Generation and characterization of polyclonal antibodies specific for human p110 sEGFR
Generation and characterization of polyclonal antibodies specific for human p110 sEGFR
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DOI：
10.1089/153685902760173908
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发表时间：
2002-06-01
期刊：
影响因子：
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通讯作者：
Maihle, NJ
中科院分区：
文献类型：
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作者：
Christensen, TA;Reiter, JL;Maihle, NJ
EGFR/ERBB受体酪氨酸激酶家族在调节细胞生长、分化和转化中起重要作用的细胞内信号转导通路。我们之前已经报道了3kb的EGFR转录本的克隆和表达，该转录本编码110 kDa形式的受体(P110 SEGFR)。该受体亚型与全长170 kDa的EGFR胞外区603个氨基酸完全相同；此外，p110 sEGFR含有78个独特的羧基末端氨基酸。在此，我们报道了针对p110 sEGFR独特的羧基末端序列的多克隆抗血清的制备和鉴定。用合成的与p110 sEGFR独特的羧基末端序列相对应的多肽免疫兔，制备了多克隆抗血清。抗血清经酶联免疫吸附试验检测为免疫反应阳性的免疫球蛋白组份，经G蛋白亲和层析和肽基层析纯化。这种亲和纯化的免疫球蛋白组分通过ELISA、免疫沉淀、免疫印迹分析和免疫细胞化学方法特异性识别p110 sEGFR。使用这些检测方法中的任何一种都没有观察到与全长P170 EGFR的交叉反应。这些新的多克隆抗体将有助于确定p110 sEGFR的表达、定位和功能，重要的是，将使我们能够区分该受体亚型和p170 EGFR的表达。
The EGFR/ERBB family of receptor tyrosine kinases mediates intracellular signal transduction pathways important in the regulation of cell growth, differentiation, and transformation. We previously have reported the cloning and expression of a 3 kb alternative EGFR transcript which encodes a 110 kDa form of the receptor (p110 sEGFR). This receptor isoform is identical to the extracellular region of the full-length 170 kDa EGFR through amino acid 603; in addition, p110 sEGFR contains 78 unique carboxy-terminal amino acids. Here, we report the generation and characterization of polyclonal antisera specific for the unique carboxy-terminal sequence of p110 sEGFR. Polyclonal antisera were generated by immunizing rabbits with synthetic peptides corresponding to peptides contained within the unique carboxy-terminal sequence of p110 sEGFR. Immunoglobulin fractions from antisera which tested positive for immune reactivity to these peptides by ELISA were affinity-purified by protein G and peptide-based chromatography. This affinity-purified immunoglobulin fraction specifically recognizes p110 sEGFR by ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemical methods. No cross-reactivity with full-length p170 EGFR is observed using any of these detection methods. These new polyclonal antibodies will be useful in determining the expression, localization, and function of p110 sEGFR, and importantly will allow us to distinguish between the expression of this receptor isoform and p170 EGFR.