IGF-I and IGF-binding protein-3 measurements on filter paper blood spots in children and adolescents on GH treatment: use in monitoring and as markers of growth performance.

BACKGROUND/AIM: In childhood an appropriate response to GH treatment is achieved by titration of growth response against dose administered, with careful observation for side-effects. In order to evaluate the potential use of IGF monitoring in children treated with GH, a cross-sectional study has been carried in 215 children and adolescents (134 with GH deficiency (GHD), 54 with Turner syndrome (TS) and 27 with non-GHD growth disorders) treated with GH for 0.2-13.7 years. METHODS: IGF-I and IGF-binding protein-3 (IGFBP-3) were measured in ELISAs, using dried capillary blood collected onto filter papers. Results were expressed as the mean S.D. range (SDS). Values of either analyte < -2 or > +2 SDS were considered abnormal. RESULTS: IGF-I and IGFBP-3 SDS were higher in the TS and non-GHD groups (mean +0.01 and +0.1 respectively) than in those with GHD (mean value -0.6). Nineteen per cent of the IGF-I values (13% low, 6% high) and 12% of IGFBP-3 values were abnormal (10% low, 2% high). Abnormalities, either low or high, were most common in the GHD group. There was a weak but significant relationship between change in height SDS over the Year up to the time of sampling in the whole group and IGF-I SDS. Satisfactory growth performance (+0.5>change in height SDS> -0.5) was found in those with high (7.2%), normal (60%) and low (9.3%) IGF-I levels. Overall, it was estimated that 26% of the tests would indicate that an adjustment to GH dose (up in 18% and down in 8%) could be considered. CONCLUSIONS: From this cross-sectional study of IGF monitoring across a broad range of diagnoses and ages, it can be concluded that the majority of children on GH have normal levels of IGF-I and IGFBP-3, but 26% of tests could suggest that a change of GH dose should be considered. Regular monitoring of IGF-I and IGFBP-3 should be considered in any child on GH treatment.

IGFBP-3 in epithelial ovarian carcinoma and its association with clinico-pathological features and patient survival.

Insulin-like growth factor binding protein-3 (IGFBP-3) regulates the mitogenic and anti-apoptotic actions of insulin-like growth factors (IGFs). To study the role of IGFBP-3 in ovarian cancer progression, we measured IGFBP-3 concentrations in tumour tissues from 147 patients with epithelial ovarian carcinoma and examined its associations with clinicopathological features of disease and patient survival. The average age of the patients was 54.6 years (range 25-88 years) and the median follow-up time was 37 months. IGFBP-3 levels were measured with a commercial immunoassay kit. Low IGFBP-3 levels were significantly associated with unfavourable prognostic features of the disease, including advanced stage (P=0.048), large size of residual tumour (P=0.007), and suboptimal debulking outcome (P=0.007). Low IGFBP-3 levels were also associated with a significantly increased risk for disease progression (RR=1.92; 95% confidence interval (CI) 1.05-3.45; P=0.034), but the association was not sustained when other clinical and pathological variables were adjusted for in the analysis. No significant associations were observed between the IGFBP-3 level and patients' overall survival and response to chemotherapy. Findings of the study indicate that IGFBP-3 may play a role in the progression of epithelial ovarian cancer, but that it has no independent value in predicting either disease prognosis or the response of patients to chemotherapy.

Free insulin-like growth factor-I and breast cancer risk.

Insulin-like growth factor-I (IGF-I) has mitogenic and anti-apoptotic effects on breast cancer cells. Epidemiologic studies have shown that high plasma levels of IGF-I and low levels of IGF binding protein (BP)-3 are associated with increased risk of breast cancer in premenopausal women. The actions of IGF-I are mediated through the IGF-I receptor (IGF-IR) and are regulated by IGFBPs. In circulation, most of the IGF-I binds to IGFBP-3, and binding of IGF-I to IGFBP-3 inhibits the actions of IGF-I. Since free IGF-I, which does not bind to IGFBPs, can readily cross the endothelial barrier to interact with IGF-IR, circulating free IGF-I is thought to be more relevant to the biologic activity of IGF-I. To examine the association of free IGF-I with breast cancer, we compared free IGF-I levels between 40 newly diagnosed breast cancer patients and 40 age- and race-matched healthy controls. Plasma levels of free IGF-I, total IGF-I and IGF-II, as well as total, intact and fragment IGFBP-3, were measured using commercial immunoassay kits. The association between IGF-I and breast cancer was examined using the conditional logistic regression analysis. Analysis of correlation (Spearman) showed that free IGF-I was correlated with total IGF-I and IGFBP-3 but not with IGF-II. The odds ratios for breast cancer patients having high plasma IGF-I (> or = median) after adjusting for menopausal status and IGFBP-3 were 2.00 (p < o r = 0.376) for total IGF-I and 6.31 (p < or = 0.047) for free IGF-I. A high ratio of IGF-I to IGFBP-3 was also associated with breast cancer (p < 0.05). No association was found for IGF-II, nor for total, intact and fragment IGFBP-3. The findings of this study suggest that measuring free IGF-I in circulation is more useful than measuring total IGF-I with respect to evaluation of an association between IGF-I and breast cancer risk.

