	US 12,104,199 B2
	Quantification of nucleosome modifications using chemically-defined recombinant nucleosomes
Martis William Cowles, Chapel Hill, NC (US); Matthew F. Whelihan, Durham, NC (US); Andrea L. Johnstone, Apex, NC (US); Michael-Christopher Koegh, Cambridge, MA (US); Zu-Wen Sun, Brentwood, TN (US); Nathan W. Hall, Chapel Hill, NC (US); and Matthew R. Marunde, Carrboro, NC (US)
Assigned to EpiCypher, Inc., Durham, NC (US)
Filed by EPICYPHER, INC., Durham, NC (US)
Filed on Jul. 24, 2020, as Appl. No. 16/938,256.
Application 16/938,256 is a continuation of application No. 16/678,686, filed on Nov. 8, 2019, granted, now 10,787,697.
Application 16/678,686 is a continuation of application No. PCT/US2019/020283, filed on Mar. 1, 2019.
Claims priority of provisional application 62/637,066, filed on Mar. 1, 2018.
Prior Publication US 2021/0010058 A1, Jan. 14, 2021
Int. Cl. C12Q 1/6804 (2018.01); C12Q 1/6811 (2018.01); G01N 33/49 (2006.01)

CPC C12Q 1/6804 (2013.01) [C12Q 1/6811 (2013.01); G01N 33/49 (2013.01); C12Y 301/31001 (2013.01)]

13 Claims

1. A method of determining the specificity of an affinity reagent for a core histone modification and/or DNA modification target epitope, the method comprising using one or more recombinant nucleosomes carrying one or more histone modification and/or DNA modification, wherein determining the specificity of an affinity reagent comprises:

a. preparing one or more recombinant mono- and/or polynucleosome sample comprising nucleosomes carrying the one or more histone modification and/or DNA modification target epitopes;

b. preparing a recombinant mono- and/or polynucleosome sample that does not comprise the one or more histone modification and/or DNA modification target epitopes;

c. providing the recombinant nucleosome sample of (a) and the recombinant nucleosome sample of (b) to create a series of reference standards;

d. adding one or more affinity reagent to the series of reference standards of (c); and

e. performing an affinity reagent-based assay to measure the amount of nucleosome modification in the series of reference standards of (c) and using this information to determine the specificity and background binding of the affinity reagent.