# US 2016/0252536 A1 · Fluorescent in-situ detection of lipid particle apolipoproteins within primary electrophoretic matrix > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2016/0252536 A1 - Document ID: US20160252536A1 - Status: Published application - Kind: A1 - Application number: 15/152,022 - Filed: 2016-05-11 - Published: 2016-09-01 - Earliest claimed: 2013-02-28 - Assignees: True Health Diagnostics, LLC - Applicants: True Health Diagnostics LLC - Inventors: Philip Guadagno; Erin Bellin - Web page: https://patentgrep.com/patent/US20160252536A1 ## CPC - G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - G01N 27/44726: {using specific dyes, markers or binding molecules} - G01N 27/44747: {Composition of gel or of carrier mixture} - G01N 27/44756: {Apparatus specially adapted therefor} - G01N 33/561: Immunoelectrophoresis - G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging} - G01N 2021/6441: {with two or more labels} - G01N 2021/7786: {Fluorescence} - G01N 2333/775: Apolipopeptides - G01N 2550/00: Electrophoretic profiling, e.g. for proteome analysis - G01N 2800/32: Cardiovascular disorders - G01N 2800/50: Determining the risk of developing a disease - G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The system according to claim 1, wherein said device quantitates the level of specific Apolipoproteins and/or lipoprotein particles based on said detecting. ### Claim 4 (depends on 1) The system according to claim 1, wherein the detectable signal is detectable by fluorometric means. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 5) The system according to claim 5, wherein the antibody is specific to Apolipoprotein B. ### Claim 7 (depends on 5) The system according to claim 5, wherein the antibody is specific to oxidized low density lipoprotein. ### Claim 8 (depends on 1) 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. ### Claim 9 (depends on 8) 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. ### Claim 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. ### Claim 11 (depends on 10) 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. ### Claim 12 (depends on 11) 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. ### Claim 13 (depends on 12) 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. ### Claim 14 (depends on 11) The method according to claim 11, wherein at least one of the two or more different lipoprotein particles or portion thereof is Apolipoprotein B. ### Claim 15 (depends on 11) 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. ### Claim 16 (depends on 11) 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. ### Claim 17 (depends on 11) 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. ### Claim 18 (depends on 17) The method according to claim 17, wherein the selected therapy regimen comprises drugs or supplements. ### Claim 19 (depends on 18) 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. ### Claim 20 (depends on 17) 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. ### Claim 21 (depends on 20) 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. ### Claim 22 (depends on 17) The method according to claim 17 further comprising: generating a report comprising a description of said selected treatment regimen. ### Claim 23 (depends on 17) The method according to claim 17 further comprising: administering said selected treatment regimen. ### Claim 24 (depends on 11) 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. ### Claim 25 (depends on 24) 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.