Insulin-like growth factor I (IGF-I) and IGF-binding protein-3 in benign prostatic hyperplasia and prostate cancer.

In view of evidence indicating significant involvement of the insulin-like growth factor (IGF) system in the pathogenesis of prostate cancer, we measured serum IGF-I and IGF-binding protein-3 (IGFBP-3) in men with benign prostatic hyperplasia (BPH; n = 75) or prostatic carcinoma (CaP; n = 84). The age-matched patient populations were selected to have circulating prostate-specific antigen (PSA), the most reliable predictor of CaP, in the overlapping diagnostic gray zone range of approximately 4--10 microg/L. Of particular interest was investigation of intact, fragment, and total IGFBP-3 levels in relation to PSA, which is also a well established IGFBP-3 protease. Among the key findings were significantly higher IGF-I and intact IGFBP-3 levels in CaP vs. BPH (P < 0.001), whereas changes in fragment and total IGFBP-3 were statistically insignificant. As expected, total PSA levels were similar in the two groups of patients (P = 0.173), whereas free PSA levels were significantly lower in those with CaP (P < 0.001). IGF-I and IGFBP-3 (intact and total) correlated significantly (P = 0.024 to <0.001) and inversely (r = -0.26 to -0.35) with free PSA in BPH, but not in CaP, and no correlations were found in comparisons involving total PSA. Statistical analysis of the various markers and their combinations indicated enhanced performance of IGF-I/free PSA [receiver operating characteristics area under the curve (AUC) = 0.728] and intact IGFBP-3/free PSA (AUC = 0.737) ratios in discriminating between BPH and CaP compared with the currently used free/total PSA ratio (AUC = 0.689). Multivariate logistic regression models confirmed the observed relationships and identified IGF-I/free PSA and intact IGFBP-3/free PSA as independent factors in predicting the presence of CaP. We conclude that increases in IGF-I and intact IGFBP-3 levels are positively associated with the presence of CaP in this group of patients with low to moderately elevated PSA, and that their measurements in relation to PSA may help improve diagnostic discrimination between BPH and prostate cancer.

Immunoassay of insulin-like growth factor-binding protein-3 (IGFBP-3): new means to quantifying IGFBP-3 proteolysis.

Posttranslational modifications, particularly proteolysis, may play a significant role in the regulation of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) physiology, and thus, measurement of modified variants of IGFBP-3 and/or their combination ratios may have important research and diagnostic relevance. Based on evaluation of a panel of monoclonal and polyclonal IGFBP-3 antibodies, we constructed three new enzyme-linked immunosorbent assays (ELISAs) using a common capture and polyclonal (ELISA-3) or monoclonal (ELISA-1 and -2) detection antibodies and evaluated them in a two-step colorimetric procedure. Evaluation of ELISA-1-3 demonstrated detection limit, dynamic range, overall precision, and recovery of the added IGFBP-3 to be generally less than 0.04 microg/L, 2-100 microg/L, less than 10%, and 91-113%, respectively. IGF-I and -II, and IGFBP-1, -2, -4, -5, and -6 did not interfere. In normal adult sera (n = 26), seminal plasma (n = 14), pregnancy sera (n = 30), and amniotic fluid (n = 30), ELISA-1-3 detected significantly different IGFBP-3 levels (by up to 6-fold, on the average), whereas levels in seminal plasma determined by ELISA-1 were undetectable. Comparison of the values obtained vs. corresponding levels by an established method (Diagnostic Systems Laboratories, Inc., active IGFBP-3 ELISA) were similarly sample dependent and, on the average, varied by up to 19-fold. Only ELISA-3 compared well with the Diagnostic Systems Laboratories, Inc., IGFBP-3 ELISA when samples from normal adults were analyzed. The observed variability could not be totally explained by 50% lower reactivity of ELISA-1-3 for glycosylated IGFBP-3 vs. the nonglycosylated form, and changes in phosphorylation had no effect on immunoreactivity. Evaluation of IGFBP-3 after proteolysis by seminal plasma, plasmin, or thrombin suggested recognition of intact IGFBP-3 by ELISA-1, whereas ELISA-3 appeared to measure intact and proteolyzed IGFBP-3 (total IGFBP-3) with similar potency. In contrast, levels determined by ELISA-2 increased severalfold, indicating preferential recognition of IGFBP-3 fragments. We propose that immunoassay capable of differential determination of IGFBP-3 variants may help better define the physiological importance and potential clinical value of IGFBP-3 measurements.

