Multiplexed immunohistochemistry using recombinant antibodies with epitope tags
The present disclosure is directed to epitope-tagged antibodies, as well as methods of employing the epitope-tagged antibodies for detecting one or more targets in a biological sample, e.g. a tissue sample.
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.
2. The method of claim 1 , wherein the sample is simultaneously contacted with the first and second anti-tag antibodies.
3. The method of claim 1 , wherein the first epitope-tagged primary antibody and the second epitope-tagged prim ay antibody are applied as a first mixture; and wherein the first and second anti-tag antibodies are applied as a second mixture.
4. The method of claim 1 , wherein the epitope tag construct has the structure (-[Spacer]-[Epitope Tag]-), wherein the structure is repeated from 1 to 12 times.
5. The method of claim 1 or 4 , wherein the method further comprises the steps of:
(d) contacting the sample with a third primary antibody specific to a third target; and
(e) contacting the sample with a third set of detection reagents for detecting binding of the third primary antibody to the sample, wherein the third set of detection reagents comprises an antibody specific for the third antibody.
6. The method of claim 5 , wherein the third primary antibody is a third epitope-tagged antibody, wherein the third epitope-tagged primary antibody comprises:
(i) a third antibody specific to the third target, and
(ii) a third epitope tag construct, wherein the third epitope tag construct comprises at least 2 repeats of a third epitope tag, wherein the repeats of the third epitope tag are separated by a spacer; and
wherein the antibody specific for the third antibody is a third anti-tag antibody, wherein the third anti-tag antibody is specific for the third epitope tag.
7. The method of claim 6 , wherein the sample is simultaneously contacted with the first epitope-tagged primary antibody, the second epitope-tagged primary antibody, and the third epitope-tagged primary antibody.
8. The method of claim 7 , wherein the sample is simultaneously contacted with the anti-tag antibodies.
9. The method of claim 8 , wherein the first epitope-tagged primary antibody, the second epitope-tagged primary antibody, and the third primary antibody are applied as a first mixture; and wherein the anti-tag antibodies are applied as a second mixture.
10. The method of claim 5 , wherein the third primary antibody does not comprise an epitope tag and has a species that is different from the first antibody and the second antibody, and wherein the antibody specific for the third antibody is an anti-species secondary antibody.
11. The method of claim 1 , wherein the first epitope tag construct is expressed at a C-terminus of a heavy chain of the first antibody; and the second epitope tag construct is expressed at a C-terminus of a heavy chain of the second antibody.
12. The method of claim 11 , wherein:
the first epitope-tagged antibody further comprises the first epitope tag construct expressed at a C-terminus of a light chain of the first antibody; and
the second antibody further comprises the second epitope tag construct expressed at a C-terminus of the light chain.
13. The method of claim 11 , wherein:
the first epitope tag construct comprises at least 2 epitope tags; and
the second epitope tag construct comprises at least 2 epitope tags.
14. The method of claim 13 , wherein:
the first epitope-tagged antibody has a configuration selected from the group consisting of H2K0, H3K0, H4K0, H5K0, H2K2, H3K3, and H4K4; and
the second epitope-tagged antibody has a configuration selected from the group consisting of H2K0, H3K0, H4K0, H5K0, H2K2, H3K3, and H4K4.
15. The method of claim 1 , wherein the first epitope tag construct is expressed at a C-terminus of a light chain of the first antibody; and the second epitope tag construct is expressed at a C-terminus of a light chain of the second antibody.
16. The method of claim 15 , wherein the first epitope-tagged antibody has a configuration H0K4; and the second epitope-tagged antibody has a configuration H0K4.