Kinetic analyses of parathyroid hormone clearance as measured by three rapid immunoassays during parathyroidectomy

BACKGROUND: Rapid intraoperative parathyroid hormone (PTH) measurements are an important prerequisite for minimally invasive parathyroidectomy, serving as a feasible marker for "cure" because of the short half-life of PTH. Because automated analysis may facilitate monitoring, two automated PTH assays were compared with an established manual method. METHODS: We collected 109 plasma samples during minimally invasive surgery on 20 patients with primary hyperparathyroidism and single-gland disease. PTH was analyzed manually with a test from Nichols and by two automated assays from Diagnostic Product Corporation (DPC) and Roche, respectively. PTH half-life and residual concentrations were calculated by two kinetic models. RESULTS: Despite good overall correlations between methods [DPC = 1.07(Nichols) - 12 ng/L; r = 0.95, S(y/x) = 26 ng/L and Roche = 1.16(Nichols) - 2.82 ng/L; r = 0.98; S(y/x) = 16 ng/L], marked interindividual differences were observed. The iterative kinetic model failed with a nonuniform PTH decrease, but the interpolative model produced valid results. The mean (SD) half-life of 3.7 +/- 1.4 min with DPC differed significantly (P <0.05) from the 4.3 +/- 1.6 min with Roche (Nichols, 4.0 +/- 1.6 min). DPC produced significantly lower mean residual PTH (15 ng/L) vs Roche (27 ng/L); Nichols results were between them (20 ng/L). However, these differences were clinically irrelevant. CONCLUSIONS: Automated methods are as suitable as the manual test. The preoperative baseline PTH is necessary but is insufficient for kinetic calculations.     

Immunoradiometric assay of parathyrin with polyclonal and monoclonal region-specific antibodies.

In this immunoradiometric assay (IRMA) of parathyrin (PTH) a polyclonal anti-amino-PTH(1-34) is the capture antibody and a radiolabeled monoclonal anti-hPTH(44-68) is the second antibody. Gel filtration of serum from a hyperparathyroid patient yielded only a single peak of PTH, corresponding to the elution position of synthetic PTH(1-84). Healthy elderly individuals (ages 78 +/- 5 y, mean +/- SD, n = 45) had PTH concentrations (21 +/- 13 ng/L) not significantly higher than those from healthy younger (38 +/- 11 y) adults (20 +/- 8 ng/L, n = 94). Assay results agreed well with those obtained with a carboxyl-terminal PTH assay both in normal subjects (r = 0.63, P less than 0.001) and in patients with primary hyperparathyroidism (r = 0.59, P less than 0.001). Both assays equally discriminated patients with surgically confirmed primary hyperparathyroidism from normal individuals, but the PTH(1-84) IRMA also allowed a nearly absolute discrimination between normal subjects and patients with primary hypoparathyroidism (undetectable serum PTH in 18 of 21 cases) and secondary hypoparathyroidism (caused by hypercalcemia that was caused by a malignant tumor, PTH 1.3 +/- 1.3 ng/L, n = 32). Moreover, the PTH(1-84) IRMA is more sensitive (detection limit in serum, 0.8 ng/L) and easier and quicker to perform than the carboxyl-terminal assay.     

Performance and diagnostic application of a two-site immunoradiometric assay for parathyrin in serum

The "N-tact" immunoradiometric assay (IRMA) from INCSTAR for parathyrin (PTH) in serum involves a 125I-labeled affinity-purified antiserum to PTH 1-34 and an affinity-purified antiserum to PTH 39-84, the latter bound to a polystyrene bead. The mean detection limit, determined in six consecutive assays, was 4 ng/L. The within-batch CV was less than 7% in the range 15 to 2135 ng/L. The between-batch CV was 11.7% and 5.3% at 30 and 371 ng/L, respectively. Serum PTH in 14 proven cases of primary hyperparathyroidism was 49-808 (median 111) ng/L, undetectable (less than 5 ng/L) in 10 cases of primary hypoparathyroidism and in 10 cases of hypercalcemia associated with malignancy, compared with 7-39 ng/L in 45 normal subjects. PTH was 9 to 19 ng/L in four patients with familial benign hypercalcemia. In 39 patients with renal failure, apparent concentrations were 14 to 857 (median 133) ng/L, but sera from these patients pre-diluted with zero standard did not parallel dilutions of the standard, PTH 1-84. PTH concentrations were not significantly decreased in blood or serum kept at 20 degrees C for up to 6 h. After successful removal of a parathyroid adenoma, the mean half-time for disappearance of PTH in vivo in five hyperparathyroid patients was 3.3 min.     

