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A LOCI Based Automated Immunoassay for the Diagnosis of Anti-Cardiolipin Antibodies

IP.com Disclosure Number: IPCOM000201302D
Original Publication Date: 2010-Nov-10
Included in the Prior Art Database: 2010-Nov-10
Document File: 2 page(s) / 111K

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Abstract

Anti-phospholipid antibodies including anti-cardiolipin antibodies are frequently detected in sera from patients with an autoimmune disease such as systemic lupus erythematosus. These autoantibodies have been associated with various venous and arterial thrombotic disorders, and are causative for the disease AntiPhospholipid Syndrome (APS), which is associated with thrombosis and fetal loss. More recently, these autoantibodies have been shown to bind to cardiolipin in combination with a protein cofactor, beta 2 Glycoprotein I (β2GP1). β2GP1 is known to bind to anionic phospolipids such as cardiolipin, hence β2GP1 binding to a surface is indicative for the presence of biochemically active cardiolipin. Heterogenous assays for the detection of antibodies to cardiolipin have already been commercialized, but so far no homogenous assay format has been described. However, there are three methods described in literature for the immobilization of cardiolipin on a solid support. These methods include coupling of selenium dioxide oxidized cardiolipin onto polystyrene microtiter plates, an attachment of a synthetic amino group-containing derivative of cardiolipin onto a carboxy-terminated solid support and coupling of periodate-permanganate oxidized cardiolipin onto amino group-containing molecules. In addition, a technique of coupling a succinyl derivative of cardiolipin onto chymotrypsin followed by liposome preparation is known.

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A LOCI Based Automated Immunoassay for the Diagnosis of Anti-Cardiolipin Antibodies

Idea: Pratap Singh, US-Newark, Delaware; Dr. Andreas Kappel, DE-Marburg

Anti-phospholipid antibodies including anti-cardiolipin antibodies are frequently detected in sera from patients with an autoimmune disease such as systemic lupus erythematosus. These autoantibodies have been associated with various venous and arterial thrombotic disorders, and are causative for the disease AntiPhospholipid Syndrome (APS), which is associated with thrombosis and fetal loss. More recently, these autoantibodies have been shown to bind to cardiolipin in combination with a protein cofactor, beta 2 Glycoprotein I (β2GP1). β2GP1 is known to bind to anionic phospolipids such as cardiolipin, hence β2GP1 binding to a surface is indicative for the presence of biochemically active cardiolipin.

Heterogenous assays for the detection of antibodies to cardiolipin have already been commercialized, but so far no homogenous assay format has been described. However, there are three methods described in literature for the immobilization of cardiolipin on a solid support. These methods include coupling of selenium dioxide oxidized cardiolipin onto polystyrene microtiter plates, an attachment of a synthetic amino group-containing derivative of cardiolipin onto a carboxy-terminated solid support and coupling of periodate-permanganate oxidized cardiolipin onto amino group-containing molecules. In addition, a technique of coupling a succinyl derivative of cardiolipin onto chymotrypsin followed by liposome preparation is known.

In the following a Luminescent Oxygen Channeling Immunoassay (LOCI) based assay is being proposed for the detection of autoantibodies to cardiolipin. For this purpose cardiolipin is immobilized onto chemibeads, and antibodies binding from a sample are detected via anti-Immunoglobulin G (IgG) or anti-Immunoglobulin M (IgM).

It is proposed to couple permanganate-oxidized cardiolipin onto ethylenediamine-modified EPRM in presence of EDC and N-hydroxysuccinimide. The resulting chemibeads show a binding to β2GP1 and furthermore allow for a discrimination of positive and negative samples containing anti-cardiolipin IgG and anti-cardiolipin IgM. The proposed procedure to couple cardiolipin onto EPRM comprises of three steps:

Preparation of EDA-EPRM: EPRM (PN 781000.301; 10 mL; 100 mg/mL) is mixed with a 10 mL solution of ethylenediamine (EDA). EDA solution is prepared by adding 0.8 mL EDA to 8 mL water containing 200 mg sodium dihydrogen phosphate, adjusting pH to 6.0 and diluting with water to a total volume of 10 mL. This light-protected mixture is shaken at room temperature for 45 min and then mixed with 4 mL of a freshly prepared solution of sodium cyanoborohydride (100 mg/mL water). Combined reaction mixture is placed in a 37°C shaker- incubator...