Selective recognition of M235T angiotensinogen variants and their determination in human plasma by monoclonal antibody-based immunoanalysis.

The common M235T mutation of human angiotensinogen has been shown to be associated with a 10-20% increase in plasma angiotensinOgen level and increased frequency of essential and pregnancy-induced hypertension. The detection of such a common factor in the plasma of individuals at risk could be a useful tool for modern molecular-based medicine. The recognition of M235T variants was investigated using four monoclonal antibodies (mAbs) directed against human angiotensinogen; two immunometric assays were developed. The first assay (using mAbS 7B2 and 4G3) allowed the direct determination of angiotensinogen concentrations and did not show a significant difference with the enzymatic measurement of angiotensinogen. The second assay (using mAbs 1H8 and 1C11) showed a fine distinction between the T235 mutant and M235 wild-type forms of angiotensinogen, with a greater affinity for the latter, as confirmed by biosensor BIAcore experiments. This assay was extremely sensitive in measuring the proportions of the M235 and T235 forms present in the test samples, the first time such a distinction has been achieved in the serpin family. The simple immunoanalysis of the plasma allowed the direct determination of the M235T genotype of the individual tested. Furthermore, it was shown that the T174M mutation, described as being in complete linkage disequilibrium with the M235T mutation, had no influence on these results. Moreover, this assay suggested the presence of the M235 and T235 angiotensinogens in approximately equal amounts in heterozygous plasmas. In conclusion, the immunometric assay described in this study should provide original tools for investigating the relationship between M235T genotype, plasma angiotensinogen levels, and regulation of blood pressure.

Immunoradiometric assay for follistatin: serum immunoreactive follistatin levels in normal adults and pregnant women.

A sensitive and specific immunoradiometric assay for follistatin was developed using antifollistatin mouse monoclonal and rabbit polyclonal antibodies. The sensitivity of the assay was 0.5 micrograms/L, and cross-reactivities with recombinant human activin A and bovine inhibin were less than 0.1%. The intra- and interassay coefficients of variation were less than 10%, and the recovery rate was about 90% in human serum. The addition of activin A to the same sample resulted in a minimal influence on follistatin recovery, indicating that this assay system can measure the total level of activin-bound and unbound follistatin. Gel filtration analysis of human serum showed that the majority of immunoreactivity was eluted in a larger molecular size position than that of free follistatin, suggesting that the large part of follistatin is bound to other proteins, presumably activins, in serum. Using this assay, immunoreactive follistatin levels in various biological fluids and human sera were examined. The dose-response curves of porcine follicular and amniotic fluids were parallel to the standard curve, and porcine follicular fluid contained extremely high follistatin immunoreactivity (5.6 mg/L). The serum follistatin level in normal human volunteers was 13.3 +/- 4.7 micrograms/L (mean +/- SD; n = 60), with a tendency to increase gradually with age. On the other hand, the serum follistatin level was remarkably elevated in pregnant women (62.7 +/- 35.3 micrograms/L; n = 57), with a positive correlation with weeks of pregnancy. These data indicated that circulating immunoreactive follistatin is detectable in human serum, and the levels vary with physiological conditions such as aging and pregnancy.

Comparison of two thyroglobulin immunoradiometric assays on the basis of comprehensive imaging in differentiated thyroid carcinoma.

The aim was to compare two thyroglobulin-immunoradiometric assays (Tg-IRMA) in the follow-up of patients with differentiated thyroid carcinoma (DTC) in order to set up interassay correlation, correlation to clinical background, and to determine whether a lower functional sensitivity (kit A: 0.5 ng/mL, kit B: 0.3 ng/mL) would allow an earlier detection of recurrences. Three hundred eight samples from 181 patients with DTC were investigated. The clinical interpretation of the Tg-IRMA results was based on comprehensive imaging and the clinical history before and during the study period. Groups were formed against this background and against the thyrotropin (TSH) levels of the samples (LT4- on and LT4-off). During a follow-up period that lasted until September 1998, the clinical situation was reevaluated in order to determine any changes in the patients' clinical status. The two assays presented a good interassay correlation of 0.838. Both assays had a high and comparably good sensitivity in the detection of recurrence of malignancy or distant metastases. Patients in remission had, in most cases, nonmeasurable or Tg values below 1 ng/mL. Kit B presented slightly measurable Tg results in a larger number of patients in remission; however, during the follow-up most of these slightly measurable Tg results were not reproducible, thus being most likely artifacts. Consequently, the functional sensitivity of 0.3 ng/mL of kit B showed no advantages in terms of an earlier tumor detection and seems to be unacceptably low. Negative consequences may be an increase in the number of investigations during the follow-up, which may be disconcerting for both the clinicians and the patients.

