WO2014029816A1

WO2014029816A1 - Anti-c1q epitope elisa

Info

Publication number: WO2014029816A1
Application number: PCT/EP2013/067405
Authority: WO
Inventors: Marten TRENDELENBURG; Dominique VANHECKE
Original assignee: Universität Basel Vizerektorat Forschung
Priority date: 2012-08-22
Filing date: 2013-08-21
Publication date: 2014-02-27

Abstract

The invention relates to a method for diagnosis of anti-C1q autoantibody-related diseases such as systemic lupus erythematosus-related nephritis and hypocomplementaemic urticarial vasculitis syndrome detecting, in a sample obtained from said patient, anti-C1q auto-antibodies. It further relates to devices and kits for detection of the indicator protein.

Description

Anti-C1 q epitope ELISA

Description

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is characterized by the occurrence of autoantibodies to a number of self-antigens, resulting in a broad spectrum of clinical and immunological manifestations. One of these self-antigens is C1 q, the first molecule of the classical complement pathway. About 20-50% of unselected SLE patients have antibodies directed against C1 q (anti-C1 q).

Anti-C1 q autoantibodies are also a diagnostic criterion for hypocomplementaemic urticarial vasculitis syndrome (HUVS) patients (see Marto N et al., Ann Rheum Dis. 2005 Mar;64(3):444-8 and references cited therein)

The C1 q molecule is a multimeric glycoprotein, composed of 18 polypeptide chains of three different types called A, B, and C. Each chain has a short N-terminal region, containing the inter-chain disulfide bonds, followed by a collagen-like region (CLR) and a C-terminal globu- lar head domain (GHR or gC1 q) (see Sellar GC et al., Biochem J. 1991 Mar 1 ;274 (Pt 2):481 -90 and references cited therein).

The sequences of the subunits A (C1 QA_HUMAN) and B (C1 QB_HUMAN) are defined by UniProt No. P02745 and UniProt No. P02746.

The importance of C1 q in the pathogenesis of SLE is underscored by the observation that loss of function mutations in C1 q, although very rare, are sufficient to cause SLE in more than 90% of affected individuals (see Botto M et al., Immunobiology. 2002 Sep;205(4-5):395- 406; Pickering MC et al., Adv Immunol. 2000;76:227-324; Schejbel L et al., Genes Immun. 201 1 Dec; 12(8):626-34 and references cited therein). This makes deficiency in C1 q the strongest known susceptibility factor for SLE. In addition, low levels of C1 q and other com- plement factors of the classical pathway are associated with active disease and the appearance of renal involvement in non-C1 q-deficient SLE patients. Reduced serum C1 q levels in SLE patients due to increased consumption or neutralization are possibly linked to the presence of anti-C1 q autoantibodies (see Fremeaux-Bacchi V et al., Lupus. 1996 Jun;5(3):216- 20.; Trouw LA et al., Clin Exp Immunol. 2003 Apr; 132(1 ):32-9 and references cited therein). Active nephritis in lupus patients has been found to be strongly associated with high levels of anti-C1 q autoantibodies (Akhter E et al., Lupus. 201 1 Oct;20(12): 1267-74; Wener MH et al., Arthritis Rheum. 1989 May;32(5):544-51 ; Siegert C et al., J Rheumatol. 1991 Feb; 18(2):230- 4; Coremans IE et al., Am J Kidney Dis. 1995 Oct;26(4):595-601 ; Trendelenburg M et al., Arthritis Rheum. 1999 Jan;42(1 ):187-8; Moroni G et al., Am J Kidney Dis. 2001 Mar;37(3):490-8; Fremeaux-Bacchi V et al., Nephrol Dial Transplant. 2002 Dec; 17(12):2041- 3; Seelen MA et al., Curr Opin Nephrol Hypertens, 2003 Nov; 12(6):619-24; Trendelenburg M et al., Nephrol Dial Transplant. 2006 Nov;21 (1 1 ):31 15-21 ; Sinico RA et al., Ann N Y Acad Sci. 2009 Sep; 1 173:47-51 and references cited therein).

However, not all patients with high levels of anti-C1 q develop lupus nephritis, suggesting that anti-C1 q antibodies are necessary but not sufficient for the development of proliferative lupus nephritis. Some studies only found a variable degree of association between anti-C1 q levels and the occurrence of lupus nephritis (see Katsumata Y et al., Arthritis Rheum. 201 1 Aug;63(8):2436-44 and references cited therein). This lack of association might be due in part to the assays used being of too low sensitivity, or to false positive test results caused by the binding of immune-complexes to the globular heads of the immobilized C1 q (see Kohro- Kawata J et al., J Rheumatol. 2002 Jan;29(1 ):84-9 and references cited therein).

Conventional anti-C1 q ELISA assays are performed in high salt buffers to avoid binding of the globular heads to immune complexes. The use of high salt buffers could interfere with the detection of particular anti-C1 q autoantibodies in patient sera.

Hence, there is a considerable medical need for an assay detecting C1 q-autoantibodies with improved sensitivity and specificity resulting in fewer false positive results. Such an assay may be helpful in i) improving risk prediction and prognostics of C1 q-autoantibody-related diseases, such as SLE, HUVS and secondary diseases, such as nephritis in SLE patients, ii) providing earlier and more reliable diagnosis, and iii) enabling monitoring of progression and regression of these diseases.

The objective of the present invention is to provide an assay for the detection of anti-C1 q autoantibodies that improves on the state of the art. This objective is attained by the subject matter of the independent claims.

The present invention was made in the context of a study to identify specific C1 q- autoantibody-binding peptides by an array technique.

