WO2012068107A2

WO2012068107A2 - Autoantibody to rna-protein complex detected by quantitative pcr

Info

Publication number: WO2012068107A2
Application number: PCT/US2011/060789
Authority: WO
Inventors: Edward K. L. Chan; Minoru Satoh; Angela Ceribelli
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2010-11-17
Filing date: 2011-11-15
Publication date: 2012-05-24
Also published as: WO2012068107A3

Abstract

The disclosure provides methods of detecting an autoantibody from a serum sample from a human or animal that is specific for an RNA:protein complex, the method comprising the steps of: (a) obtaining an immunoglobulin-RNA:protein aggregate, wherein said aggregate can comprise a target RNA:protein complex and an RNA:protein complex-specific antibody; (b) isolating the RNA from the immunoglobulin-RNA-protein aggregate; and (c) detecting the isolated RNA by quantitative reverse transcription PCR, whereby detection of the RNA can indicate the presence of the RNA:protein complex-specific antibody in a subject human or animal. One example of the method is configured for the detection of autoantibodies specifically binding to a U3 RNA: protein complex.

Description

AUTOANTIBODY TO RNA-PROTEIN COMPLEX DETECTED BY QUANTITATIVE

PCR

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. :

61 /414,537, entitled "AUTOANTIBODY TO RNA-PROTEIN COMPLEX DETECTED BY QUANTITATIVE PCR" filed on November 17, 2010, the entirety of which is hereby incorporated by reference.

TECHNICAL FIELD

The present disclosure is generally related to methods of detecting autoantibodies to

RNA:protein complexes using quantitative PCR.

BACKGROUND

Scleroderm a (Systemic Sclerosis, SSc) is an autoimmune disease characterized by vascular changes, fibrosis and presence of autoantibodies. The most commonly detected autoantibodies of SSc are anti-centromere (ACA), anti-topoisomerase I (topo I) and anti- RNA polymerase III (RNAPIII), in about 20% each (Steen V.D. (2005) Semin. Arthritis Rheum. 35: 35-42). SSc patients can be classified into subsets, associated with unique clinical features and specific autoantibodies. In particular, two major subsets are clinically recognized: limited (IcSSc) and diffuse (dcSSc) cutaneous variants. The dcSSc is frequently associated with anti-topo I, anti-RNAPI II , or anti-U3RNP (fibrillarin) while IcSSc is associated with ACA or anti-Th/To antibodies (Steen V.D. (2005) Semin. Arthritis Rheum. 35: 35-42; Satoh et al. (2007) Expert Rev. Clin. Immunol. 3: 721 -738).

Detection of autoantibodies in SSc is important. They are pathognomonic for a specific diagnosis, even before the complete clinical onset of the disease. They are also associated with unique clinical subsets, and for this reason they can be useful in monitoring and predicting SSc clinical manifestations (Steen V.D. (2005) Semin. Arthritis Rheum. 35: 35-42).

Despite the remarkable clinical importance, many SSc autoantibodies cannot be utilized clinically because of the unavailability of antibody testing (Satoh er a/. , (2009) Mod. Rheumatol. 19: 219-228). A recently developed assay is the ELISA for anti-RNAPI II antibodies. This autoantibody was originally described in 1987 as a marker of severe SSc with diffuse skin fibrosis, renal crisis and pulmonary hypertension (Okano ef a/. , (1993) Ann. Intern. Med. 1 19: 1005-1013). However, no widely available assay could identify this autoantibody, as only immunoprecipitation, performed in a few centers worldwide, was a standardized assay for anti-RNAPIII antibodies. In 2005, an ELISA kit became commercially available, allowing the detection of this autoantibody and the publication of an increasing number of studies on the significance of anti-RNAPIII antibodies in SSc (Kuwana et al. , (2005) Arthritis Rheum. 52: 2425-2432; Ceribelli et al. , (2010) J. Rheumatol. 37: 1544 ;

Cavazzana et al. , (2009) Autoimmun. Rev. 8: 580-584).

In the case of less frequent autoantibodies, such as anti-Th/To and anti-U3 (fibrillarin) , the main limitation is that they can be clearly identified only by immunoprecipitation , a technique not widely used in clinical laboratories. Even for these specificities, however, a strong link between autoantibody and clinical features has already been identified. In fact, anti-Th/To antibodies are considered to be fairly specific for SSc, because they are present in patien ts with SSc and primary Raynaud's phenomenon , but not in patients affected by other autoimmune disease (Okano & Medsger (1990) Arthritis Rheum . 33: 1822-1828) . Their prevalence varies between 2-5% in most studies (Ceribelli et al. , (2010) J. Rheumatol. 37: 574). They recognize a complex of six proteins associated with 7-2 and 8-2 RNA, which have been identified as two endonucleases, the human RNase MRP and RNase P ribonucleoprotein co mplexes (Okano & Medsger (1990) Arthritis Rheum. 33: 1822-1828; Ceribell i ef al. , (2010) J. Rheumatol. 37: 1574). The target proteins are not readily observed in protein- immunoprecipitation using standard [³⁵S]-methionine labeled cell extract, so the specificity is usually based on the detection of 7-2 and 8-2 R NA components in RNA- immunoprecipitation (Ceribelli et al. , (2010) J. Rheumatol. 37: 1 574; Gold et al. , (1998) Proc. Natl. Acad. Sci. U S A . 85: 5483-5487; Gold et al. , (1989) Science 245: 1377-1380). For the anti-U3RNP (fibrillarin) antibodies, it is well known that their frequency varies from 16-22% in SSc patients of African-American ethnicity to 4% in Caucasian SSc patients (Steen V. D. (2005) Semin. Arthritis Rheum. 35: 35-42). They are highly specific for severe dcS Sc associated with myositis, interstitial lung disease , pulmonary hypertension, small bowel involvement, renal disease and severe peripheral neuropathies (Steen V.D. (2005) Semin. Arthritis Rheum. 35: 35-42.

Another autoimmune disease in which autoantibodies to RNA-protein components play an important role is Poly/Dermatomyositis (PM/DM). The "myositis-specific antibodies" (MSAs) are markers of clinical subset, disease prognosis and treatment response for PM/DM patients, as in the case of SSc patients (Mammen A.L. (2010) Ann. N. Y. Acad. Sci. 184: 1 34-1 53).

MSAs traditionally include anti-aminoacyl tRNA synthetase and anti-signal recognition particle (SRP) antibodies. Serological testing is analytically complex in PM/DM because of the presence of many different specificities with low prevalence (1 -5%), with the only exception of anti-Jo- 1 antibodies (20%;Table 1 ). Moreover, another limitation in clinical practice is the lack of reliable and validated assays. Recently, new methods (such as ELISA, line blot assay) have been assessed for the detection of MSAs, but it is still necessary to perform validation tests before including them in laboratory diagnostics (Ghirardello et al. (2010) Rheumatology (Oxford) 49: 2370-2374). Several clinical and epidemiologic studies have shown that M SAs are associated with specific clinical characteristics (Mammen A. L. (2010) Ann. N. Y. Acad. Sci. 1 184: 134-153). Some of these associations are well-defined, as for anti-Jo-1 and the anti-synthetase syndrome, while others seem to be less convincing especially due to the small number of studies on positive patients . This is the case of anti-aminoacyl tRNA synthetase other than anti-Jo-1 and anti- SRP antibodies, which are rarely detected, and this makes difficult to study the main features of PM/DM patients with these specificities.

As mentioned before, RNA-immunoprecipitation is the test commonly used to identify antibodies to RNA:protein complexes. However, this technique is not widely available, and only a few centers are able to set up all the necessary steps to complete RNA- immunoprecipitation analyses. The detection of RNA bands is made by silver staining, a technique through which it can be difficult to obtain good quality and consistent results. Moreover, RNA-immunoprecipitation is a qualitative and not a quantitative technique, as it allows to detect the presence or absence of specific RNA bands but not to quantify the reactivity. The whole procedure has many variables that can hinder the correct detection of RNA components, such as water purity, voltage for gel electrophoresis, staining of the bands, and drying of the gel.

