Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
  • G01N33/54388—Immunochromatographic test strips based on lateral flow
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the invention relates to means for the prognosis and/or diagnosis of antiphospholipid syndrome (APS).
• APS antiphospholipid syndrome
• 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.
• aPL antiphospholipid antibodies
• 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
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• PG preferably at least PG
• 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).
• APS antiphospholipid syndrome
• 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.
• aPL antiphospholipid antibodies
• 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.
• SLE systemic lupus erythematosus
• diphosphatidylglycerol such as phosphatidylserine (PS)
• PS phosphatidylserine
• 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.
• APS 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
• 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
• ELISAs enzyme-linked immunosorbent assays
• LA lupus anticoagulant
• beta 2 glycoprotein I ( ⁇ 2 ⁇ ) is the clinically most relevant and studied autoantigenic target of aPL. 7
• 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.
• PT prothrombin
• PE phosphatidylethanolamine
• aCL and anti- ⁇ 2 ⁇ assays detect aPL to ⁇ 2 ⁇ mainly.
• Recent data suggest that pathogenic aPL mainly recognize an immunodominant epitope on domain I which is better exposed after the ⁇ 2 ⁇ interaction with negatively charged phospholipids or oxygenated solid-phases employed in state- of-the-art aPL ELISA.
• 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.
• 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
• probably most important infections can provide the required triggers for a second hit.
• current techniques included in the classification criteria have not allowed differentiating aPL in patients with APS and those in individuals
• 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.
• 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
• 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 for use in a multi-dot and/or multi-line immunoassay.
• 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 ( ⁇ 2 ⁇ ).
• 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).
• APS antiphospholipid syndrome
• 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
• PTFE polytetrafluorethylene
• PL phospholipids
• PG phosphatidylglycerol
• 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.
• 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.
• 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.
• PL in particular PG
• 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.
• 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.
• polystyrene as a hydrophobic material is excluded from the invention.
• polypropylene as a hydrophobic material is excluded from the invention.
• 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.
• 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 PVDF
• PVDF polyvinylidene difluoride
• PTFE polytetrafluorethylene
• hydrophobic membrane wherein said phospholipids (PL) comprise at least phosphatidylglycerol (PG).
• PL phospholipids
• PG phosphatidylglycerol
• 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.
• 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.
• phospholipids coated onto hydrophobic membranes seem to present ⁇ 2 ⁇ in conformational changes similar to those occurring on the oxygenated polysterene surface of ELISA.
• 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 ⁇ 2 ⁇ and eventually exposing the immunodominant epitope on domain I.
• the solid phase as described herein is characterized in that phosphatidylserine (PS) is immobilized to the hydrophobic membrane.
• PS phosphatidylserine
• the solid phase as described herein is characterized in that prothrombin (PT) is immobilized to the hydrophobic membrane.
• PT prothrombin
• the solid phase as described herein is characterized in that phosphatidylinositol (PI) is immobilized to the hydrophobic membrane.
• PI phosphatidylinositol
• the solid phase as described herein is characterized in that phosphatidic acid (PA) is immobilized to the hydrophobic membrane.
• PA phosphatidic acid
• the solid phase as described herein is characterized in that phosphatidylethanolamine (PE) is immobilized to the hydrophobic membrane.
• PE phosphatidylethanolamine
• 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.
• 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.
• the solid phase as described herein is characterized in that wherein cardiolipin (CL) is immobilized to the hydrophobic membrane.
• the solid phase as described herein is characterized in that beta2-glycoprotein I (B2GPI) is immobilized to the hydrophobic membrane.
• B2GPI beta2-glycoprotein I
• the solid phase as described herein is characterized in that phosphatic acid (PA) is immobilized to the hydrophobic membrane.
• PA phosphatic acid
• 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.
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• C cardiolipin
• B2GPI beta2-glycoprotein I
• phosphatidylglycerol phosphatidylinositol
• PI phosphatidylinositol
• PS phosphatidylserine
• PA phosphatidic acid
• PT prothrombin
• C cardiolipin
• B2GPI beta2-glycoprotein I
• 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.
• 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.
• 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.
• 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.
• a 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.
• 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.
• 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.
• 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
• kits comprising a solid phase as described herein.
• the kit of the present invention comprises human anti-lgG or anti-lgM antibodies for the detection of antibodies bound from a patient sample.
• human anti-lgG or anti-lgM antibodies for the detection of antibodies bound from a patient sample.
• the kit of the present invention comprises human anti-lgG and anti-lgM antibodies for the detection of antibodies bound from a patient sample.
• 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.
• an enzyme such as horseradish peroxidase or alkaline phosphatase
• 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).
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• the invention therefore provides means for risk stratification for subjects exhibiting aPL.
• 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.
• 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.
• the method of the present invention comprises: a Obtaining or providing a sample from a subject, preferably a blood or serum sample;
• 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);
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• PL phospholipids
• aPL directed against one or more of said PL is indicative of APS, or increased risk of APS occurring, in the subject.
• 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.
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• 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.
• CL cardiolipin
• B2GPI beta2-glycoprotein I
• 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.
• the method of the present invention comprises the diagnosis or prognosis of APS.
• the method of the present invention is characterised in that the antibodies are autoantibodies directed against the corresponding PL epitopes.
• the method comprises the determination of IgG and/or IgM antibodies.
• the antibodies are one or more of:
• aPG IgG and/or IgM antiphosphatidylglycerol antibodies
• IgG and/or IgM antiphosphatidylserine antibodies aPS
• IgG and/or IgM antiprothrombin antibodies aPT
• IgG and/or IgM antiphosphatidic acid antibodies e. IgG and/or IgM antiphosphatidic acid antibodies (aPA).
