Methods and reagents for detecting bioactive lipids
View Patent ↗Compositions and methods for producing monoclonal antibodies and their derivatives reactive against bioactive lipid targets are described. These compositions include derivatized lipids, each of which comprises a bioactive lipid that having a polar head group and at least one hydrocarbon chain (e.g., a lysolipid such as lysophosphatidic acid or sphingosine-1-phosphate) in which a carbon atom has been derivatized with a pendant reactive group; immunogens made by linking a derivatized lipid to a carrier moiety (e.g., a carrier protein, polyethylene glycol, colloidal gold, alginate, or a silicone bead); monoclonal antibodies and derivatives produced by immunizing an animal with such an immunogen; and therapeutic and diagnostic compositions containing such antibodies and antibody derivatives. Methods for making such derivatized lipids, immunogens, and monoclonal antibodies and derivatives, methods for detecting such antibodies once generated, and therapeutic and diagnostic methods for using such antibodies and derivatives, are also described.
1. A diagnostic reagent, comprising a solid support and a derivatized lipid covalently attached thereto either directly or indirectly, wherein the derivatized lipid is a derivative of a bioactive lysolipid, which lysolipid has a polar head group and a single hydrocarbon chain and is selected from the group consisting of:
(a) a lysolipid having a glycerol backbone with a polar head group at carbon sn-3 and a single hydrocarbon chain contained in a single fatty acid moiety attached at either carbon sn-1 or carbon sn-2, the carbon atom of the terminal methyl group of said hydrocarbon chain of the fatty acid having been derivatized with a sulfhydryl group by substitution of a hydrogen of the terminal methyl group with said sulfhydryl group prior to attachment to said solid support via said sulfhydryl group; and
(b) a lysolipid having a sphingoid backbone with a polar head group and having the terminal methyl group of the single hydrocarbon chain of the sphingoid backbone of the lysolipid derivatized with a sulfhydryl group by substitution of a hydrogen of the terminal methyl group with said sulfhydryl group prior to attachment to said solid support via said sulfhydryl group and wherein the lysolipid is sphingolipid or a sphingolipid metabolite.
2. A diagnostic reagent according to claim 1 , wherein the lysolipid having a glycerol backbone is selected from the group consisting of a lysophosphatidic acid, a lysophosphatidic acid precursor, and a lysophosphatidic acid metabolite.
3. A diagnostic reagent according to claim 1 wherein the sphingolipid or sphingolipid metabolite is selected from the group consisting of sphingosine-1-phosphate, sphingosine, sphingosylphosphorylcholine, dihydrosphingosine, and dihydrosphingosine-1-phosphate.
4. A diagnostic reagent according to claim 1 wherein the sphingolipid is sphingosine-1-phosphate.
5. A diagnostic reagent according to claim 1 wherein the derivatized lipid is attached directly to the solid support.
6. A diagnostic reagent according to claim 1 , wherein the derivatized lipid is covalently conjugated to a carrier moiety via said sulfhydryl group, which carrier moiety is attached to the sold support.
7. A diagnostic reagent according to claim 6 wherein the carrier moiety is an albumin molecule.