Multisite immunochemiluminometric assay for simultaneously measuring whole-molecule and amino-terminal fragments of human parathyrin.

The immunochemiluminometric assay described uses immobilized anti-human parathyrin (parathyroid hormone, hPTH)(1-44) and anti-hPTH(44-68) antisera and acridinium ester-labeled anti-hPTH(1-34) to simultaneously measure both intact hPTH and its amino-terminal fragments. Results by the assay correlate well with those by a cAMP-based bioassay and the Nichols Allegro immunoradiometric assay. The minimal detection limit is 0.08 pmol/L. The normal range is 1.0-5.0 pmol/L, and values are higher in older women. About 90% of study patients with surgically proven parathyroid adenomas had above-normal preoperative PTH concentrations, whereas patients with hypercalcemia of malignancy had normal or suppressed values. This assay was designed to detect both intact PTH and amino-terminal PTH fragments; however, chromatographic fractionation of pools of primary and secondary hyperparathyroid plasma showed virtually no amino-terminal fragment activity. Nonetheless, the design is important because the absence of carboxyl-terminal binding sites prevents interference by carboxyl-terminal fragments and because bioactive amino-terminal fragments will react in the assay if they are present in the patients' sera or if they are produced by in vitro proteolysis of intact PTH.     

Technical and clinical characterization of the Bio-PTH (1-84) immunochemiluminometric assay and comparison with a second-generation assay for parathyroid hormone

BACKGROUND: The Bio-Intact parathyroid hormone (1-84) assay (Bio-PTH), a newly developed two-site immunochemiluminometric assay, measures exclusively PTH (1-84) in contrast to second-generation "intact PTH" (I-PTH) assays. We investigated the technical performance and clinical significance of this new assay. METHODS: PTH was measured simultaneously by the Bio-PTH assay and Allegro intact PTH IRMA in sera from Japanese patients with calcium disorders. RESULTS: Measured Bio-PTH in serum was unaffected by six freeze-thaw cycles and was stable at 4 degrees C for 7 days and during storage at -20 or -80 degrees C over 28 days. The calibration curve was linear to 1800 ng/L. The detection limit was 3.9 ng/L. The intra- and interassay imprecision was <2.8% and 3.5%, respectively, for analyte concentrations spanning the range of the calibration curve. Bio-PTH was unaffected by a 1000-fold excess of PTH (7-84), although I-PTH reacted equally with PTH (7-84) and PTH (1-84). Bio-PTH was correlated with I-PTH in healthy individuals (r = 0.953; P <0.0001; n = 26) and in the full population without renal dysfunction (r = 0.994; P <0.0001; n = 62). In 72 volunteers, mean (SD) Bio-PTH was 22.2 (7.1) ng/L, or 62% of the mean I-PTH [36.1 (22.3) ng/L]. This ratio was 51% in hemodialysis patients (n = 177). Mean Bio-PTH was high in patients with primary hyperparathyroidism [121 (85) ng/L; n = 18] and hemodialysis patients [102 (104) ng/L; n = 177], low in idiopathic hypoparathyroidism [5.5 (2.8) ng/L; n = 4], and within 2 SD of the mean for healthy controls in Paget disease of the bone [34 (15) ng/L; n = 9] and bone metastasis [24 (12) ng/L; n = 8]. CONCLUSION: The Bio-PTH assay is sensitive and precise and produces expected results for patients with the studied disorders of calcium metabolism.     

Evaluation of the performance and clinical impact of a rapid intraoperative parathyroid hormone assay in conjunction with preoperative imaging and concise parathyroidectomy