SUMMARY

Briefly described, embodiments of this disclosure, among others, encompass embodiments of a method of detecting an autoantibody specific for an RNA:protein complex, comprising the steps of: (a) obtaining an immunoglobulin-RNA:protein aggregate, wherein said aggregate can comprise a target RNA:protein complex and an RNA:protein complex- specific antibody; (b) isolating the RNA from the immunoglobulin-RNA-protein aggregate; and (c) detecting the isolated RNA by quantitative reverse transcription PCR, whereby detection of the RNA can indicate the presence of the RNA:protein complex-specific antibody in a subject human or animal.

In embodiments of this aspect of the disclosure, step (a) can comprise the steps of: (i) obtaining a target RNA:protein complex; (ii) contacting the target RNA: protein complex with an isolated immunoglobulin fraction from a subject human or animal under conditions that allow the formation of an immunoglobulin-RNA:protein aggregate comprising the target RNA: protein complex and an antibody of the isolated immunoglobulin fraction, said antibody being specific for the RNA:protein complex; (iii) isolating the immunoglobulin-RNA:protein aggregate ; (iv) isolating the RNA component from the isolated immunoglobulin-RNA:protein aggregate ; (v) contacting the isolated RNA component with a system configured for the amplification of an RNA species by quantitative reverse transcription PCR; and (vi) determining the amount of the isolated RNA by quantitative reverse transcription PCR, whereby the presence of the RNA indicates that the target RNA complex was specifically bound by an RNA-protein-specific antibody of the composition, and thereby indicating the presence of the RNA:protein complex-specific antibody in the subject human or animal.

In embodiments of this aspect of the disclosure, the immunoglobulin fraction can be obtained from the serum of a subject human or animal.

In embodiments of this aspect of the disclosure, the step of isolating the

immunoglobulin fraction from the serum sample can comprise contacting the serum sample with an immunoglobulin-binding agent under conditions allowing the formation of an immunoglobuiin-immunoglobulin-binding agent complex; and isolating the immunoglobulin- immunoglobulin-binding agent complex from the serum sample.

In embodiments of this aspect of the disclosure, the immunoglobulin-binding agent can be selected from the group consisting of an anti-immunoglobulin antibody, Protein A, Protein G, Protein A/G, and Protein L.

In embodiments of this aspect of the disclosure, the i mmunoglobulin-binding agent can be immobilized on a solid substrate.

In embodiments of this aspect of the disclosure, the target RNA:protein complex can be a component of a human or animal cell, or a lysate thereof.

In embodiments of this aspect of the disclosure, the target RNA:protein complex can be a component of a cell lysate, a serum, or an isolated target RNA: protein complex.

In embodiments of this aspect of the disclosure, the RT-PCR system can comprise at least one pair of primer oligonucleotides selected for specifically amplifying an RNA species of an RNA:protein complex specifically binding to an RNA-protein-specific antibody characteristic of Scleroderma (Systemic Sclerosis, SSc), and a labeled oligonucleotide selected to specifically detect the amplification product.

In embodiments of this aspect of the disclosure, the pair of primer oligonucleotides can be for specifically amplifying an RNA species of an RNA: protein complex of U3RNP or Th/To.

In embodiments of this aspect of the disclosure, the RT-PCR system can be for specifically amplifying an RNA species of an RNA:protein complex of U3RNP, and comprises the primer oligonucleotides having the nucleotide s equences according to SEQ ID Nos.: 1 and 2, and the labeled oligonucleotide can comprise at a first terminus thereof a fluorophore and a quencher disposed at a second terminus thereof.

In embodiments of this aspect of the disclosure, the labeled oligonucleotide fu rther comprises an internal quencher.

Another aspect of the disclosure encompasses embodiments of a kit comprising at least one pair of oligonucleotide primers selected for quantitatively amplifying by reverse transcription PCR an RNA of a target RNA:protein complex specifically recognized by an autoantibody, a labeled oligonucleotide for the detection of the amplification product, and packaging comprising instructions for the detection of an autoantibody in a serum sample from a human or animal subject using said oligonucleotide primers and RT-PCR.

In embodiments of this aspect of the disclosure, the kit can further comprise at least one of the group consisting of: a target RNA:protein complex or a composition comprising said target RNA:protein complex, a composition comprising an immunoglobulin-binding agent, and a system for RT-PCR using the oligonucleotide primers specifically hybridizing to a complement of an RNA of a target RNA:protein complex recognized by an autoantibody.

In embodiments of this aspect of the disclosure, the composition comprising an immunoglobulin-binding agent can be selected from the group consisting of an anti- immunoglobulin antibody , Protein A, Protein G, Protein A/G, and Protein L.

In embodiments of this aspect of the disclosure, the immunoglobulin binding agent can be attached to a solid substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Figs. 1A-1 F shows a series of graphs illustrating the results from applying quantitative PCR (qPCR) for the detection of the Th RNA component (Figs. 1A-1 C) and U3 RNA component (Figs. 1 D-1 F) in immunoprecipitates of reference sera. Results are shown demonstrating the highly sensitive detection of specific RNA as Ct (cycle threshold) changing with serial dilutions of cell lysate (Figs. 1 A and 1 D), RNA (Figs. 1 B and 1 E) or cDNA (Figs. 1C and 1 F). Primers specifically complementing the Th RNA component or U3 RNA component were used.

Fig. 2A is a digital image of the detection of anti-Th/To antibodies by urea-PAGE analysis and silver staining.

Fig. 2B is a digital image of the detection of anti-U3 (fibrillarin) antibodies by urea- PAGE analysis and silver staining.

Fig. 3 shows a graph illustrating the results of quantitative RT-PCR (qRT-PCR) for the detection of the Th RNA component. Results are shown comparing levels for Normal Human Serum (NHS), anti-Th (positive control) and anti-U3 fibrillarin (unrelated negative control) sera. Dilutions are referred to purified RNA after the immunoprecipitation step. A ^■ "filter" method was also used for purification of RNA and was used undiluted.

Fig. 4 is a graph illustrating the screening of anti-Th/To (+) samples. Median Ct values of n=2 independent experiments ^* by one-way ANOVA with Bonferroni correction Cut-off based on the ROC curve analysis of positive and control groups Th/To, n=22; U3, n=12; La, n=12; RNAPIII (anti-RNA polymerase III), n= 15; ACA (anti-centromere), n=15; Topo I (anti-topoisomerase I), n=17; PM/Scl, n=5; NHS (normal human serum), n=15 Fig. 5 is a graph illustrating the screening of anti-Th/To (+) samples. Median Ct values of n=2 independent experiments ^* by one-way ANOVA with Bonferroni correction. Cut-off based on the ROC curve analysis of positive and control groups Th/To, n=22; U3, n=12; TMG, n=12; Topo I (anti-topoisomerase I), n=17; RNAPIII (anti-RNA polymerase III), n= 15; ACA (anti-centromere), n=15; PM/Scl, n=5; NHS (normal human serum), n=15.

Fig. 6 shows a flow-chart showing the main steps of the RNA- immunoprecipitation/Urea PAGE/silver staining procedure compared with

immunoprecipitation-qRT-PCR. After RNA extraction, reverse transcription and quantitative PCR were performed instead of running UREA-PAGE gel and silver staining.

The drawings are described in greater detail in the description and examples below.

