DESCRIPTION

The invention relates to means for the prognosis and/or diagnosis of antiphospholipid syndrome (APS). In particular, the invention enables differentiation between subjects suffering and/or at risk of suffering from APS and subjects that exhibit elevated levels of antiphospholipid antibodies (aPL) without clinical manifestations or with reduced risk of exhibiting clinical manifestations or a clinical phenotype of APS.

The invention therefore relates to a solid phase comprising a hydrophobic membrane, wherein one or more phospholipids (PL), and optionally one or more proteins, are immobilized to said hydrophobic membrane, wherein said phospholipids comprise one or more of

phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and/or prothrombin (PT), preferably at least PG, and optionally cardiolipin (CL) and/or beta2-glycoprotein I (B2GPI), for use in a multi-dot and/or multi-line immunoassay.

The invention relates further to methods for the prognosis and/or diagnosis of antiphospholipid syndrome (APS). In particular, a method is provided for differentiation between a) subjects suffering and/or at risk of suffering from APS and b) subjects that exhibit elevated levels of antiphospholipid antibodies (aPL) without clinical manifestations or with reduced risk of exhibiting clinical manifestations of APS.

BACKGROUND OF THE INVENTION

APS is a chronic systemic autoimmune disorder with disabling potential. It can occur primary or be associated with other systemic autoimmune diseases, mainly systemic lupus erythematosus (SLE). APS clinical signs are represented by recurrent arterial/venous thrombosis and/or pregnancy morbidity in the persistent presence of aPL. ¹ APS has a substantial socioeconomic impact affecting approximately 1 % of the general population at relatively young age. There is a significant female preponderance, particularly in APS secondary to SLE. Thus, APS causes a substantial socioeconomic impact requiring effective diagnostic and therapeutic approaches.

The international consensus for the classification of antiphospholipid syndrome (APS) requires clinical and laboratory criteria to be considered at an equal level for diagnosing APS. ¹ Thus, detection of antiphospholipid antibodies (aPL) being a hallmark of APS has been the object of intensive investigation over the past 40 years. Accordingly, in approaches previously described in the art, IgG and IgM to beta-2 glycoprotein I ( 2GPI) and the cardiolipin (CL)- 2GPI complex are detected by enzyme-linked immunosorbent assay (ELISA) and further the lupus anticoagulant (LA) by a functional clotting test. However, appropriate detection of aPL still remains a laboratory challenge due to their heterogeneity comprising autoantibody reactive to different phospholipid- binding plasma proteins, such as beta-2 glycoprotein I ( 2GPI) and prothrombin. ²

The relevance of aPL interacting with phospholipids other than cardiolipin (CL,

diphosphatidylglycerol), such as phosphatidylserine (PS), remains elusive with regard to the diagnosis of APS. Recently, the concept of aPL profiling has been introduced to assess the risk of thrombotic complications in patients with APS. ³ New assay techniques, apart from enzyme-linked immunosorbent assays (ELISAs) recommended by the international consensus for the classification of APS, have been proposed for multiplexing of aPL testing. ² Line immunoassays (LIAs) employing a novel hydrophobic solid phase for the simultaneous detection of different aPL have been proposed. ^4,5

The serological hallmark of APS is the persistent presence of aPL being apart from clinical criteria one of the mandatory classification criteria of APS and one of the classification criteria of SLE. aPL can be found in patients with stroke (13.5%), myocardial infarction (1 1.0%), as well as deep venous thrombosis (9.5%) and roughly 20% of patients under the age of 50 years with stroke or venous thromboembolism are diagnosed with APS. ⁶ In general, two main diagnostic techniques are recommended today to test for aPL, enzyme-linked immunosorbent assays (ELISAs) and a functional coagulation assay to detect the so called lupus anticoagulant (LA). ¹ Growing evidence supports the fact that APS-specific aPL interact with plasma cofactors of phospholipids. In this context, beta 2 glycoprotein I (β2ΘΡΙ) is the clinically most relevant and studied autoantigenic target of aPL. ⁷ However, other cofactors like prothrombin (PT) and high molecular weight kininogen have been reported to be specific targets of aPL in the anti-PT and anti- phosphatidylethanolamine (PE) assays. The relevance of aPL to pure phospholipids for the serological diagnosis in APS remains elusive. Thus, in accordance with the international consensus, only ELISAs for the detection of aPL to β2ΘΡΙ and CL are run in routine laboratories, while anti-PT and anti-PE antibodies are still matter of debate. As a matter of fact, aCL and anti- β2ΘΡΙ assays detect aPL to β2ΘΡΙ mainly. Recent data suggest that pathogenic aPL mainly recognize an immunodominant epitope on domain I which is better exposed after the β2ΘΡΙ interaction with negatively charged phospholipids or oxygenated solid-phases employed in state- of-the-art aPL ELISA.

Risk stratification is a major challenge in treating patients with APS and a potential role of aPL as risk or even prognostic factors for arterial/venous thrombosis and miscarriages is debated intensively. ^3,8 In this context, different profiles with regard to single, double, and triple positivity of aPL are detected in patients with APS, whereas in particular the latter seems to be associated with a higher risk for the appearance or recurrence of thrombotic events and miscarriages. ⁹ Furthermore, LA positivity seems to be the best predictor of clinical manifestations in APS, whereas medium/high levels of IgG to CL and β2ΘΡΙ are more indicative than low levels thereof and IgM.

There is growing evidence that aPL are pathogenic, although aPL alone are not sufficient to induce and probably perpetuate APS. A so called "second hit" is required to support these pathophysiological processes. Factors such as traditional cardiovascular risks (e.g., hypertension, diabetes mellitus, obesity), acquired thrombotic risks (e.g., smoking, oral contraception, pregnancy), genetic factors of hypercoagulation (e.g., factor V Leiden mutation, deficiency of protein C and S), and probably most important infections, can provide the required triggers for a second hit. Up to date, however, current techniques included in the classification criteria have not allowed differentiating aPL in patients with APS and those in individuals without typical manifestations of APS.

Reagents for the identification of APS-associated autoantibodies have been described in the art. For example, US 2006/0234392 describes the solid phase immobilization of phospholipids and cofactor proteins via covalent attachment. US 5,506,1 10 describes a carrier for binding anti- phospholipid antibodies and immunoassays using said carrier. The solid phases described in the art relate to Polypropylene (PP), for example in the form of a standard plastic plate surface. The use of hydrophobic membranes such as PVDF or PTFE is neither disclosed nor suggested.

US 2013/0023061 discloses a method for stabilizing glycerophospholipids and methods of their use. Phosphatidylglycerol (PG) is disclosed as a potential phospholipid capable of stabilization according to this technology. No mention is made of using a hydrophobic membrane, such as PVDF or PTFE, for the solid phase unto which the phospholipid is to be immobilized.

Further immunoassays are described in the art in which hydrophobic membranes such as PVDF or PTFE are applied as solid phases for immobilization of a PL (Reference ² , Reference ⁵ , US 201 1/0172106, US 2014/0087398, US 2009/0263825). However, none of these disclosures has suggested attachment of PG in combination with these substrates in an immunoassay suitable for detecting autoantibodies associated with APS.

In light of the existing technology available, further advances and novel products are required that enable more reliable detection of APS-associated autoantibodies and differentiation between the two patient groups of individuals with APS-associated autoantibodies, but who either do or do not show clinical manifestations of APS-syndrome. Improved diagnostic products are required in order to effectively enable risk stratification and to reduce false positive diagnoses in this medical field.

SUMMARY OF THE INVENTION

In light of the prior art the technical problem underlying the present invention is to provide improved means for the diagnosis and/or prognosis of APS. A technical problem of the invention may be described as the provision of improved or alternative means for differentiation between individuals with APS-associated autoantibodies, but who either do or do not show clinical manifestations of APS-syndrome. The technical problem of the invention may also be

represented as the provision of alternative or improved means for risk stratification of APS and/or for the reduction of false positives in APS diagnostics based on autoantibody detection.

This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

The invention therefore relates to a solid phase comprising a hydrophobic membrane, wherein one or more phospholipids (PL), and optionally one or more proteins, are immobilized to said hydrophobic membrane, wherein said phospholipids preferably comprise at least

phosphatidylglycerol (PG), for use in a multi-dot and/or multi-line immunoassay. In a preferred embodiment the invention relates to a combination of aPL antigens as described herein, preferably phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and prothrombin (PT), and optionally cardiolipin (CL) and/or beta2-glycoprotein I (β2ΘΡΙ).

In light of the technical advantages of the invention described herein, the invention provides novel means for the prognosis and/or diagnosis of antiphospholipid syndrome (APS), and the use of such means in a method for the prognosis and/or diagnosis of antiphospholipid syndrome (APS). In particular, the combination of PL with the hydrophobic membrane of the invention, represented as the solid phase described herein, and the method employing said solid phase, enables differentiation between subjects suffering and/or at risk of suffering from APS and subjects that exhibit elevated levels of antiphospholipid antibodies (aPL) without clinical manifestations or with reduced risk of exhibiting clinical manifestations or a clinical phenotype of APS (+aPL).