A bone marrow-derived IgG /lgGA antigen-binding immunoglobulin fragment (Fab) phage display library from a SLE patient with high anti-C1 q Ab titer against purified human C1 q was screened. Several monoclonal anti-C1 q Fabs were isolated (see Schaller M et al., J Immu- nol. 2009 Dec 15; 183(12):8225-31 and references cited therein). By analysing the binding characteristics of these anti-C1 q Fabs derived from the SLE patient, two oligopeptide molecules were identified that are targets of SLE anti-C1 q antibodies. Affinity and specificity of binding were confirmed using surface plasmon resonance and peptide based ELISAs. Sera from a cohort of SLE patients and a cohort of asymptomatic donors were compared for bind- ing to the identified peptide molecule or to the intact C1 q molecule that is used in conventional ELISA assays.

It was surprisingly found that the peptide ELISA has a higher sensitivity and specificity for the detection of patient anti-C1 q antibodies as compared to the classic anti-C1 q ELISA that is routinely used in the clinic for the diagnosis of active nephritis in suspected SLE patients.

Anti-C1 q Fabs generated from an SLE patient and selected for binding to C1 q (Fabs A4 and A14) were shown to bind to the CLR of the C1 qA and/or C1 qB chains of C1 q (see Bigler C et al., J Immunol. 2009 Sep 1 ; 183(5):3512-21 ; Schaller M et al., J Immunol. 2009 Dec 15; 183(12):8225-31 and references cited therein).

An array of overlapping 13mer peptide molecules representing the sequence of C1 qA and C1 qB chains was synthesized and probed with the anti-C1 q Fabs (Fig. 1 ). Two regions, one on C1 qA and one on C1 qB, showed a significant higher binding (approximately 1000-fold for the highest signal) with anti-C1 q Fabs as compared to the other peptide sequences on the array. The 13-mer peptide sequences that resulted in the highest SLE-derived Fab binding were assigned the code A08, representing an A-chain region and B78, representing a region on the B-chain.

The peptide molecules correspond to the following amino acid sequences:

A08: GRPGRRGRPGLKG (SEQ ID NO: 03) and

B78: PGKVGPKGPMGPK (SEQ ID NO: 04).

The binding of the anti-C1 q Fabs to the peptide molecules A08 and B78 has been confirmed using surface plasmon resonance analysis. As demonstrated in Fig. 2, Fabs A4 and A14, but not irrelevant polyclonal control Fabs, bind to immobilized A08 and B78 peptides. In contrast, the Fabs did not bind to a control peptide under the same conditions.

The prevalence of autoantibodies to the A08 peptide molecule was further evaluated in a cohort of SLE patients when compared to healthy donors. Serum antibodies from 61 patients with confirmed SLE and from 72 healthy donors were analysed for their binding to immobilized C1 q (conventional ELISA) or C1 q-derived A08 peptides (peptide based ELISA). In contrast to healthy individuals (NHS), serum antibodies from most SLE patients specifically bound to the A08 epitope (Fig. 5). When comparing both assays the peptide-based ELISA allowed a better discrimination between SLE patients and controls than the C1 q assay, resulting in a higher specificity compared to the conventional anti-C1 q ELISA assay.

In total 79% of the SLE patient sera scored positive when tested with the A08-ELISA compared to 62% when using the anti-C1 q ELISA. In addition, the peptide ELISA was better in discriminating non-symptomatic donors from patient sera (79% SLE versus 14% NHS) than the C1 q based ELISA (62% SLE versus 20% NHS).

Within the subgroup of patients with active nephritis at the time of sampling, more patients were found to be positive for anti-A08 than for anti-C1 q (75% versus 67%). The majority of the 13 patients that scored anti-A08 positive but anti-C1 q negative suffered from active nephritis (46%) or had had nephritis confirmed by biopsy in the past (30%).

All things considered, a major linear target of SLE associated anti-C1 q antibodies, a marker of severe lupus nephritis, has been identified.

The peptide-based assay presented here provides more specific and more sensitive results than a conventional anti-C1 q assay, and allows a better discrimination between low-level positive and true negative sera. The A08-peptide based ELISA assay represents an improved alternative assay for the detection of anti-C1 q autoantibodies in SLE.

The subject matter of the present invention allows distinguishing between SLE patients with active nephritis or past nephritis, and healthy individuals with higher specificity than the con- ventional ELISA assay. Hence, the method of the invention provides a method for the detection of SLE patients. Furthermore, the method of the invention is suitable for the detection of other C1 q-related diseases, such as HUVS.

An oligopeptide molecule in the context of this description is a linear chain of proteinogenic amino acids linked by amide bonds. Non-peptide linkages may be used to stabilize an oligo- peptide molecule, conserving the spatial arrangement of side chains making up the recognized epitope. Oligopeptide molecules may be covalently modified by acetylation, formylation or benzylation of the terminal amino group, by esterification of the terminal carboxy group or by other common modifications employed in peptide chemistry. The oligopeptide side chains, where basic or acidic, may be present in their respective salt form.

Oligopeptide sequences are given in standard one letter amino acid code (see Hausmann et al., The Cell: a molecular approach, ISBN 0-87893-214-3) and refer, where not indicated otherwise, to L-amino acids linked by peptide bonds.

Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides may be modified for the purposes of ease of conjugation to a carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cystein. In addition it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic terminus distal from the conjugated terminus of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier protein, thereby presenting the peptide in a conformation which most closely resembles that of the peptide as found in the context of the whole native molecule. According to a first aspect of the invention, an oligopeptide molecule is provided for use in a method of detection of anti-C1 q autoantibodies, the oligopeptide having a sequence length of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, and an amino acid sequence comprised as a contiguous sequence in any of the following sequences:

SEQ ID NO 01 : EAGRPGRRGRPGLKGEQ

SEQ ID NO 02: GNPGKVGPKGPMGPKGG

SEQ ID NO 03: GRPGRRGRPGLKG

SEQ ID NO 04: PGKVGPKGPMGPK

The underlined amino acids refer to the 13mer peptide molecules showing the highest affinity to the anti-C1 q Fabs as detected in the microarray (A08 [SEQ ID NO 03] and B78 [SEQ ID NO 04]).

