Fluorescent in-situ detection of lipid particle apolipoproteins within primary electrophoretic matrix

Abstract

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.

What is claimed: 
     
         1 . A gel electrophoresis system for detecting the level of specific Apolipoproteins and/or lipoprotein particles present in a biological sample comprising:
 a gel substrate to receive a biological sample;   at least two lipoprotein-binding complexes, each complex comprising an antibody that binds a lipoprotein particle or a portion thereof, wherein the antibody is bound to a signal producing molecule capable of producing or causing production of a detectable signal and wherein each detectable signal of the at least two lipoprotein-binding complexes is distinguishable from the other detectable signal; and   a device for detecting the detectable signal, wherein said detecting indicates the level of said specific Apolipoproteins and/or lipoprotein particles in the biological sample.   
     
     
         2 . The system according to  claim 1  further comprising:
 a reagent capable of interacting with the signal producing molecule, wherein the signal producing molecule produces the detectable signal upon contact with the reagent. 
 
     
     
         3 . The system according to  claim 1 , wherein said device quantitates the level of specific Apolipoproteins and/or lipoprotein particles based on said detecting. 
     
     
         4 . The system according to  claim 1 , wherein the detectable signal is detectable by fluorometric means. 
     
     
         5 . The system according to  claim 1 , wherein the lipoprotein particle or portion thereof is 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. 
     
     
         6 . The system according to  claim 5 , wherein the antibody is specific to Apolipoprotein B. 
     
     
         7 . The system according to  claim 5 , wherein the antibody is specific to oxidized low density lipoprotein. 
     
     
         8 . The system according to  claim 1  comprising three or more lipoprotein-binding complexes comprising antibodies specific to different lipoprotein particles or portions thereof, wherein each detectable signal of the three or more lipoprotein-binding complexes is distinguishable from the other detectable signal. 
     
     
         9 . The system according to  claim 8 , wherein the three or more different lipoprotein particles or portions thereof are each 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. 
     
     
         10 . A method of determining whether a subject is at increased risk for cardiovascular disease comprising:
 assessing the level of specific Apolipoproteins and/or lipoprotein particles present in a biological sample comprising:   providing a biological sample comprising lipoprotein particles;   providing at least two lipoprotein-binding complexes, each complex comprising an antibody that binds a lipoprotein particle or a portion thereof, wherein the antibody is bound to a signal producing molecule capable of producing or causing production of a detectable signal and wherein each detectable signal of the at least two lipoprotein-binding complexes is distinguishable from the other detectable signal;   contacting the biological sample with the at least two lipoprotein-binding complexes under conditions suitable to form a lipoprotein-antibody-signal producing molecule complex;   separating the lipoprotein particles present in the biological sample by depositing the biological sample on an electrophoretic gel and carrying out gel electrophoresis;   detecting the detectable signal produced by the signal producing molecule of the lipoprotein-antibody-signal producing molecule complex on the electrophoretic gel; and   determining the level of the specific Apolipoprotein and/or lipoprotein particle present in the biological sample based on said detecting and correlating the determined level of the Apolipoprotein and/or lipoprotein particle to a control or reference value to determine if the subject is at an increased risk for cardiovascular disease.   
     
     
         11 . The method according to  claim 10 , wherein
 said assessing comprises:
 forming two or more lipoprotein-antibody-signal producing molecule complexes, wherein the two or more antibodies are specific to two or more different lipoprotein particles or a portion thereof; 
 separating the lipoprotein particles present in the biological sample by depositing the biological sample on an electrophoretic gel and carrying out gel electrophoresis; 
 detecting the detectable signal produced by the signal producing molecules of the respective lipoprotein-antibody-signal producing molecule complexes on the electrophoretic gel; and 
 determining the levels of the different Apolipoproteins and/or lipoprotein particles present in the biological sample based on said detecting, and 
   said correlating comprises correlating the determined levels of the different Apolipoproteins and/or lipoprotein particles to a control or reference value to determine if the subject is at an increased risk for cardiovascular disease.   
     
     
         12 . The method according to  claim 11 , wherein the two or more different lipoprotein particles or portion thereof are 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. 
     
     
         13 . The method according to  claim 12 , wherein at least one of the two or more different lipoprotein particles or portion thereof is oxidized low density lipoprotein. 
     
     
         14 . The method according to  claim 11 , wherein at least one of the two or more different lipoprotein particles or portion thereof is Apolipoprotein B. 
     
     
         15 . The method according to  claim 11 , wherein the two or more different lipoprotein particles or portions thereof comprise at least Apolipoprotein B and low density lipoprotein and wherein an elevated level of Apolipoprotein B and low density lipoprotein particles indicates that an individual has increased risk for cardiovascular disease. 
     
     
         16 . The method according to  claim 11 , wherein the different lipoprotein particles or portions thereof comprise at least Apolipoprotein B and lipoprotein (a) isoform and wherein an elevated level of Apolipoprotein B and lipoprotein (a) isoform type indicates that an individual has increased risk for cardiovascular disease. 
     
     
         17 . The method according to  claim 11 , wherein the subject is determined to be at an increased risk for cardiovascular disease and the method further comprises: selecting a treatment regimen based on said elevated risk. 
     
     
         18 . The method according to  claim 17 , wherein the selected therapy regimen comprises drugs or supplements. 
     
     
         19 . The method according to  claim 18 , wherein the drug is selected from an anti-inflammatory agent, an antithrombotic agent, an anti-platelet agent, a fibrinolytic agent, a lipid reducing agent, a direct thrombin inhibitor, a glycoprotein IIb/IIIa receptor inhibitor, an agent that binds to cellular adhesion molecules and inhibits the ability of white blood cells to attach to such molecules, a calcium channel blocker, a beta-adrenergic receptor blocker, an angiotensin system inhibitor, or combinations thereof. 
     
     
         20 . The method according to  claim 17 , wherein the selected therapy regimen involves giving recommendations on making or maintaining lifestyle choices based on the results of said determining. 
     
     
         21 . The method according to  claim 20 , wherein the lifestyle choices involve changes in diet, changes in exercise, reducing or eliminating smoking, or a combination thereof. 
     
     
         22 . The method according to  claim 17  further comprising: generating a report comprising a description of said selected treatment regimen. 
     
     
         23 . The method according to  claim 17  further comprising: administering said selected treatment regimen. 
     
     
         24 . The method according to  claim 11 , wherein said correlating further comprises:
 assigning the subject to a risk category selected from the group consisting of high risk, intermediate risk, and low risk groups for developing or having cardiovascular disease.   
     
     
         25 . A method of monitoring the risk for developing cardiovascular disease, comprising:
 carrying out the method according to  claim 24 , wherein the risk category assigned is a first risk category;   repeating the method according to  claim 24 , wherein the risk category assigned is a second risk category;   comparing the first and second risk categories; and   determining, based on said comparing step, if the subject's risk for developing cardiovascular disease has increased or decreased, thereby monitoring the risk for developing cardiovascular disease.