The invention therefore relates to a solid phase for use in an immunoassay, comprising a porous hydrophobic membrane, preferably selected from polyvinylidene difluoride (PVDF) or

polytetrafluorethylene (PTFE), wherein one or more phospholipids (PL), and optionally one or more proteins, are immobilized to said hydrophobic membrane, wherein said phospholipids (PL) comprise at least phosphatidylglycerol (PG).

In a preferred embodiment of the invention the hydrophobic membrane relates to a material or mixture of materials exhibiting hydrophobic characteristics. The hydrophobic membrane is preferably a porous hydrophobic membrane. The hydrophobic membrane or solid phase of the invention may comprise or consist of a porous hydrophobic polymer material or comprise a surface that comprises a porous hydrophobic polymer material.

In a preferred embodiment the hydrophobic membrane is configured to enable PL immobilization to the hydrophobic membrane, wherein the hydrophobic part of the PL molecule is partially or completely hidden by its binding to the membrane pores and/or surface, for example wherein the hydrophobic part of the PL molecule is bound to the solid phase such that it is no longer accessible to antibodies, or no longer sufficiently accessible to antibodies in order to form a stable binding between antibody and the PL, when a solution comprising antibodies is brought into contact with the PL immobilized on the hydrophobic membrane. This property may be described as incorporation of the hydrophobic tail into the porous hydrophobic membrane.

As described in detail in the experimental examples, in contrast to a planar ELISA-solid phase, the porous hydrophobic membrane used in the solid phase of the present invention is able to incorporate (to hide) the hydrophobic PL tail. This shields the by far larger tail of the amphiphatic PL molecule from the reaction environment and, thus, prevents unspecific interactions. Through the unique combination of PL, in particular PG, with hydrophobic membranes that enable such hiding of hydrophobic PL tail, antibodies directed against the hydrophobic PL are typically not bound, or bound in reduced amounts compared to a planar ELISA-solid phase, thereby reducing the false positives that have plagued the technology of the prior art regarding APL diagnosis.

The invention relates therefore to the novel technical effect that APS-associated autoantibodies that are directed to the hydrophilic heads of PL are disease-indicative, whereas aPL directed against the hydrophobic PL tail of particular PL molecules represent disease-unrelated autoantibodies, that provide a relatively poor or no diagnostic statement regarding APS or risk of developing APS.

In this context, solid phases in which the hydrophobic tails of PL molecules remain sufficiently exposed upon binding to the hydrophobic material to enable stable binding of an antibody to said tail are, in some embodiments, excluded from the invention. Solid phases comprising a material such as is used in common ELISA plates, for example produced with a planar polystyrene, are not preferred. In one embodiment, polystyrene as a hydrophobic material is excluded from the invention. In one embodiment, polypropylene as a hydrophobic material is excluded from the invention. In one embodiment, polycarbonate as a hydrophobic material is excluded from the invention.

Other known solid phases used for biomolecule immobilisation may also include Anopore, Cellulose acetate, Cellulose nitrate, Nylon/polyamide, Polycarbonate, Polyethersulfone and Regenerated cellulose. However, these materials are typically present as hydrophilic membranes and are therefore not suited to the present invention. The aforementioned materials may however exhibit a suitable structure or other properties for use in the present invention. In such a case, a modification of these materials to render them hydrophobic, for example via coating of a hydrophobic substance or chemical modification, may subsequently lead to their appropriate use in the present invention.

The invention therefore relates to a solid phase for use in an immunoassay, comprising a porous hydrophobic membrane configured for incorporation of the hydrophobic tail of a phospholipid

(PL), preferably selected from polyvinylidene difluoride (PVDF) or polytetrafluorethylene (PTFE), wherein one or more PL, and optionally one or more proteins, are immobilized to said

hydrophobic membrane, wherein said phospholipids (PL) comprise at least phosphatidylglycerol (PG).

It was entirely surprising that the unique combination of PG, potentially in combination with other PLs, immobilized on a porous hydrophobic membrane, in particular a PVDF or PTFE membrane, enabled the differentiation between +aPL subjects and those with APS. PG bound upon the hydrophobic membrane creates an epitope bound by a sub-set of aPL autoantibodies, enabling identification of patients with APS from those with aPL but no clinical manifestations.

The present invention is characterised in a preferred embodiment by the particular solid phase, specially the hydrophobic membrane employed in the method. Polyvinylidene difluoride (PVDF) is a preferred embodiment, but alternatives, such as polytetrafluorethylene (PTFE), show similar properties.

According to the present invention, LIAs employing hydrophobic membranes instead of polysterene plastics as solid phase may be successfully employed for aPL profiling. Their usefulness has already been proven for the specific assessment of autoantibodies to

lipopolysaccharides and glycolipids demonstrating similar physicochemical properties to phospholipids for the serological diagnosis of patients with autoimmune peripheral neuropathies. After interaction with plasma cofactors present in patient's own serum, phospholipids coated onto hydrophobic membranes seem to present β2ΘΡΙ in conformational changes similar to those occurring on the oxygenated polysterene surface of ELISA.

Furthermore, anionic phospholipids immobilized on such membranes appear to generate a different reaction environment for the interaction with aPL, in contrast to ELISA, due to the porous structure of the membrane hiding the large hydrophobic part of the immobilized phospholipids. This seems to lead to a denser presentation of the hydrophilic moiety of phospholipids, favoring its interaction with the PL-binding site located in the fifth domain of β2ΘΡΙ and eventually exposing the immunodominant epitope on domain I.

In one embodiment of the present invention the solid phase as described herein is characterized in that phosphatidylserine (PS) is immobilized to the hydrophobic membrane.

In one embodiment of the present invention the solid phase as described herein is characterized in that prothrombin (PT) is immobilized to the hydrophobic membrane.

In one embodiment of the present invention the solid phase as described herein is characterized in that phosphatidylinositol (PI) is immobilized to the hydrophobic membrane.

In one embodiment of the present invention the solid phase as described herein is characterized in that phosphatidic acid (PA) is immobilized to the hydrophobic membrane.

In one embodiment of the present invention the solid phase as described herein is characterized in that phosphatidylethanolamine (PE) is immobilized to the hydrophobic membrane.

The combinations of PG and PS, PG and PI, PG and PT, PG and PA or PG and PE upon the hydrophobic membrane surprisingly provide statistically relevant statements regarding the presence of aPL and whether these aPL are associated with APS. The invention relates to a specific and unique selection of particular aPL antigens that enable the diagnosis and/or prognosis described herein. The selection relates to a specific disease-related sub-group of aPL antigens, including both PL and/or proteins, that could not have been predicted to show the surprising properties as described herein. Although ELISA and other immunoassays are known in the comprising the detection of aPL, none of the cited art has disclosed or indicated that the particular selection of antigens described herein could solve the technical problem underlying the present invention.

In one embodiment of the present invention the solid phase as described herein is characterized in that wherein cardiolipin (CL) is immobilized to the hydrophobic membrane.

In one embodiment of the present invention the solid phase as described herein is characterized in that beta2-glycoprotein I (B2GPI) is immobilized to the hydrophobic membrane.

In one embodiment of the present invention the solid phase as described herein is characterized in that phosphatic acid (PA) is immobilized to the hydrophobic membrane.

Further embodiments of the invention relate to a solid phase as described herein, wherein at least phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) are immobilized to the hydrophobic membrane. Further embodiments of the invention relate to a solid phase as described herein, wherein at least phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and prothrombin (PT) are immobilized to the hydrophobic membrane.

Further embodiments of the invention relate to a solid phase as described herein, wherein at least phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and prothrombin (PT), in addition to cardiolipin (CL) and/or beta2-glycoprotein I (B2GPI), are immobilized to the hydrophobic membrane.

Further embodiments of the invention relate to a solid phase as described herein, wherein at least phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA) and prothrombin (PT), in addition to cardiolipin (CL) and beta2-glycoprotein I (B2GPI), are immobilized to the hydrophobic membrane.

Further embodiments of the invention relate to the solid phase as described herein, wherein the immobilized PL and/or proteins are isolated from one another upon the solid phase. Further embodiments of the invention relate to the solid phase as described herein, wherein the immobilized PL and/or proteins are sufficiently isolated from one another in a manner to enable to enable visualization of antibodies bound to said PL and/or proteins. The structure of the solid phase is configured for simultaneous detection of signals produced from any given detection reaction generated by aPL bound to the immobilized epitopes. The spatial isolation may therefore relate to stripes, dots, arrays, or other suitable formats for analysis, preferably automatic detection.

In a preferred embodiment of the invention the solid phase described herein is configured to enable incubation between the immobilized phospholipids with a patient sample, wherein the patient sample is preferably selected from blood, serum and/or plasma.

In a preferred embodiment of the invention the solid phase described herein is configured with respect to its physical dimensions and properties for use in a multi-dot and/or multi-line immunoassay.

Further embodiments of the invention relate to the solid phase as described herein, wherein said solid phase is configured with respect to its physical dimensions for use in a (preferably automatic) plate reader, such as a dot blot analyzer, a micro-plate reader, or other plate reader or sample detection device, in which standardized micro-plates may be analyzed.

