TY - JOUR
T1 - A sandwich type acridinium-linked immunosorbent assay (ALISA) detects soluble ErbB1 (sErbB1) in normal human sera
AU - Baron, Andre T.
AU - Lafky, Jacqueline M.
AU - Connolly, Denise C.
AU - Peoples, Jennifer
AU - O'Kane, Dennis J.
AU - Suman, Vera J.
AU - Boardman, Cecelia H.
AU - Podratz, Karl C.
AU - Maihle, Nita J.
N1 - Funding Information:
We thank Dr. George Klee for his advice concerning the design of these experiments and for critically evaluating our data during the preparation of this manuscript. We also thank Mr. Benjamin Madden for his assistance with the quantitative amino acid analysis and for reading this manuscript, Dr. Jill Reiter for helpful discussions about the cloning strategy of psErbB1ECD589 and for critically reading this manuscript, Dr. Marites Buenafe for helpful discussions and for carefully reading this manuscript, and Dr. Jeffrey Salisbury for providing access to equipment and reagents within his laboratory. This work was supported by the Fraternal Order of Eagles Cancer Research Fund, and by NIH grants 1RO1 CA57534 (Truncated c-erbB Receptors in Women with Ovarian Cancer) to NJM, CA09441-13 (Multidisciplinary Basic Research Training in Cancer) to the Mayo Foundation, and 1KO7 CA76170-01A1 (Soluble ErbB1 molecules as Tumor Biomarkers) to ATB.
PY - 1998/10/1
Y1 - 1998/10/1
N2 - The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression. In addition to the ErbB 1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB 1, i.e., sErbB 1. Overexpression of ErbB 1 in a variety of tumors has led us to hypothesize that sErbB1 levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers. To address this hypothesis we have developed an acridinium- linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media. 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 (~ 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV. Our ALISA does not detect full-length ErbB2, ErbB3, or 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 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years. Additional analyses do not indicate that serum sErbB 1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA. Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a ~ 110-kDa protein. 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 tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.
AB - The epidermal growth factor receptor (ErbB1) is overexpressed in various human tumor-derived cell lines and neoplasms, where it is believed that receptor dysregulation plays a role in oncogenic transformation and tumor progression. In addition to the ErbB 1 holoreceptor, numerous studies demonstrate that cells synthesize soluble or secreted forms of ErbB 1, i.e., sErbB 1. Overexpression of ErbB 1 in a variety of tumors has led us to hypothesize that sErbB1 levels also may be altered during oncogenesis, tumor progression, and/or metastasis; and that these molecules may be useful tumor biomarkers. To address this hypothesis we have developed an acridinium- linked immunosorbent assay (ALISA) specific for the extracellular domain of ErbB1 that can be used to quantify the levels of sErbB1 molecules in body fluids and conditioned culture media. 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 (~ 1 to 4000 fmol/ml), and good reproducibility (CVs < 10%). Specificity experiments show that this ALISA detects p170 ErbB1 and soluble forms of ErbB1 that embody extracellular subdomains I through IV, but not forms of sErbB1 lacking subdomain IV. Our ALISA does not detect full-length ErbB2, ErbB3, or 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 years, differ significantly from those of healthy men (median = 24,512 fmol/ml), ranging in age from 25 to 79 years. Additional analyses do not indicate that serum sErbB 1 levels change with age in either healthy men or women. Immunoprecipitation experiments show that monoclonal antibodies specific for extracellular epitopes of ErbB1 completely neutralize the detection of sErbB1 in normal human sera by ALISA. Finally, we show by immunoprecipitation and Western immunoblot analyses with monoclonal antibodies specific for the extracellular domain of ErbB1 that normal human female and male sera contain a ~ 110-kDa protein. 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 tumor biopsy specimens and body fluids, respectively, and for determining whether sErbB1, like ErbB1, is a useful tumor biomarker.
KW - Acridinium
KW - Acridinium- linked immunosorbent assay
KW - EGFR
KW - Epidermal growth factor receptor
KW - ErbB1
KW - Soluble ErbB1
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U2 - 10.1016/S0022-1759(98)00129-X
DO - 10.1016/S0022-1759(98)00129-X
M3 - Article
C2 - 9831386
AN - SCOPUS:0032192335
SN - 0022-1759
VL - 219
SP - 23
EP - 43
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -