Generation and characterization of polyclonal antibodies specific for human p110 sEGFR.
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.
Christensen
Trace A
TA
Tumor Biology Program, Mayo Clinic, Rochester, MN 55905, USA.
Reiter
Jill L
JL
Baron
Andre T
AT
Maihle
Nita J
NJ
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D011233:Precipitin Tests; D011817:Rabbits
D000906:Antibodies; D066246:ErbB Receptors