US 12,258,587 B2
Compositions and methods for internal controls of microvesicle isolations
Georg Stoll, Munich (DE); Daniel Enderle, Martinsried (DE); Mikkel Noerholm, Martinsried (DE); and Johan Karl Olov Skog, Lincoln, MA (US)
Assigned to Exosome Diagnostics, Inc., Waltham, MA (US)
Appl. No. 17/295,287
Filed by Exosome Diagnostics, Inc., Waltham, MA (US)
PCT Filed Nov. 20, 2019, PCT No. PCT/US2019/062429
§ 371(c)(1), (2) Date May 19, 2021,
PCT Pub. No. WO2020/106853, PCT Pub. Date May 28, 2020.
Claims priority of provisional application 62/769,745, filed on Nov. 20, 2018.
Prior Publication US 2021/0403881 A1, Dec. 30, 2021
Int. Cl. C12N 15/10 (2006.01); C12N 7/00 (2006.01); C12Q 1/6806 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/70 (2006.01)
CPC C12N 7/00 (2013.01) [C12N 15/1003 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6851 (2013.01); C12Q 1/70 (2013.01); C12N 2770/24251 (2013.01); C12N 2770/24351 (2013.01)] 19 Claims
 
1. A method comprising:
(a) adding a known quantity of control particles comprising at least one control nucleic acid and/or at least one control protein to a biological sample, wherein the control particles comprise a non-human animal enveloped virus, wherein the non-human animal enveloped virus is selected from mouse hepatitis virus (MHV), bovine diarrhea virus (BVDV) and transmissible gastroenteritis coronavirus (TGEV);
(b) isolating microvesicles and the control particles from the biological sample;
(c) extracting nucleic acids and/or protein from the microvesicles and the control particles;
(d) assaying the amount of the at least one control nucleic acid and/or the at least one control protein recovered, thereby determining the amount of control particles recovered;
(e) determining that the amount of control particles calculated in step (d) is within a predetermined range of values; and
(f) subjecting the nucleic acids and/or protein from step (c) to further analysis if the amount of control particle calculated in step (d) is within the predetermine range of values;
wherein the isolating of the microvesicles and control particles from the biological sample comprises contacting the biological sample with a capture surface under conditions sufficient to retain cell-free DNA, microvesicles, and control particles on the capture surface, and
wherein the capture surface comprises one or more beads that are positively charged, one or more beads that are an anion exchanger functionalized with quaternary ammonium (R—CH2—N+(CH3)3), or one or more beads that are positively charged and functionalized with quaternary ammonium.