The details of some exemplary embodiments of the methods and systems of the present disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent to one of skill in the art upon examination of the following description, drawings, examples and claims. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended clai ms.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disc losure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "includi ng," and the like; "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

The term "autoantibody" as used herein refers to an antibody manufactured by the immune system that is directed against one or more of an individual's own antigens such as an epitope of a protein, a peptide, or a non-protein epitope. Many autoimmune diseases, notably lupus erythematosus, scleroderma, and polymyositis/dermatomyositis associated with autoantibodies.

The term "autoantibody specific for an RNA:protein complex" as used herein refers to an antibody that specifically binds to an epitope of an RNA:protein complex. The epitope may be, but is not limited to, a region of the RNA, the protein complexed with the RNA species or an epitope formed by the combination of the RNA and the protein.

The term "immunoglobulin-RNA:protein aggregate" as used herein refers to the complex formed when an autoantibody specifically binds to an RNA:protein complex. Such binding may take place in vivo or in vitro.

The term "target RNA: protein complex" as used herein refers to an RNA protein complex for which it is desired to determine whether a serum sample from a human or animal subject includes an autoan tibody species specific thereto.

The term "isolated RNA component" as used herein refers to the RNA obtained from an RNA:protein complex after said complex has been substantially purified or isolated from a serum sample. It is contemplated that substantially purified RNA may be a homogeneous , or a heterogeneous combination of a plurality of species.

The terms "complementarity" or "complementary" as used herein refer to a sufficient number in the oligonucleotide of complementary base pairs in its sequence to interact specifically (hybridize) with the target nucleic acid sequence to be amplified or detected. As known to those skilled in the art, a very high degree of complementarity is needed for specificity and sensitivity involving hybridization, although it need not be 100%. Thus, for example, an oligonucleot ide that is identical in nucleotide sequence to an oligonucleotide disclosed herein, except for one base change or substitution, may function equivalently to the disclosed oligonucleotides. A "complementary DNA" or "cDNA" gene includes recombinant genes synthesized by reverse transcription of messenger RNA ("mRNA").

The term "cyclic polymerase-mediated reaction" as used herein refers to a biochemical reaction in which a template molecule or a population of template molecules is periodically and repeatedly copied to create a complementary template molecule or complementary template molecules, thereby increasing the number of the template molecules over time.

The term "denaturation" of a template molecule as used herein refers to the unfolding or other alteration of the structure of a template so as to make the template accessible to duplication. In the case of DNA, "denaturation" refers to the separation of the two complementary strands of the double helix, thereby creating two complementary, single stranded template molecules. "Denaturation" can be accomplished in any of a variety of ways, including by heat or by treatment of the DNA with a base or other denaturant.

A "detectable amount of product" as used herein refers to an amount of amplified nucleic acid that can be detected using standard laboratory tools. A "detectable marker" refers to a nucleotide analog that allows detection using visual or other means. For example, fluorescently labeled n ucleotides can be incorporated into a nucleic acid during one or more steps of a cyclic polymerase-mediated reaction, thereby allowing the detection of the product of the reaction using, e.g. fluorescence microscopy or other fluorescence- detection instrumentation.

The term "detectably labeled" as used herein refers to a fragment or an

oligonucleotide contains a nucleotide that is radioactive, or that is substituted with a fluorophore, or that is substituted with some other molecular species that elicits a physical or chemical response that can be observed or detected by the naked eye or by means of instrumentation such as, without limitation, scintillation counters, colorimeters, UV spectrophotometers and the like. As used herein, a "label" or "tag" refers to a molecule that, when appended b y, for example, without limitation, covalent bonding or hybridization, to another molecule, for example, also without limitation, a polynucleotide or polynucleotide fragment, provides or enhances a means of detecting the other molecule. A fluorescence or fluorescent label or tag emits detectable light at a particular wavelength when excited at a different wavelength. Essentially any fluorophore may be used , including BODIPY, fluoroscein, fluoroscein substitutes (Alexa Fluor dye, Oregon green dye, and the like), long wavelength dyes, and UV-excited fluorophores. These and additional fluorophores are listed in Fluorescent and Luminescent Probes for Biological Activity, A Practical Guide to

Technology for Quantitative Real-Time Analysis, Second Ed. W. T. Mason, ed. Academic Press (1999) (incorporated herein by reference). In embodiments of the disclosure an advantageous fluorophore can be, but is not limited to, 6-carboxyfluorescein (FAM) having an excitation range of about 460 to about 500 nm.

The term "quencher" as used herein refers to a molecule that absorb s the energy of an excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher . By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon , thereby preventing fluorescence. Quenchers may be categorized as non-fluorescent and fluorescent quenchers. Non-fluorescent quenchers are capable of quenching the fluorescence of a wide variety of fluorophores. Generally, non-fluorescent quenchers absorb energy from the fluorophore and release the energy as heat. Examples of non-fluorescent quenchers include 4-{4'-dimethylaminophenylazo)benzoic acid) (DABCYL), QSY-7, QSY-33, or such as the dark quencher IOWA BLACK FQ- 3'.RTM (Integrated DNA Technologies, Inc., Coralville, Iowa, U.S.A.). A quencher may also be an internal quencher such as, but not limited to the ZEN. RTM quencher (Integrated DNA Technologies, Inc. , Coralville, Iowa, U.S.A.).

When choosing a fluorophore, a quenche r, or where to pos ition these molecules, it is important to consider, and preferably to test, the effect of the fluorophore or quencher on the enzymatic activity of the nucleic acid enzyme. Also, it is advantageous that the fluorophore display a high quantum yield and energy transfer efficiency. Long-wavelength (excitation and emission) fluorophores are preferred because of less interference from other absorbing species. The fluorophore should also be less sensitive to pH change or to non-specific quenching by metal ions or other species.

The term "DNA" refers to the polymeric form of deoxyribonucleotides (adenine, guanine , thymine, or cytosine) in either single stranded form, or as a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found , inter alia, in linear DNA molecules (e.g. , restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e. , the strand having a sequence homologous to the mRNA).

The term "DNA amplification" as used herein refers to any process that increases the number of copies of a specific DNA sequence by enzymatically amplifying the nucleic acid sequence. A variety of processes are known. One of the most commonly used is the polymerase chain reaction (PCR), which is defined and described in later sections below. The PCR process of Mullis is described in U.S. Pat. Nos. 4,683, 195, and 4,683,202. PCR involves the use of a thermostable DNA polymerase, known sequences as primers, and heating cycles, which separate the replicating deoxyribonucleic acid (DNA), strands and exponentially amplify a gene of interest. Any type of PCR, such as quantitative PCR, RT- PCR, hot start PCR, LAPCR, multiplex PCR, touchdown PCR, etc. , may be used.

Advantageously, RT-PCR is used. In general, the PCR amplification process involves an enzymatic chain reaction for preparing expo nential quantities of a specific nucleic acid sequence. It requires a small amount of a sequence to initiate the chain reaction and oligonucleotide primers that will hybridize to the sequence. In PCR the primers are annealed to denatured nucleic acid followed by extension with an inducing agent (en zyme) and nucleotides. This results in newly synthesized extension products. Since these newly synthesized sequences become templates for the primers, repeated cycles of denaturing, primer annealing, and extension results in exponential accumulation of the specific sequence being amplified. The extension product of the chain reaction will be a discrete nucleic acid duplex with a termini corresponding to the ends of the specific primers employed.

The terms "enzymatically amplify" or "amplify" as used herein refer to DNA amplification, i.e. , a process by which nucleic acid sequences are amplified in number.

There are several means for enzymatically amplifying nucleic acid sequences. Currently the most commonly used method is the polymerase chain reaction (PCR).

The term "fragment" of a molecule such as a protein or nucleic acid as used herein refers to any portion of the amino acid or nucleotide genetic sequence.