The high molecular weight insulin-like growth factor-binding protein complex: epitope mapping, immunoassay, and preliminary clinical evaluation.

Measurements of insulin-like growth factor I(IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.

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.

Filter paper blood spot assay of human insulin-like growth factor I (IGF-I) and IGF-binding protein-3 and preliminary application in the evaluation of growth hormone status.

To facilitate broader applications of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) analysis, we developed procedures for their measurements in extracts of whole blood dried on filter paper. A single 8-mm diameter filter paper disc containing about 13 microL blood was used. IGFBP-3 was efficiently extracted in a buffer within 1 h of incubation. IGF-I extraction involved incubation in buffer followed by acidification and neutralization steps. Blood spot assays showed intra- and interassay coefficients of variation (including interspot variations) of 5.4-16.7% for IGF-I and 6.6-11.7% for IGFBP-3; recoveries were 97 +/- 7.1% and 101 +/- 8.7%, respectively. Recoveries of IGF-I and IGFBP-3 in response to 4- to 8-fold variations in extraction buffer volume were 97 +/- 8.2% and 107 +/- 6.1%, respectively. Dried blood spot IGF-I and IGFBP-3 showed greater than 1-month stability at -20 C, 4 C, and room temperature and retained more than 65% of the immunoreactivity after approximately 1 month at 37 C. Both IGF-I and IGFBP-3 were contained within the plasma fraction of whole blood, and variations (mean +/- SD) in IGF-I (204 +/- 29 micrograms/L) and IGFBP-3 (4.4 +/- 0.48 mg/L) measured in extracts of dried blood spot with adjusted hematocrit of 0.2-0.62 were acceptable. IGF-I and IGFBP-3 in paired plasma and dried blood spot extracts of random samples (n = 46) showed excellent correlation (r > 0.94) with slopes of near unity. Compared to conventional methods, the filter paper procedures were equally effective in distinguishing IGF-I and IGFBP-3 levels in untreated GH receptor-deficient (n = 11) and age-matched normal controls (n = 16). We conclude that blood collected on filter paper is ideal for IGF-I and IGFBP-3 analysis and may find applications in pediatric and large scale infant screening programs.

Rapid, convenient radioimmunoassay of estrone sulfate.

We developed a specific, simple, and rapid RIA for the direct quantification of estrone sulfate (E1S) and established its performance characteristics. The assay has a dynamic range of 0.05-90 micrograms/L with a detection limit of 0.009 microgram/L. Intraassay CVs were 9.2%, 4.5%, and 4.6% at 0.35, 9.0, and 60 micrograms/L, respectively. Interassay CVs were 8.8%, 5.1%, and 5.5% at 0.076, 0.5, and 12 micrograms/L, respectively. Linearity of dilution studies showed values of 80-105% of expected, and recovery of E1S added to serum samples ranged from 82% to 102%. Cross-reactivities with structurally related estrogens were < 5%. When compared with a conventional assay (involving hydrolysis of E1S and indirect measurement of estrone), the present RIA showed excellent correlation (r = 0.99, slope = 1.54, Sy/x = 2.14, n = 71). Mean E1S concentrations measured with this RIA for normal men (n = 20) and women in follicular (n = 20) and luteal (n = 25) phases of their menstrual cycle were 0.96, 0.96, and 1.74 microgram/L, respectively. Mean E1S concentrations for oral contraceptive users (n = 20) and postmenopausal women without hormone replacement therapy (n = 21) or on hormone replacement therapy (n = 22) were 0.74, 0.13, and 2.56 micrograms/L, respectively. Serum concentrations of E1S in pregnant women in their first (n = 14), second (n = 17), and third (n = 15) trimesters were 20, 66, and 105 micrograms/L, respectively. Availability of this simple RIA should provide a useful tool for the assessment of estrogen status in women.

