A SANDWICH-TYPE ACRIDINIUM-LINKED IMMUNOSORBENT-ASSAY (ALISA) DETECTSSOLUBLE ERBB1 (SERBB1) IN NORMAL HUMAN SERA

The epidermal growth factor receptor (ErbB1) is overexpressed in vario us human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation a nd tumor progression. In addition to the ErbB1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB1, i.e., sErbB1. Overexpression of ErbB1 in a variety of tumors h as led us to hypothesize that sErbB1 levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these mol ecules may be useful tumor biomarkers. To address this hypothesis we h ave developed an acridinium-linked immunosorbent assay (ALISA) specifi c for the extracellular domain of ErbB1 that can be used to quantify t he levels of sErbB1 molecules in body fluids and conditioned culture m edia. This assay can also detect full-length ErbB1 in cell and tissue extracts. Our ALISA is characterized by high sensitivity (intra-assay LLD < 1 fmol/ml), a broad linear range (similar to 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show th at this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embod y extracellular subdomains I through IV, but not forms of sErbB1 lacki ng subdomain TV. Our ALISA does not detect full-length ErbB2, ErbB3, o r ErbB4; or p105 soluble ErbB2. We report that serum sErbB1 levels of healthy women (median = 3716 fmol/ml), ranging in age from 43 to 76 ye ars, differ significantly from those of healthy men (median = 24,512 f mol/ml), ranging in age from 25 to 79 years. Additional analyses do no t indicate that serum sErbB1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal ant ibodies specific for extracellular epitopes of ErbB1 completely neutra lize the detection of sErbB1 in normal human sera by ALISA. Finally, w e show by immunoprecipitation and Western immunoblot analyses with mon oclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a similar to 110-kDa protei n. We conclude that our ALISA is measuring the relative levels of this p110 sErbB1 analog in normal human sera. Our ALISA, therefore, should be useful for measuring the levels of ErbB1 and sErbB1 molecules in t umor biopsy specimens and body fluids, respectively, and for determini ng whether sErbB1, like ErbB1, is a useful tumor biomarker. (C) 1998 E lsevier Science B.V. All rights reserved.