Immunoenzymatically determined pepsinogen C concentration in breast tumor cytosols: an independent favorable prognostic factor in node-positive patients.

The aim of this study was to determine the concentration and to evaluate the prognostic value of pepsinogen C (PepC) in breast cancer patients. PepC is an aspartic proteinase that is involved in the digestion of proteins in the stomach and is also synthesized by a subset of human breast tumors. PepC concentrations were measured with a highly sensitive immunofluorometric assay, which uses two monoclonal antibodies that are specific for PepC and has a detection limit of 0.1 ng/ml. Breast tumor cytosols from 151 patients (median follow-up, 67 months), stratified according to nodal status, were evaluated. An optimal cutoff value, equal to 1.75 ng/mg of extracted protein, was first defined by statistical analysis. PepC status was then compared with other established prognostic factors, in terms of disease-free survival (DFS) and overall survival (OS). High PepC concentrations were found in small (P = 0.003) and well-differentiated tumors (P = 0.042) as well as in stage I (P = 0.003) and node-negative patients (P = 0.040). Statistically significant associations of PepC concentration with patient age and estrogen receptor and progesterone receptor status were not observed. In univariate Cox regression analysis of the entire cohort of patients, negative PepC proved to be a significant predictor of reduced DFS (P = 0.0086) and OS (P = 0.025). Multivariate analysis in subgroups of patients defined by nodal status indicated that PepC status was a strong predictor of DFS (P = 0.0039) and the strongest factor for predicting OS (P = 0.0046) in node-positive but not in node-negative patients. Our results suggest that PepC may be used as an independent favorable prognostic factor in node-positive breast cancer patients because there were no significant associations between PepC and the other prognostic factors evaluated in this group of patients.

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.

Associations between insulin-like growth factors and their binding proteins and other prognostic indicators in breast cancer.

Recent studies have suggested that insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs) may be implicated in the development and progression of breast cancer. Prostate-specific antigen (PSA), a serine protease, may play a role in the regulation of IGFs' function through cleavage of IGFBP-3, resulting in release of active IGFs from IGFBP-3. As IGFs, IGFBPs and PSA are all present in breast cancer, possible associations among these proteins were speculated. In this study, we have measured PSA, IGF-I, IGF-II, IGFBP-1 and IGFBP-3 in tumour tissue cytosols from 200 women with primary breast cancer, and have examined relationships between IGFs or IGFBPs and PSA along with other markers, including p53 protein, steroid hormone receptors (oestrogen and progesterone), cathepsin-D, epidermal growth factor receptor, Her-2/neu protein, S-phase fraction and DNA ploidy. Correlations or associations between PSA and IGF-I, IGF-II, IGFBP-1 or IGFBP-3 were not observed. IGF-II was positively correlated with both IGFBP-3 and IGFBP-1. IGF-I was not associated with either of the two binding proteins, nor with IGF-II. Both IGF-II and IGFBP-3 were inversely associated with the oestrogen receptor, and IGFBP-3 was also positively associated with S-phase fraction. Our finding of IGF-II and IGFBP-3 in association with unfavourable prognostic indicators of breast cancer suggests that IGFs may be involved in the progression of breast cancer.

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.

Hapten-heterologous conjugates evaluated for application to free thyroxine immunoassays.

We evaluated the effect of hapten heterology on free thyroxine (FT4) immunoassays involving the biotin-streptavidin system and time-resolved fluorometry. We compared protein derivatives of thyroxine (T4) and triiodothyronine (T3) as solid-phase antigen or biotinylated protein-tracer conjugate for competitive (or sequential) binding to a mouse anti-T4 monoclonal antibody. In both one- and two-step assays, the heterologous combination of the antibody and T3 conjugates showed superior standard curve sensitivity but up to eightfold lower zero standard signal (B(o)) when the same amounts of antibody and conjugates were used. The improved sensitivity was not altered when the amount of coupled T3 was increased to obtain a B(o) value similar to that of the homologous combination of antibody and T4 conjugates. In the two-step format, the sensitivity of the homologous assay was insufficient for routine use, consistent with displacement of bound T4 during the antibody back-titration step (demonstrated in the T4 displacement experiment with excess conjugate). Results from the one-step (labeled antibody) heterologous assay for approximately 85 clinical samples correlated well with those from an immunofluorometric assay and a two-step radioimmunoassay. The assay was not affected by a wide variation in endogenous serum concentrations of T4-binding globulin and albumin.

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.

Ultrasensitive thyrotropin immunoassay based on enzymatically amplified time-resolved fluorescence with a terbium chelate.

We describe an ultrasensitive, enzymatically amplified time-resolved fluorescence immunoassay of thyrotropin (thyroid-stimulating hormone) in serum with use of a terbium chelate as the detectable moiety. In this assay, thyrotropin is first simultaneously reacted with a solid-phase (microtiter well) monoclonal antibody and a soluble biotinylated monoclonal detection antibody. After washing, a streptavidin-alkaline phosphatase conjugate is added, followed by another washing. Alkaline phosphatase acts on the substrate 5-fluorosalicyl phosphate (FSAP) to produce 5-fluorosalicylic acid (FSA). FSA, but not FSAP, can then form with Tb3+ and EDTA a highly fluorescent ternary complex of long fluorescence lifetime. This complex is quantified with time-resolved fluorometry. The thyrotropin assay is highly sensitive (detection limit approximately 0.003 milli-int. unit/L when a total assay time of 85 min is used), precise, and accurate. The thyrotropin assay can also be completed in less than 30 min (detection limit 0.013 milli-int. unit/L), thus making this procedure a candidate technology for high-throughput automated analyzers.

Sensitive time-resolved fluorescence immunoassay of somatotropin in serum.

