Modified cardiolipin and uses therefor
Inventors
Castro, Arnold R. • Wang, Huiying
Assignees
Centers of Disease Control and Prevention CDC • US Department of Health and Human Services
Publication Number
US-7888043-B2
Publication Date
2011-02-15
Expiration Date
2026-11-17
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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 in subjects, for example, when used in lateral flow devices. Lateral flow devices 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 compositions, methods, and devices for the detection of anti-lipoidal antibodies and the diagnosis of diseases such as syphilis. It focuses particularly on oxidized cardiolipins, which are modified to present terminal carboxyl groups on fatty acid side chains, enabling their conjugation with attachment molecules like BSA, KLH, biotin, among others. This conjugation facilitates the immobilization of cardiolipin on solid supports like microporous membranes, allowing for reliable use in immunoassays and immunoassay devices, including ELISA and lateral flow devices. The ability to maintain antigenicity and specificity of cardiolipin toward anti-lipoidal antibodies despite these modifications is a key aspect.
The problem being solved arises from the difficulty of attaching hydrophobic cardiolipin molecules to solid substrates for immunoassays, due to their small size and hydrophobic fatty acid side chains. Traditional usage of cardiolipin in non-treponemal syphilis tests involves natural cardiolipin, cholesterol, and lecithin in solution-based assays, which require laboratory processing and delay diagnosis. The challenge of developing point-of-care devices for detecting anti-lipoidal antibodies stems from cardiolipin's poor binding to polar surfaces such as nitrocellulose and the loss of antigenicity upon conjugation to larger molecules.
The invention addresses these issues by oxidizing the fatty acid side chains of cardiolipin to introduce terminal carboxyl groups, enabling covalent conjugation to polypeptide carriers that readily attach to solid supports. This modification retains the immunogenic glycerol moiety of cardiolipin, preserving its antigenicity. The oxidized cardiolipin-attachment molecule complexes can be immobilized on solid phases and used in immunoassays and immunoassay devices that provide rapid, on-site detection of anti-lipoidal antibodies, with embodiments also enabling concurrent detection of treponemal antibodies for syphilis diagnosis.
Claims Coverage
The patent contains one independent claim defining a method for preparing oxidized cardiolipin for immunoreactivity and conjugation to proteins.
Preparation of oxidized cardiolipin with terminal carboxyl groups
The method involves reacting cardiolipin with a periodate salt and a permanganate salt to oxidize fatty acid side chains, producing terminal carboxyl groups.
Quenching oxidation and retaining immunogenicity
After oxidation, a reducing agent is added to quench oxidation and reduce a β-ketone in the central glycerol moiety to a β-hydroxyl group, preserving immunogenicity.
Activation of carboxyl groups for protein conjugation
The terminal carboxyl groups of oxidized cardiolipin are activated, e.g., via carbodiimide chemistry (such as EDC and NHS), to covalently attach a protein carrier to at least one carboxyl group.
Specific reaction conditions for oxidation
Oxidation occurs in an alcohol solvent or under an argon atmosphere, with periodate added before permanganate; for example, sodium m-periodate and potassium permanganate with defined molar ratios to cardiolipin.
Types of proteins for conjugation and subsequent processing
Proteins like bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or avidin can be conjugated to the oxidized cardiolipin, with phase separation and recovery steps included.
The independent claim covers a multi-step oxidation and conjugation method that produces immunoreactive oxidized cardiolipin with terminal carboxyl groups, suitable for protein conjugation and attachment to solid substrates, enabling improved immunoassays targeting anti-lipoidal antibodies.
Stated Advantages
Enables reliable attachment of cardiolipin antigen to solid supports while maintaining antigenicity.
Facilitates development of non-solution-based immunoassays such as ELISA and rapid lateral flow devices for point-of-care testing.
Allows for concurrent detection of non-treponemal and treponemal antibodies in a single immunoassay device.
Improves sensitivity and specificity of anti-lipoidal antibody detection in biological samples for syphilis diagnosis.
Documented Applications
Detection of anti-lipoidal antibodies in biological samples for diagnosis of syphilis.
Use in lateral flow devices for rapid, on-site testing of anti-lipoidal and treponemal antibodies.
Development of enzyme-linked immunosorbent assays (ELISA) using oxidized cardiolipin conjugates immobilized in microtiter plates.
Diagnosis of lupus through detection of anti-lipoidal antibodies using the disclosed immunoassay devices.
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