotin. In one or more aspects, the kit additionally comprises a solid support. In one or more aspects, the solid support is modified with a reactive functional group In one or more aspects the solid support is coated with avidin or streptavidin. In one or more aspects, the solid support is beads, particles, paramagnetic particles, microtiter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or avidin or modified to enable formation of a covalent bond with DNA or protein. In one or more aspects, the kit additionally comprises a ligase. In one or more aspects, the kit additionally comprises a DNA polymerase. The DNA polymerase is sufficient to extend the 3'-terminus of the single stranded oligonucleotide or ssDNA using hybridized circular DNA as a template. The DNA polymerase is not capable of causing extension-dependent strand displacement.

In a preferred embodiments described herein, the invention provides for a kit for detecting the presence autoantibodies to dsDNA in a sample material. The kit comprises at least a dsDNA stably attached to a solid support. In one or more aspects, the kit additionally comprises an antibody. The antibody is labeled with a detectable group and the antibody recognizes autoantibodies present in a biological sample. The autoantibodies are IgG or IgM. In one or more aspects, the biological sample comprises a body fluid or tissue sample. The sample may be, for example, blood, blood products, urine, saliva, cerebrospinal fluid or lymph.

These and other aspects and embodiments of the invention are also described in the following sections of the application, which are provided to highlight specific embodiments of the invention and are not intended to limit the invention, the scope of which is limited only by the issued claims. Brief Description of the Figures and Tables

Figure 1 is a schematic drawing of the procedure for producing dsDNA stably attached to a solid support.

Figure 2 is a drawing of a magnetic particle coated with the hypothetical dsDNA structure formed following the described procedure for producing dsDNA stably attached to a solid support.

Figure 3 shows the DNA sequences used for the invention and for the control reactions of the different steps of the procedure for producing dsDNA stably attached to a solid support. Table 1 is Relative Light Unit (RLU) results from the detection of autoantibodies against dsDNA with a chemiluminescent assay using the dsDNA coupled beads of the invention compared with an ELISA assay. 17 positive samples, 11 negative samples from rheumatoid arthritis (RA) patients, 20 healthy controls and 11 discrepant samples with other method were analyzed. Table 2 is RLU results from the detection of autoantibodies against dsDNA with a chemiluminescent assay using the dsDNA coupled beads of the invention. 52.6% of patients diagnosed with systemic lupus erythematosus (SLE) are positive while patients affected by other diseases and healthy donors are negative.

Detailed Description

Referring to Figure 1, to make the solid support with dsDNA stably attached or immobilized thereon a ssDNA oligonucleotide is bound to a magnetic particle (MP). The binding is through a modification of the 5 '-terminus of the ssDNA oligonucleotide. The modification can be the attachment of biotin or a reactive group capable of forming a covalent bond. The MP is modified or coated with avidin, streptavidin or an appropriate reactive group. The circularizable oligonucleotide is shown hybridized to the ssDNA oligonucleotide. In the next step, ligase is added to catalyze the formation of a phosphodiester bond between the 5 '-phosphate (plain end) and the 3'-hydroxyl (arrow end) of the circularizable oligonucleotide. After addition of the ligase, the circularizable oligonucleotide is shown as a closed circle of DNA catenated to the ssDNA oligonucleotide (depicted as squiggly line portion). In the next step, polymerase and deoxynucleotides are added to extend the 3 '-end (arrow end) of the ssDNA oligonucleotide. The polymerase is not capable of causing extension-dependent strand displacement. The resulting structure is shown comprising partially single stranded, partially double stranded DNA attached to a magnetic particle.

In Figure 2 it is contemplated that a magnetic particle is coated with multiple dsDNA structure formed following the described procedure for producing dsDNA stably attached to a solid support.