We describe a new "sandwich"-type non-isotopic immunoassay for human somatotropin (GH, growth hormone) in serum. In the assay, GH is captured by a monoclonal antibody immobilized in a white microtiter well and simultaneously reacted with a second biotinylated monoclonal antibody. The degree of binding of biotinylated antibody, which increases with increasing amount of GH in the sample, is quantified by adding streptavidin labeled with the europium chelate of 4.7 - bis(chlorosulfophenyl) - 1.10 - phenanthroline - 2.9 - dicarboxylic acid. The fluorescent complex on the solid phase is then measured by excitation at 337.1 nm (nitrogen laser) and monitoring the emission at 615 nm in a gated fluorometer/analyzer. The proposed procedure has short incubation times (less than 4 h protocol), uses only 25 microL of serum per microtiter well, and gives precise and accurate results. The method was clinically evaluated with samples obtained from pediatric patients undergoing investigation for growth abnormalities and from a patient with acromegaly.

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).

Time-resolved immunofluorometric assay of human pancreatic isoamylase in serum, with use of two monoclonal antibodies.

This new method for determining pancreatic isoamylase (EC 3.2.1.1) in serum involves two monoclonal antibodies: one immobilized in a microtitration well (the capture antibody), the other biotinylated. After the sample is incubated with the two antibodies, the captured immunocomplex is quantified by adding streptavidin labeled with a europium chelator and measuring the specific Eu3+ fluorescence in a time-resolved mode. Three assay protocols are proposed, involving incubation times of 90, 45, or 25 min. The assay has low (0.005%) cross-reactivity with the salivary isoenzyme. Analytical performance was satisfactory. Results correlate well with results obtained by measuring total amylase activity or by measuring pancreatic isoamylase activity after immunoinhibition. Unlike numerous current amylase assays, this method measures enzyme mass rather than enzyme activity. Potentially, this is a highly specific assay.

Digoxin immunoassay with monoclonal and polyclonal antibodies using time-resolved fluorometry.

We describe a nonisotopic heterogeneous competitive immunoassay of digoxin in serum using either Fab fragments of a polyclonal antibody or a high-affinity monoclonal antibody. In the assay, digoxin competes with immobilized digoxin (digoxin:thyroglobulin conjugate) for binding to a biotinylated immunoreactant (Fab or monoclonal). The amount of biotinylated moiety bound to the solid phase (white polystyrene microtiter wells), which is inversely related to the amount of digoxin in the sample, is then quantified by adding streptavidin labeled with the europium chelator 4,7-bis(chlorosulfophenyl)-1,10- phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The fluorescent immunocomplex formed is measured directly on the dry solid phase by time-resolved fluorometry. The assay is simple to perform and its characteristics are similar to those of other currently used immunoassay techniques. The Fab fragments and the monoclonal antibody procedure performed equally well on the system. Our results suggest that monoclonal antibodies with high affinity for digoxin can be used for the routine determination of the drug in serum.

Digoxin immunoreactivity in cord and maternal serum and placental extracts. Partial characterization of immunoreactive substances by high-performance liquid chromatography and inhibition of Na+, K+-ATPase.

Digoxin-like immunoreactive substances (DLIS) have been successfully extracted and concentrated from cord serum, mixed (cord and maternal) serum and placentas. Similar substances have also been extracted from normal adult serum, but DLIS in this medium are present in much lower concentrations. Concentrated DLIS have been separated into several immunoreactive fractions by use of reverse-phase high-performance liquid chromatography. Immunoreactive fractions were tested for their ability to inhibit the Na+,K+-ATPase by measuring the 86Rb-uptake of red blood cells in the presence of these fractions. A potent inhibitor was identified in an immunoreactive fraction which also contains progesterone, but the results suggest that the pump inhibitor is not progesterone. Cross-reactivity studies utilising fluorescence polarization immunoassay have shown that cortisone is the most potent immunoreactive substance of cord serum.

Are immunoassays for digoxin reliable?

The specificity of radioimmunoassay and fluorescence polarization immunoassay procedures for the measurement of digoxin has been assessed. Many steroids and lipids have been found to interfere and give false positive results for digoxin under the conditions of the study. Caution is recommended in the interpretation of digoxin measurements made by immunoassay procedures.

Enzymic measurement of primary bile acids and the primary bile acid ratio in serum with the IL-Multistat III Fluorescence Light-Scattering Centrifugal Analyzer.

Enzymic fluorimetric methods are described for the determination of primary bile acids and of chenodeoxycholic acid (CDC) and cholic acid (C) in serum. Bile acids are extracted from 0.3 mL of serum in a simple 5-min step with use of Sep-Pak C cartridges. Total primary bile acids are measured by an equilibrium technique after reaction with beta-NAD in the presence of 7 alpha-hydroxysteroid dehydrogenase. Chenodeoxycholic acid (and its conjugates) is measured by a reaction-rate technique employing the same reaction as above but under different experimental conditions. A small contribution of cholic acid (and its conjugates) to the reaction rate is eliminated by simple calculations. Cholic acid is calculated by difference of the two determinations. In both assays NADH fluorescence is measured with the Multistat centrifugal analyzer. Absolute recovery of bile acids from serum was about 87%. Day-to-day standard deviations for CDC and C were 1.6 and 2.0 mumol/L at serum concentrations of 22.1 and 24.1 mumol/L respectively. Comparison data with a cholylglycine RIA procedure gave the following correlation coefficients (x = RIA, y = proposed method): r = 0.980 (RIA vs total primary bile acids), r = 0.918 (RIA vs CDC) and r = 0.989 (RIA vs C). The methods described appear more practical for use on a routine basis than methods in the literature for the calculation of the primary bile acid ratio.

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.