The solid phase of the invention is characterised in a preferred embodiment by its dimensions and suitability for use in a multi-dot and/or multi-line immunoassay, or for its employment in a standardized plate-reader or dot blot analyzer or other similar detection device.

A strip comprising the solid phase of the present invention is intended in one embodiment of the present invention, for example as is shown in the examples.

In one embodiment, the invention is characterised in that the solid phase is configured to a length and breadth essentially that of a micro-plate, for example a 96 well microtiter plate. The hydrophobic solid phase could be formatted to be used in automatic analysis or in existing plate reader apparatuses. For example, the system may be exhibit a length or breadth of approximately 100 mm greater than the length or breadth of any given standardised microtiter plate, preferably 80 mm, or more preferably 50 mm or 20 mm greater than the length or breadth of a standardised microtiter plate. For example, 96 well microtiter plates are known to a skilled person and are approximately 128 mm x 86 mm in length and breadth. A microtiter plate may also be referred to as a micro-plate. The micro-plates used herein may relate to any given size or design. Preferably standard micro-plates are used according to ANSI/SLAS 1-2004 (formerly recognized as

ANSI/SBS 1 -2004).

A further aspect of the invention relates to a kit, comprising a solid phase as described herein. In a preferred embodiment the kit of the present invention comprises human anti-lgG or anti-lgM antibodies for the detection of antibodies bound from a patient sample. In a preferred

embodiment the kit of the present invention comprises human anti-lgG and anti-lgM antibodies for the detection of antibodies bound from a patient sample.

In further preferred embodiments the kit of the present invention is characterised in that the human anti-lgG and anti-lgM antibodies are labeled with (coupled to) an enzyme, such as horseradish peroxidase or alkaline phosphatase, for the detection of antibodies bound from a patient sample. In a preferred embodiment the kit comprises a substrate solution, such as tetrmethyl benzidine or nitroblue tetrazolium with bromo-chloroindolyl-phoshate for color development.

A further aspect of the invention relates to a method for differentiation between (a) one or more subjects suffering and/or at risk of suffering clinical manifestations of APS and (b) one or more subjects that exhibit elevated levels of antiphospholipid antibodies (aPL) compared to control subjects without clinical manifestations and/or with reduced risk of exhibiting clinical

manifestations of APS compared to (a), comprising the identification of antibodies that bind one or more of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and/or prothrombin (PT).

The invention therefore provides means for risk stratification for subjects exhibiting aPL. In many cases, the identification of aPL is alone not sufficient to accurately predict whether the subject will develop the disease itself. The detection of aPL as a means for APS diagnosis/prognosis is therefore plagued by false positives, creating significant discomfort for the individual and cost to the health system.

In a preferred embodiment the method of the invention comprises the provision and/or use of a solid phase and/or kit as described herein.

A further aspect of the invention relates to a method for the diagnosis of aPL comprising the use of the solid phase as described herein. The features directed to the solid phase as described herein may also be employed with respect to particular embodiments of the method, without having to explicitly recite every possible combination of PL with each solid phase in the context of the method.

In a preferred embodiment the method of the present invention comprises: a Obtaining or providing a sample from a subject, preferably a blood or serum sample;

b Contacting said sample to a porous hydrophobic membrane, preferably PVDF or PTFE, wherein one or more phospholipids (PL), and optionally one or more proteins, are immobilized to said hydrophobic membrane, wherein said phospholipids comprise one or more of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and/or prothrombin (PT);

c. Detecting antibodies from said sample that are bound to the one or more phospholipids (PL), and optionally one or more proteins, immobilized to said hydrophobic membrane.

d Wherein the presence of aPL directed against one or more of said PL is indicative of APS, or increased risk of APS occurring, in the subject.

According to the present invention the method may be characterised in that the presence, or elevated levels relative to a control, of antibodies that bind one or more of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and/or prothrombin (PT) from the subject's sample indicate the presence and/or increased risk of developing clinical manifestations of APS in said subject. Appropriate controls are disclosed in the examples herein.

The method of the present invention may in some embodiments encompass the use of cardiolipin (CL) and/or beta2-glycoprotein I (B2GPI) in order to determine the presence and/or amount of antibodies directed to said targets.

The identification and/or detection of antibodies can be carried out according to the knowledge of a skilled person. For example, anti-lgG or anti-lgM antibodies, preferably coupled with a fluorescent label or enzyme capable of producing a signal from a substrate, are incubated with the solid phase after incubation between the solid phase and sample. Any appropriate washing step could also be carried out as appropriate. The signal can then be used to determine the presence, or increased levels compared to a control, of an antibody bound to the immobilized aPL ligand.

In one embodiment the method of the present invention comprises the diagnosis or prognosis of APS. In one embodiment the method of the present invention is characterised in that the antibodies are autoantibodies directed against the corresponding PL epitopes. In preferred embodiments of the invention the method comprises the determination of IgG and/or IgM antibodies.

According to some embodiments of the method of the present invention, the antibodies are one or more of:

a. IgG and/or IgM antiphosphatidylglycerol antibodies (aPG);

b IgG and/or IgM antiphosphatidylinositol antibodies (aPI);

c. IgG and/or IgM antiphosphatidylserine antibodies (aPS); d. IgG and/or IgM antiprothrombin antibodies (aPT); and/or

e. IgG and/or IgM antiphosphatidic acid antibodies (aPA).

As can be seen from the experimental examples below, IgG and/or IgM may directed to the same epitope may exhibit distinct profiles and propensities with respect to certain patient groups. The kit, and method, of the invention may therefore specifically encompass the use of either both or one of IgM or IgG depending on the relevance of each aPL sub-type in any given patient group.

A further embodiment of the invention relates to a method as described herein, wherein the one or more subjects (b) that exhibit elevated levels of antiphospholipid antibodies (aPL) compared to control subjects without clinical manifestations and/or with reduced risk of exhibiting clinical manifestations of APS compared to (a) have tested positive to the Venereal Disease Research Laboratory test.

The experiments disclosed herein provide support that infectious diseases, for example Venereal diseases, also lead to +aPL detection in patients who do not exhibit APS or APS syndromes. The means and method of the present invention therefore enable differentiation between subjects with infectious diseases, such as venereal diseases, who show aPL but no APS syndromes or no significant risk of developing APS, and those subjects with or at risk of having APS:

A further aspect of the invention relates to a method for differentiation between (a) one or more subjects suffering and/or at risk of suffering clinical manifestations of APS with venous and/or arterial thrombosis and (b) one or more subjects suffering and/or at risk of suffering clinical manifestations of APS having at least one obstetrical manifestation, comprising the determination of higher amounts of IgG and/or IgM antibodies directed against cardiolipin (CL) in (a) compared to (b).

Surprisingly, the present invention enables the differentiation between subjects with thrombotic risk factors and those with an obstetrical manifestation. No evidence has been described previously that aCL could be a defining factor in identifying the pathological manifestation of an aPL-associated disorder. The present invention therefore will enable identification of subject groups in pregnancy and appropriate monitoring and/or therapeutic approaches.

A further aspect of the invention relates to a method for differentiation between (a) one or more subjects suffering and/or at risk of suffering clinical manifestations of APS with intrauterine growth retardation, intrauterine death of fetuses, early pregnancy loss, premature birth and/or eclampsia/preemclampsia and (b) one or more subjects that exhibit elevated levels of antiphospholipid antibodies (aPL) compared to control subjects, comprising the determination of lower amounts of IgM and/or IgG antibodies directed against PA, PS and/or B2GPI in (a) compared to (b). Surprisingly, the present invention enables the differentiation between subjects with APS combined with pregnancy manifestations and those APS, or +aPL. No evidence has been described previously that relatively low levels of IgM and/or IgG antibodies directed against PA, PS and/or B2GPI could be a defining factor in identifying subjects with aPL and pregnancies at risk of adverse outcomes. The present invention therefore will enable identification of subject groups in pregnancy and appropriate monitoring and/or therapeutic approaches. The present invention also encompasses a corresponding treatment of APS in a subject, preferably human subject, for example after the outcome of the diagnostic method has been obtained, or after the use of the solid phase or kit described herein, depending on the outcome of the method (diagnosis).

Possible treatments are known to a person skilled in the art. For example, APS is treated by prescribing aspirin or other blood thinners to inhibit platelet activation, and/or warfarin as an anticoagulant. It is not usually carried out in patients who have had no thrombotic symptoms, but can be carried out. Anticoagulation appears to prevent miscarriage in pregnant women with APS. In pregnancy, low molecular weight heparin and low-dose aspirin are used. Women with recurrent miscarriage are often advised to take aspirin and to start low molecular weight heparin treatment after missing a menstrual cycle.

The present invention further relates to a method for the treatment of APS in a human subject, comprising:

i. having a sample of biological fluid obtained from the subject,

ii. having an assay conducted on the sample, said assay comprising

- Contacting said sample to a porous hydrophobic membrane, preferably PVDF or PTFE, wherein one or more phospholipids (PL), and optionally one or more proteins, are immobilized to said hydrophobic membrane, wherein said phospholipids comprise one or more of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS) and/or prothrombin (PT);

- Detecting antibodies from said sample that are bound to the one or more phospholipids (PL), and optionally one or more proteins, immobilized to said hydrophobic membrane.

iii. treating the subject for APS.