Analytical performance of CA 19.9, CA 125 and CA 15.3 assays as observed through an external quality assessment program.

Immunoassays of the tumor markers CA 19.9, CA125 and CA 15.3 are generally acknowledged to be a useful tool in the management of cancer patients. As a consequence, many methods developed by different companies are now commercially available. However, discrepancies have been described in the results of marker determinations even when the same monoclonal antibody was used. An external quality assessment (EQA) was carried out; starting from 1989 about 110 laboratories participated; since December 1991 the program was linked with the interlaboratory program Oncocheck organized by the Service de Radiopharmacie et Radioanalyse, University of Lyon. At present more than 200 laboratories of many European countries are involved: cumulatively 47 quality control samples have been prepared and sent to the participants. This manuscript is a report on data collected for CA 19.9, CA 125, and CA 15.3.

Immunoradiometric assay of chromogranin A in the diagnosis of small cell lung cancer: comparative evaluation with neuron-specific enolase.

The aims of our work were 1) to determine the diagnostic performance of an immunoradiometric assay of chromogranin A (CgA) in small cell lung cancer and 2) to compare its discriminatory power with that of neuron-specific enolase (NSE), the marker currently used for SCLC. We selected 166 cases of small cell (64) and non-small cell (102) lung cancer and 106 cases of non-malignant lung diseases as controls. Both CgA and NSE were assayed by immunoradiometric methods and cutoff values were established on the basis of a pre-fixed specificity of 95% in non-malignant lung diseases. The CgA assay showed better diagnostic sensitivity than NSE in SCLC (61% versus 57%), especially in limited disease, and a low positivity rate in NSCLC with respect to NSE (14% versus 22%). By contrast, NSE reflected disease extent more accurately than CgA (U test: CgA p<0.05, NSE p<0.001). Finally, we found that the CgA assay was not affected by hemolysis whereas NSE serum levels greatly increased in hemolyzed sera. In conclusion, CgA assaying by an IRMA method is a reliable procedure in the diagnosis of SCLC. NSE remains the marker of choice in staging and monitoring of the disease. Further studies are needed to evaluate the prognostic significance of the marker and its role in therapy monitoring and patient follow-up.

Serum TPS versus TPA in Egyptian bladder cancer patients.

This study included 328 cases (106 with bladder cancer, 152 with non-malignant urinary tract diseases and 70 healthy controls). Serum TPA was determined using the Prolifigen TPA IRMA kit supplied by AB Sangtec Medical, Bromma, Sweden and serum TPS was determined using the TPS IRMA kit supplied by Beki Diagnostics AB, Bromma, Sweden. The results of this study revealed that serum TPA had better sensitivity than serum TPS while no marked difference was found in the false-positivity rates in the non-malignant urinary tract diseases. A correlation coefficient of 0.83 was found between serum TPA and TPS. No relation was found between either TPA or TPS and histopathological stage, grade or association of the tumor with bilharziasis. As regards the histopathological type of the tumor, serum TPS was slightly higher in squamous cell than transitional cell carcinoma but TPA showed no difference. In the follow-up of bladder cancer patients after surgery both TPA and TPS showed an excellent concordance with the clinical state of the patients. In conclusion, TPS does not seem to be an optimal test in Egyptian patients with bladder cancer but serial determinations of one of the two markers can be used in the follow-up of these patients after surgery.

No evidence of the hook effect in peritoneal fluid CA 125 measurement using immunoenzymatic second generation assays: comparison with immunoradiometric assays.

OBJECTIVE: To monitor the occurrence of the hook effect in measurements of peritoneal fluid CA 125 levels using two different immunoenzymatic second generation assays (ETI-II and EIA-II), and to compare these results with those obtained using the respective immunoradiometric versions of the assays (IRMA-II and ELSA-II). STUDY DESIGN: CA 125 levels were determined in peritoneal fluid and serum samples obtained from 45 women with gynecological diseases. The assays were carried out using IRMA-II and ETI-II (Sorin Biomedica) and ELSA-II and EIA-II (CIS Bio International) assays. Occurrence of the hook effect and linearity of the assays were evaluated. Statistical analyses were performed by Wilcoxon's test and linear regression analysis. RESULTS: Undiluted peritoneal fluids, assayed for their CA 125 content, showed falsely underestimated values of the antigen when IRMA-II and ELSA-II assays were performed. The phenomenon disappeared only at high dilutions of the sample (> 50). Conversely, immunoenzymatic ETI-II and EIA-II assays performed on undiluted peritoneal fluids did not show underestimated CA 125 values. CA 125 values obtained by immunoenzymatic assay were lower than those obtained using their respective immunoradiometric versions at a dilution of 1:100 (P < 0.001). A good correlation was observed between ELSA-II and EIA-II (r = 0.929) CA 125 values. CONCLUSION: The EIA-II immunoenzymatic assay appears to be more suitable for CA 125 measurement in peritoneal fluid in that it is not subject to the hook effect and its results correlated well with those obtained via its immunoradiometric version.