Based on amino acid substitutions it was found that five amino acid residues (GRRGR) within the target sequence of A08 play an important role in binding of SLE patient sera. Based on this core sequence the present inventors tested variant peptides that include this core se- quence. One of these peptides A08-3-4-8 (GSGGRRGRGRERARGGS) [SEQ ID NO 05] showed a similar specificity as the original A08 with specific binding of SLE sera but not sera from non-symptomatic donors. However the signal intensity obtained with the new peptide is higher than that obtained with A08 in a peptide ELISA.

Said oligopeptide molecule is contacted with a patient sample obtained from said patient, and it is determined whether said oligopeptide molecule is specifically bound by an antibody comprised in said sample.

In some embodiments, the oligopeptide has a sequence length of 13 amino acids. In some embodiments, the oligopeptide's amino acid sequence is comprised as a contiguous sequence in SEQ ID NOs 03 or 04.

The method of the invention encompasses diagnosing anti-C1 q-related diseases, such as SLE-related nephritis or HUVS in a patient. It also relates to assessing a patient's risk of developing or having developed an anti-C1 q-related disease.

In one embodiment the method is performed ex vivo on a sample obtained from the patient.

A variety of methods to obtain samples offer themselves to practise the invention. In some embodiments, a blood sample, particularly a peripheral blood sample, is used for said method. In the context of the present invention, the term "amount" also refers to concentration. It is evident that the concentration of the compound can be calculated from the total amount of a compound of interest in a sample of known size, and vice versa.

In the context of the present invention the term "sample" refers to any component of the sample independently of the degree of separation of the components.

The method of the present invention comprises the steps of providing an oligopeptide molecule having a sequence length of 5 to 20 amino acids, and having an amino acid sequence comprised as a contiguous sequence in SEQ ID NO 01 or SEQ ID NO 02, contacting said oligopeptide molecule with a patient sample suspected of comprising anti-C1 q antibody mol- ecules ex vivo under conditions allowing for the specific binding of an antibody to an epitope, and determining whether said oligopeptide molecule is specifically bound by an anti-C1 q antibody molecule comprised in said patient sample.

Conditions allowing for the specific binding of an antibody to an epitope generally include aqueous buffer systems or aqueous solutions at physiologic pH and ionic strength. Such buffers are, by way of non-limiting example, carbonate buffer, phosphate buffered saline, sodium phosphate buffer systems, Tris/HCI buffer, glycine buffer or acetate buffer. The pH of the buffer should range between 5 and 10. Salt concentrations are defined between 0 and 250 mmol/l using sodium chloride or an equivalent salt. Buffers may be supplemented with high salt concentrations up to 1 M to avoid unwanted interactions.

Any known method may be used for the detection of peptide binding antibodies in a sample such as body fluids. Methods considered are, by way of non-limiting example, chromatography, mass spectrometry (and combinations thereof), enzymatic assays, electrophoresis and antibody-based assays (by way of non-limiting example, ELISA, RIA, EIA, CLIA (see Zhou et al, J Zhejiang Univ Sci B. 2005 Dec;6(12):1 148-52 and references cited therein), CEDIA (see Hamwi et al., Am J Clin Pathol. 2000 Oct;1 14(4):536-43 and references cited therein), CMIA(see Berry et al., Clin Chem. 1988 Oct;34(10):2087-90 and references cited therein), MEIA (see Rodrigues et al., Mem Inst Oswaldo Cruz. 2009 May; 104(3):434-40 and references cited therein), FPIA (see Stricher et al., Biochem J. 2005 Aug 15;390(Pt 1 ):29-39 and references cited therein), GLORIA (see Armenta et al., Trends in Analytical Chemistry, Vol. 27, No. 4, 2008 and references cited therein), microarray analysis, fully-automated or robotic immunoassays and latex agglutination assays).

According to one embodiment of the invention, the method comprises the steps of contacting the sample with an oligopeptide or a plurality of oligopeptides having the lengths and sequences indicated above, resulting in antibody molecules specific for said oligopeptides bind- ing to the oligopeptides, and determining, in a quantification step, the amount of antibodies bound to the peptide molecule. According to one embodiment of the invention, the oligopeptide, or a plurality of oligopeptides having the lengths and sequences indicated above, is attached to a surface such as, by way of non-limiting example, the surface of a microtiter well, a gold or glass surface, a lab- on-a-chip device (LOC, microfluidics analysis), a microarray or the matrix material of a lateral flow immunoassay.

An oligopeptide is attached, in the context of the present invention, if the oligopeptide is bound to the surface by covalent or non-covalent bonds so that the attachment withstands repeated washing steps with aqueous buffer of pH 5 - 8. One example is a covalent bond, for example by ester or amide linkage or disulfide bridge; another is the streptavidin-biotin bond, avidin-biotin bond or neutravidin-biotin bond or simple adsorption of proteins to polymer surfaces. Oligopeptides may be attached to gold surfaces by sulfide groups.

In some embodiments the surface is the surface of a plasmon resonance or quartz microbal- ance chip, which will allow direct measurement of the amount of indicator protein attached to the ligand by means of the changes effected in the physical properties of the chip. The amount of the bound auto-immune antibody is thus determined directly.

Other ways to practise the invention are the attachment of the oligopeptide molecules to a glass or plastic slide or bead, a silicon wafer, a magnetic bead, a polystyrene or sepharose bead, a cellulose or polypropylene membrane.

In some embodiments, the quantification step comprises contacting, in a second contacting step, said antibody with a ligand specifically reactive to said antibody and determining an amount of said ligand bound to said antibody.