BACKGROUND: (99m)Tc-sestamibi scans and rapid, intraoperative intact parathyroid hormone (PTH) assays allow preoperative identification of diseased glands and intraoperative confirmation of diseased gland removal, respectively. Use of these two new technologies may facilitate simpler, more concise surgery, shorter hospital stays, and decreased costs for frozen-section analysis. One major drawback to this new strategy has been the high cost of rapid point-of-care PTH assays. METHODS: We performed rapid PTH assays with the DPC Turbo PTH assay on the DPC IMMULITE automated analyzer. The number of intraoperative frozen sections, type of anesthesia, surgical approach, length of hospital stay, and pre- and postoperative calcium values were compared between a group of 49 patients undergoing parathyroidectomy where the intraoperative PTH assay was used in conjunction with preoperative imaging, and a historical control group of 55 patients before the use of these two technologies in our institution. RESULTS: Comparison of the Turbo PTH assay to the standard IMMULITE PTH assay gave the following: y = 1.08 x - 4.36 (r = 0.97; n = 48). For the 49 patients, the median turnaround time for each intraoperative PTH determination was 19 min (range, 14-40 min). The median decrease in PTH values from baseline was 88% (range, 33-99%). Thirty-seven patients required two PTH determinations, 7 required three, 4 had four, and 1 required five determinations. The average laboratory cost for the rapid intraoperative PTH assays was < $100 per patient (range, $55 to $113). Compared with the control group, the experimental group had significantly fewer frozen sections (1.4 vs 2.5; P < 0.0001), shorter hospital stays (17 discharged on the day of surgery vs none discharged on the day of surgery; P < 0.0001), greater use of local anesthesia (33% vs 0%; P < 0.001), and more unilateral, rather than bilateral neck explorations (65% vs 0%; P < 0.001). CONCLUSIONS: The combination of intraoperative Turbo PTH assay and preoperative (99m)Tc-sestamibi scans can lead to significant decreases in laboratory and surgical pathology costs, hospital stays, and exposure to general anesthesia by facilitating concise parathyroidectomy surgery.     

A competitive ELISA for lipoprotein(a).

A competitive ELISA for lipoprotein(a) (Lp(a)) is described. The method uses a commercially available polyclonal anti-Lp(a) antibody and an IgG biotinstreptavidin-horseradish peroxidase detection system. The method is simple and robust with an assay sensitivity of 0.7 ng/well (1.4 micrograms/l). The antibody cross-reactivity was 0.14% against LDL and 0.70% against plasminogen. The coefficients of variation obtained with control sera of 266 and 552 mg/l were: 5.0% and 4.6% (n = 6), respectively for the intraassay; and 10.8% and 9.5% (n = 16), respectively for the interassay. The method showed an excellent correlation with a commercial immunoradiometric assay (IRMA), y (ELISA) = 0.94x (IRMA) - 8, (r = 0.98). A recovery study in which a 200 mg/L standard and four plasma samples were diluted with different proportions of a low plasma sample, gave linear relationships and also confirmed the specificity of the antibody.     

Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia

We have developed a highly sensitive, two-site immunoradiometric assay (IRMA) for human parathyrin (PTH) that is specific for the intact, secreted, biologically active 84-amino-acid peptide. This assay has several technical advantages: it does not detect even high concentrations of inactive carboxyl-terminal fragments, results are available within 24 h, and the detection limit for intact hormone is low (1 ng/L). The assay readily measures concentrations of PTH in all healthy subjects and distinguishes these values from low or undetectable PTH values observed in clinical situations in which PTH secretion is expected to be suppressed. We found complete separation of results from 37 patients with surgically proven hyperparathyroidism and those from 23 patients with hypercalcemia associated with malignancy, the latter having PTH values at or below the lower limits of normal for this assay. The sensitivity, specificity, and rapid turnaround time of this two-site IRMA should advance the laboratory evaluation of patients with disorders of calcium metabolism.     

Determination of intact parathyrin by immunoradiometric assay evaluated in normal children and in patients with various disorders of calcium metabolism

We report the reference values for intact parathyrin (PTH) measured by a two-site immunoradiometric assay (IRMA) during childhood. The study has been carried out in 215 healthy children and adolescents, ages 2.0 to 18.7 years. Some patients with altered mineral homeostasis were also studied to assess the sensitivity of the method in a clinical setting. Mean intact PTH concentrations were 30.8 (SD 9.6) ng/L; the median was 28.5 ng/L. Normal reference values were 16.0-59.0 ng/L (95% confidence interval). The distribution of intact PTH values was nongaussian. We found no significant variations between males and females and no age-related variations. The IRMA used was sufficiently sensitive to detect differences in PTH concentrations between healthy children and patients with hypocalcemia or hypercalcemia.     

Clinical performance of parathyroid hormone immunometric assays.