Applicability of short-lived radiometallic nuclide for high sensitivity two-site "sandwich" immunoradiometric assay: human growth hormone assay.

The sensitivity of the IRMA method is limited by the specific activity (SA) of the conventionally employed radioisotropic label and high sensitivity radioimmunoassay should theoretically be attained by the use of short-lived radiometallic nuclides. Our group have achieved radiolabeling of high SA IgG by using the radiometal, gallium-67 (67Ga) with a short half-life (T1/2 = 78 h) and deferoxamine (DF), a bifunctional chelating agent bound through a multispacer (dialdehyde starch, DAS) as the linker (J Nucl Med 32:825, 1991). In the present work, the application of the approach is attempted by employing a two-site IRMA for human growth hormone (hGH); the monoclonal antibody to hGH (MAB2) is bound to DF via DAS and the coupled DF-DAS-MAB2 is radiolabeled with 67Ga. The 67Ga-DF-DAS-MAB2 of high SA (4,884 MBq/mg versus 370-518 MBq/mg calculated for radioiodinated MAB2) was thus used for the two site 'sandwich' 67Ga-IRMA. Excellent correlation with the 125I-IRMA was registered, and higher detection capability obtained by using 67Ga over the 125I in the hGH IRMA offered a good basis for the exploitation of short-lived radio-nuclides in the IRMA system.

[The value of the parathyrin-related protein (PTH-RP) in the diagnosis of cancer-associated hypercalcemia]. / Valor de la proteína relacionada con la paratirina (PTH-RP) en el diagnóstico de la hipercalcemia asociada al cáncer.

BACKGROUND: The parathyrine related protein (PTH-RP) is very similar, both in structure and in function, to the PTH and is considered as a mediator in humoral hypercalcemia in cancer. The aim of this study was to know the clinical value of PTH-RP measurement. METHODS: Serum PTH-RP concentrations were studied in 22 healthy subjects, 13 patients with primary hyperparathyroidism, 9 patients with solid neoplasms and normocalcemia, 26 patients with solid neoplasms and hypercalcemia and 4 patients with hematologic neoplasms and hypercalcemia. The PTH-RP was quantified by a competitive radioimmunoassay technique using a specific antibody of the PTH-RP 1-40 fragment. Intact parathyrine (i-PTH) was quantified by an IRMA method using 2 polyclonal antibodies (INCSTAR). RESULTS: Fifteen (68%) of the healthy controls presented undetectable serum PTH-RP concentrations. The serum PTH-RP concentration was normal in all those patients with hyperparathyroidism. Elevated serum PTH-RP values were not found in patients with solid neoplasms and normocalcemia or in those with hematologic neoplasms and hypercalcemia. High values of PTH-RP were observed in 8 out of 9 (88%) of the patients with solid neoplasms and hypercalcemia with bone metastasis and in 7 out of 11 (63%) of the patients with bone involvement. CONCLUSIONS: Serum parathyrine-related protein was found to be high in a large proportion of patients with solid neoplasms and hypercalcemia. Serum PTH-RP determination is useful in the clinical investigation of patients with hypercalcemia. Even in patients with bone metastasis, hypercalcemia may have a humoral background.

Serum insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) in healthy Thai children and adolescents: relation to height, weight, and body mass index.

The objective of this study was to evaluate the correlation between serum concentrations of insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3) and growth parameters (height, weight, and body mass index) in 260 healthy children and adolescents aged 5-20 years. The subjects were divided into 2 groups according to the age achieving final height. Group 1 included children with active growth consisting of girls aged under 14 years (N = 80) and boys aged under 16 years (n = 74). Group 2 included adolescents who achieved final height consisting of females aged at and over 14 years (n = 82), and males aged at and over 16 years (n = 24). In group 1, the serum concentrations of IGF-1 and IGFBP-3 were significantly positive correlated with all growth parameters. In group 2, although the correlation was insignificant, the concentrations of IGF-1 and IGFBP-3 seemed to be greater in individuals who were relatively taller and had lean body mass than those who were relatively short and over average body mass.

