Assays for detecting modified compounds
Provided are methods and compositions which are useful for separating, isolating, detecting, and quantifying compounds of interest which have been modified chemically, enzymatically or catalytically from other compounds which have not been so modified. The modifications may take the form of functional groups which are gained, lost or retained by the compounds of interest.
1. A method for detecting or quantifying an activity in a sample that causes a compound to gain a functional group, the method comprising the steps of:
(a) providing:
(i) a compound which can gain a functional group, the compound comprising at least one non-radioactive signaling moiety;
(ii) a chemical source of the functional group which can be gained by the compound; and
(iii) a sample;
wherein the compound is a lipid, and
wherein the functional group is a glycosyl group;
(b) forming a mixture comprising the compound, the source of the functional group and the sample, and incubating the mixture under conditions suitable for the compound to gain the functional group; and
(c) capturing any compound that has gained the functional group on a matrix comprising a glycosyl group-binding lectin and separating said compound which has gained the functional group from any compound which has not gained the functional group; and
(d) detecting or quantifying the non-radioactive signaling moiety of any captured separated compounds having the functional group, thereby detecting or quantifying the activity in the sample.
2. The method of claim 1 , wherein the non-radioactive signaling moiety comprises a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chromogenic compound, a chelating compound, an electron dense compound, a magnetic compound, an energy transfer member or pair, an intercalating compound, an antibody, an antigen, a hapten, a receptor, a hormone, a ligand or an enzyme, or any combination thereof.
3. A method for detecting or quantifying an activity in sample which removes a functional group from a compound, the method comprising the steps of:
(a) providing:
(i) a compound comprising a functional group and at least one non-radioactive signaling moiety; and
(ii) a sample;
wherein the compound is a lipid, and
wherein the functional group is a glycosyl group;
(b) forming a mixture comprising the compound and the sample and incubating the mixture under conditions suitable for the compound to lose the functional group; and
(c) capturing on a matrix comprising a glycosyl group-binding lectin and separating any compound that has the functional group following the incubation from any compound which has lost the functional group; and
(d) detecting or quantifying the non-radioactive signaling moiety of any captured separated compounds having the functional group, thereby detecting or quantifying the activity in the sample.
4. The method of claim 3 , wherein the non-radioactive signaling moiety comprises a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chromogenic compound, a chelating compound, an electron dense compound, a magnetic compound, an energy transfer member or pair, an intercalating compound, an antibody, an antigen, a hapten, a receptor, a hormone, a ligand or an enzyme, or any combination thereof.