The term "hybridization" as used herein refers to the process of association of two nucleic acid strands to form an antiparallel duplex stabilized by means of hydrogen bonding between residues of the opposite nucleic acid strands. "Hybridizing" and "binding", with respect to polynucleotides, are used interchangeably. The terms "hybridizing specifically to" and "specific hybridization" and "selectively hybridize to," as used herein refer to the binding, duplexing, or hybridi zing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions.

By way of example, a nucleotide sequence of the present disclosure may be identical to a reference sequence, that is be 100% identical , or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group including at least one nucleotide deletion, substitut ion, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminus positions of the reference nucleotide sequence or anywhere between those terminus positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in the reference nucleotide by the numerical percent of the respective percent identity (divided by 100) and subtracting that product from said total number of nucleotides in the reference nucleotide.

The term "immobilized on a solid support" as used herein refers to a fragment, primer or oligonucleotide is attached to a substance at a particular location in such a manner that the system containing the immobilized fragment, primer or oligonucleotide may be subjected to washing or other physical or chemical manipulation without being dislodged from that location. A number of solid supports and means of immobilizing nucleotid e-containing molecules to them are known in the art. For example, and not intended to be limiting, immobilization onto a solid support may be by directly chemically cross-linking a molecular species to an underlying support material. Alternatively, and especially useful for linking an immunoglobulin-binding agent to a solid support as used in the methods of the disclosure, an immunoglobulin-binding agent such as an anti-immunoglobulin antibody may have a biotin moiety attached thereto. In this system, the solid support may have such as streptavidin, which has an affinity for biotin, bound to the solid support; any of these supports and means may be used in the methods of this invention. It is also understood that a solid support can be such as, but not limited to, beads, plastic surfaces, or any other surface to which an immunoglobulin-binding agent can be bound such as, but not intended to be limiting, SEPHAROSE .RTM, agarose, polyacrylamide and the like.

The term "immunoglobulin-binding agent" as used herein refers to proteins such as Protein A, a surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It has been used in biochemical research because of its ability to bind

immunoglobulins from many of mammalian species, most notably, with the Fc region of immunoglobulins ( IgGs) through interaction with the heavy chain. Protein A binds with high affinity to human lgG1 and lgG2 as well as mouse lgG2a, lgG2b, and lgG2c (previously, lgG2a of b allotype). Protein A binds with moderate affinity to human IgM, IgA and IgE as well as to mouse lgG3 and lgG1 . It does not react with human lgG3 or IgD, nor will it react to mouse IgM , IgA or IgE. Other antibody-binding proteins include, but are not limited to, such as Protein G, Protein A/G and Protein L, or anti-immunoglobulin antibodies and the like, all commonly used to purify, immobilize or detect immunoglobulins.

The term "melting temperature" as used herein refers to the temperature at which hybridized duplexes dehybridize and return to their single-stranded state. Likewise, hybridization will not occur in the first place between two oligonucleotides, or, herein, an oligonucleotide and a fragment, at temperatures above the melting temperature of the resulting duplex. It is presently advantageous that the difference in melting point temperatures of oligonucleotide-fragment duplexes of this invention be from about 1 °C to about 10 °C so as to be readily detectable.

The term "nucleotide monomer" as used herein refers to a molecule which is not incorporated in a larger oligo- or poly-nucleotide chain and which corresponds to a single nucleotide sub-unit; nucleotide monomers may also have activating or protecting groups, if such groups are necessary for the intended use of the nucleotide monomer. The terms "nucleotide" , "nucleotide monomer" and a "nucleotide moiety" as used herein refer to a sub- unit of a nucleic acid (whether DNA or RNA or an analogue thereof) which may include, but is not limited to, a phosphate group, a sugar group and a nitrogen containing base, as well as analogs of such sub-units. Other groups (e.g., protecting group s) can be attached to the sugar group and nitrogen containing base group.

It will be appreciated that, as used herein, the terms "nucleotide" and "nucleoside" will include those moieties which contain not only the naturally occurring purin e and pyrimidine bases, e.g. , adenine (A), thymine (T), cytosine (C), guanine (G) , or uracil (U), but also modified purine and pyrimidine bases and other heterocyclic bases which have been modified (these moieties are sometimes referred to herein, collectively , as "purine and pyrimidine bases and analogs thereof). Such modifications include, e.g. , diaminopurine and its deravitives, inosine and its deravitives, alkylated purines or pyrimidines, acylated purines or pyrimidines thiolated purines or pyrimidines, and the like, or the addition of a protecting group s uch as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9- fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, Ν, Ν-diphenyl carbamate, or the like. The purine o r pyrimidine base may also be an analog of the foregoing; suitable analogs wi ll be known to those skilled in the art and are described in the pertinent texts and literature. Common analogs include, but are not limited to, 1 -methyladenine, 2- methyladenine, N6-methyladen ine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, Ν, Ν-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1 -methylguanine, 2-methylguanine, 7-methylguanine, 2,2- dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguan ine, 8- thioguanine, 5 -fluorou racil, 5-bromouracil, 5-chlorouracil, 5-iodou racil, 5-ethyluracil, 5- propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil , 5-(carboxyhydroxymethyl)uracil, 5- (methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2- thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1 -methylpseudouracil, queosine, inosine, 1 -methylinosine,

hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopuri ne, and 2,6- diaminopurin e.

The terms "oligonucleotide" as used herein refers to any polyribonucleotid e or polydeoxribonucleotide t hat may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single - and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The terms "nucleic acid," "nucleic acid sequence, " or "oligonucleotide" also encompass a polynucleotide as defined above .

The term "polymerase" as used herein refers to an enzyme that catalyzes the sequential addition of monomeric units to a polymeric chain, or links two or more monomeric units to initiate a polymeric chain. In advantageous embodiments of this invention, the "polymerase" will work by adding monomeric units whose identity is determined by and which is complementary to a template molecule of a specific sequence. For example, DNA polymerases such as DNA pol 1 and Taq polymerase add deoxynbonucleotides to the 3' end of a polynucleotide chain in a template-dependent manner, thereby synthesizing a nucleic acid that is complementary to the template molecule. Polymerases may be used either to extend a primer once or repetitively or to amplify a polynucleotide by repetitive priming of two complementary strands using two primers.

The term "polymerase chain reaction" or "PCR" as used herein refers to a thermocyclic, polymerase-mediated, DNA amplification reaction. A PCR typically includes template molecules, oligonucleotide pri mers complementary to each strand of the template molecules, a thermostable DNA polymerase, and deoxynbonucleotides, and involves three distinct processes that are multiply repeated to effect the amplification of the original nucleic acid. The three processes (denaturation, hybridization, and primer extension) are often performed at distinct temperatures, and in distinct temporal steps. In many embodiments , however, the hybridization and primer extension processes can be performed concurrently. The nucleotide sample to be analyzed may be PCR amplification products provided using the rapid cycling techniq ues described in U. S. Pat. Nos. 6,569,672; 6,569,627; 6,562,298; 6,556, 940; 6,569,672 ; 6,569,627; 6,562,298; 6,556,940; 6,489,1 12; 6,482 ,615; 6,472, 156; 6,413, 766; 6,387,621 ; 6,300, 124; 6,270,723; 6,245,514; 6,232,079; 6,228 ,634; 6,218,193 ; 6,210, 882; 6, 197,520; 6,174,670; 6, 132,996; 6,126,899; 6, 124,138; 6,074 ,868; 6,036,923 ; 5,985,651 ; 5,958,763; 5,942,432; 5,935,522; 5,897,842; 5,882,918; 5,840 ,573; 5,795,784 ; 5,795,547; 5,785,926; 5,783,439; 5,736, 106; 5,720,923; 5,720,406; 5,675 ,700; 5,616,301 ; 5,576,218 and 5,455,175 , the disclosures of which are incorporated by reference in their entireties. Other methods of amplification include, without limitation, NASBR, SDA, 3SR, TSA and rolling circle replication. It is understood that, in any method for producing a polynucleotide containing given modified nucleotides, one or several polymerases or amplification methods may be used. The selection of optimal polymerization conditions depends on the applicat ion.