[The prognostic significance of the measurement of Ca 15-3 in the fluid of large breast cysts]. / Significato prognostico del dosaggio del Ca 15-3 nel liquido di grosse cisti della mammella.

Ca 15-3 was assayed in serum and in breast cysts fluid of 78 non neoplastic patients presenting Gross Cysts (GCD). In apocrine cysts and in the mixed type (serous and apocrine, class III) relapse was indicated by high serum and intracystic levels of the marker. Ca 15-3, therefore, may discriminate in a group of cysts a higher cellular resistance as well as an increased cell proliferation. Results suggest an important role of the marker in the follow up of patients with GCD and for the early detection of cyst relapse.

Chromogranin-A and N-terminal pro-brain natriuretic peptide: an excellent pair of biomarkers for diagnostics in patients with neuroendocrine tumor.

PURPOSE: For the last decade chromogranin-A (CgA) has been a well-established marker for neuroendocrine tumor (NET), and N-terminal pro-brain natriuretic peptide (NT-proBNP) has been a useful marker for left ventricular dysfunction. This study examined the diagnostic value of CgA and NT-proBNP for carcinoid heart disease (CHD), and their prognostic value for overall survival in NET patients. PATIENTS AND METHODS: Serum samples were obtained and cardiac ultrasound studies performed in 102 NET patients. The criterion for mild and severe CHD was tricuspid regurgitation stage I/II and III/IV, respectively. Proportional odds and Cox proportional hazards models were constructed respectively to identify the association between CHD and overall survival with patient characteristics and the two markers. RESULTS: Severe CHD was found in 15 (15%) of 102 patients, 13 of whom had elevated NT-proBNP levels. In the univariate proportional odds model CHD was correlated with age (P = .007), CgA (P = .002), and NT-proBNP (P < .001), whereas in the multivariate model NT-proBNP and CgA were significantly associated with CHD (P < .001 and P = .01). In the univariate Cox models, age (P = .04), sex (P = .03), CgA (P = .003), and NT-proBNP (P = .04) were related to overall survival, and in the multivariate model CgA and NT-proBNP remained significantly related to overall survival (P = .002 and P = .04, respectively). CONCLUSION: NT-proBNP and CgA are very important markers in the diagnosis of CHD in patients with NET. Furthermore, patients with elevated NT-proBNP in addition to elevated CgA levels showed worse overall survival than patients with elevated CgA alone.

Immunoradiometric assay (IRMA) for human follicle stimulating hormone (FSH) and luteinizing hormone (LH) using common avidin solid phase.

This paper describes the use of avidin-biotin interaction as an affinity system, wherein avidin immobilized magnetizable particles (cellulose) are used as a common separation system in immunoradiometric assay (IRMA) for hormones of the human reproductive system, human follicle stimulating hormone (FSH), and luteinizing hormone (LH). Biotinylated probe was prepared by biotinylation of specific monoclonal antibody for respective antigen using the caproyl derivative of biotin N-hydroxysuccinimide. The detector antibody for the respective antigen was radiolabelled with 125I by a chloramine-T oxidation method and purified by gel filtration. In the IRMA procedure, standard/sample, respective biotinylated, and radiolabelled antibody as a single reagent, and avidin solid phase were added simultaneously to the assay tubes. After incubation for 3 h with shaking, the bound complex was quantitated for its radioactivity associated with the common avidin solid phase. Results showed that the developed assay protocol is applicable to IRMA of FSH and LH with good precision (intra and inter assay CV less than 8% and 11%, respectively), good assay range (0-200 mIU/mL) and analytical recovery (87-110%). The assay could detect 0.5 mIU/mL and 0.9 mIU/mL of FSH and LH, respectively, and showed good correlation with commercially available kits (FSH y = 0.98x + 0.21 and LH y = 0.99x + 0.18).

Heterologous double-determinant immunoradiometric assay CA 125 II: reliable second-generation immunoassay for determining CA 125 in serum.