DETAILED DESCRIPTION OF THE INVENTION

Antiphospholipid syndrome (APS or APLS), or Hughes syndrome, is an autoimmune,

hypercoagulable state caused by antiphospholipid antibodies. APS symptoms include blood clots (thrombosis) in both arteries and veins as well as pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, and severe preeclampsia. The presence of

antiphospholipid antibodies (aPL) in the absence of blood clots or pregnancy-related

complications does not however indicate APS in all subjects.

Antiphospholipid syndrome can cause symptoms such as arterial or venous blood clots, in any organ system, or pregnancy-related complications. In APS patients, the most common venous event is deep vein thrombosis of the lower extremities, and the most common arterial event is stroke. In pregnant women affected by APS, there is an increased risk of recurrent miscarriage, intrauterine growth restriction, and preterm birth. A frequent cause of such complications is placental infarctions. In some cases, APS seems to be the leading cause of mental and/or development retardation in the newborn, due to an aPL-induced inhibition of trophoblast differentiation. The antiphospholipid syndrome responsible for most of the miscarriages in later trimesters seen in concomitant systemic lupus erythematosus and pregnancy.

Other clinical manifestations or symptoms of APS relate to low platelet count and heart valve disease. There are also associations between antiphospholipid antibodies and headaches, such as migraines.

The term subject suffering and/or at risk of suffering clinical manifestations of APS encompasses subjects with aPL and the presence of one or more of the above mentioned APS symptoms, or with aPL and an elevated risk of developing one or more symptoms. For example, the presence of the aPL against PG on the hydrophobic membrane described herein may indicate also an increased risk of obtaining APS.

The term subject that exhibits elevated levels of antiphospholipid antibodies (aPL) compared to control subjects without clinical manifestations and/or with reduced risk of exhibiting clinical manifestations of APS encompasses +aPL subjects, with aPL but without APS, according to the symptoms described above. A control subject may be a healthy individual. The absence of the molecular indicators described herein, such as the absence of the aPL directed against PG immobilized on the hydrophobic membrane described herein, may also indicate a reduced risk of obtaining APS. The method and use of the solid phase described herein may therefore relate to diagnostic or prognostic effects.

Polyvinylidene fluoride, or polyvinylidene difluoride (PVDF) is a non-reactive thermoplastic fluoropolymer produced by the polymerization of vinylidene difluoride. In the biomedical sciences PVDF is used in immunoblotting as an artificial membrane, usually with 0.22 or 0.45 micrometres pore sizes, on which proteins or lipids are transferred. PVDF membranes may be used in various biomedical applications as part of a membrane device, for example in the form of a filter. The various properties of this material such as heat resistance, resistance to chemicals, corrosion and low protein binding properties make this material valuable in the biomedical sciences.

Polytetrafluoroethylene (PTFE) is a synthetic fluoropolymer of tetrafluoroethylene that has numerous applications and may be applied similarly to PVDF.

A porous hydrophobic membrane configured for incorporation of the hydrophobic tail of a phospholipid (PL) can be assessed using methods described herein, for example by testing for the binding of antibodies directed against particular portions of a PL molecule (tail or head). In a further example, a comparative test can be carried out, in which a classical polystyrene ELISA plate is compared to the porous hydrophobic membrane under question with respect to its properties regarding the incorporation of the hydrophobic tail of the PL into the membrane structure.

The term "diagnosing" includes the use of the devices, methods, and systems, of the present invention to determine the presence or absence or likelihood of presence or absence of a medically relevant disorder in an individual. The term also includes devices, methods, and systems for assessing the level of disease activity in an individual. One skilled in the art will know of other methods for evaluating the severity of APS in an individual. The comparative analysis described herein between autoantibody binding to different aPL is a preferred method of the present invention. Direct comparison based on autoantibody binding as measured in the same experiment may be used. For this embodiment the amount of aPL provided for the experiment should be controlled carefully to enable direct comparative analysis. Alternatively, or in combination, control values or standards may be used that provide samples with autoantibodies or represent control amounts thereof, as have already been obtained from previous analytical tests. It is possible to use control values having been generated by the testing of cohorts or other large numbers of subjects suffering from any given disease or control group. Appropriate statistical means are known to those skilled in the art for analysis and comparison of such data sets. Control samples for positive controls (such as disease sufferers) or negative controls (from healthy subjects) may be used for reference values in either simultaneous of non- simultaneous comparison.

The invention also encompasses use of the method for disease monitoring, also known as monitoring the progression or regression of the autoimmune disease. The term "monitoring the progression or regression of the autoimmune disease" includes the use of the devices, methods, and systems of the present invention to determine the disease state (e.g., presence or severity of the autoimmune disease) of an individual. In certain instances, the results of a statistical algorithm (e.g., a learning statistical classifier system) are compared to those results obtained for the same individual at an earlier time. In some aspects, the devices, methods, and systems of the present invention can also be used to predict the progression of the autoimmune disease, e.g., by determining a likelihood for the autoimmune disease to progress either rapidly or slowly in an individual based on the presence or level of at least one marker in a sample. In other aspects, the devices, methods, and systems of the present invention can also be used to predict the regression of the autoimmune disease, e.g., by determining a likelihood for the autoimmune disease to regress either rapidly or slowly in an individual based on the presence or level of at least one marker in a sample. Therapy monitoring may also be conducted, whereby a subject is monitored for disease progression during the course of any given therapy.

The term "individual," "subject," or "patient" typically refers to humans, but also to other animals including, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like. As used herein, the term "antibody" includes a population of immunoglobulin molecules, which can be polyclonal or monoclonal and of any isotype, or an immunologically active fragment of an immunoglobulin molecule. Such an immunologically active fragment contains the heavy and light chain variable regions, which make up the portion of the antibody molecule that specifically binds an antigen.

In one advantageous embodiment an immunoassay assay is employed in the detection of antibodies, to which end binding of the PL antigen to a solid phase is envisaged. Following addition of sample solution, the patient's antibody included therein binds to the PL antigen. The antibody which is obtained e.g. from the serum or stool of a patient and bound to PL is subsequently detected using a label, or labelled reagent and optionally quantified. Thus, according to the invention, detection of the antibodies in this method is effected using labelled reagents according to the well-known ELISA (Enzyme-Linked Immunosorbent Assay) technology. ELISA detection technology may also be applied to the LIA approaches described herein, and/or to the hydrophobic membrane-based approaches described herein.

Labels according to the invention therefore comprise enzymes catalyzing a chemical reaction which can be determined by optical means, especially by means of chromogenic substrates, chemiluminescent methods or fluorescent dyes. In another preferred embodiment the

autoantibodies are detected by labelling with weakly radioactive substances in

radioimmunoassays ( IA) wherein the resulting radioactivity is measured.

As examples of means for detecting the label in the method of the present invention, a variety of immunoassay techniques can be used to determine the presence or level of one or more markers in a sample (see, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996)). The term

immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), antigen capture ELISA, sandwich ELISA, IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated.

In another preferred embodiment of the invention, solid phase-bound PL molecules are used to bind the antibodies. In a second reaction step, anti-human immunoglobulins are employed, preferably selected from the group comprising anti-human IgA, anti-human IgM and/or anti- human IgG antibodies, said anti-human immunoglobulins being detectably labelled conjugates of two components which can be conjugated with any conventional labelling enzymes, especially chromogenic and/or chemiluminescent substrates, preferably with horseradish peroxidase, alkaline phosphatase. The advantage of this embodiment lies in the use of ELISA-similar technology usually available in laboratory facilities so that detection according to the invention can be established in a cost-effective manner. In another preferred embodiment of the invention the antibody bound to PL reacts with anti-human immunoglobulins, preferably selected from the group comprising anti-human IgA, anti-human IgM and/or anti-human IgG antibodies, detectably coupled to fluorescein isothiocyanate (FITC). Much like the above-mentioned ELISA, the FITC technology represents a system that is available in many places and therefore allows smooth and low-cost establishment of the inventive detection in laboratory routine.

According to the present invention a multi-dot and/or multi-line immunoassay is envisaged. Such an assay relates for example to a dot blot assay or similar, in which the PL antigen is spotted or sprayed in adjacent positions to one another in isolated fashion upon the solid phase comprising PVDF or PTFE, positioned such to allow detection using the immunoassay detections means described herein.

FIGURES

The figures provided herein represent potential and/or preferred embodiments of the present invention and are not considered limiting in nature to the scope of the invention. Figure 1 : Reactivity of antibeta2-glycoprotein I (aB2GPI) minibody (A) and monoclonal antibody (mAb) HCAL (B) with phospholipids and cofactors by line immunoassay (LIA) according to example 2:

Minibody (0.1 mg/L) and mAb HCAL (0.02 mg/L) were in run LIA alone or together with serum, β2ΘΡΙ, blood donor serum, and bovine serum albumin (BSA).

