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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.

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.

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.

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.

To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.

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.

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.

Many companies manufacture and distribute immunoassay kits for research applications. A number of research-oriented immunoassay kits are expensive and are not suitable for studies in which relatively substantial numbers of samples are analyzed. For these reasons, many researchers frequently consider setting up in-house immunoassay methodologies. The set-up of a particular immunoassay method could take a very short or a very long time depending on the availability and quality of the necessary reagents, especially of the antibodies. The researcher must also define the needs and set-up goals and objectives for the problem at hand. Immunoassays can be homogeneous or heterogeneous. Many manufacturers make excellent products, most composed of polystyrene of high protein-binding capacity. In many cases, the polystyrene proteinbinding capacity is increased by irradiation. For absorption and fluorescence spectroscopy, clear wells are used. For chemiluminescence and for time-resolved fluorescence immunoassays based on reflectance measurements, opaque, white, or black microtiter wells are used. Microtiter plates are the easiest to handle and use for in-house development because of the availability of a wide range of auxiliary equipment. The principles behind the set-up of immunological assays are simple. Proper selection of the assay design allows the researcher to set up assays without the need to perform relatively cumbersome procedures.

Development of an ultrasensitive immunoassay for serum PSA involving conventional detection probes.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.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 were0.012 micrograms/L in the majority of patients (53.8%) who had undergone radical prostatectomy.This assay is well suited for post-surgical monitoring of PSA in patients with prostate cancer.

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.

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 Eu 3+ chelator 4,7- bis (chlorosulfophenyl)-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.

We developed a direct competitive-type immunoassay for progesterone in serum that combines the advantages of the biotin-streptavidin system with the antibody-immunobilization approach. We synthesized biotinylated progesterone derivatives of five different proteins and, after initial evaluation of the conjugates, selected biotinylated bovine IgG-progesterone as the most suitable tracer. Progesterone released from binding proteins with danazol competes with the biotinylated tracer conjugate for binding to a limited amount of a mouse anti-progesterone monoclonal antibody in microtitration wells coated with a goat anti-mouse IgG antibody. The binding of the biotinylated tracer is then monitored by reaction with a streptavidin-based universal detection reagent developed for time-resolved fluorometry. The assay demonstrated typical performance characteristics with respect to the dynamic range, detection limit, and precision. Recovery averaged 99.3% (SD 8.3%) and dilution experiments showed good linearity. Measurements correlated well with those from three commercially available direct immunoassays for progesterone.

To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.

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).

Multiple fluorescence labeling with conventional probes like fluorescein, to improve the detection limit of labeled reactants, is not usually successful because of fluorescence quenching. In contrast, we found that the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) can be incorporated into proteins at very high molar ratios. Working with thyroglobulin as a model protein, we found that when 160 BCPDA molecules are incorporated into one thyroglobulin molecule, the fluorescence emitted by the labeled protein in the presence of excess Eu3+, is equivalent to that emitted by approximately 900 molecules of unconjugated BCPDA:Eu3+ complexes. We took advantage of the lack of any quenching effects and of the enhancement observed with the multiply labeled protein, to develop a universal reagent system consisting of (a) streptavidin covalently coupled to BCPDA labeled thyroglobulin and (b) excess Eu3+. With this approach, streptavidin is heavily labeled through thyroglobulin and retains its full biotin binding activity. We used the reagent to develop a highly sensitive time-resolved heterogeneous immunofluorometric assay of alpha-fetoprotein (AFP) in serum, using monoclonal antibodies. One antibody is immobilized in white microtitration wells (solid-phase) and the other is biotinylated. We demonstrate that this assay, using the newly developed reagent, is 25-fold more sensitive than the one using directly BCPDA labeled antibody and 5-fold more sensitive than an assay that uses BCPDA-labeled streptavidin. The detection limit of the assay with the new reagent was down to 60 amol of AFP per well. We conclude that multiple fluorescence labeling with europium chelators is an effective method of extending the sensitivity of currently used fluorescence immunoassay procedures.

We describe a new non-isotopic immunoassay for the quantitation of ferritin in serum. Ferritin is first immunoextracted from serum with a monoclonal antibody immobilized in white microtiter wells. A second biotinylated antibody is then added. The bound biotinylated antibody is quantified by a bridge reaction with streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in the presence of excess Eu3+. The fluorescence on the bottom of the dry microtiter well consisting of monoclonal antibody-ferritin-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+ is then measured with a time-resolved fluorometer. The assay is sensitive (0.5 microgram/l), precise (CV's 3-9%) and accurate (average recovery 102%). Results compared well (r = 0.99) with those of a widely used RIA procedure.

