Multiplex immunohistochemical assay using primary antibodies of the same host species

Patent metadata

Field	Value
Publication number	US-9874571-B2
Application number	US-201715585115-A
Country	US
Kind code	B2
Filing date	May 2, 2017
Priority date	Aug 28, 2013
Publication date	Jan 23, 2018
Grant date	Jan 23, 2018

Abstract

Disclosed herein are methods and systems for detecting two different epitopes in the same sample using immunohistochemical methods using primary antibodies from the same host species and secondary antibodies immunoreactive with antibodies of the host species of the primary antibodies. Methods to denature and block the first primary antibody contacted with the sample are provided.

First claim

We claim:

1. A method comprising:
contacting a sample with a buffer comprising Tris and a preservative, wherein the pH of the buffer is about 8-9;
contacting the sample with a first primary antibody, wherein the first primary antibody is immunoreactive with a first epitope;
contacting the sample with a first secondary antibody having a first label conjugated thereto, wherein the first secondary antibody is immunoreactive with the first primary antibody;
contacting the sample with a set of reagents reactive with the first secondary antibody to generate a first detectable signal in proximity to the first epitope in the sample;
denaturing the sample by incubating the sample at 80° C.-100° C. for at least 5 minutes;
contacting the sample with a blocking antibody;
contacting the sample with the buffer;
contacting the sample with a second primary antibody, wherein the second primary antibody is immunoreactive with a second epitope different from the first epitope, and further wherein the first primary antibody and the second primary antibody are from the same host species;
contacting the sample with a second secondary antibody having a second label conjugated thereto, wherein the second labeled secondary antibody is immunoreactive with the second primary antibody; and
contacting the sample with a set of reagents reactive with the second label to generate a second detectable signal in proximity to the second epitope in the sample, wherein the first detectable signal and the second detectable signal are different.

2. The method of claim 1 , wherein the sample is contacted with the buffer for at least 30 minutes prior to contacting the sample with the first primary antibody.

3. The method of claim 1 , wherein the sample is contacted with the buffer for 40 minutes prior to contacting the sample with the first primary antibody.

4. The method of claim 1 , wherein the sample is contacted with the first primary antibody for at least 5 minutes at 37° C.

5. The method of claim 1 , wherein the sample is contacted with the first primary antibody for 8 minutes at 37° C.

6. The method of claim 1 , wherein the denaturing the sample comprises incubating the sample at 95° C. for at least 8 minutes.

7. The method of claim 1 , wherein the blocking antibody comprises an antibody reactive with the host species of the first primary antibody.

8. The method of claim 1 , wherein the method further comprises contacting the sample with a reaction buffer comprising Tris for at least 20 minutes to remove residual blocking antibody.

9. The method of claim 1 , wherein:
the first label comprises a hapten, and the set of reagents reactive with the first label comprises: a tertiary antibody conjugated to a first enzyme, wherein the tertiary antibody is immunoreactive with the hapten, and one or more reagents that produce a first detectable reaction product in the presence of the enzyme, wherein the detectable reaction product generates the first detectable signal; and
the second label comprises a second enzyme, and the set of reagents reactive with the second label comprises one or more reagents that produce a second detectable reaction product in the presence of the second enzyme, wherein the second detectable reaction product generates the second detectable signal.

10. The method of claim 1 , wherein:
the first label comprises a first enzyme, and the set of reagents reactive with the first label comprises one or more reagents that produce a first detectable reaction product in the presence of the first enzyme, wherein the second detectable reaction product generates the second detectable signal; and
the second label comprises a hapten, and the set of reagents reactive with the second label comprises: a tertiary antibody conjugated to a second enzyme, wherein the tertiary antibody is immunoreactive with the hapten, and one or more reagents that produce a second detectable reaction product in the presence of the enzyme, wherein the detectable reaction product generates the second detectable signal.

11. The method of claim 1 , wherein the sample is a formalin fixed, paraffin embedded (FFPE) sample.

12. The method of claim 1 , wherein the sample is a cancer sample.

13. The method of claim 1 , wherein:
the first primary antibody is an anti-human CD3 monoclonal antibody, and the second primary antibody is an anti-human CD16 monoclonal antibody; or
the first primary antibody is an anti-human CD16 monoclonal antibody, and the second primary antibody is an anti-human CD3 monoclonal antibody.

14. The method of claim 13 , wherein:
the label of the secondary antibody that is immunoreactive with the anti-CD3 monoclonal antibody comprises a hapten selected from the group consisting of fluorescein, dinitrophenyl, biotin, and 3-hydroxyquinoxaline-2-carboxylic acid (HQ), and the set of reagents reactive with the label of the secondary antibody immunoreactive with the anti-CD3 monoclonal antibody comprises: a tertiary antibody comprising a detectable label, wherein the tertiary antibody is immunoreactive with the hapten;
the label of the secondary antibody that is immunoreactive with the anti-CD16 monoclonal antibody comprises an alkaline phosphatase (AP) multimer, and the set of reagents reactive with the label of the secondary antibody that is immunoreactive with the anti-CD16 monoclonal antibody comprises: napthol, an alkaline phosphatase enhancer, and fast red.

15. The method of claim 13 , wherein the CD3 antibody and the CD16 antibody are from the same host species, the host species being a mouse or a rabbit.

16. The method of claim 13 , further comprising scoring the presence of CD3 protein and CD16 protein.

17. The method of claim 16 , wherein the sample comprises a tumor sample, and scoring the presence of CD3 protein and CD16 protein comprises:
a) determining an absolute number of cells staining with the CD3 antibody in the sample using a 5×5 ocular grid with an area of 0.25 square millimeter, determining an absolute number of cells staining with the CD16 antibody in the sample using a 5×5 ocular grid with an area of 0.25 square millimeter, extrapolating the absolute number of cells staining with the CD3 antibody to a number of cells in a 1 square millimeter region, and extrapolating the absolute number of cells staining with the CD16 antibody to a number of cells in a 1 square millimeter region, thereby generating a score of CD3 protein and CD16 protein; or
b) determining the area of staining with the CD3 antibody within the intratumoral and contiguous peri-tumoral stroma of the sample, determining the area of staining with the CD16 antibody within the intratumoral and contiguous peri-tumoral stroma of the sample, dividing the area of staining with the CD3 antibody in the intratumoral and contiguous peri-tumoral stroma by the area of total stroma, thereby generating a score of CD3 protein for the stroma, and dividing the area of staining with the CD16 antibody in the intratumoral and contiguous peri-tumoral stroma by the area of total stroma, thereby generating a score of CD3 protein for the stroma; or
c) a combination of a and b.

18. The method of claim 17 , wherein 1 to 15 randomly selected regions of the sample containing the tumor are selected for scoring.

19. The method of claim 17 , wherein the region of the tumor consists of tumor cells only, stroma only or tumor and stroma.

20. The method of claim 16 , wherein the method further comprises producing an output of the number of CD3 cells and CD16 cells.

21. The metho

Assignees

• Ventana Med Syst Inc

Inventors

• Smith Jacquelyn
• Hnatyszyn Harry James
• Olson Erik
• Theiss Noah
• Yates Alton
• Kaldjian Eric
• Vennapusa Bharathi

Classifications

• G01N33/5759 Primary

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• G01N1/312

  for samples mounted on planar substrates · CPC title

• G01N33/6872 Primary

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• G01N2001/302

  Stain compositions · 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