A 1 : 0.1 mg/L aB2GPI minibody

2: 0.1 mg/L aB2GPI minibody + 10 mg/L β2ΘΡΙ

3: 0.1 mg/L aB2GPI minibody + 10 mg/L BSA

4: 0.1 mg/L aB2GPI minibody + 30 μί serum (1/33)

5: 30 [ L serum (1 /33)

B 1 : 0.02 mg/L aB2GPI HCAL

2: 0.02 mg/L aB2GPI HCAL + 10 mg/L B2GPI

3: 0.02 mg/L aB2GPI HCAL + 10 mg/L BSA

4: 0.02 mg/L aB2GPI HCAL + 30 μί serum (1/33)

5: 30 [ L serum (1 /33)

Figure 2: Reactivity of antiphospholipid (aPL) monoclonal antibody (mAb) RR7F with phospholipids and cofactors by line immunoassay (LIA) according to example 2:

mAb RR7F (10.0 mg/L) was in run LIA alone or together with serum, B2GPI, blood donor serum, and bovine serum albumin (BSA).

1 : 10.0 mg/L RR7F

2: 10.0 mg/L RR7F + 10 mg/L B2GPI

3: 10.0 mg/L RR7F + 10 mg/L BSA

4: 10.0 mg/L RR7F + 30 μί serum (1 /33)

5: 30 [ L serum (1 /33)

Figure 3: Reactivity of a nti prothrombin (aPT) (A) and antiannexin V (aAnV) (B) polyclonal antibodies with phospholipids and cofactors by line immunoassay (LIA) according to example 2:

A 1 : 1 .0 mg/L aPT

2: 1 .0 mg/L aPT + 10 mg/L B2GPI

3: 1 .0 mg/L aPT + 10 mg/L BSA

4: 1 .0 mg/L aPT + 30 μί serum (1/33)

5: 30 [ L serum (1 /33) B 1 : 5.0 mg/L aAnV

2: 5.0 mg/L aAnV + 10 mg/L β2ΘΡΙ

3: 5.0 mg/L aAnV + 10 mg/L BSA

Figure 4: Inhibition of antibeta2-glycoprotein I (aB2GPI) minibody and monoclonal antibody (HCAL) as well as antiphospholipid (aPL) monoclonal antibody (RR7F) by cardiolipin (CL) micelles in line immunoassay (LIA) according to example 2:

1 : 0.1 mg/L aB2GPI minibody

2: 0.1 mg/L aB2GPI minibody + CL micelles

3: 0.02 mg/L aB2GPI monoclonal antibody (HCAL)

4: 0.02 mg/L aB2GPI monoclonal antibody (HCAL) + CL micelles

5: 10.0 mg/L aPL RR7F

6: 10.0 mg/L aPL RR7F + CL micelles

EXPERIMENTAL EXAMPLES

The examples provided herein represent potential and/or preferred embodiments of the present invention and are not considered limiting in nature to the scope of the invention. Alternative embodiments to those listed in the examples that fall within the spirit of the present invention and within the capabilities of a skilled person are also considered potential embodiments of the invention.

EXAMPLE 1 :

IgG and IgM antibodies to phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), phosphatidylcholin, phosphatidylethanolamine, phosphatic acid (PA), phosphatidylglycerol (PG), annexin V (AnV), prothrombin (PT), and beta2-glycoproteinl (B2GPI) were analyzed by LIA and ELISA (aCL and B2GPI only) in a pilot study comprising analysis of sera of 15 patients with APS, 15 individuals with aPL positivity and 15 blood donors (BD; control).

Material and Subjects:

Briefly, the phospholipids and the proteins were sprayed onto PVDF membrane in lines in a "strip" format for immobilization. After blocking, strips were incubated with sera diluted 1 in 100 for 120 minutes at 4° C while shaking. Subsequently, strips were washed three times and incubated with polyclonal antihuman IgG or IgM antibodies labeled with horseradish peroxidase for 60 min at 4°C while shaking. After washing, precipitating tetramethyl benzidine was added as substrate for color development. The reaction was stopped after 10 min at room temperature by a further washing step with distilled water. Processed strips were read out densitometrically employing a scanner with the evaluation software Dot Blot Analyzer (GA Generic Assays GmbH, Dahlewitz, Germany). Analysis was carried out as described in references 4 and 5. Results:

aPL profiling comprising multiplex detection of aPL by LIA with the hydrophobic membrane can differentiate between patients suffering from APS and individuals just bearing elevated aPL without any clinical phenotype of APS (+aPL) .

Testing of aPL by LIA and ELISA demonstrated no statistical difference according to McNemar's test scoring one positive aPL as a positive diagnostic criterion (Table 1 ) (difference: 4.26%; 95% CI: -5.21 % to 8.40%; P = 0.6250). Thus, aPL analysis by LIA covering 10 aPL is not less specific than ELISA testing for aCL and aB2GPI only. Strength of agreement between both methods was very good (Cohen's kappa = 0.805, standard error 0.093, 95% CI: 0.623 to 0.987).

Both LIA and ELISA demonstrated significant higher prevalences of aPL IgG and/or IgM in APS patients compared to BD (P < 0.001 , respectively). However, aPG IgG (10/15 vs 2/15) and aPG IgM (8/15 vs 1/15) by LIA demonstrated a significantly higher prevalence in APS patients compared to the +aPL group (P = 0.008, 0.014, respectively). The same phenomenon was observed for aPI IgG (8/15 vs 2/15, P = 0.050), aPS IgG (13/15 vs 6/15, P = 0.021 ), aPS IgM (12/15 vs 5/15, P = 0.025) and aPT IgG (7/15 vs 1/15, P = 0.035). In contrast aCL and B2GPI determined by either ELISA or LIA did not demonstrate significantly different prevalences in patients with APS and +aPL individuals (P > 0.05, respectively). Neither did aPA IgG and IgM detected by LIA.

In conclusion, determination of aPG, aPI, aPS and/or aPT in single or in combination thereof can be employed for the serological differentiation of aPL positive individuals with clinical symptoms of APS (APS patients) and those without.

EXAMPLE 2:

Data was obtained from a study employing an LIA based on a novel hydrophobic solid phase for the simultaneous detection of multiple aPL in a well-defined cohort of 61 APS patients with thrombotic and obstetrical manifestations and 146 controls including in particular 24 aPL+ subjects.

Methods employed in the examples:

Patients and controls

In total, 207 individuals were enrolled into a study comprising 61 patients with APS diagnosed in accordance with the international APS classification criteria and 146 controls (Table 3). Patients with APS were further classified as primary APS (PAPS) with arterial and/or venous thrombosis in the absence of any other related disease, secondary APS (SAPS) in which APS occurs along with another autoimmune diseases, obstetric APS (OAPS) with pregnancy-related complications listed in the classification criteria (early pregnancy loss, intrauterine death, premature birth, pre/eclampsia, intrauterine growth retardation). Further, 24 individuals were included with no clinical APS manifestations but being positive for at least one of aPL detected by classification- criteria recommended assays (lupus anticoagulant, aCL and anti-B2GPI autoantibodies). As disease controls 73 patients were included who suffered from infectious diseases (3 patients infected with Eppstein-Barr virus, 14 with Toxoplasma gondii, 24 with Cytomegalovirus, 8 with Rubella virus, 1 with Hepatitis C and 23 with Treponema palladium demonstrating a positive VDRL test). All sera had been stored at -20°C.

Monoclonal aPL and polyclonal anticofactor antibodies

To investigate the interaction with β2ΘΡΙ in the novel assay environment, a chimeric IgG monoclonal antibody (mAb) HCAL was employed, comprising human κ and γ constant regions and variable regions from the mouse monoclonal 2GPI-dependent anti-CL WBCAL-1. To determine β2ΘΡΙ domain reactivity a human minibody was tested containing a single chain fragment variable fused to lgG1 CH2-CH3 domain that recognizes domain I of human β2ΘΡΙ. The human mAb RR7F interacting with PL in ELISA was used to analyze the reactivity to PL immobilized on the hydrophobic membrane employed in LIA.

An anti-PT (aPT) mAb (Kerafast, Boston, United States) and an anti-AnV (aAnV) (Cusabio, Wuhan, China) polyclonal antibody were employed to investigate the binding to PT and AnV. To reveal their specific binding, polyclonal anti-mouse and anti-rabbit IgG labeled with peroxidase were used as secondary antibodies, respectively.

Interaction of cof actors with PL

For cofactor binding to PL, human 2GPI purified from pooled plasma, purified human PT (Arotec Diagnostics, Wellington, New Zeeland), and recombinant human AnV (Diarect, Freiburg, Germany) was used.

Inhibition of aPL reactivity by CL micelles

For aPL inhibition experiments, CL micelles were employed. Briefly, aPL containing solutions were incubated with a suspension of CL micelles for 1 h at 37°C on a rotator and subsequently overnight at 4°C. After ultracentrifugation at 16000 rpm for 45 minutes, the supernatant was collected and the aPL reactivity determined in LIA.

ELISA for the detection of autoAb to CL and β2βΡΙ

For the detection of aCL and aB2GPI in the patient sera, commercially available solid-phase ELISA employing purified human B2GPI in complex with cardiolipin and human B2GPI, respectively, as solid-phase antigens were employed (GA Generic Assays GmbH, Dahlewitz, Germany). Assessment of aPL was conducted according to the instructions of the manufacturer. Sera were considered positive when their concentration exceeded the cut-off of 10 GPU or MPU for IgG and IgM, respectively.