In some embodiments of this aspect of the invention the method thus comprises the following steps:

- a surface is provided, where the oligopeptide, or a plurality of oligopeptides having the lengths and sequences indicated above is attached;

- blood or other fluid sample obtained from a patient is contacted, optionally after a work-up for removal of irrelevant components, with the surface under conditions allowing for the specific binding of an antibody to an epitope;

- a ligand specifically reactive to IgG is contacted with the bound anti-C1 q antibody molecule, and the amount of the bound antibody molecule is determined;

- optionally, a washing step is inserted between the first and the second step to remove any sample components that did not interact with the surface. Another washing step may follow the second contacting step and precede the determination.

In one embodiment of the invention the ligand specifically reactive to the peptide-bound anti- body is a polyclonal or monoclonal antibody specific for the whole anti-C1 q antibody or frag- merits of this antibody such as the Fc or Fab regions. In one embodiment of the invention the antibody specifically reactive to the peptide-bound antibody is attached or specifically attachable to an enzyme activity or a detectable label. Typically the ligand specifically reactive to the anti-C1 q antibody is an antibody specifically reactive to the Fc part of human IgG or IgM and is conjugated directly or indirectly via biotin-Streptavidin/Avidin/Neutravidin to horse radish peroxidase or Alkaline phosphatase or a fluorescent label.

In some embodiments, a ligand specific for the constant region of IgG molecules is attached to a surface, and a patient sample is brought into contact with said surface, resulting in any IgG molecules present in the sample becoming attached to the surface. Subsequently, the oligopeptide molecule, or a plurality of oligopeptide molecules having the lengths and sequences indicated above, is brought into contact with the surface, the oligopeptide being modified by a means of detection such as an optically active chemical group or other detectable label.

A ligand according to any aspect or embodiment of the invention may be any molecule that binds to serum antibody molecules with high affinity and specificity.

High affinity in the context of the present specification refers to the dissociation constant of the binding of the ligand to the C1 q-antibody, wherein the dissociation constant is 10^"7, 10^"8 or 10^"9 mol/l or less and wherein the ligand does not bind to control proteins with unrelated structural features.

High specificity in the context of the present specification refers to the ratio of properly detected indicator protein and the sum of all detected oligopeptides, wherein the ratio is 80%, 85%, 90%, 95%, 99% or equal or greater than 99.9%.

In some embodiments the ligand reactive to the peptide bound antibody is a Fab fragment of an antibody. A Fab fragment is the antigen-binding fragment of an antibody, specifically reac- tive to the anti-C1 q antibody.

In some embodiments the ligand is a single-domain antibody (sdAb or Nanobody), for instance a single-chain variable fragment (scFv) antibody selected by phage display methods, or a camelide antibody, specifically reactive to the indicator protein.

Alternatively, a ligand employed in practising the invention may be a molecule engineered or selected to demonstrate a high specificity and affinity for the peptide-bound antibody. Such engineered or selected molecule may be an antibody-like oligopeptide or synthetic antibody selected by phage-display (see Pini et al., J.Biol. Chem. (1998) 273, 21769-21776) and references cited therein, or by other evolutionary selection methods). Similarly, molecules such as protein-G, Protein-A or Protein-L (optionally conjugated to enzymes, dyes or other labels) can be used to detect the bound antibodies. In some embodiments, the ligand has a fluorescent or luminescent quality and determining is performed by fluorescence-activated cytometry. A fluorescent or luminescent quality in the context of the present specification refers to the ability of the ligand to emit light after excitation by light of a shorter wavelength.

In some embodiments, the method according to the invention will involve the comparison of the determined amount of peptide-bound antibody molecules with a standard. An absolute measurement may achieve the desired accuracy and validity, for example if a reproducible determination of binding can be attained, as is the case for surface plasmon resonance (SPR) assays with a properly calibrated SPR device, or spectroscopic read-outs in HPLC, where the integral of the chromatogram is proportional to the amount of the analyte.

In some embodiments the detection of the peptide-bound antibody is performed in a homogenous immunoassay, such as EMIT (see Beresini et al., Clin Chem. 1993 Nov;39(1 1 Pt 1 ):2235-41 and references sited therein).

In some embodiments the quantification step further comprises the use of a second ligand with specificity for the peptide bound antibody. The second ligand binds to a different surface section of the peptide-specific antibody than the part involved in binding to the peptide so as not to interfere with antibody-peptide binding.

The second ligand may be attached to an enzymatic activity or label, such as a peroxidase, to allow for fitting the method into the well-established ELISA formats. Other labels may be, but are not limited to, a phosphatase or luciferase activity or the detectable label is a radioactive label or a gold particle.

Likewise, the second ligand may have an optically detectable quality such as a colour label, a fluorescent, a phosphorescent or luminescent quality.

In general, suitable labels are chromogenic labels, i.e. enzymes which can be used to con- vert a substrate to a detectable colored or fluorescent compound, spectroscopic labels, e.g. fluorescent labels or labels presenting a visible color, affinity labels which may be developed by a further compound specific for the label and allowing easy detection and quantification, or any other label suitable to standard ELISA formats. Examples for fluorescent labels or labels presenting a visible color include, without being restricted to, fluorescein, rhodamine, fluorescein isothiocyanate (FITC), Rhodamine (TRITC, TAMRA), Allophycocyanin (APC), Phycoeythrin (PE), Alexa Fluors, Dylight fluors, ATTO Dyes and BODIPY Dyes.

An optically detectable label or enzyme activity may be detected on a surface.

In some embodiments, the peptide molecule and the second ligand are attached to the respective components of a system that allows to determine a quantitative measure of proximi- ty-paired partners, such as the "Amplified Luminescent Proximity Homogeneous Assay (AL- PHA)" commercialized by Perkin Elmer, whereby singlet oxygen transferred from a donor partner to an acceptor results in emission of light if the two partners are within 200 nm distance.

