Oxidized cardiolipin and uses to detect cardiolipin antibodies

摘要

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.

主权项

1. A kit for diagnosing syphilis, comprising:
(a) multi-well plate, 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; andactivating 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; (b) labeled anti human IgM in a container; (c) one or more of a negative control serum, a positive control serum, labeled anti human IgG, wash buffer, and dilution buffer, each in a separate container; and (d) optionally, a calibration curve for non-treponemal IgG and/or IgM.