LIA for the detection of aPL antibodies

AutoAb to CL, phosphatidic acid (aPA), phosphatidylcholine (aPC), phosphatidylethanolamine (aPE), phosphatidylglycerol (aPG), phosphatidylinositol (aPI), PS (aPS) as well as to the proteinious cofactors B2GPI, AnV and PT in patient sera were detected simultaneously. Briefly, CL, PA, PC, PE, PG, PI, PS, as well as B2GPI, AnV and PT were sprayed onto a (polyvinylidene difluoride) PVDF membrane in lines for immobilization as described for glycolipids. Processed strips were read out densitometrically employing a scanner with the evaluation software Dr. Dot Line Analyzer (GA Generic Assays GmbH).

Statistical analysis and determination of assay performance characteristics

Fisher's exact test with two-tailed probability was used to test the differences between groups. Inter-rater agreement statistics was applied for comparison of classifications. Assay performance characteristics were determined by using Medcalc statistical software (Medcalc, Mariakerke, Belgium). Intra- and inter-assay coefficients of variations (CV) were calculated.

Results:

Reactivity patterns of monoclonal aPL to cof actor proteins in LIA

To analyze the reactivity of proteinous autoantigenic targets employed in the LIA, aB2GPI mAb and polyclonal antibodies to PT and AnV were tested alone and either in the presence of the respective antigenic proteins or serum as a source thereof. As expected, aB2GPI minibody and monoclonal aB2GPI IgG (HCAL) alone reacted only with immobilized B2GPI on the surface of the membrane in LIA (Fig. 1 ). Likewise, polyclonal antibodies to PT and AnV also bound specifically with their corresponding immobilized antigens (Fig. 3).

Coincubation of the aB2GPI mAb and the minibody with serum revealed additional positive bands indicating reactivity with immobilized CL, PA, PS, PG and PI. In contrast, such additional bands were not detected for the simultaneous incubation of serum with the polyclonal aPT and aAnV (Fig. 3). The additional bands detected for the aB2GPI mAb and minibody incubation with serum could be reproduced by incubating the immobilized PL on the strips either prior or simultaneously with B2GPI (Fig. 1 ). B2GPI interacted with the immobilized negatively charged CL, PA, PS, PG and PI in a dose-dependent manner and was recognized by the aB2GPI monoclonal IgG and minibody subsequently. The latter has been reported to interact with domain 1 of the B2GPI molecule hinting at the accessibility of domain I epitopes on the PL-bound B2GPI in the novel LIA reaction environment.

In contrast, incubation of PT with the immobilized PL and cofactors followed by aPT mAb and of AnV followed by polyclonal aAnV did not reveal additional bands, indicating no reactivity of PT and AnV with the immobilized PL (Fig. 3). The addition of Ca ²⁺ ions up to a concentration of 20 mM/L did not effect this reactivity pattern.

The human monoclonal aPL RR7F (IgG) known to be reactive with several PL in ELISA were employed to investigate the reactivity of immobilized PL in LIA. It showed reactivity with CL, PA, and PS.

Coincubation of B2GPI did not interfere with this aPL reactivity and revealed additional bands for B2GPI and PG (Fig. 2). Interestingly, the reactivity of RR7F to PL could be blocked by CL micelles completely (Fig. 4). In contrast, the aB2GPI reactivity of the minibody and the HCAL were not affected by coincubation with CL micelles (Fig. 4).

Comparison of aPL testing by LIA and ELISA To detect aPL profiles and analyze possible differences in aPL detection, we tested 61 sera from patients with APS and 146 controls including 24 asymptomatic aPL+ individuals as well as disease and healthy controls by classical ELISA and novel LIA (Table 4).

Comparing both techniques for the detection of aPL regarding the positivity of at least one aPL, there was a fair agreement between both assays (Cohen's kappa = 0.594, 95% CI: 0.486 -

0.701 ; Table 3). In accordance with McNemar's test, the difference between both techniques was significant (9.6%, 95% CI: 3.23 - 14.67; p = 0.0034). This was due to a significant difference of aPL positive samples detected by LIA compared with ELISA in patients with OAPS (18/22 vs 12/22, p = 0.0313) and infectious diseases (16/50 vs 4/50, p = 0.0050). The significant difference in patients with infectious diseases was alone brought about by the high number of false-positive aAnV IgM since all patients with cytomegalovirus infections scored positive (n = 10). All other remaining control cohorts did not reveal significant differences for both methods (McNemar's test, p > 0.05, respectively).

Comparison of both methods regarding aPL recommended by the international classification criteria revealed good agreement for aB2GPI IgG and/or IgM as well as aCL IgG and/or IgM (Cohen's kappa = 0.784, 95% CI: 0.640 - 0.879; 0.720, 95% CI: 0.623 - 0.817, respectively; Table 5).

A significant difference in the study cohorts was revealed for aCL IgG and/or IgM in asymptomatic aPL+ individuals (McNemar's test, 33.33%, 95% CI: 4.55 - 40.28, p = 0.0215). This was due to a significantly higher prevalence of aCL IgG and/or IgM positive samples detected by ELISA compared with LIA in this group (17/24 vs 9/24, p = 0.0415).

Comparison of aPL testing in APS patients and controls

As expected, patients suffering from APS (n = 61 ) demonstrated significantly higher prevalences of aCL and aB2GPI IgG as well as IgM detected by ELISA compared with those in IDC and HS (p < 0.05, respectively). Comparing APS patients with VDRL+ patients via ELISA, onlz aB2GPI IgG and IgM were significantly more prevalent in APS patients (p = 0.0023, 0.0001 , respectively).

Asymptomatic aPL+ patients did not demonstrate significantly different prevalences of all aPL detected by ELISA compared with patients suffering from APS (p > 0.05, respectively).

Furthermore, even the prevalence of at least one aPL positivity detected by ELISA (aB2GPI IgG/lgM and/or aCL IgG/lgM positive) in patients with APS was not significantly different from the cohorts with asymptomatic aPL+ and VDRL+ samples.

Similar to aPL detected by ELISA, aPL positivity of at least one of the 20 different aPL analyzed by LIA (Table 4) was significantly more prevalent in patients with APS compared with IDC and HS (p < 0.0001 , respectively). In contrast to ELISA, however, the prevalence of aPL positivity by LIA was also significantly higher in APS patients compared with VDRL+ individuals (p = 0.0023).

Indeed, not only aB2GPI IgG and IgM analyzed by LIA showed a significantly lower prevalence in VDRL+ individuals compared with APS patients (p < 0.0001 , p = 0.0009, respectively) as has also been demonstrated by ELISA, but, in contrast to ELISA, also IgG to the B2GPI-binding CL analyzed by LIA did (p = 0.0004). As a fact, this result is supported by significantly lower prevalences of aPA IgG and IgM (p < 0.0001 , p = 0.001 1 , respectively), aPG IgG (p = 0.0021 ) and IgM, aPI IgG and IgM (p = 0.0001 , p = 0.0321 , respectively), as well as aPS IgG and IgM (p < 0.0001 , p = 0.001 1 , respectively) in VDRL+ individuals. All the latter aPL react with PL that bind B2GPI like CL does. Noteworthy, neither aPL to other cofactors like AnV and PT did demonstrate such a significantly elevated prevalence of their positivity in APS patients compared to VDRL+ individuals nor did aPL to PL not interacting with B2GPI such as PC and PE (p > 0.05, respectively).

Remarkably, in contrast to aPL testing by ELISA, aPL analyzed by LIA demonstrated further a significantly diminished prevalence thereof in asymptomatic aPL+ individuals compared with APS patients. Albeit the prevalence of aPL positivity of at least one of the 20 aPL analyzed by LIA was not significantly different for this cohort comparison, aCL and aPS IgG (p < 0.0001 , respectively) as well as aPI IgG (p = 0.0426) and aPG IgG and IgM (strong tendency; p = 0.05 or similar) demonstrated a higher prevalence in patients with APS in contrast to asymptomatic aPL+ individuals.

IgG to the non-B2GPI binding PE showed also a significantly higher prevalence in patients with APS (p = 0.0294). Furthermore, in contrast to ELISA analysis, aB2GPI IgG demonstrated a significantly diminished prevalence in asymptomatic aPL+ individuals, too (p = 0.0284).

Notably, LA analysis, like ELISA testing, did not reveal significant differences in the prevalence of aPL comparing APS patients with asymptomatic aPL+ individuals. Altogether, only aPL analysis by LIA discriminated APS patients from aPL+ individuals.

Comparison of aPL testing in APS patient subgroups

Comparing patients suffering from PAPS/T, OAPS, and SAPS a significantly higher prevalence of aCL determined by ELISA was found in patients with PAPS/T (27/29 vs 1 1/22, p = 0.0001 ). Further, the prevalence of at least one aPL positivity detected by ELISA (aB2GPI IgG/lgM and/or aCL IgG/lgM positive) was also significantly higher in PAPS/T patients compared to those with OAPS (27/29 vs 10/22, p = 0.0021 ). In contrast, there was no significant difference regarding the prevalence of at least one of the 20 different aPL analyzed by LIA (p > 0.05). However, aCL and aPS IgG demonstrated a significant higher prevalence in patients with PAPS/T compared to those with OAPS (p = 0.04973, 0.01 154, respectively).

