	US 11,988,673 B2
	Multiplexed immunohistochemistry using recombinant antibodies with epitope tags
Michael Farrell, Tucson, AZ (US); Antony Hubbard, Tucson, AZ (US); Eric May, Oro Valley, AZ (US); Lei Tang, Oro Valley, AZ (US); Tsu-Shuen Tsao, Tucson, AZ (US); and Wenjun Zhang, Tucson, AZ (US)
Assigned to Ventana Medical Systems, Inc., Tucson, AZ (US)
Filed by Ventana Medical Systems, Inc., Tucson, AZ (US)
Filed on Jul. 30, 2021, as Appl. No. 17/389,757.
Application 17/389,757 is a division of application No. 16/123,750, filed on Sep. 6, 2018, granted, now 11,112,413.
Application 16/123,750 is a continuation of application No. PCT/US2017/021157, filed on Mar. 7, 2017.
Claims priority of provisional application 62/461,651, filed on Feb. 21, 2017.
Claims priority of provisional application 62/418,667, filed on Nov. 7, 2016.
Claims priority of provisional application 62/305,440, filed on Mar. 8, 2016.
Prior Publication US 2022/0057408 A1, Feb. 24, 2022
Int. Cl. G01N 33/68 (2006.01); C07K 16/18 (2006.01); C07K 16/28 (2006.01); C07K 16/32 (2006.01)

CPC G01N 33/6878 (2013.01) [C07K 16/18 (2013.01); C07K 16/2809 (2013.01); C07K 16/2815 (2013.01); C07K 16/2827 (2013.01); C07K 16/2887 (2013.01); C07K 16/2896 (2013.01); C07K 16/32 (2013.01); C07K 2319/40 (2013.01); C07K 2319/42 (2013.01); G01N 2570/00 (2013.01)]

16 Claims

1. A multiplex immunohistochemical method for detecting a plurality of targets in a sample, wherein the method comprises the steps of:

(a) simultaneously contacting the sample with:

(a1) a first epitope-tagged primary antibody, wherein the first epitope-tagged primary antibody comprises:

(i) a first antibody specific to a first target, and

(ii) at least a first epitope tag construct, wherein the first epitope tag construct comprises alternating spacers and a first epitope tag; and

(a2) a second epitope-tagged primary antibody, wherein the second epitope-tagged primary antibody comprises:

(i) a second antibody specific to a second target that is different from the first target and wherein the first antibody and the second antibody are same-species antibodies; and

(ii) at least a second epitope tag construct, wherein the second epitope tag construct comprises alternating spacers and a second epitope tag, and wherein the second epitope tag is different from the first epitope tag; and

(b) contacting the sample with a first set of detection reagents for detecting binding of the first epitope-tagged primary antibody to the sample, wherein the first set of detection reagents comprises a first anti-tag antibody, wherein the first anti-tag antibody is specific for the first epitope tag; and

(c) contacting the sample with a second set of detection reagents for detecting binding of the second epitope-tagged primary antibody to the sample, wherein the set of detection reagents comprises a second anti-tag antibody, wherein the second anti-tag antibody is specific for the second epitope tag.