Materials and methods for performing histochemical assays for human pro-epiregulin and amphiregulin
The invention provides anti-human pro-epiregulin and anti-human amphiregulin antibodies and methods of using the same. Anti-EREG antibodies raised against amino acids 148-169 and 156-169 of the human EREG protein, and anti-AREG antibodies raised against amino acids 238-252 of the human AREG protein are disclosed. Methods of using these antibodies to detect EREG and AREG and kits and other products for performing such methods are also disclosed.
1. An antibody or antibody fragment capable of specifically binding to amino acids 238-252 of SEQ ID NO: 36, wherein said antibody comprises:
a heavy chain variable domain (VH) amino acid sequence comprising the following heavy chain hypervariable regions (HVR-Hs):
(a) an HVR-H1 comprising SEQ ID NO: 37;
(b) an HVR-H2 comprising SEQ ID NO: 38; and
(c) an HVR-H3 comprising SEQ ID NO: 39; and
a light chain variable domain (VL) amino acid sequence comprising the following light chain hypervariable regions (HVR-Ls):
(d) an HVR-L1 comprising SEQ ID NO: 44;
(e) an HVR-L2 comprising SEQ ID NO: 45; and
(f) an HVR-L3 comprising SEQ ID NO: 46.
2. The antibody or antibody fragment of claim 1 , wherein the VH amino acid sequence further comprises the following variable domain framework regions (FRs):
(g) FR-H1 comprising SEQ ID NO: 40;
(h) FR-H2 comprising SEQ ID NO: 41;
(i) FR-H3 comprising SEQ ID NO: 42; and
(j) FR-H4 comprising SEQ ID NO: 43.
3. The antibody or antibody fragment of claim 1 , wherein the VL amino acid sequence further comprises the following variable domain framework regions (FRs):
(k) FR-L1 comprising SEQ ID NO: 47;
(l) FR-L2 comprising SEQ ID NO: 48;
(m) FR-L3 comprising SEQ ID NO: 49; and
(n) FR-L4 comprising SEQ ID NO: 50.
4. The antibody or antibody fragment of claim 1 , wherein the VH amino acid sequence has at least 95% sequence identity to SEQ ID NO: 51.
5. The antibody or antibody fragment of claim 4 , wherein the VH amino acid sequence comprises SEQ ID NO: 51.
6. The antibody or antibody fragment of claim 4 , wherein the VL amino acid sequence comprises SEQ ID NO: 52.
7. The antibody or antibody fragment of claim 4 , wherein the VH amino acid sequence comprises SEQ ID NO: 51 and the VL amino acid sequence comprises SEQ ID NO: 52.
8. The antibody of claim 1 , wherein the antibody is a monoclonal antibody.
9. The antibody of claim 8 , wherein the monoclonal antibody is a rabbit monoclonal antibody.
10. The antibody of claim 8 , wherein the monoclonal antibody is an IgG antibody.
11. The antibody fragment of claim 1 , wherein the antibody fragment is selected from the group consisting of Fab, single chain variable fragment (scFv), Fv, Fab′, Fab′-SH, F(ab′)2, and diabody.
12. The antibody or antibody fragment of claim 1 , wherein the VL amino acid sequence has at least 95% sequence identity to SEQ ID NO: 52.
13. The antibody or antibody fragment of claim 1 , wherein the VH amino acid sequence has at least 95% sequence identity to SEQ ID NO: 51, and the VL amino acid sequence has at least 95% sequence identity to SEQ ID NO: 52.
14. A method of detecting the presence or expression level of human amphiregulin in a biological sample comprising:
contacting the biological sample with the antibody or antibody fragment of claim 1 ; and
detecting the presence of the bound antibody or antibody fragment.
15. The method of claim 14 , wherein the detecting is by immunohistochemistry, immunofluorescence, or immunoblot.
16. The method of claim 14 , wherein the biological sample comprises a fixed tissue.
17. The method of claim 16 , wherein the fixed tissue is a formalin-fixed paraffin embedded (FFPE) tissue.
18. The method of claim 14 , wherein the biological sample is from a subject having or predisposed to cancer.
19. The method of claim 18 , wherein the cancer is colon cancer, breast cancer, or lung cancer.