Figure 3 shows the DNA sequences used for the invention and for the control reactions of the different steps of the procedure for producing dsDNA stably attached to a solid support. TlCl-b is a single-stranded, linear DNA molecule comprising, from 5 '-end to 3 '-end, biotin, a region complementary to a forward PCR primer (shaded box), another region complementary to a reverse PCR primer (shaded underlining bar), and a 3'- hydroxyl group. TlCl-b is also depicted as attached to an MP (curved line). T1C1- NH2 is the same single-stranded, linear DNA molecule as TlCl-b, except that instead of biotin, the 5 '-terminus is modified with an amine group. P1C1 is a circularizable oligonucleotide that is a single-stranded, linear DNA molecule comprising, from 5' end to 3' end, a 5' phosphate group, a first target probe portion (shown hybridized to T1C1) including the 5' phosphate group, a second target probe portion (shown hybridized to T1C1), and including a 3' hydroxyl group. In addition PI CI contains a region complementary to a forward PCR primer (shaded box), another region complementary to a reverse PCR primer (shaded underlining bar). The forward PCR primers are shown as ssDNA F and the reverse PCR primers are shown as ssDNA R.

ssD A/Single Stranded Oligonucleotides

Referring to Figure 3, a ssD A or single stranded oligonucleotide according to the invention is a single-stranded DNA molecule of any length. A ssDNA or single stranded oligonucleotide may contain between 40 to 1000 nucleotides, preferably between about 20 to 200 nucleotides, and most preferably between about 25 to 150 nucleotides. The ssDNA or single stranded oligonucleotide has a 5' phosphate group and a 3' hydroxyl group. The 5 '-phosphate is modified with biotin or with a reactive group capable of forming a covalent bond with another, appropriate reactive group. The 3' hydroxyl group is used for the polymerization reactions described herein. It is preferred that ssDNA or single stranded oligonucleotides do not have any sequences that are self- complementary. The ssDNA or single stranded oligonucleotides are considered not to have any self-complementary sequences if the ssDNA or single stranded oligonucleotides have no complementary regions greater than six nucleotides long without a mismatch or gap. The ssDNA or single stranded oligonucleotides according to the invention are designed to contain regions that are complementary to circularizable oligonucleotides.

In Figure 3, TlCl-b is a single-stranded, linear DNA molecule comprising, from 5 '-end to 3 '-end, biotin, a region complementary to a forward PCR primer (shaded box), another region complementary to a reverse PCR primer (shaded underlining bar), and a 3'-hydroxyl group. TlCl-b is also depicted as attached to an MP (curved line). T1C1- NH2 is the same single-stranded, linear DNA molecule as TlCl-b, except that instead of biotin, the 5 '-terminus is modified with an amine group. A particularly preferred embodiment is a single stranded oligonucleotide of 25 to 150 nucleotides with biotin covalently attached at the 5'-terminus.

Circularizable Oligonucleotides

Refeixing to Figure 3, a circularizable oligonucleotide according to the invention is a single-stranded DNA molecule of any length. A circularizable oligonucleotide may contain between 40 to 1000 nucleotides, preferably between about 50 to 200 nucleotides, and most preferably between about 55 to 150 nucleotides. The circularizable oligonucleotide has a 5' phosphate group and a 3' hydroxyl group. The 5'- phosphate and 3 '-hydroxyl groups allow the ends of the circularizable oligonucleotide to be ligated to each other using a DNA ligase. It is preferred that circularizable oligonucleotides do not have any sequences that are self-complementary. The circularizable oligonucleotide is considered not to have any self-complementary sequences if the circularizable oligonucleotide has no complementary regions greater than six nucleotides long without a mismatch or gap. In Figure 3, P1C1 is a circularizable oligonucleotide that is a single-stranded, linear DNA molecule comprising, from 5' end to 3' end, a 5' phosphate group, a first target probe portion (shown hybridized to T1C1) including the 5' phosphate group, a second target probe portion (shown hybridized to T1C1), and including a 3' hydroxyl group. In addition PI CI contains a region complementary to a forward PCR primer (shaded box), another region complementary to a reverse PCR primer (shaded underlining bar). The forward PCR primers are shown as ssDNA F and the reverse PCR primers are shown as ssDNA R. The DNA sequences shown in Figure 3 are from Crithidia htciliae, however the sequence of the DNA of the ssDNA, singled stranded oligonucleotides and circularizable oligonucleotides is not critical to the invention and any sequences designed as described herein can be used to make the modified solid support of the invention (i.e., a solid support with dsDNA stably bound thereto. Likewise, any sequences designed as described herein can be used in the methods of making and methods of using the modified solid support of the invention.