The new CA 125 II (Centocor) serum assay utilizing the M11 mouse monoclonal antibody as capture antibody and OC125 as tracer antibody, was investigated for its technical and clinical performance against the original CA 125 assay. The CA 125 II test revealed a quadruple increase in signal-to-noise ratio, good intra- and interassay precision (with CVs < 5% and 7%, respectively), improved dilution linearity, and a minimal detectable dose of 0.38 units/mL. Sera were obtained from healthy females (n = 192), women with benign conditions (n = 208), and patients with various cancers (n = 379). Both assays measured highly similar CA 125 distributions with equal reference ranges and nearly identical positivity (> 35 units/mL) rates, resulting in similar receiver-operating characteristic curves and monitoring graphs. Linear regression analysis of results by the two assays (CA 125 = x, CA 125 II = y) in ovarian cancer patients showed, for CA 125 assay values between 0 and 1000 units/mL, a slope of 1.00 and a y-intercept of 12.6 (n = 254, r = 0.8617, P < 0.0001). The heterologous CA 125 II assay appeared to be more accurate in patients who had human anti-mouse antibodies after immunoscintigraphy. The CA 125 II immunoradiometric assay is sensitive and reliable for measuring serum CA 125, and fully retains the cutoff values of 35 and 65 units/mL that were defined with the original CA 125 immunoradiometric assay.

Two bone alkaline phosphatase assays compared with osteocalcin as a marker of bone formation in healthy elderly women.

Serum bone alkaline phosphatase (ALP; EC 3.1.3.1) was measured with a wheat germ agglutinin (WGA) precipitation assay and with a new IRMA in a group of healthy elderly women. Both assays were correlated with serum total ALP activity and with osteocalcin. The two bone ALP assays have comparable within- and between-run imprecisions (WGA assay within-run CVs 2.6-5.4% and between-run, 4.0-5.1%; IRMA within-run CV 5.0% and between-run, 3.2%). Comparison of the WGA precipitation assay (x) with the IRMA (y) demonstrated a correlation coefficient of 0.87 [Deming regression equation: y = (0.58 +/- 0.02)x - (4.62 +/- 0.45); n = 101; Sy/x = 1.26; P < 0.001). Correlation studies with osteocalcin and total ALP showed correlation coefficients (all P < 0.001) of 0.34 and 0.65, respectively, for the WGA precipitation assay and of 0.36 and 0.68, respectively, for the IRMA. We conclude that the two bone ALP assays have similar imprecision and that neither can be given preference over the other as a marker of bone turnover.

New decision limits and quality-control material for detecting human chorionic gonadotropin misuse in sports.

Male athletes may administer human chorionic gonadotropin (hCG) to enhance performance. Despite imposing a ban on hCG doping, the International Olympic Committee has no recommended procedure to confirm the presence of hCG. Our aim was to establish a limit for hCG in urine above which a sample may be considered positive. We measured hCG concentrations in urine samples from 1400 men with the Serono MAIAclone IRMA. Statistical evaluation of the results gave a "far outside" value [75th percentile + (3 x interquartile range)] of 5.0 IU/L; greater values are extremely unusual. Immunoreactive material in urine samples from a reference group of 120 noncompeting individuals was concentrated about sevenfold by centrifugal ultrafiltration; all these concentrated samples gave assay values < 5.0 IU/L. To ensure with the greatest possible degree of certainty that no false-positive result is reported, we propose a decision limit of 10 IU/L in nonconcentrated ultrafiltered samples. We also prepared quality-control material to contain hCG at 10 IU/L. This material provides a decision limit above which, after confirmatory procedures, a sample should be considered positive. This should help establish comparability of results among laboratories testing for sports drugs.

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen.

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.

Clinical and analytical evaluation of an immunoradiometric assay for corticotropin.

We evaluated a new IRMA developed commercially for the measurement of corticotropin (ACTH) in human plasma. The assay involves purified polyclonal goat capture antibodies specific for ACTH 26-39 and an 125I-labeled monoclonal signal antibody specific for ACTH 1-17. CVs for intraassay and total precision at ACTH concentrations between 9 and 801 ng/L ranged from 2.5% to 4.7% and from 3.3% to 9.3%, respectively, with an assay detection limit of 1.7 ng/L. The reference interval determined for adults with the new method (16-52 ng/L) differed significantly (P < 0.05) from that for an established ACTH IRMA (9-54 ng/L). Method comparison with clinical samples (n = 179) revealed a correlation coefficient of 0.970 and a best-fit equation of y (new IRMA) = (1.011 +/- 0.019)x + (4.17 +/- 3.31) with Sylx = 40.2. Both methods showed equivalent clinical sensitivity in evaluating Cushing disease, adrenal tumors, ectopic ACTH-producing tumors, hypopituitarism, steroid suppression, surgical adrenalectomy, Nelson syndrome, Addison disease, and corticotropin-releasing hormone stimulation. We conclude that the new IRMA is technically simple to perform and provides a specific and sensitive method for evaluating of adrenocortical function.