[196] Development of New Rabbit Monoclonal Antibody to Estrogen Receptor alpha (Clone EP1) and HER2/ERBB2 (Clone EP3) for Immunohistochemical Application

Aihua Li, Hongyang Pan, Nenghui Jiang, Zhiqiang Liu, Zhiling Fang, Maria Frolkis, Weimin Zhu, Taiying Chen. Epitomics, Inc., Burlingame, CA

Background: Rabbit monoclonal antibodies (RabMAbs) are known for their superior sensitivity and specificity in the immunohistochemical (IHC) detection of antigens in formalin-fixed paraffin-embedded (FFPE) tissue compared to mouse monoclonal and rabbit polyclonal antibodies. A RabMAb against ER alpha (ER) or HER2 has been shown to improve the IHC test quality in breast cancer diagnosis. However, the cross reactivity of ER with lung adenocarcinomas or ER beta protein is still a concern. The cross reactivity of anti-HER2 with HER4 protein may lead to false positive HER2 testing results in breast cancer patients. As a resolution, we developed a new RabMAb ER alpha using an immunogen that resides on the N-terminal portion of the ER protein, which shows no reactivity with ER beta. We have also produced a new HER2 RabMAb without HER4 immunoreactivity.
Design: Rabbits were immunized with recombinant human ER protein or a HER2 peptide corresponding to residues in human HER2 protein. Initial antibody characterization was performed by ELISA, differential western blot (WB) and IHC. Antibodies suitable for IHC were further validated with specific target tissues as well as normal, tumor and breast cancer tissue arrays. For ER, the performance was further compared with current ER standard clone SP1. Positive and negative staining status was scored according to ASCO/CAP guideline. A tumor with positive staining was determined when 1% or more cells were stained. The specificity, sensitivity and concordance of EP1 versus SP1 were analyzed.
Results: For ER antibody, clone EP1, WB results show that EP1 has no cross reactivity with ER beta. IHC analysis shows that EP1 labels the nucleus of target cells in breast, cervix and uterus in normal tissue arrays. No staining was observed in other normal tissues. In a breast cancer array, there is a high concordance between EP1 and SP1: the positive, negative and overall agreements are 95%, 96% and 98% respectively. For HER2 antibody, clone EP3, WB showed no reactivity with the HER4 protein. IHC testing shows that EP3 only labels the membrane of breast cancer and gastric cancer cells. No staining was observed in other tumor and normal tissues tested.
Conclusions: RabMAbs anti-ER alpha, clone EP1 and anti-HER2, clone EP3 are specific and sensitive in the detection of target proteins by IHC in FFPE tissues. EP1 is highly concordant with SP1. EP1 is useful in the immunohistochemical assessment of hormone receptor status in breast cancer. Compare to current HER2 antibodies, EP3 may be a potentially better tool for HER2 IHC testing.
Category: Breast

Wednesday, March 21, 2012 1:00 PM

Poster Session VI # 32, Wednesday Afternoon


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