In some embodiments a FRET-based detection system may be used to determine peptide- specific antibodies, where the energy of an excited dye (donor) is transferred to a second dye (acceptor).

Such arrangements allow for the practice of the invention in solution.

In some embodiments, a liquid volume of sample is placed onto a sample area composed in such way as to provide capillary forces that act to transport at least a part of the sample to- wards an analysis area. Between the sample area and the analysis area, or as part of the sample area, a conjugate area is provided. According to the invention oligopeptide molecules specific for the anti-C1 q-antibodies are provided, without attachment to the matrix, in the conjugate area, and upon binding to peptide specific antibodies in the sample, these oligopeptide molecules are drawn with the capillary flow into the analysis area. The oligopeptide mol- ecules according to this embodiment of the invention are conjugated to latex or gold micro- particles or enzymatically active proteins. Further ligands specific for the oligopeptide are attached to the analysis area, forming a line. Upon binding of anti-C1 q antibodies (with peptide molecules attached) to the ligands, the resulting "sandwiches" of ligand, peptide molecule and anti-C1 q-antibody form a line, which may be visible as such (if particles are at- tached to the first ligands), or may be rendered visible by providing a substrate for an enzymatic activity attached to the first ligand. This embodiment is referred to as a lateral flow assay.

In some embodiments, detection is based on competition between the serum antibodies and a labelled anti-oligopeptide antibody. Binding of the serum antibodies prevents binding of the labelled antibody to an immobilised oligopeptide.

In one embodiment, the method of detection employs antibody-based proximity ligation (Gullberg et al., PNAS 2004 vol. 101 , 8420-8424).

According to some embodiments of any of the method aspects of the present invention, the level of anti-peptide antibodies in the sample is compared to the corresponding levels in (a) sample(s) of (a) healthy subject(s). An altered level compared to the level in the sample of the healthy subject is indicative of a disease or of the risk to develop the disease.

According to one embodiment of the present invention, the measured level of anti-peptide antibodies indicates whether an individual is suffering from anti-C1 q antibody related diseases or has an increased risk of developing such disease. The terms used in this context, i.e. "non-increased level", "not elevated level", "increased levels" and "decreased levels" are known to, or can be determined by the person skilled in the art according to, for example, the procedure outlined in the following paragraphs.

In another embodiment of the invention the level of the indicator protein is compared to a defined threshold. A lowest threshold is currently not known. Other thresholds are the detec- tion limit or the quantitation limit (LOD or LOQ). In the examples shown herein, the cut-off for a positive signal was used to evaluate the binding of patient sera as determined as the interquartile mean (IQM) of the values obtained with the control sera (NHS) plus three times the standard deviation. A trimmed mean like the IQM was used since it gives a much more robust estimation (an estimation not greatly affected by outliers) of the average than the arith- metic mean.

The person skilled in the art is able to determine actual values corresponding to "non- increased level", "not elevated level", "increased level" and "decreased level" for the relevant biochemical markers. For example, the levels may be assigned according to percentiles of the levels observed in a representative sample of apparently healthy individuals, typically below an age of 50 years. The sample should be of sufficient size to yield statistically significant outcomes (for example, at least 100, more preferably at least 500, most preferably at least 1000 individuals). A non-increased level may correspond, by way of example, to the maximum level observed in the 97.5% percentile of healthy individuals. Alternatively, the levels may be determined as "normal ranges" as known to the skilled statistician.

The levels may also be determined or further refined by studies performed on individuals by comparing levels of the indicator proteins of apparently healthy individuals with individuals suffering from active lupus nephritis or HUVS. Such studies may also allow to tailor the levels according to the type of disease or/and certain patient sub-groups, e.g. elderly patients, patients undergoing medication or patients with a certain lifestyle.

The value of the levels considered as "increased" or "decreased" may also be chosen according to the desired sensitivity or specificity (stringency) of exclusion. The higher the desired sensitivity, the lower is the specificity of exclusion and vice versa. In the above example, the higher the percentile chosen to determine each level, the more stringent is the exclusion criterion, i.e. fewer individuals would be considered "individuals at risk".

The method according to the present invention also allows the determination of the risk or the likelihood, respectively, of an individual of suffering from anti-C1 q antibody-related diseases, such as SLE or HUVS. In the context of the present invention, the terms "risk" or "likelihood" relate to the probability of a particular incident to develop anti-C1 q antibody-related diseases. The grade of risk can be decreased, non-increased, increased, or highly in- creased. "Non-increased risk" or "small likelihood" means that there is apparently no or very little risk of suffering from anti-C1 q antibody-related diseases compared to the average risk of a healthy individual.

The degree of risk is associated with the level of anti-C1 q antibodies. A non-altered level of anti-C1 q antibodies indicates no increased risk, an altered level of anti-C1 q antibodies indi- cates an increased risk.

If the level of anti-C1 q antibodies is altered, the patient might be recommended to undergo a primary treatment or secondary interventions like the initiation of therapeutic life-style changes, drug therapy depending on risk factor constellation and reviews at regular intervals. If the methods according to the present invention indicate an increased risk, that information will preferably have consequences for the further treatment of the individual.

Hence, the term "diagnosis" in the context of the present invention includes embodiments comprising (by non-limiting example) diagnosis of acute disease, determination of risk to develop or to be diagnosed with acute disease, and monitoring of the state of disease during or after treatment.

According to a second aspect of the invention, a device for diagnosis of anti-C1 q autoantibody related diseases such as SLE-related nephritis or HUVS is provided. This device comprises a surface comprising an oligopeptide molecule specifically reactive to anti-C1 q antibodies. Such surface may be an ELISA plate, a LOC device, a surface plasmon resonance chip, a surface of a quartz microbalance sensor or the matrix material of a lateral flow immu- noassay.

