Polymer stabilization of chromogen solutions

Patent metadata

Field	Value
Publication number	US-10775281-B2
Application number	US-201715483589-A
Country	US
Kind code	B2
Filing date	Apr 10, 2017
Priority date	Jan 23, 2012
Publication date	Sep 15, 2020
Grant date	Sep 15, 2020

Abstract

Disclosed embodiments concern a composition comprising DAB chromogen, and/or derivative thereof, a stabilizer, and polymer capable of preventing or reducing DAB precipitation relative to a composition that does not comprise the polymer. Also disclosed herein is a method for using the disclosed composition and embodiments of a kit.

First claim

We claim:

1. A method for detecting a target in a tissue sample with a diaminobenzidine (DAB) chromogen or a derivative thereof, comprising:
contacting the tissue sample with a specific binding moiety, the specific binding moiety being specific to the target and being conjugated to a hapten,
labeling the specific binding moiety with an enzyme conjugate, wherein the enzyme conjugate comprises an antibody specific to the hapten conjugated to the specific binding moiety,
contacting the tissue sample with a composition for chromogenic immunohistochemistry, the composition comprising a solvent, a DAB chromogen or a derivative thereof, a stabilizer, and a polyethyleneimine polymer soluble in the solvent, wherein the composition is configured to reduce precipitation of the DAB chromogen or the derivative thereof under storage conditions but maintain precipitation of the DAB chromogen or the derivative thereof under detection conditions,
contacting the tissue sample with an oxidant, wherein a reaction between the oxidant, the composition for chromogenic immunohistochemistry, and the enzyme conjugate causes the DAB chromogen or the derivative thereof to deposit proximally to the target,
contacting the tissue sample with a counterstain, and
detecting the target in the tissue sample by locating the DAB chromogen or the derivative thereof.

2. The method of claim 1 , wherein the hapten conjugated to the specific binding moiety is selected from the group consisting of oxazole, a pyrazole, a thiazole, a nitroaryl compound other than dinitrophenyl, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin, a cyclolignan, a heterobiaryl, an azo aryl, and a benzodiazepine.

3. The method of claim 1 , wherein the hapten is selected from the group consisting of 3-hydroxy-2-quinoxalinecarbamide (HQ), digoxigenin (DIG), 2,4-dinitrophenyl (DNP), 2-acetamido-4-methyl-5-thiazolesulfonamide (TS), 5-nitro-3-pyrazolecarbamide (NP), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxyylic acid (DCC), and biotin.

4. The method of claim 1 , wherein the polyethyleneimine polymer has an average molecular weight of between 1 kDa and about 500 kDa.

5. The method of claim 1 , wherein the polyethyleneimine polymer is about 0.05 percent to about 10 percent by weight of the composition for chromogenic immunohistochemistry.

6. The method of claim 1 , wherein the composition for chromogenic immunohistochemistry further comprises an enhancer selected from the group consisting of imidazole, 2-hydroxypyridine and combinations thereof.

7. The method of claim 1 , wherein the stabilizer is sodium metabisulfate.

8. The method of claim 1 , wherein the enzyme is a peroxidase.

9. The method of claim 8 , wherein the peroxidase is horseradish peroxidase.

10. The method of claim 1 , wherein the tissue sample is a formalin-fixed, paraffin-embedded tissue sample.

11. The method of claim 1 , wherein storage conditions include a sealed storage container not in fluid communication with other reagents and detection conditions include the oxidant and the composition for chromogenic immunohistochemistry deposited on the tissue sample and in contact with the enzyme.

12. The method of claim 1 , wherein the specific binding moiety is an antibody or a nucleic acid probe.

13. The method of claim 1 , wherein contacting the tissue sample with the composition for chromogenic immunohistochemistry comprises adding a first component solution comprising the DAB chromogen or the derivative thereof, a second component solution comprising the stabilizer, and a third component solution comprising the polymer to the tissue sample to form the composition for chromogenic immunohistochemistry while in contact with the tissue sample.

14. A method for detecting a target in a tissue sample with a DAB chromogen or a derivative thereof, comprising:
contacting the tissue sample with a specific binding moiety, the specific binding moiety being specific to the target,
labeling the specific binding moiety with an enzyme conjugate,
contacting the tissue sample with a composition for chromogenic immunohistochemistry, the composition comprising a DAB chromogen, a stabilizer, and a polymer; wherein the stabilizer is sodium metabisulfite or sodium bisulfite, and the polymer is polyethyleneimine, wherein the composition is configured to remain stable under storage conditions so that the polymer complexes the DAB chromogen or the derivative thereof so as to maintain the amount of the DAB chromogen or the derivative thereof in the solution, and wherein the composition is configured so that the DAB precipitates under detection conditions to produce a signal suitable for chromogenic immunohistochemistry,
contacting the tissue sample with an oxidant, wherein a reaction between the oxidant, the composition for chromogenic immunohistochemistry, and the enzyme conjugate causes the DAB chromogen or the derivative thereof to deposit proximally to the target,
contacting the tissue sample with a counterstain, and
detecting the target in the tissue sample by locating the DAB chromogen or the derivative thereof.

15. The method of claim 14 , wherein the DAB chromogen is 3,3′-diaminobenzidine, and the stabilizer is sodium metabisulfite.

16. The method of claim 15 , wherein the composition comprises about 1 to about 15 mM 3,3′-diaminobenzidine, about 0.1 to about 6 mM sodium metabisulfite and about 0.05% to about 0.5% (w/w) polyethyleneimine.

Assignees

• Ventana Med Syst Inc

Inventors

• Bieniarz Christopher
• Kelly Brian D
• Kosmeder Ii Jerome W
• May Eric J
• Morrison Larry

Classifications

• C12Q1/28 Primary

  involving peroxidase · CPC title

• G01N33/535

  with enzyme label {or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates} · CPC title

• G01N1/30 Primary

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title

• G01N33/532

  Production of labelled immunochemicals · CPC title

• G01N33/54393

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title