High affinity monoclonal antibody for recognizing the estrogen receptor (ER) and method for creating the antibody
High affinity monoclonal antibodies for recognizing estrogen receptor (clone SP1) with immunohistochemistry and methods for creating such an antibody are disclosed. The lagomorph derived ER antibody provides a significant advantage over the currently available mouse ER antibodies in that there is no need for target retrieval when performing immunohistochemistry. Furthermore, the very low background when the lagomorph derived ER antibody is used in immunohistochemistry is also impressive. The immunohistochemistry comparative study with about fifty clinical specimens showed that the new ER (clone SP1) antibody had favorable results when compared to mouse monoclonal ER antibodies (clone 1D5). The lagomorph derived ER antibody may prove of great value in the assessment of ER status in human breast cancer. Humanized versions of the ER antibody may also provide therapeutic benefits.
1. A rabbit monoclonal antibody having a specific affinity for human estrogen receptor that is higher than that of murine monoclonal antibody 1D5, wherein the rabbit monoclonal antibody specifically binds an epitope within amino acids 578–595 of the estrogen receptor alpha protein present in a fresh tissue section and present, in the absence of a tissue section processing step involving proteolytic digestion or heat treatment for antigen retrieval, in a formalin-fixed, paraffin-embedded tissue section after processing steps of deparaffinization and rehydration.
2. The antibody of claim 1 , wherein the specific affinity for the estrogen receptor is over eight times higher than that of the murine monoclonal antibody 1D5.
3. A method for performing immunohistochemistry for the human estrogen receptor on a formalin-fixed, paraffin-embedded tissue section after processing steps of deparaffinization on and rehydration in the absence of a tissue section processing step involving proteolytic digestion or heat treatment for antigen retrieval, comprising the step of contacting the section with the antibody of claim 1 to specifically detect the estrogen receptor.
4. A method for performing immunohistochemistry for the human estrogen receptor on a formalin-fixed, paraffin-embedded tissue section after processing steps of deparaffinization and rehydration in the absence of a tissue section processing step involving proteolytic digestion or heat treatment for antigen retrieval, comprising the step of contacting the section with the antibody of claim 2 to specifically detect the estrogen receptor.
5. A method for selecting a monoclonal antibody for immunohistochemistry analysis of a human estrogen receptor, said method comprising the steps of:
a) contacting a rabbit monoclonal antibody of a plurality of rabbit monoclonal antibodies with a formalin-fixed, paraffin-embedded tissue section that contains the estrogen receptor, in the absence of a tissue section processing step involving proteolytic digestion or heat treatment for antigen retrieval, and after processing steps of deparaffinization and rehydration;
b) detecting specific binding of the monoclonal antibody to the estrogen receptor in the absence of antigen retrieval; and
c) selecting the monoclonal antibody from the plurality of monoclonal antibodies for immunohistochemistry analysis of the estrogen receptor.
6. The method of claim 5 , further comprising prior to the contacting step, the step of performing an ELISA with supernatants from hybridomas to identify the recited plurality of rabbit monoclonal antibodies.
7. The method of claim 5 , further comprising after the selecting step, the step of humanizing the rabbit monoclonal antibody.
8. The method of claim 4 , wherein the rabbit monoclonal antibody specifically binds an epitope within amino acids 578–595 of the estrogen receptor alpha protein present in the formalin-fixed, paraffin-embedded tissue section without antigen retrieval.
9. The method of claim 5 , wherein the rabbit monoclonal antibody specifically binds an epitope within amino acids 578–595 of the estrogen receptor alpha protein present in the formalin-fixed, paraffin-embedded tissue section without antigen retrieval, and the specific affinity for the estrogen receptor is over eight times higher than that of murine monoclonal antibody 1D5.