According to yet another aspect of the invention, a kit of parts is provided for diagnosis of anti-C1 q autoantibody related diseases, or for assessing a patient's risk of developing or having developed such a disease, said kit comprising oligopeptide molecule specifically reactive, both ligands being specifically reactive to anti-C1 q antibodies.

According to one embodiment of this aspect of the invention, the oligopeptide molecule is attached to the surface of a microtiter plate, and the ligand comprises an enzymatic activity or a luminescent activity.

In some embodiments, the ligands are monoclonal antibodies, single chain antibodies or single domain antibodies.

Brief description of the figures

Fig. 1 : The results of a micro-array based peptide scan to screen an overlapping peptide library representing CLR sequences of C1 q-A and C1 q-B are presented. The peptide number on the X-axis and the relative signal intensity on the Y-axis are shown. The signal corresponds to the binding of anti-C1 q Fab A4 generated from a SLE patient to C1 q-A CLR (peptides A05 to A08) and C1 q-B CLR (peptides B77-B78). Relative signal intensities are the mean value from triplicate fluorescence signals.

The surface plasmon resonance (Biacore) of anti-C1 q SLE Fabs A4 and A14 with time in seconds on the X-axis and the response units (RU) on the Y-axis for biotinylated synthetic A08 or B78 peptide molecules immobilized on strep- tavidin coated biosensor chips is shown. Association and dissociation constants of the two anti-C1 q Fabs (A4 and A14) against the two peptides (A08 and B78) are shown under the figure (ka = association rate constant, k_d = dissociation rate constant, K_A = equilibrium association constant, K_D = equilibrium dissociation constant).

shows (A) Serial dilutions of Fab A14 (starting 200microgram/ml) or SLE patient serum (SLE1 , starting dilution 1 :400) added to immobilized A08 or B78 peptides in ELISA. Binding was observed to A08 but not to B78; (B and C) Binding of SLE serum to A08 peptide molecule after pre-incubation with increasing amounts of soluble A08 or a control peptide in an ELISA assay is shown. The signal obtained from incubating antibodies to an irrelevant biotinylated peptide was considered as background and this O.D. value was subtracted from the specific signal. The data on the Y-axis is expressed as relative level of expression (per-cent signal compared to control).

(A) This figure shows the binding of patient serum (1 :100) to native C1 q and denatured C1 q (56 °C) after incubation with soluble A08 peptides in an ELISA assay. Figure 4a: 20 micrograms peptides/100 microliter were used, resulting in a molar excess of about 200x. Binding levels are per-cent signal compared to binding of the same serum to native C1 q in the presence of a control peptide. Per-cent inhibition to either C1 q or denatured C1 q (indicated above the corresponding bar) was calculated as the reduction in binding in the presence of A08 peptide relative to binding of the same sera in the presence of the control peptide. Figure 4b: Sera of 38 SLE patients (1 :100) tested for binding to C1 q or denatured C1 q, in the presence of A08 or control peptide (40 micrograms peptides/100 microliter, resulting in a molar excess of about 400x). The median value for the different data sets is shown.

Binding to immobilized A08 peptide and C1 q was determined for serum samples from a cohort of 72 healthy donors (NHS) and a cohort of 61 SLE patients (SLE), diluted 1 :50 for the anti-C1 q ELISA and 1 :800 for the anti-A08 peptide ELISA. Antibody binding is expressed in relative units based on the binding of a positive SLE serum that was included as standard on each ELISA plate. The median value for the different data sets is shown.

Fig. 6: Correlation between C1 q and A08 binding for sera of SLE and control (NHS) cohorts described in Fig. 5. Crossed-bars indicate the cut-off values for anti-C1 q and anti-A08 as described in the methods. Fractions (%) of each cohort that scored positive or negative for anti-C1 q or anti-A08 are shown top-right of each graph.

Examples

Example 1 :

The peptide sequence of C1 q that is recognized by SLE Fabs has been identified in a pep- tide-based microarray. The Fab clones have been identified using anti-C1 q Fabs generated from an SLE patient and selected for binding to immobilized C1 q interacting with the CLR of the C1 qA and/or C1 qB chains of C1 q. A peptide scan was performed to detect the peptide sequence that is recognized on C1 qA and/or C1 qB (Fig. 1 ). Two regions, one on C1 qA and one on C1 qB, were showing a significant higher binding (approximately 1000-fold for the highest signal) with SLE Fab A4 as compared to the other peptide sequences.

The 13-mer peptide sequences that resulted in the highest binding by SLE Fabs were assigned as A08 (representing an A-chain region) corresponding to "GRPGRRGRPGLKG" peptide sequence and B78 (representing a region on the B-chain) corresponding to "PGKVGPKGPMGPK".

Example 2:

Binding of anti-C1 q Fab clones to the target peptides A08 and B78 was analysed by surface plasmon resonance analysis (Biacore). As shown in Fig. 2, Fab clones A4 and A14, but not irrelevant polyclonal control Fabs, bind to immobilized A08 and B78 peptides. In contrast, the Fabs did not bind to a control peptide under the same conditions. The association was relatively rapid for the four interactions tested (k_a of 2,04 and 3,43 x 10⁴ 1/Ms) and the formed complexes were relatively stable (k_d of 6,49 and 15,4 x 10^"4 1/s), giving a nano-molar affinity (K_D of 3, 18 and 4,49 x 10^"8 M). These affinities are comparable to the affinities of Fab binding to C1 q CLR determined in a previous study (K_D of 5.16 x 10^"8 M and 8.35 x 10^"8 M for Fab clone A4 and Fab clone A14 respectively, see Schaller M et al., J Immunol. 2009 Dec 15; 183(12):8225-31 and references cited therein) Example 3:

To further analyse the binding of anti-C1 q antibodies to the peptides and to be able to analyse large number of different SLE sera, a peptide based ELISA has been developed. Bioti- nylated A08-peptides were bound via coated Neutravidin on ELISA plates. Anti-C1 q Fabs (clone A14) and a control SLE patient serum bound to the A08 peptide but not to Neutravidin only or Neutravidin conjugated to a control biotinylated peptide.