Target Probe Portions

As depicted in Figure 3, there are two target probe portions on each circularizable oligonucleotide, one at each end of the circularizable oligonucleotide. The target probe portions can each be any length that supports specific and stable hybridization between the target probes and the ssD A or single stranded oligonucleotides.. The target probe portions may each be a length of 8 to 35 nucleotides, for example, with target probe portions 10 to 30 nucleotides long being most preferred. The target probe portion at the 5' end of the circularizable oligonucleotide is referred to as the first target probe, and the target probe portion at the 3' end of the circularizable oligonucleotide is referred to as the second target probe. These target probe portions are also referred to herein as first and second target probes or first and second probes. The target probe portions are complementary to a ssDNA or single stranded oligonucleotide immobilized on a solid support..

As shown in Figure 3, the target probe portions are complementary to the ssDNA or single stranded oligonucleotide, such that upon hybridization, the 5' end of the first target probe portion and the 3' end of the second target probe portion are base-paired to nucleotides in the ssDNA or singled stranded oligonucleotide that are contiguous with one another, such that a nick in present in the double stranded portion of the complex. A nick is a discontinuity in a double stranded DNA molecule where there is no phosphodiester bond between adjacent nucleotides of one strand. As shown in Figure 1 and as described herein, the nick is "repaired" (i.e., a phosphodiester bond is formed between the 5' phosphate of the first target probe portion and the 3' hydroxyl of the second target probe portion) via a ligation reaction., Effective ligation may only occur when (1) the target probe portions are hybridized to the ssDNA or single stranded oligonucleotide with no mismatches between the target probe portions and the ssDNA or single stranded oligonucleotide and (2) no nucleotides are present between where the first target probe is bound and where the second target probe is bound

A particularly preferred embodiment is a circularizable oligonucleotide of 50 to 150 nucleotides including a first target probe of 18 nucleotides and a second target probe of 19 nucleotides. The first target probe and second target probe hybridize to the ssDNA or single stranded oligonucleotide.

Oligonucleotide Synthesis

Circularizable oligonucleotides, ssDNA, single stranded oligonucleotides, PCR primers, and any other oligonucleotides can be synthesized using established oligonucleotide synthesis methods. Methods to produce or synthesize oligonucleotides are well known in the ait. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (for example, Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, Mass. or ABI Model 380B). Synthetic methods useful for making oligonucleotides are also described by Ikuta et al.

Many of the oligonucleotides described herein are designed to be complementary to certain portions of other oligonucleotides or nucleic acids such that stable complexes can be formed between them. The stability of these complexes can be calculated using methods well known in the art.

Surface Functionalization of Solid Supports

Different types of solid supports are commercially available. Some solid supports have different surface chemistries allowing distinct covalent bondings with modified DNA. Some solid supports are covalently bound to streptavidin or avidin allowing for biotin binding.

Nucleic acids can be covalently attached to solid supports with any of several methods know in the art. Carboxyl and amino groups are the most common reactive groups for attaching oligonucleotides to surfaces. These groups are very stable over time, and their chemistries are well understood. Examples of surface chemistry modifications of solid supports for covalent attachment of ssDNA or single stranded oligonucleotides include carboxylic acid (-COOH), primary aliphatic amine (-KNH2), aromatic amine (-ArNH2), chloromethyl (vinyl benzyl chloride (-ArCH2Cl), amide (-CONH2), hydrazide (- CONHNH2), aldehyde (-CHO), hydroxyl (-OH), thiol (-SH), and epoxy (-COC-).

In a particularly preferred embodiment solid supports are coated with a monolayer of recombinant streptavidin covalently coupled to the surface by Life Technologies. In another preferred embodiment the solid support is coated with a monolayer of recombinant avidin covalently coupled to the surface.

Modification of ssDNA or Single Stranded Oligonucleotides

A single stranded oligonucleotide or ssDNA is attached to a solid support through a covalent bond or through a biotin streptavidin/avidin binding.

A primary amine can be used to covalently attach a variety of products to an

oligonucleotide, including fluorescent dyes biotin, alkaline phosphatase, EDTA, or to a solid support

5' amino modifiers are -cyanoethyl phosphoramidites which, when activated with 1H tetrazole, can couple to the 5' terminus of the oligonucleotide in the same time frame and with similar efficiency as nucleoside phosphoramidites. 5^f amino modifiers can include simple amino groups with a carbon spacers of various lengths. Varying the length of spacers at the 5' end can extend the dsDNA complexes formed by the methods of the invention away from the solid support to optimize autoantibody binding by eliminating possible steric hindrances.

