US 9,810,702 B2
HDL-associated protein extraction and detection
Cory Bystrom, Beachwood, OH (US); and Timothy Collier, Cleveland, OH (US)
Assigned to THE CLEVELAND HEARTLAB, INC., Cleveland, OH (US)
Filed by Cleveland HeartLab, Inc., Cleveland, OH (US)
Filed on Sep. 18, 2015, as Appl. No. 14/858,111.
Claims priority of provisional application 62/052,854, filed on Sep. 19, 2014.
Prior Publication US 2016/0084856 A1, Mar. 24, 2016
Int. Cl. G01N 33/92 (2006.01)
CPC G01N 33/92 (2013.01) [G01N 2333/775 (2013.01)] 13 Claims
 
1. A method of extracting and detecting at least one HDL-associated protein 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; and
g) subjecting at least a portion of said bottom aqueous layer to a detection assay such that at least one of the following HDL-associated proteins is detected: Apolipoprotein A-I (APOAI), 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 C1 (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), and Apolipoprotein D (APOD).