Detection of different estrogen receptor forms in breast cancer cytosol by enzyme immunoassay.
Estrogen receptors (ER) are routinely measured in tissue extracts from breast cancer using a radioligand binding assay (RBA) and an enzyme immunoassay (EIA). Although good correlation was found between the two methods, they are expected to measure, at least in part, different ER amounts in individual samples, because the RBA should detect unfilled ER only, whereas EIA should recognize both unfilled receptors and those filled by endogenous estrogens. The purpose of the present investigation was to evaluate if ER-EIA mainly detects ER filled by endogenous estrogens when using an estrogen-free buffer to dilute cytosol samples. Indeed, the commercially available EIA assay kit (ER-EIA; Abbott) is equipped with a sample dilution buffer containing a high concentration of 17beta-estradiol which should allow for the saturation of all the ERs. ER was measured in 57 cytosol samples from primary breast cancer with RBA and ER-EIA. In the latter case, samples were diluted using both the estradiol-rich dilution buffer of the kit and an estrogen-free low salt phosphate buffer. RBA and ER-EIA showed tightly correlated results. However, ER-EIA detected higher ER levels than RBA in the majority of cases. Results obtained by low salt ER-EIA were also correlated to both RBA and ER-EIA, showing, however, lower ER concentrations. ER levels measured by ER-EIA were not significantly different from the sum of ER concentrations found by RBA and low salt ER-EIA. These findings suggest that ER-EIA detects ER only in the conformational status that is achieved after saturation by estrogens. These findings were confirmed by sedimentation shift experiments, which showed that the monoclonal antibody D547 used in the kit binds ER in the occupied form only. This leads to the conclusion that ER-EIA detects functioning (in terms of binding with estradiol) ERs. From the present investigation, we suggest that it is possible and probably worthwhile to optimize the EIA method by using different buffers to measure: (a) the total number of ERs capable of binding estradiol; (b) the ER filled by endogenous estrogens; and (c) by difference, the unfilled ER concentrations.
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Centro Regionale Specializzato per lo Studio degli Indicatori Biochimici di Tumore, Ospedale Civile, Campo SS. Giovanni e Paolo, Venezia, Italy.
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Amoroso
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D000911:Antibodies, Monoclonal / Q000276:immunology; D001943:Breast Neoplasms / Q000737:chemistry*; D002021:Buffers; D003600:Cytosol / Q000737:chemistry*; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D009363:Neoplasm Proteins / Q000032:analysis* / Q000145:classification / Q000276:immunology / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay; D011933:Reagent Kits, Diagnostic; D011960:Receptors, Estrogen / Q000032:analysis* / Q000145:classification / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D002021:Buffers; D009363:Neoplasm Proteins; D011933:Reagent Kits, Diagnostic; D011960:Receptors, Estrogen; D004958:Estradiol; D011189:Potassium Chloride