The attachment of an amino-modified oligonucleotide to a surface or another molecule requires an acylating reagent. Depending on which acylating reagent is used,

carboxamides, sulfonamides, ureas or thioureas are formed upon reaction with the amine moiety. The kinetics of the reaction depends on the reactivity and concentration of both the acylating reagent and the amine. Buffers that contain free amines such as Tris and glycine must be avoided when using any amine-reactive reagent. Examples of acylating agents used for amino modified oligonucleotides for linkage to molecules or surfaces include carbodiimide to form carbonyl amide linkages, isothiocyanate to form thiourea linkages, sulfonyl chloride to form sulfonamide linkages and succinimidyl esters (NHS-ester) to form carboxamide linkages.

5' thiol modification of ssDNA or single stranded oligonucleotides also enables attachment to solid surfaces (e.g. CPG-SH; Pierce) via a disulphide bond or maleimide linkages. The incorporation of a thiol group at trie end of an oligonucleotide is achieved with S-trityl-6- mercaptohexyl derivatives.

The cross-linkers used to attach thiol-modified oligonucleotides to solid supports are typically heterobifunctional, meaning that they possess functional groups capable of reaction with two chemically distinct functional groups, e.g. amines and thiols. The linkers serve two purposes: to covalently bind two distinct chemical entities which otherwise would remain un-reactive toward each other and as a physical spacer which provides greater accessibility and or freedom to each of the linked biomolecules.

A number of heterobifunctional cross linkers have been developed for covalent attachment of thiol-modified DNA oligomers to aminosilane monolayer films [35], These cross linkers combine groups reactive toward amines such as N-hydroxy- succinimidyl esters and groups reactive toward thiols such as maleimide or alpha- haloacetyl moieties. One such cross linker is succinimidyl 4- [maleimidophenyljbutyrate (SMPB), which is used to link a thiol-modified oligonucleotides to an amine derivatized solid support.

Small molecule attachment chemistry(acrydites), developed by Apogent Discoveries, enables covalent attachment of oligonucleotides to surfaces via acrylic linkages. An oligonucleotide derivatized with an Acrydite group can polymerize with free acrylic acid monomer to form polyacrylamide or can react with SH or silane surfaces. This chemistry will also work with plastic and other polymer surfaces.

An acrylic acid group can be directly attached to the 5 '-end of an oligo nucleotide (with a 6-carbon linker arm) at the time of synthesis using Acrydite, an acrylic- phosphoramidite developed by Mosaic Technologies.

The Acrydite-acrylamide group can be used to immobilize oligonucleotides, which are fully available for hybridization when bound to the solid support. In addition, the acrylamide group is thermostable.

Ligases

A "ligase" is an enzyme that is capable of covalently linking the 3' hydroxyl group of a nucleotide to the 5' phosphate group of a second nucleotide. Any DNA ligase is suitable for use in the methods according to the invention. Preferred ligases are those that preferentially form phosphodiester bonds at nicks in double-stranded DNA. That is, ligases that fail to ligate the free ends of single-stranded DNA at a significant rate are preferred. Thermostable ligases are especially preferred. Many suitable ligases are known, such as T4 DNA ligase, E. coli UNA ligase, AMPLIGASE™, Taq DNA ligase (Barany, Proc. Natl. Acad. Sci. USA 88:189-193 (1991), Thermus t ermophilus DNA ligase (Abbott laboratories), Thermus scotoductus DNA ligase and Rhodotherm s marinus DNA ligase.

Polymerases

A "polymerase" is an enzyme that is capable of incorporating nucleotides to extend a 3' hydroxy! terminus of a "primer molecule." In the methods of the invention described herein, the ssDNA or single stranded oligonucleotide contains a 3'hydroxyl terminus and thus functions as a "primer molecule".. Examples of DNA polymerases that can be used in accordance with the methods described herein are included below. Preferably, a polymerase that is not capable of causing extension-dependent strand displacement of hybridized circular DNA is used to avoid the synthesis of new single stranded DNA. One preferred polymerase is Deep Vent_R DNA Polymerase, a high-fidelity thermophilic DNA polymerase made with recombinant technology and available from New England Biolabs. The fidelity of Deep Vent_R DNA Polymerase is derived in part from an integral Ύ→ 5' proofreading exonuclease activity. Deep Vent_R is stable at temperatures of 95 to 100°C, but does not have extension-dependent strand displacement activity.

