Modified cardiolipin and uses therefor
AssigneeThe United States of America as represented by the Secretary of the Department of Health and Human Services, Centers for Disease Control and Prevention
InventorsArnold R. Castro, Huiying Wang
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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.
Claims19
A solid support, comprising:
- amine molecules;
- oxidized cardiolipin attached to the amine molecules, thereby covalently attaching oxidized cardiolipin to the solid support, wherein the oxidized cardiolipin is immunoreactive with anti-lipoidal antibodies, comprises a central glycerol moiety and fatty acid side chains, and is generated by a method comprising:
- reacting cardiolipin with a periodate salt and a permanganate salt to oxidize alkene groups of fatty acid side chains of the cardiolipin to provide terminal carboxyl groups on one or more of the fatty acid side chains, thereby producing a cardiolipin suspension;
- adding a reducing agent to the cardiolipin suspension after reacting the cardiolipin with the periodate salt and the permanganate salt to quench oxidation of the cardiolipin and to reduce a β-ketone formed in the central glycerol moeity to a β-hydroxyl group to retain immunogenicity of the central glycerol moiety, thereby generating an oxidized cardiolipin;
- and
- activating the terminal carboxyl groups of the oxidized cardiolipin, thereby generating activated terminal carboxyl groups to permit attachment of the oxidized cardiolipin to the amine molecules.
An enzyme-linked immunosorbent assay (ELISA) multi-well plate, comprising:
- oxidized cardiolipin-protein conjugates covalently attached to the ELISA multi-well plate, wherein the oxidized cardiolipin-protein conjugates are immunoreactive with anti-lipoidal antibodies, comprise a central glycerol moiety and fatty acid side chains, and are generated by a method comprising:
- reacting cardiolipin with a periodate salt and a permanganate salt to oxidize alkene groups of fatty acid side chains of the cardiolipin to provide terminal carboxyl groups on one or more of the fatty acid side chains, thereby producing a cardiolipin suspension;
- adding a reducing agent to the cardiolipin suspension after reacting the cardiolipin with the periodate salt and the permanganate salt to quench oxidation of the cardiolipin and to reduce a β-ketone formed in the central glycerol moeity to a β-hydroxyl group so as to retain immunogenicity of the central glycerol moiety, thereby generating an oxidized cardiolipin;
- activating the terminal carboxyl groups of the oxidized cardiolipin, thereby generating activated terminal carboxyl groups;
- and
- covalently attaching a protein to at least one of the activated terminal carboxyl groups, thereby generating oxidized cardiolipin-protein conjugates.
Dependencies are inferred from the claim text.