A. Papanastasiou-Diamandi

Insulin-like growth factor–binding protein-3 and breast cancer survival

Insulin‐like growth factors (IGFs) are potent mitogens involved in the regulation of cell proliferation and apoptosis. The action of IGFs is mediated through a specific cell membrane receptor (IGF‐IR), and the interactions between IGFs and this receptor are regulated by IGF‐binding proteins (IGFBPs). IGFBP‐3 is one such protein which either suppresses or enhances the actions of IGFs. Findings from most in vitro studies suggest that IGFBP‐3 inhibits breast cancer cell growth and facilitates apoptosis, but clinical studies have found that high levels of IGFBP‐3 in breast cancer tissues are associated with unfavourable prognostic indicators of the disease, such as large tumour size, low levels of steroid hormone receptors, elevated S‐phase fraction and DNA aneuploidy. To further examine the role of IGFBP‐3 in breast cancer recurrence and survival, we conducted the following nested case‐control study. From a cohort of 1,000 women treated surgically for primary breast cancer, we consecutively selected 100 patients who developed recurrent disease after surgery and 100 age‐ and year of diagnosis‐matched patients who had no relapse. Concentrations of IGFBP‐3 in breast tissue extracts were determined with an ELISA. Inverse correlations of IGFBP‐3 were revealed with estrogen receptor expression and patient age but not with tumour size or S‐phase fraction. Levels of IGFBP‐3 in breast tissues were slightly higher in the recurrent patients than in controls, but the differences were not statistically significant. No significant association was found between IGFBP‐3 and breast cancer recurrence. Survival analysis, however, indicated that the risk of death was increased with higher IGFBP‐3 levels, and the association was independent of other prognostic markers. In conclusion, our results demonstrate that high levels of IGFBP‐3 are associated with unfavourable prognostic features of breast cancer. Int. J. Cancer (Pred. Oncol.) 79:624–628, 1998.

AN ULTRASENSITIVE IMMUNOASSAY FOR PROSTATE-SPECIFIC ANTIGEN BASED ON CONVENTIONAL COLORIMETRIC DETECTION

OBJECTIVE Development of an ultrasensitive immunoassay for serum PSA involving conventional detection probes. DESIGN AND METHODS The assay involves a polyclonal antibody immobilized in microtitration wells and a monoclonal antibody labeled with horseradish peroxidase. In a one-step assay, the enzymatic activity of the bound detection antibody is monitored by the addition of hydrogen peroxide/tetramethylbenzidine substrate reagent followed by spectrophotometric quantification of the conversion product. RESULTS The assay has a lower detection limit of 0.003 micrograms/L, biological detection limit of 0.009 micrograms/L, and intra- and interassay CVs of 8.2% and 10.5% at PSA concentrations of 0.022 and 0.065 micrograms/L, respectively. The recovery of the assay averaged 104% and it demonstrated a dilution linearity down to at least 0.01 micrograms of PSA/L. Results of comparison data correlated well with those obtained by a well established enzyme immunoassay. The serum PSA concentrations were < 0.012 micrograms/L in the majority of patients (53.8%) who had undergone radical prostatectomy. CONCLUSIONS This assay is well suited for post-surgical monitoring of PSA in patients with prostate cancer.

Immunoassay of triiodothyronine in serum by time-resolved fluorometric measurement of europium-chelate complexes in solution

We describe the development of a competitive immunoassay for triiodothyronine (T3) in serum. The assay combines immobilized antigens in microtitration wells with a biotinylated monoclonal anti-T3 antibody and a streptavidin-based universal detection reagent labeled with the Eu3+ chelator 4,7-bis(chloro-sulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid (BCPDA). In the assay, T3 released from binding proteins by thimerosal competes with immobilized antigen for binding to a limited amount of antibody. The bound biotinylated antibody is identified by a subsequent reaction with the detection reagent, and fluorescence of the final complex is then quantified in solution after it has been dissociated from the solid support by the addition of a detergent solution. Evaluation of the method demonstrated good overall precision and appropriate detection limit (0.2 nmol/L) and dynamic range. Analytical recovery averaged 99.9%, and results of dilution experiments were in agreement with the expected values. Measurements by the present method correlated well with those by a commonly used radioimmunoassay.

Triiodothyronine Uptake Measurement in Serum by Time-Resolved Fluorescence Immunofluorometry

A solid phase competition-type fluoroimmunoassay for triiodothyronine (T3) uptake in serum is described. In the assay, exogenous free T3 binds to the unoccupied binding sites on serum thyroxine binding proteins while the remaining unbound T3 competes with immobilized T3 for binding to a soluble biotinylated anti-T3 monoclonal antibody. The bound biotinylated antibody is quantitated by the addition of streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl-1,10 phenanthroline-2,9-bicarboxylic acid (BCPDA) in the presence of excess europium. The fluorescence signal of the final complex, which is directly proportional to the number of unoccupied binding sites on thyroxine binding proteins, is then measured on the dried solid-phase with a pulsed-laser time-resolved fluorometer. The assay requires a 10 microliters serum sample and a total incubation time of 90 minutes. The coefficients of variation for within-run and between-run assays ranged from 2.0 to 5.7%. Results obtained by the present method compared well with those determined by two commercial radioimmunoassays (r greater than 0.9).

Direct enzymic fluorimetric method for the determination of individual bile acids in bile

We describe a new, simple, fluorimetric enzymic method for the determination of individual bile acids in bile. Bile is diluted 4000-fold with water and 3 alpha- and 7 alpha-hydroxy bile acids are determined by equilibrium methods and chenodeoxycholic acid is determined by a differential kinetic method, without any prior separation and preparatory step. The equilibrium methods are based on the reaction of 3 alpha- or 7 alpha-hydroxy bile acids with beta-NAD+ in the presence of the enzyme 3 alpha- or 7 alpha-hydroxysteroid dehydrogenase (HSD). The kinetic method is based upon the reaction of chenodeoxycholic acid with beta-NAD+ in the presence of the enzyme 7 alpha-HSD. All measurements are monitored fluorimetrically. Cholic and deoxycholic acid are calculated by difference. Recovery experiments gave satisfactory results. Gallbladder bile from patients was analysed for the three major bile acids. The proposed method is suitable for clinical use.