Antibodies

As used herein, the terms "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies (in one aspect, a bird (for example, a duck or goose), in another aspect, a shark or whale, in yet another aspect, a mammal, including a non- primate (for example, a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, mouse, etc) and a non-human primate (for example, a monkey, such as a cynomologous monkey, a chimpanzee, etc), recombinant antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, single domain antibodies, Fab fragments, F(ab')₂ fragments, disulfide-rinked Fv (sdFv), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the present invention), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA and IgY), class (for example, IgGi, IgG₂, IgG₃, IgG₄, IgAi and IgA₂) or subclass.

Antibodies of the invention can be modified with enzymes, such as horseradish peroxidase (HRP), alkaline phosphatase (AP) or glucose oxidase. These enzymes allow for detection often because they produce an observable color change in the presence of certain reagents. In some cases these enzymes are exposed to reagents which cause them to produce light. Flourogenic reporters like phycoerythrin can also be attached to antibodies of the invention. In a preferred embodiment, labels that work on an chemiluminescent principle, in which the label emits detectable light in response to chemical energy, are used. In the preferred embodiment isoluminol is the label used. Oxidation of isoluminol by hydrogen peroxide in the presence of a catalyst produces light emission at a certain wave length.

A preferred antibody or antibodies of the invention is one that specifically bind to autoantibodies, which in turn bind to dsDNA. Autoantibodies are antibodies that act against the body's own molecules and tissues and may cause an autoimmune disorder. Preferred antibodies of the invention include anti-IgG and anti-IgM antibodies where the IgG and IgM are autoantibodies that bind to dsDNA. Preferably, the antibodies are detectably labled for use in an immunoassay. Labels are typically chemically linked or conjugated to the desired antibody through methods known in the art.

Kits

Any combination of the materials useful according to the methods of the invention can be packaged together as a kit for performing the methods according to the invention. In particular, circularizable probes, single stranded oligonucleotides, ssDNA, PCR primers, solid supports, single stranded oligonucleotides with a modified 5 '-terminus (e.g., a reactive group or biotin), ssDNA with a modified 5 '-terminus (e.g., a reactive group or biotin), solid supports modified with a reactive group, solid supports coated with avidin or streptavidin, and solid supports with dsDNA stably attached thereto are useful components of such kits. Enzymes necessary for the methods of the invention are also preferred components of such kits. Detectably-labeied antibodies are also preferred components of such kits.

A preferred kit includes at least dsDNA stably attached to a solid support. Another preferred kit includes at least dsDNA stably attached to a solid support and detectably- labeled antibodies. Method of Making a Solid Support with dsDNA Stably Attached

The method of making a solid support with dsDNA stably attached thereto involves providing a solid support functionalized to immobilize an appropriately modified ssDNA or single stranded oligonucleotide. As discussed above, the solid support can be functionalized with a reactive group capable of forming a covalent bond or with avidin or streptavidin. The ssDNA or single stranded oligonucleotide modified with an appropriate reactive group or biotin is mixed with or contacted with the functionalized solid support under appropriate conditions to effect attachment. Then a circularizable oligonucleotide is added to a solution containing the solid support with attached single stranded oligonucleotide or ssDNA under appropriate conditions for the first and second target probe portions to hybridize to the complementary sequences of the ssDNA or single stranded oligonucleotide. After hybridization a ligase is added to the mixture to form a covalent phosphodiester bond between the 5' phosphate and 3' hydroxyl of the circularizable oligonucleotide to obtain a circularizable DNA stabilized to the single stranded oligonucleotide or ssDNA. by formation of a catenated structure. The single- stranded oligonucleotides or ssDNA are designed to have a 3 'terminus which is used by a DNA polymerase to extend the circle. A DNA polymerase is added to the reaction mixture that is not capable of causing extension-dependent strand displacement the of hybridized circular DNA to avoid the synthesis of new single stranded DNA. The polymerization step after the ligation of the circularizable oligonucleotide forms a double stranded DNA structure which is very stable and resistant to denaturation due to the formation of a concatenated structure.