Acid-labile subunit of human insulin-like growth factor-binding protein complex: measurement, molecular, and clinical evaluation.

Although the acid-labile subunit (ALS) of the approximately 150-kDa insulin-like growth factor (IGF)-binding protein (IGFBP) complex was described over a decade ago, details of ALS physiology have remained largely uncertain. We evaluated antibodies to synthetic human ALS and constructed a noncompetitive ALS enzyme-linked immunosorbent assay. Whereas uncomplexed ALS is directly measured, determination of total levels required sample pretreatment with SDS, which was found to optimally dissociate complexed ALS and significantly enhance ALS immunoreactivity. ALS in random adult sera was approximately 50% uncomplexed, and samples devoid of complexed ALS by immunoaffinity separation contained about 54% of the total levels. Serum ALS fractionated by gel filtration high performance liquid chromatography eluted in a single peak at approximately 150 kDa with IGF-I and IGFBP-3, but appeared at about 400-500 kDa after sample acidification and fractionation under acidic condition. The unexpected shift in ALS immunoreactivity remained unchanged when acid-neutralized or SDS-treated samples were fractionated under neutral pH and was reproducible when the 150-kDa complex was isolated, treated with acid or SDS, and rechromatographed. ALS in adult sera more tightly correlated with IGFBP-3 than IGF-I or IGF-II. The total levels (mean +/- SD) were 16.7 +/- 3.7 mg/L in 22 normal subjects, 28.3 +/- 8.1 mg/L in 20 acromegalic patients, and 9.5 +/- 3.8 in 32 GH-deficient adults. Little or no ALS was detectable in amniotic fluid, cerebrospinal fluid, seminal plasma, or milk, whereas high levels were present in synovial fluid. The development of ALS enzyme-linked immunosorbent assay should greatly facilitate further investigations of this unique glycoprotein.

Immunoassay of insulin-like growth factor binding protein-1.

Accurate measurement of insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is important for precise definition of its physiological roles and potential diagnostic values. Because altered phosphorylation results in altered IGFBP-1 immunoreactivity, current assays may significantly underestimate or fail to detect physiological changes in the IGFBP-1 concentrations. We developed three ELISAs (ELISA 1-3) using a common capture but three different detection antibodies. IGFBP-1 in serum, synovial fluid (SF), cerebrospinal fluid (CSF), and amniotic fluid (AF) were measured before and after treatment with alkaline phosphatase (ALP). Among the methods, only ELISA-1 was unaffected by IGFBP-1 phosphorylation and generated identical results before and after ALP treatment. The serum and SF values by ELISA-2 and -3 were lower by approximately 4- to 10-fold, but increased after ALP treatment to within 66-98% of those by ELISA-1. The medians in AF, and to a lesser extent in CSF, by all methods were similar and did not change significantly after dephosphorylation. ELISA-1 showed excellent correlation with ELISA-2, ELISA-3, and a commercial IGFBP-1 IRMA only after ALP-treated samples were analyzed by the comparative methods. ELISA-1 is highly specific for IGFBP-1 and demonstrated acceptable analytical performance characteristics.

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.

Noncompetitive ELISA for human serum insulin-like growth factor-I.

Measurement of serum insulin-like growth factor I (IGF-I) is important in assessing the growth hormone/IGF axis. Competitive immunoassay methods for IGF-I are complicated by substantial interference from IGF-binding proteins (IGFBPs) due to the relatively low ratio of specific antibody to IGFBPs in the sample, even after standard acid-ethanol sample extraction. We report of development of a noncompetitive ELISA for human IGF-I that avoids this problem by utilizing excess amounts of capture and detection antibodies. Serum samples were prepared by using an abbreviated acid-ethanol extraction method. Neutralized supernatant was added to microwells coated with IGF-I capture antibody; horseradish peroxidase-labeled detection antibody was then added, incubated for 2 h, and then developed. Compared with the "gold standard" method of acid-column chromatography, the simplified acid-ethanol extraction yields a mean +/- SD recovery of 103% +/- 5.5% despite the presence of residual IGFBPs in the extracted sample. Comparisons with a centrifugal filtration sample extraction method are also shown. The ELISA is specific for IGF-I with an absolute sensitivity of 0.03 microgram/L and inter- and intraassay CVs of 3.9-8.8% and 2.6-6.7%, respectively. The availability of a rapid IGF-I ELISA combined with a simple and reliable sample preparation procedure should facilitate clinical and research studies of this important growth factor.

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).