The term "primer oligonucleotide" as used herein refers to an oligonucleotide, the sequence of at least a portion of which is complementary to a segment of a template DNA which to be amplified or replicated. Typically primers are used in performing the polymerase chain reaction (P CR). A primer hybridizes with (or "anneals" to) the template DNA and is used by the polymerase enzyme as the starting point for the replication/amplification process. By "complementary" is meant that the nucleotide sequence of a primer is such that the primer can form a stable hydrogen bond complex with the template; i.e. , the primer can hybridize or anneal to the template by virtue of the formation of base-pairs over a length of at least ten consecutive base pairs.

The primers herein are selected to be "substantial ly" complementary to different strands of a particular target DNA sequence . This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non- complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non- complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

The term "probe" as used herein refers to oligonucleotides nucleic acid sequences of variable length, used in the detection of identical, similar, or complementary nucleic acid sequences by hybridization. An oligonucleotide sequence used as a detection probe may be labeled with a detectable moiety. Various labeling moieties are known in the art. Said moiety may, for example, either be a radioactive compound, a detectable enzyme (e.g. horse radish pe roxidase (HRP)) or any other moiety capable of generating a detectable signal such as a calorimetric, fluorescent, chem iluminescent or electrochemiluminescent signal. The detectable moiety may be detected using known methods.

The term "lysate" as used herein refers to a suspension of isolated cells that have had their cell membranes disrupted chemically, physically, enzymatically, or by a

combination thereof. The cells may be lysed in a buffer, the disruption in the cell membranes releasing to the surroundi ng buffer a m ix of proteins and other cell constituents. The lysis may be total, where all cells in the treated cell population pro cedure release their intracellular contents, or partial where at least 50%, advantageou sly, at least 75%, more advantageously at least 90%, and most advantageously 100% of the cells in a population of isolated cells are disrupted and relea se their intracellular contents into a suspension buffer.

The term "protein" as used herein refers to a large molecule composed of one or more chains of amino acids in a specific order. The order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has a unique function.

The term "reverse transcription polym erase chain reaction (RT-PCR)" as used herein refers to a variant of polymerase chain reaction (PCR) where an RNA strand is first reverse transcribed into its DNA complement (cDNA) using the enzyme reverse transcriptase, and the resulting c DNA is amplified using traditional or real-time PCR. In RT-PCR reverse transcription (RT) , in which RNA is reverse transcribed to cDNA uses reverse transcriptase . The RT step can be performed either in the same tube with PCR (one-step PCR) or in a separate one (two-step PCR). Real-time RT-PCR provides a method in which the amplicons can be visualized as the amplification progresses using a fluorescent reporter molecule. There are three major kinds of fluorescent reporters used in real time RT-PCR, which are general non -specific DNA Binding Dyes such as SYBR Green I, TAQMAN.RTM Probes and Molecular Beacons. The fluorescence increases as the amplification progresses and the instrument performs data acquisition during the annealing step of each cycle. The number of amplicons will reach the detection baseline after a specific cycle, which depends on the initial concentration of the target DNA sequence. The cycle at which the instrument can discriminate the amplification generated fluorescence from the background noise is called the threshold cycle (Ct). The higher the initial DNA concentration, the lower its Ct will be.

The term "template" as used herein refers to a target polynucleotide strand, for example, without limitation, an unmodified naturally-occurring DNA strand, which a polymerase uses as a means of recognizing which nucleotide it should next incorporate into a growing strand to polymerize the complement of the naturally-occurring strand. Such DNA strand may be single-stranded or it may be part of a double-stranded DNA template. In applications of the present invention requiring repeated cycles of polymerization, e.g. , the polymerase chain reaction (PCR), the template strand itself may become modified by incorporation of modified nucleotides, yet still serve as a template for a polymerase to synthesize additional polynucleotides.

The term "thermocyclic reaction" as used herein refers to a multi-step reaction wherein at least two steps are accomplished by changing the temperature of the reaction.

The term "thermostable polymerase" as used herein refers to a DNA or RNA polymerase enzyme that can withstand extremely high temperatures, such as those approaching 100 °C. Often, thermostable polymerases are derived from organisms that live in extreme temperatures, such as Thermus aquaticus. Examples of thermostable polymerases include Taq, Tth, Pfu, Vent, deep vent, UITma, and variations and derivatives thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

Further definitions are provided in context below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitabl e methods and materials are described herein.

Whichever probe sequences and hybridization methods are used, one skilled in the art can readi ly determine suitable hybridization conditions, such as temperature and chemical conditions. Such hybridization methods are well known in the art. For example, for applications requiring high selectivity, one will typically desire to employ relatively stringent conditions for the hybridization reactions, e.g. , one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02M to about 0.10M NaCI at temperatures of about 50 °C to about 70 °C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand, and are particularly suitable for detecting specific SNPs according to the present invention. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide. Other variations in hybridization reaction conditions are well known in the art (see for example, Sambrook et a/. , Molecular Cloning; A Laboratory Manual 2d ed. (1989)) .

Nucleic acid molecules that differ from the sequences of the primers and probes disclosed herein, are intended to be within the scope of the invention. Nucleic acid sequences that are complementary to these sequences, or that are hybridizable to the sequences described herein under conditions of standard or stringent hybridization, and also analogs and derivatives are also intended to be within the scope of the invention.

Advantageously, such variations will differ from the sequences described herein by only a small number of nucleotides, for example by 1 , 2, or 3 nucleotides.

The primers and probes described herein may be readily prepared by, for example, directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production. Methods for making a vector or recombinants or plasmid for amplification of the fragment either in vivo or in vitro can be any desired method, e.g., a method which is by or analogous to the methods disclosed in, or disclosed in documents cited in: U.S. Patent Nos. 4,603,1 12 ; 4,769,330; 4,394,448;

4,722, 848; 4,745,051 ; 4,769,331 ; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744, 141 ; 5,756, 103; 5,762,938 ; 5,766, 599; 5,990,091 ; 5, 174,993; 5,505,941 ; 5,338 ,683; 5,494,807 ;

5,591 ,639; 5,589,466 ; 5,677, 178; 5,591 ,439; 5,552, 143; 5,580,859; 6, 130 ,066; 6,004,777;

6,130, 066; 6,497,883 ; 6,464,984; 6,451 ,770; 6,391 ,314; 6,387,376; 6,376 ,473; 6,368,603 ;

6,348, 196; 6,306,400 ; 6,228,846; 6,221 ,362; 6,217,883; 6,207,166; 6,207, 165; 6, 159,477 ;

6,153, 199; 6,090,393 ; 6,074,649; 6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882 ; 6,312,682; 6,348,450 and 6; 312,683; U.S. patent application Serial No. 920, 197, filed

October 16, 1986; WO 90/01543; W091 /1 1525; WO 94/16716; WO 96/39491 ; WO 98/33510 ;

EP 265785; EP 0 370 573; Andreansky et a/. , (1996) Proc. Natl. Acad. Sci. U. S.A. 93: 1 1313-1 1318; Ballay er a/. , (1993) EMBO J. 4: 3861 -65; Feigner et a/. , (1994) J. Biol. Chem. 269: 2550-2561 ; Frolov er a/. , (1996) Proc. Natl. Acad. Sci. U. S.A. 93: 1 1371 -1 1377;

Graham (1990) Tibtec 8: 85-87; Grunhaus et al. , (1992) Sem. Virol. 13: 237-52; Ju et al. , (1998) Diabetologia 41 : 736-739; Kitson er a/. , (1991 ) J. Virol. 65: 3068-3075; McClements er a/. , (1996) Proc. Natl. Acad. Sci. U. S.A. 93: 1 1414-1 1420; Moss(1996) Proc. Natl. Acad. Sci. U. S.A. 93: 1 1341 -1 1348; Paoletti(1996) Proc. Natl. Acad. Sci. U.S.A. 93: 1 1349-1 1353; Pennock et al. , (1984) Mol. Cell. Biol. 4: 399-406; Richardson (Ed), Methods in Molecular Biology 1995;39, "Baculovirus Expression Protocols," Humana Press Inc.; Smith er a/. (1983) Mol. Cell. Biol. 3: 2156-2165; Robertson er a/. , (1996) Proc. Natl. Acad. Sci. U. S.A. 93: 1 1334-1 1340; Robinson et al. , (1997) Sem. Immunol. 9: 271 ; and Roizman (1996) Proc. Natl. Acad. Sci. U. S.A. 93: 1 1307-1 1312.