Noteworthy, such significant difference in the prevalence of aPL comparing patients with PAPS/T and OAPS was not established for LA analysis.

Association of aPL with thrombosis in patients with APS

Patients demonstrating either arterial (n = 15) or venous thrombosis (n = 12) only were selected to analyze the association of aPL with thrombotic events. Thus, APS patients with thrombosis and concomitant obstetrical manifestations were excluded from the analysis. There was no significantly different prevalence of aPL determined by ELISA, LIA or LA testing regarding the type of thrombosis. However, comparison of the 27 APS patients with either venous or arterial thrombosis with patients without thrombosis and having at least one obstetrical manifestation (n = 22) revealed a significantly higher prevalence of aCL IgG and IgM detected by ELISA in the former cohort (27/27 vs 1 1/22, p < 0.0001 ; 18/27 vs 8/22, p = 0.0467; respectively). Regarding aPL testing by LIA, the same significant difference was found for aCL IgG (24/27 vs 13/22, p < 0.0212) and, interestingly, for aB2GPI IgG (22/27 vs 10/22, p = 0.0149). There was no such significant difference for LA (p < 0.05).

Association of aPL with pregnancy morbidity in patients with APS

Patients suffering from APS (n = 22) were stratified in accordance with the type of pregnancy- related complication (Table 3). Likewise, APS patients with thrombosis and concomitant obstetrical manifestations were excluded from the analysis. Detection of aPL by ELISA, LIA, and LA testing did not reveal a significantly higher prevalence of any aPL for one of the pregnancy- related complications (p < 0.05).

However, there was a significantly lower prevalence of in particular aPL IgM in several subgroups. Thus, aCL and aB2GPI IgM by ELISA was significantly less prevalent in APS patients with intrauterine growth retardation (1/9 vs 25/40, p = 0.0081 ; 1/9 vs 23/40, p = 0.0232;

respectively) and in patients with eclampsia/preemclampsia (0/6 vs 26/43, p = 0.0072; 0/6 vs 24/43, p = 0.0223; respectively). aCI IgM by ELISA was significantly less prevalent in patients with intrauterine death of fetuses and premature birth (6/14 vs 32/35, p = 0.0007; 0/5 vs 26/44, p = 0.0176; respectively).

Remarkably, this phenomenon was confirmed by LIA results. Thus, aPA, aPS and aB2GPI IgM by LIA was significantly less prevalent in APS patients with intrauterine growth retardation (0/9 vs 20/40, p = 0.0067; 1/9 vs 21/40, p = 0.0305; 1/9 vs 22/40, p = 0.0256; respectively) and aPS in patients with eclampsia/preemclampsia (0/6 vs 22/43, p = 0.0265). There was only a tendency for a reduced prevalence of aPS and aB2GPI IgM by LIA in patients with premature birth (0/5 vs 22/22, p = 0.0561 ; 0/5 vs 23/44, p = 0.0521 ; respectively). In contrast to ELISA, patients with intrauterine death of fetuses demonstrated a significantly reduced prevalence of IgG to PS and a tendency for aB2GPI IgG (6/14 vs 29/35, p = 0.01 17; 6/14 vs 26/35, p = 0.0505; respectively). Furthermore, there was a significantly reduced prevalence of aPA and aB2GPI IgG in patients suffering from early pregnancy loss (2/1 1 vs 23/38, p = 0.0181 ; 4/1 1 vs 28/38, p = 0.0330;

respectively)

In contrast to aPL analysis by ELISA and LIA, LA testing did not reveal significant differences in APS patients with pregnancy morbidity (p > 0.05, respectively).

Summary of Example 2:

The persistent presence of aPL is the serological hallmark of APS and represents one of the mandatory classification criteria of APS. Furthermore, the detection of aPL belongs to the classification criteria of SLE, thus APS can occur as a secondary manifestation. Approximately 20% of patients under the age of 50 years with stroke or venous thromboembolism are diagnosed with APS. It is a commonly accepted consensus that APS-specific circulating aPL interact with plasma cofactors such as in particular 2GPI interacting with negatively charged PL.

Furthermore, cofactor-PL complexes like PT/PS and high molecular weight kininogen/PE have been reported as aPL targets. This supports the concept of aPL profiling for the risk assessment of thromboembolic complications in patients with APS rather than single aPL analysis for diagnostic purposes alone.

Risk stratification is a major challenge in treating patients with APS and a potential role of aPL as risk or even prognostic factors for arterial/venous thrombosis and miscarriages is debated intensively. In general, ELISA and a functional coagulation assay to detect the so called LA are employed to test for aPL in routine clinical diagnostics. In this context, LA positivity seems to be the best predictor of clinical manifestations in APS, whereas medium/high levels of IgG to CL and β2ΘΡΙ are more indicative than low levels thereof and IgM.

However, 1 to 5% of healthy individuals demonstrate circulating aPL detectable with the currently recommended aPL assays. This raises the question of pathogenic aPL and their appropriate analysis. Recent data suggests that epitopes on domain I of the β2ΘΡΙ molecule which are better exposed after the interaction with anionic PL or bacterial proteins like protein H of Streptococcus pyogenes generate the main targets for pathogenic aPL. Thus, assay techniques addressing the aforementioned requirements play a pivotal role in the analysis of pathogenic aPL.

Thus, example 2 of the present invention provides data using a LIA employing a novel hydrophobic solid phase for the simultaneous detection of multiple aPL in a well-defined cohort of 61 APS patients with thrombotic and obstetrical manifestations and 146 controls including in particular 24 aPL+ subjects. In this study evaluating at least 1 positive aPL, the LIA demonstrated a sensitivity of 86.9% compared to 75.4% by ELISA and 85.2% by LA analysis.

In contrast to a planar ELISA-solid phase, the porous hydrophobic membrane used in LIA is assumed to incorporate the hydrophobic PL tail. This shields the by far larger tail of the amphiphatic PL molecule from the reaction environment and, thus, prevents unspecific interactions. Furthermore, a high density of the hydrophilic 2GPI-binding site on the LIA membrane mimicking the native structure of PL in the cellular lipid bilayer is brought about. Of note, the human mAb RR-7F interacting with anionic PL only in ELISA also bound readily immobilized anionic PL in the investigated LIA. This reactivity was completely inhibited by CL micelles that expose only hydrophilic CL heads on their surface in aqueous solutions.

Consequently, this confirms the interaction of RR-7F with the hydrophilic heads of PL on the PVDF membrane.

Investigating the interaction of cofactors with the PL heads at high-density on the LIA membrane, the human a 2GPI minibody recognizing an epitope on the domain I of β2ΘΡΙ readily detected its immobilized target on the PVDF membrane. This demonstrates the accessibility of this important pathogenic epitope-bearing domain in the LIA reaction environment. Interestingly, the addition of serum or purified β2ΘΡΙ to the minibody revealed different binding characteristics of the immobilized anionic PL to this cofactor in the novel reaction environment. Diphosphatidylglycerol, also referred to as CL, binds β2ΘΡΙ better than its monomeric variant PG. Otherwise, PS bearing only one phosphatic group has a better binding than PI or PG. This supports the assumption that the number, orientation, and accessibility of anionic phosphatic groups in the hydrophilic PL heads determine the binding of 2GPI and most importantly the conformation thereof. Of note, we could not determine the binding of either serum or purified PT with immobilized anionic PL, particularly with PS, in the multiplex LIA environment as shown for β2ΘΡΙ. Even in the presence of Ca ⁺² ions which are required for the aPT/PS ELISA reaction environment, no binding was detected. Of note, there was a significant prevalence difference in aPT and aPS IgG (p < 0.0001 ) as well as IgM-positive APS patients (p = 0.0017) by LIA.

Regarding aPL analysis by LIA, the novelty of this aPL assay is the binding of the patient's own serum β2ΘΡΙ to the immobilized PL. This is in contrast to the ELISA reaction environment where purified β2ΘΡΙ from pooled human serum is employed as autoantigenic target for aPL analysis in general. Growing evidence indicates a conformational change of the circular β2ΘΡΙ in the serum binding to membrane-bound targets which leads to the exposure of hidden, most probably immunodominant, epitopes in particular on domain I. Patients with APS bearing aPL to domain I seem to have a higher risk of developing thromboembolic events and asymptomatic aPL+ subjects lack a polarized profile toward domain I reactivity in the framework of aPL profiling to β2ΘΡΙ domains. After binding of β2ΘΡΙ to negatively charged surfaces like immobilized anionic PL by domain V, domain I forms the top of the induced fish-hook like β2ΘΡΙ structure

predisposed to interact with aPL. Given the pathogenic characteristics and LA activity of the 8β2ΘΡΙ minibody, its strong interaction with such PL-bound β2ΘΡΙ in the LIA supports this assumption. Indeed, there is a significant difference in the analysis of aPL binding to anionic Ρί/β2ΘΡΙ complexes in LIA compared with ELISA in VDRL+ patients and in particular in asymptomatic aPL+ individuals. Thus, the significantly lower prevalence of aPL detected by LIA in the latter cohort indicates a more specific detection of pathogenic aPL by LIA. Remarkably, aPL analysis by LA testing and ELISA did not reveal such significant differences which suggests the detection of aPL to non-pathogenic β2ΘΡΙ epitopes by these methods. Since the aPL reactivity to domains IV and V in ELISA has been reported not to be associated with

thromboembolism our data hint at probably less accessibility of those domains in LIA. Assuming a high density of the hydrophilic PL heads on the LIA membrane, membrane-close β2ΘΡΙ domains like IV and V could be indeed covered due the conformational change and the high density of β2ΘΡΙ both induced by PL binding on the membrane. The varying aPL reactivity detected in LIA could be explained either by the different level of β2ΘΡΙ binding to the immobilized PL or by conformational differences in the fish-hook like structure of β2ΘΡΙ interacting with differently shaped PL heads.