We describe a new "sandwich"-type immunofluorometric assay for thyroxine-binding globulin (TBG) in serum. The assay involves a solid-phase monoclonal antibody immobilised in white microtiter wells, and a soluble biotinylated monoclonal antibody that reacts with the captured TBG molecules. Addition of streptavidin labeled with the europium chelator, BCPDA (4,7-bis(chlorosulfophenyl)-1,10 phenanthroline-2,9-dicarboxylic acid), and excess europium results in the formation of a highly fluorescent product. The fluorescence signal of the final complex is quantitated on the dried solid-phase with a pulsed-laser time-resolved fluorometer. The assay requires a 151-fold sample pre-dilution and a total incubation time of 90 minutes. It has a broad dynamic range of 0-100 mg/L and a minimum detection limit of 0.4 mg/L. The coefficients of variation for within-run and between-run assays averaged 4.5% and 5.4%, respectively. The mean analytical recovery of TBG added to serum was 103%. Results obtained by this method correlated well with those determined by a commercial radioimmunoassay (r = 0.96, n = 112) and by an immunoradiometric procedure (r = 0.95, n = 131).

In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.

We describe a new "sandwich"-type non-isotopic immunoassay for human choriogonadotropin (hCG) in serum. In the assay, hCG is captured by a beta-subunit-specific monoclonal antibody, which is immobilized in a white microtiter well. The sandwich is completed by adding a second biotinylated monoclonal antibody specific for the whole hCG molecule. The degree of binding of biotinylated antibody, which is proportional to the amount of hCG present in the sample, is quantified by adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The fluorescent complex formed on the solid-phase [monoclonal antibody-hCG-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+] is measured by excitation at 337.1 nm with a nitrogen laser and monitoring the emission at 615 nm in a specially designed gated fluorometer working in a time-resolved mode. A two-step procedure is proposed for routine use to avoid the "high-dose hook effect" of the simpler and faster one-step procedure. The hCG assay described has a dynamic range of 1 to 500 int. units/L, and is precise and accurate. Results agree well with those obtained with a commercially available immunoradiometric and a time-resolved immunofluorometric procedure.

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.

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.

We describe a new "sandwich"-type non-isotopic immunoassay for human choriogonadotropin (hCG) in serum. In the assay, hCG is captured by a beta-subunit-specific monoclonal antibody, which is immobilized in a white microtiter well. The sandwich is completed by adding a second biotinylated monoclonal antibody specific for the whole hCG molecule. The degree of binding of biotinylated antibody, which is proportional to the amount of hCG present in the sample, is quantified by adding streptavidin labeled with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA), in the presence of excess Eu3+. The fluorescent complex formed on the solid-phase [monoclonal antibody-hCG-monoclonal antibody-biotin-streptavidin-BCPDA-Eu3+] is measured by excitation at 337.1 nm with a nitrogen laser and monitoring the emission at 615 nm in a specially designed gated fluorometer working in a time-resolved mode. A two-step procedure is proposed for routine use to avoid the "high-dose hook effect" of the simpler and faster one-step procedure. The hCG assay described has a dynamic range of 1 to 500 int. units/L, and is precise and accurate. Results agree well with those obtained with a commercially available immunoradiometric and a time-resolved immunofluorometric procedure.

In this new immunofluorometric method for quantification of lutropin in serum, the "sandwich" principle is combined with time-resolved fluorescence measurements, with the europium chelate 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) used as label. A monoclonal antibody to the alpha-subunit of lutropin is adsorbed onto the walls of white-opaque microtiter wells to form the solid-phase capture antibody, and a biotin-labeled soluble monoclonal antibody is used for antigen quantification. The detection system is completed with streptavidin, which has been linked to a protein bulking agent labeled with multiple BCPDA residues. In the presence of excess europium, the fluorescence of the final complex attached to captured lutropin molecules is measured on the dried solid phasse with an automated time-resolved fluorometer. The assay can be performed as a rapid (less than 60 min incubation) or regular (150 min incubation) procedure. The rapid assay is well-suited for routine daily monitoring of increasing or ovulatory lutropin concentrations; the regular assay, with its greater sensitivity (0.5 int. unit/L), is a practical procedure for lutropin measurements in hyposecretory states. The assay measures up to 240 int. units/L, and results compare well with those by a commercially available radioimmunoassay, an immunoradiometric assay, and another time-resolved immunofluorometric procedure.

By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.