Oxidized cardiolipin and uses to detect cardiolipin antibodies
Inventors
Castro, Arnold R. • Mody, Himanshu Champaklal
Assignees
Arlington Scientific Inc • US Department of Health and Human Services
Publication Number
US-9081009-B2
Publication Date
2015-07-14
Expiration Date
2026-11-17
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Compositions, methods and devices for the detection of anti-lipoidal antibodies and the diagnosis of disease, for example, syphilis, are described. In particular, oxidized cardiolipins, which may be conjugated with a variety of attachment molecules, such as BSA, KLH, biotin, synthetic protein MAPS, IgY, streptavidin, or avidin, are described. Such oxidized cardiolipin, alone or complexed with one or more attachment molecules, are useful to detect anti-lipoidal antibodies (such as IgG and IgM antibodies) in subjects, for example, when used in ELISA plates. ELISA plates are described that permit the detection of anti-lipoidal antibodies and that permit the co-detection of nontreponemal and treponemal antibodies in biological samples.
Core Innovation
The invention provides oxidized cardiolipin compositions and methods for immobilizing oxidized cardiolipin to solid supports, enabling immunoassays such as ELISA to detect anti-lipoidal antibodies (IgG and IgM) and facilitate diagnosis of diseases like syphilis. The oxidized cardiolipin may be conjugated to various attachment molecules (e.g., BSA, KLH, IgY, avidin) or directly attached to solid substrates with amine groups, thereby maintaining immunogenicity and specificity for anti-lipoidal antibodies.
A central aspect of the invention is the method of oxidizing cardiolipin by reacting it with a periodate salt and a permanganate salt to oxidize the fatty acid side chains to terminal carboxyl groups, followed by quenching oxidation with a reducing agent, preserving the β-hydroxyl group in the central glycerol moiety. Activated carboxyl groups on oxidized cardiolipin are then covalently attached to protein carriers or amine groups on solid supports, allowing stable, antigenically active immobilization for use in immunoassays.
The problem solved arises from the difficulty in attaching cardiolipin, a hydrophobic and small molecule antigen, to solid substrates for immunoassays to detect anti-lipoidal antibodies. Traditional non-treponemal tests rely on naturally occurring cardiolipin antigen suspensions and have limitations including poor binding to polar surfaces and loss of antigenicity when conjugated to larger molecules. This invention overcomes these issues by chemically modifying cardiolipin to introduce reactive groups for covalent conjugation without altering the antigenic epitope.
Claims Coverage
The patent includes independent claims directed to kits for diagnosing syphilis comprising multi-well plates with oxidized cardiolipin immobilized via specific oxidation and conjugation methods, as well as components for antibody detection.
Oxidized cardiolipin preparation and immobilization on multi-well plates
Oxidized cardiolipin is generated by reacting cardiolipin with a periodate salt and a permanganate salt to oxidize alkene groups in fatty acid side chains to terminal carboxyl groups, quenching oxidation with a reducing agent to retain immunogenicity of the central glycerol moiety, followed by activation of carboxyl groups to permit covalent attachment to amine molecules on solid supports such as multi-well plates.
Composition of kits including assay reagents and controls
Kits comprise multi-well plates with amine molecules covalently attached to oxidized cardiolipin prepared as above, labeled anti-human IgM, optional controls (negative and positive sera, labeled anti-human IgG), wash and dilution buffers, and optional calibration curves for non-treponemal IgG and/or IgM.
Use of carbodiimide and N-hydroxysuccinimide (NHS) for carboxyl activation
Activation of terminal carboxyl groups of oxidized cardiolipin is performed by reacting with a carbodiimide such as 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC), optionally followed by reaction with N-hydroxysuccinimide (NHS) to form amine-reactive esters for conjugation.
Optimum conditions for oxidation and immobilization
The oxidation is performed in an alcohol solvent, preferably t-butanol, under an argon atmosphere, with specific molar ratios of sodium m-periodate to cardiolipin (about 4:1 to 5:1) and potassium permanganate to cardiolipin (about 0.5:1 to 1:1), and sodium bisulfite as the reducing agent to quench oxidation and restore β-hydroxyl groups.
Optionally including treponemal antigen for concurrent antibody detection
The multi-well plates can further include treponemal antigens capable of reacting with antibodies to Treponema pallidum to allow concurrent detection of non-treponemal and treponemal antibodies in the same device.
The claims focus on kits comprising multi-well plates with oxidized cardiolipin covalently attached via controlled oxidation and conjugation processes, including assay reagents for detection of anti-lipoidal antibodies and optional components enabling combined non-treponemal and treponemal antibody detection for syphilis diagnosis.
Stated Advantages
Permits automated and objective detection of non-treponemal antibodies with reduced interpretational error compared to conventional methods.
Enables quantitative detection of nontreponemal IgG and IgM antibodies, which is not available in commercial enzyme immunoassays.
Facilitates attachment of immundgenically active cardiolipin to solid supports, overcoming previous challenges due to cardiolipin’s small size and hydrophobicity.
Allows development of immunoassay devices (e.g., ELISA plates, lateral flow devices) capable of concurrently detecting both non-treponemal and treponemal antibodies rapidly and on-site.
Supports monitoring of syphilis treatment efficacy and determination of disease stage through differential detection of antibody isotypes.
Documented Applications
Detection of anti-lipoidal antibodies (IgG and IgM) for diagnosis of syphilis infection.
Concurrent detection of non-treponemal and treponemal antibodies in biological samples for improved syphilis screening and diagnosis.
Monitoring efficacy of syphilis therapy via quantification of antibody levels.
Determining the stage of syphilis infection through isotype-specific antibody detection.
Immunoassays for detecting anti-cardiolipin autoimmune diseases, such as lupus, particularly with the use of cofactors like β2-glycoprotein I.
Interested in licensing this patent?