The Ligation Step

A circularizable oligonucleotide, is incubated with a solid support with a ssDNA or single stranded oligonucleotide attached thereto under suitable hybridization conditions, and then ligated to form a covalently closed circle. The ligation step results in the circularizable oligonucleotide being catenated to the ssDNA or single stranded oligonucleotide. Suitable ligases for the ligation operation are described above.

Ligation conditions are generally known. Most ligases require Mg²⁺. There are two main types of ligases, those that are ATP-dependent and those that are NAD-dependent. ATP or NAD, depending on the type of ligase, should be present during ligation. The ligase and ligation conditions may be optimized to limit the frequency of ligation of single-stranded termini. Such ligation events do not depend on the presence of the hybridized complex (i.e., the circularizable oligonucleotide hybridized to the ssDNA or single stranded oligonucleotide). In the case of AMPLIGASE™-catalyzed ligation, which takes place at 60^°C, it is estimated that no more than 1 in 1,000,000 molecules with single-stranded DNA termini will be ligated. This estimate is based on the level of non-specific amplification seen with the AMPLIGASE™ ligase in the ligase chain reaction. Any higher nonspecific ligation frequency would cause enormously high background amplification in the ligase chain reaction.

The Replication Step

The ligated circularizable oligonucleotides serve as substrates for replication. A DNA polymerase is added to extend the 3'-hydiOxyl terminus of the ssDNA or single stranded oligonucleotide. The DNA polymerase with no strand displacement activity catalyzes extension, but terminates when free 3'hydroxyl groups are no longer available because strand displacement does not occur.

Detection of Autoantibodies to dsDNA

Anti-double-stranded DNA autoantibodies are diagnostic for systemic lupus erythematosus (SLE) and have been implicated in the pathogenesis of the SLE renal disease and other manifestations. Lupus is a chronic, inflammatory autoimmune disorder that may affect the skin, joints, blood cells, and internal organs, especially the kidneys, heart, and lungs. An abnormal titre of anti-dsDNA antibodies is a well- established criterion of the ACR (American College of Reumathology) classification for SLE. In prospective studies it has been shown that the level of anti-dsDNA antibody has prognostic value and titres of anti-dsDNA are an excellent measure of disease activity in some patients. During lupus flares anti-dsDNA antibody levels frequently decrease, possibly due to immune complex formation and antibody deposition in tissues, specifically the kidneys.

Since anti-dsDNA antibodies were first described, a number of techniques have been utilized for their detection. Earlier methods such as complement fixation and haemagglutination are rarely used today. Nowadays, the most commonly used assays include immunofluorescent assays (IFA) on Crithidia luciliae, enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays ( IA).

The solid supports with immobilized dsDNA made by the methods of the current invention are a powerful diagnostic tool for use in assays for detecting anti-dsDNA antibodies.

EXAMPLES

Example 1: dsDNA BEADS COUPLING PROTOCOL This example illustrates one embodiment of the invention. According to the methods of the invention, dsDNA is stably attached to a solid support. In this example, dsDNA is stably attached to paramagnetic particles.

1) Reagents:

- Paramagnetic particles (DynaBeads M280)

- Single Stranded Oligonucleotide: TICI-b

- Circularizable probe : P 1 CI

- 2x Washing buffer (W/B):

- 10 mM Tris-HCl pH 7.5

- 1 mM EDTA

2M NaCI

Ligase reaction mix (Ampligase© Thermostable DNA Lig

Epicentre® (an Illumina© company):

- 50 ih Ampligase reaction buffer 1 Ox

- 5 μ L Ampligase DNA ligase (5U/mL)

- 25 μ L on 500 nM circularizable probe

- 320 μ L H₂0

- Amplification reaction mix (Vent ®DNA Polymerase, New England

BioLabs):

50 μ L Thermopol buffer 1 Ox

- 10 μ L d TPs lO mM

- 5 μ L of Vent r DNA polymerase (2U/ μ£)

- 435 μ L ¾0

2) DNA sequences TlCI-b: 5' bio TTT TTT TTT AATTAGTAGCTCATCCAG TGG TTG CCC TCA ATG CGT G TCG TTG TAC TCG TAG C GG (SEQ ID NO: 1) P1CI: 5' Phos ACG CAT TGA GGG CAA CCA GAC CTT CCC GAT CCC GCG CAT GAC

GAA CAA CGA GAT GGC CGA CAA GTA CCG CTA CGA GTA CAA CGA C (SEQ ID NO: 2)

Procedures

Oligonucleotide immobilization

500 μ L of beads modified with streptavidin (3x10⁸ beads) were washed

3 times using W/B 2x solution.

