Flourescent in-situ detection of lipid particle apolipoproteins within primary electrophoretic matrix
View Patent ↗The present invention relates to, among other things, a gel electrophoresis system for detecting the level of specific Apolipoproteins and/or lipoprotein particles present in intact lipid particles in a biological sample. The system includes a gel substrate to receive a biological sample, at least two lipoprotein-binding complexes. Each complex includes an antibody that binds a lipoprotein particle or a portion thereof, which is bound to a signal producing molecule capable of producing or causing production of a detectable signal. The system also includes a device for detecting the detectable signal. The present invention also relates to methods of assessing the level of specific Apolipoproteins and/or lipoprotein particles present in a biological sample, determining whether a subject is at increased risk for cardiovascular disease, and monitoring the risk for developing cardiovascular disease.
1. A method of assessing a level of specific lipoprotein particles present in a biological sample comprising:
providing a biological sample comprising lipoprotein particles;
providing a first and a second lipoprotein-binding complex, wherein the first lipoprotein binding complex comprises an antibody that binds to a first lipoprotein particle or portion thereof wherein the antibody is bound to a first signal producing molecule capable of producing or causing production of a first detectable signal and the second lipoprotein-binding complex comprises a second antibody that binds to a second lipoprotein particle or portion thereof, wherein the second antibody is bound to a second signal producing molecule capable of producing or causing production of a second detectable signal and wherein the first and the second detectable signals are distinguishable from each other when detected in a single lane of an electrophoretic gel;
separating the lipoprotein particles present in the biological sample by depositing the biological sample on an electrophoretic gel and carrying out gel electrophoresis;
probing the lipoprotein particles or portions thereof with the first and second lipoprotein-binding complexes to produce the first and the second detectable signals;
detecting the first and the second detectable signals on the electrophoretic gel; and
determining the level of the first and the second lipoprotein particles or portions thereof present in the biological sample based on said detecting.
2. The method of claim 1 , wherein the first and second lipoprotein particles or portion thereof are independently selected from the group consisting of Apolipoprotein A, Apolipoprotein B, Apolipoprotein C, Apolipoprotein D, Apolipoprotein E, Apolipoprotein H, lipoprotein (a), high density lipoprotein, intermediate density lipoprotein, low density lipoprotein, very low density lipoprotein, Chylomicrons, Lipoprotein X, oxidized variants and mixtures thereof.
3. The method according to claim 2 , wherein the first lipoprotein particle or portion thereof comprises Apolipoprotein B.
4. The method according to claim 2 , wherein the second lipoprotein particle or portion thereof comprises oxidized low density lipoprotein.
5. The method according to claim 1 , wherein said determining step comprises: quantitating the level of lipoprotein particles based on said detecting.
6. The method according to claim 5 further comprising: determining relative ratio(s) of lipoprotein particles present in the biological sample based on said quantitating.
7. The method according to claim 1 , wherein the detectable signal is detectable by fluorometric means.
8. The method of claim 1 , wherein the first and second lipoprotein particles or portions thereof are independently selected from the group consisting of Apolipoprotein A, Apolipoprotein B, Apolipoprotein C, Apolipoprotein D, Apolipoprotein E, Apolipoprotein H, lipoprotein (a), high density lipoprotein, intermediate density lipoprotein, low density lipoprotein, very low density lipoprotein, Chylomicrons, Lipoprotein X, oxidized variants and mixtures thereof.
9. The method of claim 8 wherein the apolipoprotein A comprises apolipoprotein A-I, apolipoprotein A-II or apolipoprotein A-IV.
10. The method of claim 8 wherein the apolipoprotein B comprises apolipoprotein B48 or apolipoprotein B100.
11. The method of claim 8 wherein the apolipoprotein C comprises apolipoprotein C-1, apolipoprotein C-II, apolipoprotein C-III or apolipoprotein C-IV.
12. The method of claim 8 wherein the apolipoprotein E comprises apolipoprotein E-2, apolipoprotein E-3 or apolipoprotein E-4.