• 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.
• aPL antiphospholipid antibodies
• 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.
• infectious diseases for example Venereal diseases
• 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).
• 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).
• the present invention enables the differentiation between subjects with APS combined with pregnancy manifestations and those APS, or +aPL.
• 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).
• 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:
• 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);
• PG phosphatidylglycerol
• PI phosphatidylinositol
• PS phosphatidylserine
• PT prothrombin
• PL phospholipids
• Antiphospholipid syndrome (APS or APLS), or Hughes syndrome, is an autoimmune,
• 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.
• blood clots thrombosis
• pregnancy-related complications such as miscarriage, stillbirth, preterm delivery, and severe preeclampsia.
• aPL antiphospholipid antibodies
• Antiphospholipid syndrome can cause symptoms such as arterial or venous blood clots, in any organ system, or pregnancy-related complications.
• APS patients the most common venous event is deep vein thrombosis of the lower extremities, and the most common arterial event is stroke.
• 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.
• 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.
• 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.
• PVDF polyvinylidene fluoride
• PVDF polyvinylidene difluoride
• 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.
• PTFE Polytetrafluoroethylene
• 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).
• 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.
• 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.
• 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.
• the results of a statistical algorithm e.g., a learning statistical classifier system
• 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.
• 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.
• 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.
• an immunoassay assay is employed in the detection of antibodies, to which end binding of the PL antigen to a solid phase is envisaged.
• 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.
• 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.
• the enzymes catalyzing a chemical reaction which can be determined by optical means, especially by means of chromogenic substrates, chemiluminescent methods or fluorescent dyes.
• 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)).
• 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.
• EIA enzyme multiplied immunoassay technique
• ELISA enzyme-linked immunosorbent assay
• MAC ELISA enzyme-linked immunosorbent assay
• MEIA microparticle enzyme immunoassay
• CEIA capillary electrophoresis immunoassays
• RIA radioimm
• solid phase-bound PL molecules are used to bind the antibodies.
• 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.
• 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).
• 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) detectably coupled to fluorescein isothiocyanate (FITC).
• FITC fluorescein isothiocyanate
• 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.
• 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
• mAb HCAL 0.02 mg/L
• serum ⁇ 2 ⁇
• BSA bovine serum albumin
• a 1 0.1 mg/L aB2GPI minibody
• 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).
• 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:
• FIG. 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:
• 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).
• 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) .
• LIA and ELISA demonstrated significant higher prevalences of aPL IgG and/or IgM in APS patients compared to BD (P ⁇ 0.001 , respectively).
• 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.
• a chimeric IgG monoclonal antibody (mAb) HCAL was employed, comprising human ⁇ and ⁇ constant regions and variable regions from the mouse monoclonal 2GPI-dependent anti-CL WBCAL-1.
• mAb monoclonal antibody
• 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.
• aPT anti-PT
• AAnV anti-AnV
• polyclonal anti-mouse and anti-rabbit IgG labeled with peroxidase were used as secondary antibodies, respectively.
• 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.
• 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.
• aB2GPI minibody and monoclonal aB2GPI IgG (HCAL) alone reacted only with immobilized B2GPI on the surface of the membrane in LIA (Fig. 1 ).
• polyclonal antibodies to PT and AnV also bound specifically with their corresponding immobilized antigens (Fig. 3).
• 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.
• 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).
• aPL positivity of at least one of the 20 different aPL analyzed by LIA was significantly more prevalent in patients with APS compared with IDC and HS (p ⁇ 0.0001 , respectively).
• aPL analyzed by LIA demonstrated further a significantly diminished prevalence thereof in asymptomatic aPL+ individuals compared with APS patients.
• aCL and aPS IgG p ⁇ 0.0001 , respectively
• aPI IgG 0.0426
• 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.
• LA testing did not reveal significant differences in APS patients with pregnancy morbidity (p > 0.05, respectively).
• APS 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.
• LA positivity seems to be the best predictor of clinical manifestations in APS, whereas medium/high levels of IgG to CL and ⁇ 2 ⁇ are more indicative than low levels thereof and IgM.
• 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.
• the LIA demonstrated a sensitivity of 86.9% compared to 75.4% by ELISA and 85.2% by LA analysis.
• 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.
• 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.
• the human a 2GPI minibody recognizing an epitope on the domain I of ⁇ 2 ⁇ 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.
• the addition of serum or purified ⁇ 2 ⁇ 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 ⁇ 2 ⁇ better than its monomeric variant PG. Otherwise, PS bearing only one phosphatic group has a better binding than PI or PG.
• the novelty of this aPL assay is the binding of the patient's own serum ⁇ 2 ⁇ to the immobilized PL. This is in contrast to the ELISA reaction environment where purified ⁇ 2 ⁇ from pooled human serum is employed as autoantigenic target for aPL analysis in general.
• Growing evidence indicates a conformational change of the circular ⁇ 2 ⁇ in the serum binding to membrane-bound targets which leads to the exposure of hidden, most probably immunodominant, epitopes in particular on domain I.
• 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).
• ELISA enzyme-linked immunosorbent assays
• APS antiphospholipid syndrome
• BD blood donors
• aB2GPI antibeta2-glycoprotein I
• aCL anticardiolipin
• aPA antiphosphatidic acid
• aPC antiphosphatidylcholine
• aPE antiphosphatidylethanolamine
• 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).
• ELISA enzyme-linked immunosorbent assays
• APS antiphospholipid syndrome
• BD blood donors
• 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
• 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+ Ven
• 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
• 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.

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