Oligonucleotide sequences used as primers or probes according to the present invention may be labeled with a detectable moiety. As used herein the term "sensors" refers to such primers or probes labeled with a detectable moiety. Various labeling moieties are known in the art. Said moiety may be, for example, a radiolabel (e.g. , ³H, ¹²⁵l, ³⁵S, ⁴C, ³²P, etc.), detectable enzyme (e.g. horse radish peroxidase (HRP), alkaline phosphatase ere), a fluorescent dye (e.g. , fluorescein isothiocyanate, Texas red, rhodamine, Cy3, Cy5, Bodipy, Bodipy Far Red, Lucifer Yellow, Bodipy 630/650-X, Bodipy R6G-X and 5-CR 6G, and the like), a colorimetric label such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.), beads, or any other moiety capable of generating a detectable signal such as a colorimetric, fluorescent, chemiluminescent or electrochemiluminescent (ECL) signal.

Primers or probes may be labeled directly or indirectly with a detectable moiety, or synthesized to incorporate the detectable moiety. In one embodiment, a detectable label is incorporated into a nucleic acid during at least one cycle of a cyclic polymerase-mediated amplification reaction. For example, polymerases can be used to incorporate fluorescent nucleotides during the course of polymerase-mediated amplification reactions. Alternatively, fluorescent nucleotides may be incorporated during synthesis of nucleic acid primers or probes. To label an oligonucleotide with the fluorescent dye, one of conventionally-known labeling methods can be used (Nature Biotechnology, 14, 303-308, 1996; Applied and

Environmental Microbiology, 63, 1 143-1 147, 1997; Nucleic Acids Research, 24, 4532-4535, 1996). An advantageous probe is one labeled with a fluorescent dye at the 3' or 5' end and containing G or C as the base at the labeled end. If the 5' end is labeled and the 3'end is not labeled, the OH group on the C atom at the 3'-position of the 3' end ribose or deoxyribose may be modified with a phosphate group or the like although no limitation is imposed in this respect. Spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means can be used to detect such labels. The detection device and method may include, but is not limited to, optical imaging, electronic imaging, imaging with a CCD camera , integrated optical imaging , and mass spectrometry. Further, the amount of labeled or unlabeled probe bound to the target may be quantified. Such quantification may include statistical analysis. In other embodiments the detection may be via conductivity differences between concordant and discordant sites, by quenching, by fluorescence perturbation analysis, or by electron transport between donor and acceptor molecules.

Description

The methods of the present disclosure encompass the use of quantitative RT-PCR

(qRT-PCR) for the detection of the RNA component of an RNA:protein complex

immunoprecipitated by a serum autoantibody. A positive detection, which may also be a quantified determination, supports the inference that a sample fluid from a subject animal or human included an amount of the autoantibody characterized as specifically recognizing and binding the RNA:protein complex. The methods of the disclosure can detect and quantify the amount of a target autoantibody of interest that is capable of specifically recognizing and binding to a particular RNA.protein complex independentl y of whether the autoantibody is recognizing and binding to an epitope unique to the RNA component, the protein component, or formed from a combination thereof, of the complex.

The methods of the disclosure can be adjusted to resolve and detect closely related autoantibodies by selecting suitable PCR primers that can distinguish one species of target RNA from another similar RNA such as, for example, one species of tRNA from another. Accordingly, the methods of the present disclosure are considered to be useful for detecting a broad spectrum of RNA:protein complex-binding autoantibodies, and also able to identify the presence of a particular autoantibody in a serum having a heterogeneous mix of autoantibodies recognizi ng and binding to similar RNA:protein complexes.

In the methods of the disclosure, a sample serum obtained from a human or animal subject, and which is suspected of including at least one species of an autoantibody, is contacted with an agent that can bind non-specifically to immunoglobulin (antibody) chains. Suitable immunoglobulin-binding agents for use in the methods of the present disclosure include, but are not limited to, an immunoglobulin-specific antibody, an immunoglobulin- binding agent derived from a bacterial source such as Protein A, Protein G, Protein A/G, and Protein L, and the like.

The complex formed between the immunoglobulins and the immunog lobulin-binding agent may be employed to isolate the immunoglobulins from the serum sample. For example, and not intending to be limiting, the immunoglobulin-binding agent may be bound to a solid support such as, for example, SEPHAROSE CL4B .RTM. After binding of the immunoglobulin to the solid support-bound immunoglobulin-binding agent, the complexes and the underlying support material can be physically separated from the serum sample by methods such as centrifugation, sedimentation, or the washing of a column of the complex.

It is further contemplated, but not intended to be limiting, that the immunoglobulin binding agent may be bound to a solid surface such as, but not limited to, an internal surface of a plastic multi-well plate or a planar surface such as a sheet of polymer, plastic, glass, or the like. Attaching the immunoglobulin binding agent to such a surface and at a plurality of locations thereon can allow the methods of the present disclosure to be readily adapted for the determination of anti-RNA:protein complex antibodies in a plurality of serum samples. Thus, for example, if the immunoglobulin binding agent is attached to the internal base surface of the wells of a multi-well plate, a plurality of samples may be independently assayed. After capturing the immunoglobulin fraction of the serum samples by the immobilized immunoglobulin binding agent, the wells can be washed, cell lysate added, and the RNA isolated without cross-contamination from adjacent samples. It will be recognized that such systems may be readily adapted for automation for liquid delivery, and removal of the RNA for subsequent qRT-PCR, by equipment and methods well known in the art.

The immunoglobulins bound to the capturing immunoglobulin-binding agent, and hence to the solid support, may then be mixed with a cell lysate comprising a heterogeneous population of potential target RNA: protein complexes, at least one of which can be specifically recognized and bound by the target autoantibody, if said autoantibody is among the population of immunoglobulins captured by the immobilized immunoglobulin-binding agent. Accordingly, an RNA:protein complex specifically recognizable by an autoantibody of the serum of the subject human or animal will have been captured. The RNA component of the complex may then be isolated and used as a template in a quantitative-PCR reaction after conversion to a cDNA, using oligonucleotide pri mers complementary for said RNA nucleotide sequence, thereby indirectly detecting the presence of the autoantibody among the population of antibodies that had been isolated from the sample serum.

Results shown in Figs. 2 and 3 support the utility of quantitative (q)RT-PCR to amplify target RNA component isolated from an RNA:protein complex immunoprecipitated by binding to an anti-RNA: protein complex autoantibody attached to a solid support material. Quantification of the autoantibody by quantifying the amount of the target cDNA (and hence RNA template) is also possible. For example, the efficacy of the test has been shown for the qualitative and quantitative detection of the Th and U3 RNA components in reference sera, using primers specific for the Th RNA component or the U3 RNA component. Moreover, results confirm that even when using 8-fold lower amount of cell lysate, RNA or cDNA in a titration experiment. This highly sensitive detection, therefore, can allow the detection of low-titer antibodies and the use of a significantly lower amount of cell lysate, thereby reducing the costs of an individual assay for an antibody.