Specificity of binding to A08 by patient polyclonal serum was confirmed by inhibition of this binding by increasing amounts of soluble A08 peptide. The same amounts of a control peptide had no effect on binding of the antibodies to immobilized A08 (Fig. 3).

Example 4:

To evaluate the contribution of the identified epitopes to the binding of patient sera to C1 q, the sera have been pre-incubated with excess amounts of A08 peptides to block binding of the antibodies to polystyrene-bound C1 q. Results obtained for sera of three patients, of which the binding to immobilized A08 could be differentially inhibited by soluble A08, are shown in Fig. 4 a. Binding of polyclonal non-purified IgG to immobilized native C1 q could be blocked by soluble A08 and this inhibition was even more pronounced for binding of the sera to more linear epitopes, exposed after heat denaturing C1 q at 56°C. Same amounts of control peptides did not affect binding to C1 q. As summarized in Fig. 4b, inhibition of binding was observed for 33 of 38 SLE sera tested. Depending on the sera, inhibition of binding to dena- tured C1 q ranged from 10 to 90% (average of 40%). These data suggest that A08 represents a major linear C1 q epitope for SLE autoantibodies.

Example 5:

Using the A08-peptide ELISA the prevalence of autoantibodies to this C1 q epitope in a cohort of SLE patients compared to healthy donors has been evaluated. Serum antibodies of 61 patients with confirmed SLE and 72 healthy donors were analysed for their binding to immobilized C1 q or C1 q-derived A08 peptides. In contrast to healthy individuals (NHS), serum antibodies from most SLE patients specifically bound to the A08 epitope (Fig. 5). When comparing both assays, the peptide-based ELISA allowed a better discrimination between SLE patients and controls than the C1 q assay suggesting that the peptide ELISA has a higher specificity.

For most sera, binding to A08 correlated with its binding to native C1 q since those that contained anti-C1 q antibodies also bound the A08 peptide (Fig. 6). Statistical analysis of the two assays showed a significant correlation (Spearman r=0.3344, p=0.0084) with linear regression (p=0.036). In total 79% of the SLE patient sera scored positive when tested with the A08-ELISA compared to 62% when using the anti-C1 q ELISA. In addition, the peptide ELISA was better in discriminating non-symptomatic donors from patient sera (79% SLE versus 14% NHS) than the C1 q based ELISA (62% SLE versus 20% NHS).

Since sensitivity and specificity are cut-off dependent, receiver operating characteristic (ROC) curves for the discrimination between SLE patients and normal donors were generated. The area under the curve for the A08-peptide ELISA was better than for the conventional anti-C1 q ELISA (0.86 versus 0.78 with p<0.0001 for both).

Within the subgroup of patients with active nephritis at the time of sampling more patients were found to be positive for anti-A08 than for anti-C1 q (75% versus 67%). The majority of the 13 patients that scored anti-A08 positive but anti-C1 q negative suffered from active nephritis (46%) or had nephritis confirmed by biopsy in the past (30%).

Material and methods

Serum antibodies and Fabs

Anti-C1 q Fabs from an SLE patient were generated as described (Schaller M et al., J Immunol. Dec 15 2009; 183(12):8225-8231 ). Control Fabs were polyclonal Fab prepared from human IgG from healthy donors.

Human serum/plasma was obtained from 61 SLE patients who fulfilled at least 4 criteria for the classification of SLE and from 72 healthy blood donors. The majority of patients (36 out of 61 , i.e. 59%) had biopsy-confirmed active renal disease at the moment of sampling and another 21 % (13/61 ) had had lupus nephritis confirmed by renal biopsy in the past. Most "active nephritis" patients were biopsy confirmed class-Ill and/or -IV. Four patients had class II nephritis and two patients class V.

Peptide scan

Anti-C1 q Fabs that were previously shown to bind to the CLR of the A and/or B chain of C1 q were used to screen an overlapping peptide library representing CLR sequences of C1 q-A and C1 q-B, consisting of 95 and 97 amino acids respectively. Peptide synthesis and Fab binding was performed by AbD Serotec, Germany. Briefly, 13-meric peptides overlapping by 1 1 amino acid residues, resulting in 42 (C1 q-A) + 43 (C1 q-B) peptides were printed onto glass slides in triplicates. These micro-array slides were incubated with anti-C1 q Fab followed by a fluorescently labelled secondary antibody. The Fab clones A4 and A14 were each analysed on separate micro-arrays. The means of the triplicate fluorescence signals are expressed as relative signal intensities. C1 q peptides

Biotinylated and non-biotinylated peptides with >95 % purity were synthesized by GenScript USA Inc. Peptide A08 (GRPGRRGRPGLKG; SEQ ID NO 03) and B78 (PGKVGPKGPMGPK SEQ ID NO 04) are derived from the C1 q-A and C1 q-B chain respectively. Peptide A08-C (GAPGKDGYDGLPG SEQ ID NO 06), derived from the C1 q-C chain is located at the N- terminal region homologous to A08 and was used as a negative control peptide.

