Modified cardiolipin and uses therefor
AssigneeThe United States of America as represented by 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.
Claims16
A method for preparing oxidized cardiolipin that is immunoreactive with anti-lipoidal antibodies, and capable of linking to a protein for attachment to a substrate, wherein the cardiolipin comprises a central glycerol moiety and fatty acid side chains, the method comprising:
- reacting the cardiolipin with a periodate salt and a permanganate salt to oxidize fatty acid side chains of the cardiolipin to provide terminal carboxyl groups on one or more of the side chains;
- 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;
- and
- activating the carboxyl groups of the oxidized cardiolipin to covalently attach a protein carrier to at least one of the carboxyl groups.
A method for preparing oxidized cardiolipin that is immunoreactive with anti-lipoidal antibodies, and capable of linking to a protein for attachment to a substrate, wherein the cardiolipin comprises a central glycerol moiety and fatty acid side chains, the method comprising:
- reacting the cardiolipin with sodium m-periodate and potassium permanganate in a t-butanol solvent under an argon atmosphere to oxidize the cardiolipin and provide terminal carboxyl groups on one or more of the fatty acid side chains;
- adding sodium bisulfite in aqueous solution to the cardiolipin suspension after reacting the cardiolipin with the sodium m-periodate and the potassium permanganate 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;
- and
- activating the terminal carboxyl groups of the oxidized cardiolipin to covalently attach a protein carrier to at least one of the carboxyl groups, wherein activating the carboxyl groups comprises reacting the oxidized cardiolipin with EDC and then NHS;
- and
- conjugating the protein to one or more of the activated carboxyl groups.
Dependencies are inferred from the claim text.