To the best of the inventor's knowledge, this is the first disclosure reporting a diagnostic assay discriminating patients with APS from asymptomatic bearers of aPL. Both the ELISA and LIA data confirm the association of aCL IgG with thrombotic events in patients with APS. Another interesting result is the diminished prevalence of several aPL IgM in APS patients with obstetrical manifestations. In conclusion, aPL detection by LIA is an effective tool with a novel reaction environment for the detection of pathogenic aPL which can be employed for the differentiation of patients with APS from asymptomatic individuals with aPL. Furthermore, this novel assay will aid in evaluating aPL profiling for risk analysis of APS manifestations. Table 1 : aPL antibody testing by enzyme-linked immunosorbent assays (ELISA) and line immunoassay LIA in 15 patients with antiphospholipid syndrome (APS), 15 individuals with positive antiphospholipid antibody detected by ELISA (+aPL) and 15 blood donors (BD). Abbreviations: aB2GPI, antibeta2-glycoprotein I; aCL, anticardiolipin; aPA, antiphosphatidic acid; aPC, antiphosphatidylcholine; aPE, anti phosphatidylethanolamine; aPG, antiphosphatidylglycerol; aPI, antiphosphatidylinositol; aPS, antiphosphatidylserine; aAnV, anti annexin V; aPT, antiprothrombin.

Table 2: Contingency table of aPL antibody testing by enzyme-linked immunosorbent assays (ELISA) and line immunoassay LIA in 15 patients with antiphospholipid syndrome (APS), 15 individuals with positive antiphospholipid antibody detected by ELISA (+aPL) and 15 blood donors (BD).

Table 3: Characteristics of 61 patients with antiphospholipid syndrome and 146 controls enrolled in the study

HS, healthy subjects; IDC, infectious diseases controls; OAPS, obstretic antiphospholipid syndrome; PAPS, primary antiphospholipid syndrome; SAPS, secondary antiphospholipid syndrome; VDRL+, Venereal Disease Research Laboratory test positive

* obstetric patients may have more than one clinical manifestation indicated Table 4: Antiphospholipid antibody (aPL) positive sera tested by enzyme-linked immunosorbent immunoassay (ELISA) and line immunoassay (LIA) in 61 patients with antiphospholipid syndrome (APS) and 146 controls

^* significant aPL prevalence difference of APS patients (n = 61 ) with the respective control cohort, p < 0.05

^** significant aPL prevalence difference of APS patients (n = 61 ) with the respective control cohort, p < 0.0001

* ^** strong tendency for aPL prevalence difference of APS patients (n = 61 ) with the respective control cohort, p = 0.05 or similar

^§ aPL prevalence comparison of PAPS/T patients (n = 29) with SAPS or OAPS patients, p < 0.05

aAnV, antiannexin V; aB2GPI, antibeta2-glycoprotein I; aCL, anticardiolipin; aPA, antiphosphatidic acid; aPC, antiphosphatidylcholine; aPE, antiphosphatidylethanolamine; aPG, antiphosphatidylglycerol; aPI, antiphosphatidylinositol; aPL+, asymptomatic patients with autoantibodies to phospholipids; aPS, antiphosphatidylserine; aPT, antiprothrombin; HS, healthy subjects; IDC, infectious diseases controls; LA, lupus anticoagulant; nd, not determined; OAPS, obstetric antiphospholipid syndrome; PAPS, primary antiphospholipid syndrome; PAPST, primary antiphospholipid syndrome with thrombotic events; PAPS/TO, primary antiphospholipid syndrome with thrombotic and obstetric manifestations; SAPS, secondary antiphospholipid syndrome; VDRL+, Venereal Disease Research Laboratory test-positive.

Table 5: Comparison of antiphospholipid antibody (aPL) testing by line immunoassay (LIA) and enzyme-linked immunosorbent assay (ELISA) in patients with APS and controls.

ELISA LIA Difference 95% CI P ^*

[%]

APS+controls

n = 207 Pos neg

1 aPL pos pos 72 11 9.66 3.23 - 14.67 0.003 neg 31 93 aCL pos 62 21 7.25 2.02 - 10.79 0.007 neg 6 118

3β2ϋΡΙ pos 49 10 0.97 -3.35 - 4.95 0.814 neg 8 140

PAPS

n = 34 Pos neg 1 aPL pos pos 29 2 0.00 -9.81 -9.81 1.000 neg 2 1 aCL pos 28 3 5.88 6.98-10.80 0.625 neg 1 2

3β2ϋΡΙ pos 24 1 8.82 -6.23-13.73 0.375 neg 4 5

OAPS

n = 22 pos neg

1 aPL pos pos 12 0 27.27 2.21 -27.27 0.031 neg 6 4 aCL pos 12 3 13.64 -5.32-13.64 0.250 neg 0 7

3β2ϋΡΙ pos 11 3 13.64 -5.32-13.64 0.250 neg 0 8

+aPL

n = 24 pos neg

1 aPL pos pos 15 2 0.00 13.90-13.90 1.000 neg 2 5 aCL pos 8 9 33.33 4.55-40.28 0.022 neg 1 6

3β2ϋΡΙ pos 11 3 4.17 -14.42-18.13 1.000 neg 2 8

IDC

n = 50 pos neg

1 aPL pos pos 14 2 24.00 7.46-31.01 0.004 neg 2 32 aCL pos 0 4 8.00 1.56 - 8.00 0.125 neg 0 46

3β2ϋΡΙ pos 0 0 2.00 -1.34 - 2.00 1.000 neg 1 46

VDRL+

n = 23 pos neg

1 aPL pos pos 12 3 13.04 -5.09 - 13.04 0.250 neg 0 8 aCL pos 11 1 4.35 -10.03 - 1 1.62 1.000 neg 2 9

3β2ϋΡΙ pos 1 1 0.00 -7.28 - 7.28 1.000 neg 1 20

HS

n = 49 pos neg

1 aPL pos pos 0 2 8.16 -4.88 - 15.04 0.289 neg 6 41 aCL pos 0 2 0.00 -6.81 - 6.81 1.000 neg 2 45

3β2ϋΡΙ pos 0 0 np np np neg 0 49

^* McNemar test

aB2GPI, antibeta2-glycoprotein I; aCL, anticardiolipin; aPL+, asymptomatic patients with autoantibodies to phospholipids; HS, healthy subjects; IDC, infectious diseases controls; np, not performable; OAPS, obstetric antiphospholipid syndrome; PAPS, primary antiphospholipid syndrome; VDRL+, Venereal Disease Research Laboratory test-positive. Reference List

1. Miyakis S, Lockshin MD, Atsumi T et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4: 295-306.

2. Roggenbuck D, Egerer K, von Landenberg P et al. Antiphospholipid antibody profiling - Time for a new technical approach. Autoimmun Rev 2012; 1 1 : 821-6.

3. Otomo K, Atsumi T, Amengual O et al. Efficacy of the antiphospholipid score for the

diagnosis of antiphospholipid syndrome and its predictive value for thrombotic events. Arthritis Rheum 2012; 64: 504-12.

4. Roggenbuck D, Egerer K, Feist E, Burmester GR, Dorner T. Antiphospholipid antibody profiling: association with the clinical phenotype of antiphospholipid syndrome? Comment on the article by Otomo et al. Arthritis Rheum 2012; 64: 2807-8.

5. Egerer K, Roggenbuck D, Buettner T et al. Single-step autoantibody profiling in

antiphospholipid syndrome using a multi-line dot assay. Arthritis Res Ther 201 1 ; 13: R1 18.

6. Nalli C, Andreoli L, Casu C, Tincani A. Management of recurrent thrombosis in

antiphospholipid syndrome. Curr Rheumatol Rep 2014; 16: 405.

7. Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: understanding the antibodies. Nat Rev Rheumatol 201 1 ; 7: 330-9.

8. Andreoli L, Tincani A. Beyond the "Syndrome": antiphospholipid antibodies as risk factors.

Arthritis Rheum 2012; 64: 342-5.

9. Pengo V, Banzato A, Denas G et al. Correct laboratory approach to APS diagnosis and monitoring. Autoimmun Rei 2013; 12: 832-4.

10. Conrad K, Schneider H, Ziemssen T et al. A new line immunoassay for the multiparametric detection of antiganglioside autoantibodies in patients with autoimmune peripheral neuropathies. Ann N Y Acad Sci 2007; 1 109: 256-64.