The quantitative RT-PCR methods of the disclosure incorporate the use of a labeled oligonucleotide probe having at one terminus (either the 5' or the 3' end) a fluorophore, and at the opposing end a quencher moiety, the sequence of the oligonucleotide probe having complementarity with a region of the nucleotide sequence amplified by the PCR procedu re. It is further contemplated that the probe may comprise a second internal quencher to enhance the quenching of fluorescence emitted by the fluorophore before hybridization of the probe to a target sequence, and thereby providing reduced background fluorescence signal.

The usefulness of the methods of the present disclosure further resides in its ability to use any method of RNA extraction, using both phenol/chloroform/isoamyl alcohol and filter systems that are commercially available, and well known in the art. A schematic flow-chart showing the main steps of the assay methods of the present disclosure , and illustrating the main differences between RNA-immunoprecipitation methods known in the art and the qRT- PCR method of the present disclosure are presented in Fig. 6.

Embodiments of the immunoprecipitation-qRT-PCR methods of the disclosure has the following advantages when compared with standard RNA-IP gel electrophoresis method: the qRT-PCR-based method allows the detection of antibodies to RNA-protein components while avoiding using an UREA-PAGE gel and silver staining that requires the use of toxic reagents (bis-acrylamide and silver staining) and is technically demanding; the qRT-PCR method can be configured as a high-throughput assay system performed for a high number of samples whereas the conventional RNA-immunoprecipitation method is limited to testing only about 10-16 samples/UREA-PAGE gel; the qRT-PCR method of the disclosure can provide more consistent results than can silver staining ; the qRT-PCR method may be readily adapted to allow quantitative or semi-quantitative detection of an autoantibody, while the RNA-immunoprecipitation-UREA-PAGE gel method is typically qualitative; the qRT-PCR method can resolve RNA components having limited sequence differences using specific primers whereas, for example, individual RNA species such as tRNAs associated with PM/DM autoantibodies would otherwise co-immunoprecipitate and/or co-migrate and not be distinguishable with the UREA-page gel system; the qRT-PCR methods allow to significantly reduce the amount of cells necessary for the lysate, from about 0.5-1 .0 x 10⁶ cells per sample to about 0.1 -1 .0 x 10⁶ cells per sample.

One aspect of the disclosure, therefore, encompasses embodiments of a method of detecting an autoantibody specific for an RNA: protein complex, comprising the steps of: (a) obtaining an i mmunoglobulin-RNA:protein aggregate, wherein said aggregate can comprise a target RNA: protein complex and an RNA: protein complex-specific antibody; (b) isolating the RNA from the immunoglobulin -RNA-protein aggregate; and (c) detecting the isolated RNA by quantitative reverse transcription PCR, whereby detection of the RNA can indicate the presence of the RNA:protein complex-specific antibody in a subject human or animal.

In embodiments of this aspect of the disclosure, step (a) can comprise the steps of: (i) obtaining a target RNA:protein complex; (ii) contacting the target RNA:protein complex with an isolated immunoglobulin fraction from a subject human or animal under conditions that allow the formation of an immunoglobulin-RNA:protein aggregate comprising the target RNA:protein complex and an antibody of the isolated immunoglobulin fraction, said antibody being specific for the RNA:protein complex; (iii) isolating the immunoglobulin-RNA:protein aggregate; (iv) isolating the RNA component from the isolated immunoglobulin-RNA: protein aggregate; (v) contacting the isolated RNA component with a system configured for the amplification of an RNA species by quantitative reverse transcription PCR; and (vi) determining the amount of the isolated RNA by quantitative reverse transcription PCR, whereby the presence of the RNA indicates that the target RNA complex was specifically bound by an RNA-protein -specific antibody of the composition, and thereby indicating the presence of the RNA:protein complex-specific antibody in the subject human or animal.

In embodiments of this aspect of the disclosure, the step of isolating the

immunoglobulin fraction from the serum sample can comprise contacting the serum sample with an immunoglobulin-binding agent under conditions allowing the formation of an immunoglobulin-immunoglobulin-binding agent complex; and isolating the immunoglobulin- immunoglobulin-binding agent complex from the serum sample.

In embodiments of this aspect of the disclosure, the immunoglobulin-binding agent can be immobilized on a solid substrate.

In embodiments of this aspect of the disclosure, the RT-PCR system can comprise at least one pair of primer oligonucleotides selected for specifically amplifying an RNA species of an RNA:protein complex specifically binding to an RNA-protein-specific antibody characteristic of Scleroderma (Systemic Sclerosis, SSc), and a labeled oligonucleotide selected to specifically detect the amplification product. In embodiments of this aspect of the disclosure, the pair of primer oligonucleotides can be for specifically amplifying an RNA species of an RNA:protein complex of U3RNP or Th/To.

In embodiments of this aspect of the disclosure, the RT-PCR system can be for specifically amplifying an RNA species of an RNA: protein complex of U3RNP, and comprises the primer oligonucleotides having the nucleotide sequences according to SEQ ID Nos.: 1 and 2, and the labeled oligonucleotide can comprise at a first terminus thereof a fluorophore and a quencher disposed at a second terminus thereof.

In embodiments of this aspect of the disclosure, the kit can further comprise at least one of the group consisting of: a target RNA.protein complex or a composition comprising said target RNA: rotein complex, a composition comprising an immunoglobulin-binding agent, and a system for RT-PCR using the oligonucleotide primers specifically hybridizing to a complement of an RNA of a target RNA: protein complex recognized by an autoantibody.

In embodiments of this aspect of the disclosure, the composition comprising an immunoglobulin-binding agent can be selected from the group consisting of an antiimmunoglobulin antibody , Protein A, Protein G, Protein A/G, and Protein L.

The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any "preferred" embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and the present disclosure and protected by the following claims.

The following exa mples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, efc), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity , and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of "about 0.1 % to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1 .1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1 %, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, +9%, or ±10%, or more of the numerical value(s) being modified.

EXAMPLE

Example 1

IP-qPCR method: 25 μΙ of Protein A-Sepharose CL4B (PAS, Pharmacia) beads were incubated with 8 μΙ of serum and 500 μΙ of 0.5 M NaCI NET/NP40. Samples were incubated 1 hr, in rotation, at 4 °C.

Cell extracts from K562 cells were prepared with the concentration of 0.1 -1 x 10⁶ cells used for each sample. For immunoprecipitation of 24 samples, one tube containing 25 x 10⁶ K562 cells was used: 2.5 ml of 0.15 M NaCI NET/NP40, 25 μΙ of PMSF (1 :100), and 25 μΙ of Aprotinin (1 : 100) were added to the cells. Cells were then sonicated for 45 sees, placed on ice for 1 min and sonicated 45 sees again. The cell lysate was then centrifuged for 30 mins at 13,200 rpm in microcentrifuge in cold room.

During this step, PAS beads were washed twice with 1 ml of 0.5 M NaCI NET/NP40. The supernatant was carefully collected avoiding the pellet and 100 μΙ/sample of cell extract was added to the beads. Samples and cell extract rotate at 4 °C for 1 hr. Beads were then washed four times with 0.5 M NaCI NET/NP40 and once with 0.15 M NaCI NET/NP40. After the last wash, the supernatant was aspirated completely and a mix containing 400 μΙ of: 0.15 M NaCI NET/NP40.16 μΙ 25% SDS, 40 μΙ 3M Na acetate, pH 5.2, was prepared. For each sample, 456 μΙ of this mix and 400 μΙ of phenol/chloroform/isoamyl alcohol are added.

