Modified cardiolipin and uses therefor
View Patent ↗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.
1. 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.
2. The solid support of claim 1 , wherein reacting the cardiolipin occurs in an alcohol solvent and in an argon atmosphere.
3. The solid support of claim 2 , wherein the alcohol is one or more of t-butanol, ethanol, propanol, or methanol.
4. The solid support of claim 1 , wherein activating the carboxyl groups comprises reacting the oxidized cardiolipin with a carbodiimide.
5. The solid support of claim 4 , wherein activating carboxyl groups further comprises reacting the carboxyl groups with an N-hydroxysuccinimide (NHS) after reacting the carboxyl groups with the carbodiimide.
6. The solid support of claim 4 , wherein the carbodiimide comprises 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC).
7. The solid support of claim 1 , wherein the cardiolipin is reacted with the periodate salt before the cardiolipin is reacted with the permanganate salt.
8. The solid support of claim 1 , wherein the periodate salt is sodium m-periodate and the permanganate salt is potassium permanganate.
9. The solid support of claim 8 , wherein the molar ratio of sodium m-periodate to cardiolipin is about 4:1 to about 5:1.
10. The solid support of claim 8 , wherein the molar ratio of potassium permanganate to cardiolipin is about 0.5:1 to about 1:1.
11. The solid support of claim 1 , wherein the reducing agent is sodium bisulfite.
12. The solid support of claim 2 , wherein the alcohol is t-butanol.
13. The solid support of claim 1 , further comprising a treponemal antigen that reacts with antibodies to T. pallidum.
14. The solid support of claim 1 , wherein the solid support comprises a multi-well plate or latex microparticle.
15. 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.
16. The ELISA multi-well plate of claim 15 , wherein activating the terminal carboxyl groups comprises activating at least one terminal carboxyl group by reacting the oxidized cardiolipin with a carbodiimide and then an NHS to provide a product with activated terminal carboxyl groups.
17. The ELISA multi-well plate of claim 16 , wherein covalently attaching the protein to at least one of the activated terminal carboxyl groups comprises conjugating the product with the protein, thereby producing the cardiolipin-protein conjugates.
18. The ELISA multi-well plate of claim 15 , wherein the protein comprises bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), IgY, or avidin.
19. The ELISA multi-well plate of claim 15 , further comprising a treponemal antigen that reacts with antibodies to T. pallidum .