The beads were resuspended in 500 μ L of W/B l solution.

500 μ L of the biotinylated oligonucleotide TICI-bat a concentration of

25 μ M was added to the resuspended beads.

- The mixture was incubated for 20 min 25°C with rolling at 700 rpm.

- The beads were washed 3 times using 1 mL of the W/B lx solution.

- The beads were resuspended in 100 μ L of ¾0.

Ligation reaction

- 400 μ L of Ligase reaction mix was added to the resuspended beads.

- The ligase reaction mix and resuspended beads mixture was incubated for 10 min @ 55°C.

- The beads were washed twice using 500 μ L of W/B lx solution.

Polymerization reaction

- The washed beads were resuspended in 500 of Amplification mix and incubated for 10 min @ 55°C.

- The beads were washed twice using 500 μ L of W/B l solution.

- The beads were resuspended with PBS (phosphate buffered saline) to the desired bead concentration. For example, in aliquots containing 5 mg of beads (3xl0⁸).

Control reactions

After each of the ligation reaction and polymerization reaction, real time-PCR amplification reactions were performed using the forward and reverse PCR primers described herein. The products were evaluated to determine the effectiveness of the ligation and polymerization reactions.

Example 2: Detection of anti-dsDNA Autoantibodies

The performance of the dsDNA-coated magnetic particles produced in Example 1 were evaluated in a chemiluminescent immunoassay with detection on the BIO-FLASH automated analyzer.

- 30 μΐ of a biological sample (1 :10 diluted) was mixed with 20 μΐ of dsDNA coated beads and 110 μΐ of assay buffer.

- The mixture was incubated for 9.30 min at 37°C. The beads were washed 2 times with 600 μΐ of washing buffer.

130 μΐ of a reaction buffer containing chemiluminescent labeled - IgG/IgM was added and the mixture was mixed.

- The mixture was again incubated for 9.30 min at 37°C.

- The beads were washed 3 times with 600 μΐ of washing buffer.

- The washing buffer was aspirated and

- 200 μΐ of each trigger reagent was added and the RLU was measured on the BIO-FLASH automated analyzer.

The results are shown in Tables 1 and 2 below.

Table 1 is Relative Light Unit (RLU) results from the detection of autoantibodies against dsDNA with a chemiluminescent assay using the dsDNA coupled beads of the invention compared with an ELISA assay. 17 positive samples (dsDNAl - dsDNAl 7), 11 negative samples from rheumatoid arthritis (RA) patients (dsDNAl 8 - dsDNA28), 20 healthy controls (dsDNA29 - dsDNA48) and 11 discrepant samples (NEUSS samples) were analyzed. Sample dsDNAl 1 was found to be positive for anti-dsDNA autoantibodies by an ELISA method, however the results were negative using the assay of the invention and negative results were also obtained using a third method (results not shown). The NEUSS samples described as discrepant with other method refer to samples that gave different results when analyzed by ELISA versus the third method. Table 2 is RLU results from the detection of autoantibodies against dsDNA with a chemiluminescent assay using the dsDNA coupled beads of the invention. 52.6% of patients diagnosed with systemic lupus erythematosus (SLE) are positive (shaded values) while patients affected by other diseases, i.e. infections, inflammatory bowel or rheumatoid arthritis, and healthy donors are negative.

All publications cited herein are hereby incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. Double stranded DNA stably attached to a solid support, comprising a partially single-stranded DNA portion and partially double-stranded DNA portion and wherein said double-stranded DNA is circular.

2. The stably attached dsDNA according to claim 1, wherein the ssDNA portion is attached to said solid support through a covalent bond.

3. The stably attached dsD A according to claim 1, wherein the ssD A portion is attached to said solid support through biotin-streptavidin or biotin-avidin binding.