Each sample was vortexed for 1 min and centrifuged at 13, 200 rpm for 1 min, at room temperature. 300 μΙ of supernatant (aqueous phase) was harvested and transferred to a new set of tubes. 900 μΙ of 100% ethanol was added, mixed by inversion and stored overnight at -80 °C. Samples are centrifuged at 13,200 RPM for 15 mins at 4 °C. Liquid was then aspirated and samples (now containing only RNA pellet) were left to dry.

The RNA pellet was resuspended in 30 μΙ of DEPC treated nuclease-free water. For reverse transcription (RT) , 10 μΙ of each sample are added to 10 μΙ of RT Master Mix (High Capacity cDNA Reverse Transcription kit, Applied Biosystems, Inc. (ABI)). For each sample, the 10 μΙ of RT Master Mix contained: 2 μΙ of 10X RT buffer, 0.8 μΙ of 25x dNTP Mix, 2 μΙ of 10x RT random primers, 1 μΙ of Multiscribe Reverse Transcriptase, 1 μΙ of RNase inhibitor , and 3.2 μΙ of nuclease-free water. The thermal cycler for RT was set with the following temperatures and timings: 10 mins at 25 °C, 120 mins at 37 °C, 5 sees at 85 °C and 4 ^CC until the use of the cDNA samples for quantitative PCR (qPCR) .

After RT, the TaqMan Fast Universal PCR Master Mix (by ABI) was prepared, and 8 μΙ of this mix are added to 2 μΙ of cDNA for each sample. For each sample, the 8 μΙ of qPCR Fast Master Mix contained: 5 μΙ of TaqMan Fast + 0.5 μΙ of primer + 2.5 μΙ of nuclease-free water.

The oligonucleotide used for Th/To 7-2 RNA (RM RP) detection was the

"Hs03298751_s1 " primer by ABI (Carlsbad, CA) .

The oligonucleotides used for U3RNA (SNORD3A) detection had the following sequences:

Probe (terminal quencher lABkFQ.RTM and internal quencher ZEN .RTM):

5'-/56-FAM/CCAAGCAAC/ZEN/GCCAGAAAGCCG/3IABkFQ/-3'

Primer 1 : (forward) 5'-TGTAGAGCACCGAAAACCAC-3' (SEQ ID No. : 1 )

Primer 2: (reverse) 5'-TCCCTCTCACTCCCCAATAC-3' (SEQ I D No.: 2)

Samples were tested in duplicate in a 96-well plate. PCR was performed using a StepOne cycler (ABI) for 40 cycles, and results were evaluated as Ct values.

Example 2

After optimizing the conditions of the assay through the titration experiments previously described, we performed the screening of 22 anti-Th/To (+) samples. The aim was to underline the significant Ct difference with other control samples with known SSc autoantibodies. The statistical significance between the Th group and all the other groups is maintained despite using 1 x10⁶ K562 cells/sample, which is ten times lower amount than in standard IP. Primer RMRP for the Th 7-2 RNA component was used, as shown in Fig. 4. Example 3

After optimizing the conditions of the assay through the titration experiments previously described, we performed the screening of 12 anti-U3 (+) samples. The aim was to underline the significant Ct difference with other control samples with known SSc autoantibodies. The statistical significance between the U3 group and all the other groups is maintained despite using 1x10⁶ K562 cells/sample, which is ten times lower amount than in standard IP. Primer SNORD3A for the U3 RNA component was used, as shown in Fig. 5.

Claims

CLAIMS We claim:

1 . A method of detecting an autoantibody specific for an RNA: protein complex, comprising the steps of:

(a) obtaining an immunoglobulin-RNA:protein aggregate, wherein said aggregate comprises a target RNA:protein complex and an RNA:protein complex-specific antibody;

(b) isolating the RNA from the immunoglobulin-RNA-protein aggregate; and

(c) detecting the isolated RNA by quantitative reverse transcription PCR, whereby detection of the RNA indicates the presence of the RNA:protein complex-specific antibody in a subject human or animal.

2. The method according to claim 1 , wherein step (a) comprises the steps of:

(i) obtaining a target RNA:protein complex;

(ii) contacting the target RNA:protein complex with an isolated immunoglobulin fraction from a subject human or animal under conditions that allow the formation of an immunoglobulin-RNA:protein aggregate comprising the target RNA:protein complex and an antibody of the isolated immunoglobulin fraction, said antibody being specific for the RNA:protein complex;

(iii) isolating the immunoglobulin-RNA:protein aggregate;

(iv) isolating the RNA component from the isolated immunoglobulin-RNA:protein aggregate ;

(v) contacting the isolated RNA component with a system configured for the amplification of an RNA species by quantitative reverse transcription PCR; and

(vi) determining the amount of the isolated RNA by quantitative reverse transcription PCR, whereby the presence of the RNA indicates that the target RNA complex was specifically bound by an RNA-protein -specific antibody of the composition, and thereby indicating the presence of the RNA:protein complex-specific antibody in the subject human or animal.

3. The method according to claim 1 , wherein the immunoglobulin fraction is obtained from the serum of a subject human or animal.

4. The method according to claim 3, wherein the step of isolating the immunoglobulin fraction from the serum sample comprises contacting the serum sample with an

immunoglobulin-binding agent under conditions allowing the formation of an

immunoglobulin-immunoglobulin-binding agent complex; and isolating the immunoglobulin- immunoglobulin-binding agent complex from the serum sample.

5. The method according to claim 4, wherein the immunoglobulin-binding agent is selected from the group consisting of an anti-immunoglobulin antibody, Protein A, Protein G, Protein A/G, and Protein L.

6. The method according to claim 4, wherein the immunoglobulin-binding agent is immobilized on a solid substrate.

7. The method according to claim 1 , wherein the target RNA:protein complex is a component of a human or animal cell, or a lysate thereof.

8. The method according to claim 2, wherein the target RNA:protein complex is a component of a cell lysate, a serum, or an isolated target RNA:protein complex.

9. The method according to claim 2, wherein the RT-PCR system comprises at least one pair of primer oligonucleotides selected for specifically amplifying an RNA species of an RIMA:protein complex specifically binding to an RNA-protein-specific antibody characteristic of Scleroderma (Systemic Sclerosis, SSc), and a labeled oligonucleotide selected to specifically detect the amplification product.

10. The method according to claim 9, wherein the pair of primer oligonucleotides is for specifically amplifying an RNA species of an RNA:protein complex of U3RNP or Th/To.

11 . The method according to claim 10, wherein the RT-PCR system is for specifically amplifying an RNA species of an RNA:protein complex of U3RNP, and comprises the primer oligonucleotides having the nucleotide sequences according to SEQ ID Nos.: 1 and 2, and the labeled oligonucleotide comprises at a first terminus thereof a fluorophore and a quencher disposed at a second terminus thereof.

12. The method of claim 1 1 , wherein the labeled oligonucleotide further comprises an internal quencher.

13. A kit comprising at least one pair of oligonucleotide primers selected for quantitatively amplifying by reverse transcription PCR an RNA of a target RNA: protein complex specifically recognized by an autoantibody, a labeled oligonucleotide for the detection of the

amplification product, and packaging comprising instructions for the detection of an autoantibody in a serum sample from a human or animal subject using said oligonucleotide primers and RT-PCR.

14. The kit according to claim 13, further comprising at least one of the group consisting of: a target RNA: protein complex or a composition comprising said target RNA:protein complex, a composition comprising an immunoglobulin-binding agent, and a system for RT-PCR using the oligonucleotide primers specifically hybridizing to a complement of an RNA of a target RNA: protein complex recognized by an autoantibody.

15. The kit according to claim 14, wherein the composition comprising an immunoglobulin- binding agent is selected from the group consisting of an anti-immunoglobulin antibody,

Protein A, Protein G, Protein A/G, and Protein L.

16. The kit according to claim 15, wherein the immunoglobulin binding agent is attached to a solid substrate.