US 11,940,452 B2
HDL-associated protein extraction and detection
Cory Bystrom, Beachwood, OH (US); and Timothy Collier, Cleveland, OH (US)
Assigned to Cleveland Heartlab, Inc., Cleveland, OH (US)
Filed by CLEVELAND HEARTLAB, INC., Cleveland, OH (US)
Filed on Oct. 29, 2019, as Appl. No. 16/667,016.
Application 15/788,876 is a division of application No. 14/858,111, filed on Sep. 18, 2015, granted, now 9,810,702.
Application 16/667,016 is a continuation of application No. 15/788,876, filed on Oct. 20, 2017, granted, now 10,466,260.
Claims priority of provisional application 62/052,854, filed on Sep. 19, 2014.
Prior Publication US 2020/0116744 A1, Apr. 16, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. G01N 33/92 (2006.01)
CPC G01N 33/92 (2013.01) [G01N 2333/775 (2013.01)] 11 Claims
 
1. A method of extracting and detecting two or more HDL-associated proteins comprising:
a) mixing, in a first container, a separating solution and a serum or plasma sample to generate a first mixed sample, wherein said separating solution comprises a strong organic acid and a hydrophilic organic solvent, and wherein said separating solution makes up 55%-93% of said first mixed sample;
b) exposing said first mixed sample to centrifugal force such that said first mixed sample separates into a pellet and supernatant;
c) transferring at least a portion of said supernatant to a second container such that it is separated from said pellet;
d) adding a non-polar organic solvent to said second container at a ratio of greater than 1:1 to generate a second mixed sample;
e) exposing said second mixed sample to centrifugal force such that said second mixed sample separates into an upper organic solvent layer and a bottom aqueous layer;
f) transferring at least a portion of said bottom aqueous layer to a third container such that it is separated from said upper organic solvent layer;
g) adding a protease to at least a portion of said bottom aqueous layer, wherein the protease is selected from trypsin, LysC, GluC, ArgC, AspN, chymotrypsin, and pepsin; and
h) subjecting at least a portion of said bottom aqueous layer to a detection assay such that at least two or more HDL-associated proteins are detected, wherein a first HDL-associated protein is Apolipoprotein A-I (APOAI) and a second HDL-associated protein is selected from Apolipoprotein A-II (ApoA2), Alpha-1-antitrypsin (SERPINA1), Apolipoprotein C-III (APOC3), Apolipoprotein C-I (APOCI), Apolipoprotein A-IV (APOA4), Apolipoprotein C-II (APOC2), Angiotensinogen (AGT), Complement C3 (C3), Complement C4-B (C4B), Serum amyloid A-4 protein (SAA4), Transthyretin (TTR), Pigment epithelium-derived factor (SERPINF1), Apolipoprotein L1 (APOL1), Apolipoprotein F (APOF), Apolipoprotein M (APOM), Apolipoprotein Cl (APOC1), Fibrinogen alpha chain (FGA), Apolipoprotein E (APOE), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Serum amyloid A-1 protein (SAA1), Alpha-2-HS-glycoprotein (AHSG), Complement C4-A (C4A), Retinol-binding protein 4 (RBP4), or Apolipoprotein D (APOD).