Measurement of binding of anti-C1 q Fabs to immobilized peptides by surface plasmon resonance

The interaction of two anti-C1 q Fab clones, A4 and A14, with three peptides (A08, B78 and an irrelevant control peptide) was analysed using surface plasmon resonance technology with Biacore2000 equipment. Biotinylated peptides were bound to streptavidin SA-chip. Purified Fabs were used as analyte at concentrations of 170 nM, 85 nM, 42.5 nM, 21.3 nM, 10.6 nM and were flowed at 10 μΙ/min in PBS. The surface was regenerated with 0.5 M NaCI 0.05 M NaOH. Data was analyzed using BIAevaluation software and the data from the negative control peptide flow cell was subtracted.

Peptide and C1 q ELISA

Purified C1 q or Neutravidine proteins were coated on NUNC Maxisorp microplates. After washing, Neutravidine plates were incubated with biotinylated peptides. Non-bound peptides were removed by washing before adding serum samples. The optimal serum dilution was found to be 1/50 for the anti-C1 q ELISA and 1/800 for the peptide ELISA. Washed C1 q- coated plates were incubated with serum samples diluted in high salt buffer (0.05% Tween20/PBS supplemented with 1 M NaCI), whereas sera were diluted in 0.05% Tween20/PBS for the peptide ELISA.

After washing, bound antibody to the peptides was detected by incubation with a rabbit anti- human IgG-AP (alkaline phosphatase)-antibody for IgG or anti-human Fab-AP for detection of bound Fab. For C1 q ELISA the same secondary antibodies were used but diluted in high salt buffer. After washing, colour development with AP substrate was performed. The reaction was stopped by addition of a same volume of NaOH and colour development was read at 405 nm. For peptide ELISA the signal obtained from incubating antibodies to an irrelevant biotinylated peptide was considered background and this O.D. value was subtracted from the specific signal. For C1 q ELISA, background binding of the AP-conjugated secondary reagent to C1 q was subtracted from the specific binding. The data is either expressed as O.D. values or as relative level of expression (per-cent signal compared to control).

For the analysis of cohorts of healthy and SLE individuals, the experiments have been standardized by expressing the data in units relative to the O.D. values obtained from refer- ence SLE sera (set as 1000 units) that show high levels of binding in the peptide ELISA or in the C1 q ELISA and that were included in each experiment/plate.

The cut-off for a positive signal to evaluate the binding of patient sera was determined as the inter-quartile mean (IQM) of the values obtained with the control sera (NHS) plus three times the standard deviation. A trimmed mean like the IQM was used since it gives a much more robust estimation (an estimation not greatly affected by outliers) of the average than the arithmetic mean.

Samples were tested in duplicate or triplicate within a single experiment, and experiments were performed twice or more.

Competition ELISA

Binding of sera to A08 or C1 q was performed as described earlier except that sera were diluted in 0.05% Tween20/PBS for both peptide and C1 q ELISAs and were pre-incubated with soluble, non-biotinylated peptides as indicated for 3 hours at RT prior to binding to A08 or C1 q.

An amount of peptide was used to reach a molar excess of at least 200x relative to the average amount of IgG present in normal human serum (10-15 mg/ml IgG). For ELISA experiments using denatured C1 q, the stock solution (1 mg/ml) was heated for 30 minutes at 56°C before dilution and coating on ELISA plates.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 4.03 (Graphpad Software, USA). Differences in antibody titers were analysed by a two-sided Mann-Whitney test and a Spearman rank-correlation coefficient.

Claims

1. A method for diagnosis of anti-C1 q autoantibody-related diseases, in particular

systemic lupus erythematosus-related nephritis and hypocomplementaemic urticarial vasculitis syndrome, comprising the steps of:

- providing an oligopeptide molecule having a sequence length of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, said oligopeptide molecule comprising an amino acid sequence comprised as a contiguous sequence in SEQ ID NO 05, SEQ ID NO 01 or SEQ ID NO 02;

- contacting said oligopeptide molecule with a sample obtained from said patient, and

- determining whether said oligopeptide molecule is specifically bound by an antibody comprised in said sample.

2. A method according to claim 1 comprising determining an amount of said antibody.

3. A method according to claim 2, further comprising comparing said amount to a

standard.

4. The method according to any of the claims 1 to 3, wherein said oligopeptide molecule is attached to a surface.

5. A method according to any of the previous claims, wherein said quantification step comprises contacting, in a second contacting step, said antibody with a ligand specifically reactive to said antibody and determining an amount of said ligand bound to said antibody.

6. A method according to claim 5, wherein said ligand is attached or specifically

attachable to an enzyme activity or a detectable label.

7. A method according to claim 6, wherein said detectable label is an optical label.

8. A method according to any of the previous claims wherein an amount of said antibody above a determined threshold level is assigned to a low or a high probability of a systemic lupus erythematosus patient developing or having developed nephritis.

9. A device comprising a surface, said surface comprising an oligopeptide molecule having a sequence length of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, said oligopeptide molecule comprising an amino acid sequence comprised as a contiguous sequence in SEQ ID NO 01 or SEQ ID NO 02.

10. The use of a device according to claim 9, for the diagnosis of anti-C1 q autoantibody- related diseases, in particular systemic lupus erythematosus-related nephritis and hypocomplementaemic urticarial vasculitis syndrome.

1 1 . A kit of parts comprising

- a device comprising a surface, said surface comprising an oligopeptide molecule having a sequence length of 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, said oligopeptide molecule comprising an amino acid sequence comprised as a contiguous sequence in SEQ ID NO 05, SEQ ID NO 01 or SEQ ID NO 02, and said oligopeptide molecule being specifically bound by an antibody, and

- a ligand reactive to said antibody

for the diagnosis of anti-C1 q autoantibody-related diseases such as systemic lupus erythematosus-related nephritis and hypocomplementaemic urticarial vasculitis syndrome.

The use of a kit of parts according to claim 1 1 , for the diagnosis of anti-C1 q autoantibody-related diseases, in particular systemic lupus erythematosus-related nephritis and hypocomplementaemic urticarial vasculitis syndrome.