4. The stably attached dsDNA according to claim 1, wherein said solid support is selected from the group consisting of beads, micro-spheres, particles, paramagnetic particles, microtiter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or avidin or modified to enable formation of a covalent bond with DNA or protein.

5. A method of producing a dsDNA stably attached to a solid support comprising the steps of: attaching a ssDNA or single stranded oligonucleotide to a solid support, hybridizing a circularizable oligonucleotide to said ssDNA or single stranded oligonucleotide, ligating the circularizable oligonucleotide, and extending the ssDNA or single stranded oligonucleotide to .form circular dsDNA.

6. The method according to claim 5, wherein the step of attaching of said ssDNA or single stranded oligonucleotide to the solid support comprises covalent bond formation.

7. The method according to claim 5, wherein the step of attaching of the ssDNA or single stranded oligonucleotide to the solid support comprises biotin-streptavidin or biotin-avidin binding.

8. The method according to claim 5, wherein said solid support is selected from the group consisting of beads, micro-spheres, particles, paramagnetic particles, microtiter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin, avidin or modified to enable formation of a covalent bond with DNA or protein.

9. The method according to claim 5, wherein the step of extending of the ssDNA to form circular dsDNA comprises adding a DNA polymerase that is not capable of causing extension-dependent strand displacement.

10. A method of detecting autoantibodies against dsDNA comprising the steps of: providing dsDNA stably attached to a solid support, wherein said DNA is partially single-stranded and partially double-stranded and said double-stranded DNA is circular; contacting a biological sample from a patient with the dsDNA stably attached to a solid support; and detecting binding of autoantibodies to dsDNA.

11. The method according to claim 10, wherein the ssDNA portion is attached to the solid support through a covalent bond.

12. The method according to claim 10, wherein the ssDNA portion is attached to the solid support through biotin-streptavidin binding or biotin-avidin binding.

13. The method according to claim 10, wherein said solid support is selected from the group consisting of beads, micro-spheres, particles, paramagnetic particles, microliter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or modified to enable formation of a covalent bond with DNA or protein.

14. The method according to claim 10, wherein said biological sample is blood, sera, plasma, urine or saliva.

15. The method according to claim 10, wherein the step of detecting binding of autoantibodies to dsDNA is via luminescence, colorimetry or fluorescence.

16. Use of dsDNA stably attached to a solid support for detecting binding of autoantibodies to dsDNA in a biological sample.

17. The use according to claim 16, wherein said DNA is partially single-stranded and partially double-stranded and said double-stranded DNA is circular.

18. The use according to claim 17, wherein the ssDNA portion is attached to the solid support through a covalent bond.

19. The use according to claim 17, wherein the ssDNA portion is attached to the solid support through biotin-streptavidin binding or biotin-avidin binding.

20. The use according to claim 16, wherein said solid support is selected from the group consisting of beads, micro-spheres, particles, paramagnetic particles, microliter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or modified to enable formation of a covalent bond with DNA or protein.

21. The use according to claim 16, wherein said biological sample is blood, sera_¾ plasma, urine or saliva.

22. The use according to claim 16, wherein the binding of autoantibodies to dsDNA is detected via luminescence, colorimetry or fluorescence.

23. A kit comprising dsDNA stably attached to a solid support and detectably-labeled antibodies.

24. The kit according to claim 23, wherein said DNA is partially single-stranded and partially double-stranded and said double-stranded DNA is circular.

25. The kit according to claim 24, wherein the ssDNA portion is attached to the solid support through a covalent bond.

26. The kit according to claim 24, wherein the ssDNA portion is attached to the solid support through biotin-streptavidin binding or biotin-avidin binding.

27. The kit according to claim 24, wherein said solid support is selected from the group consisting of beads, micro-spheres, particles, paramagnetic particles, microliter plates, membranes, tubes, slides and any solid support that can be coated with streptavidin or modified to enable formation of a covalent bond with DNA or protein.

28. Use of the kit according to claims 23 for detecting binding of autoantibodies to dsDNA in a biological sample.

29. Use of the kit according to claims 23 for diagnosing systemic lupus erythematosus in a patient.

30. Use of the kit according to claims 23 for providing a disease prognosis for a patient diagnosed with systemic lupus erythematosus.