Three immunometric assays of parathyroid hormone (PTH)--a commercial immunoradiometric assay, an in-house immunoradiometric assay, and an immunochemiluminometric assay--were evaluated in 50 patients with surgically proven primary hyperparathyroidism. Of these patients, 43 had increased values with the commercial assay (sensitivity, 86%), whereas 45 patients had increased concentrations with both the in-house immunoradiometric and the in-house immunochemiluminometric assays (sensitivities, 90%). Because of the results of this comparison study, we confidently chose the immunochemiluminometric assay as our routine assay; this assay was evaluated retrospectively in 361 patients with surgically proven primary hyperparathyroidism. In 45 patients, PTH values were below the upper limit of normal (sensitivity, 88%). The results indicate that the sensitivities of current immunometric assays are approximately 90%. Twenty patients who had hypercalcemia associated with malignant involvement were assessed with the immunochemiluminometric assay. Of these 20 patients, 19 had subnormal PTH values, and 1 had a value within the normal range. In contrast, in the past, PTH values determined with radioimmunoassays have often been in the normal range for such patients. Thus, an immunometric PTH assay is superior to a radioimmunoassay in the differential diagnosis of hypercalcemia associated with malignant disease.     

Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays

BACKGROUND: The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. METHODS: We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. RESULTS: The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P <0.001). Regression and Bland-Altman analyses suggested that the LIAISON 25OHD assay reads lower than the DiaSorin RIA at low concentrations but higher at high concentrations. However, the cutoff (50 nmol/L) used in our laboratories to define vitamin D insufficiency with the DiaSorin RIA is applicable to the LIAISON 25OHD assay. In 927 healthy individuals, the 3rd-97th percentile intervals were 3-80 ng/L and 13-151 nmol/L for the LIAISON PTH and 25OHD concentrations, respectively. However, 506 individuals (54.6%) were vitamin D-insufficient; we therefore considered only the 421 individuals with a LIAISON 25OHD >50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). CONCLUSIONS: Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.     

Total urinary hydroxyproline determined with rapid and simple high-performance liquid chromatography.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).     

Evaluation of a radioimmunoassay for estradiol in unextracted serum

We describe the performance of a commercial (Steranti/EIR) RIA reagent kit for measuring 17 beta-estradiol directly in serum. Day-to-day precision data for control sera were as follows: mean = 102.8 ng/L, CV = 6.8%, n = 20; mean = 231.1 ng/L, CV = 5.3%, n = 21; mean = 747.7 ng/L, CV = 9.4%, n = 21. Analytical recovery of added estradiol from seven different serum pools from men, to which three different concentrations of estradiol had been added, was (mean +/- SD): 98.6 +/- 7.0% at 107.5 ng/L added; 98.8 +/- 4.7% at 322.5 ng/L added; 108.2 +/- 4.8% at 645 ng/L added. Overall recovery of estradiol in these experiments (mean +/- SD for 21 determinations) averaged 101.9 +/- 7.0%. Assay of 32 serum specimens from women by both the direct (y) and an extraction method (x) gave the following linear regression statistics: y = 1.12x - 1.3, r = 0.998, Sy/x = 30.2 ng/L, mean y = 438.2 ng/L, mean x = 391.4 ng/L. Hemoglobin, bilirubin, and moderate lipemia do not interfere. Sensitivity of the direct assay was 2.6 ng/L. Compared with the extraction assay, the direct estradiol assay has advantages of speed and simplicity.     

术中快速测定甲状旁腺素的临床意义

目的:介绍术中快速测定甲状旁腺素(PTH)在甲状旁腺功能亢进(HPT)中的应用方法、治愈标准、临床特征。方法:3例HPT患者,于麻醉后切开皮肤前抽血,切除异常旁腺后20 min抽血,并进行术中快速测定PTH,手术成功标准是PTH下降比切除前大于50%。结果:3例患者中,1例PTH下降大于50%、1例正常、1例三发性甲状旁腺功能亢进(ⅢHPT)降至正常下线(在切除2个旁腺后PTH变化不大,切除第3个后PTH降至正常下线)。术后均出现低血钙,补钙后症状消失,随访半年血钙及PTH正常。结论:术中快速测定PTH是甲状旁腺术判断所有亢进旁腺是否切除的有效方法。     

Method for the determination of total homocysteine in plasma and urine by stable isotope dilution and electrospray tandem mass spectrometry.

BACKGROUND: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. METHODS: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 microL of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d(4) was 1.5 min in a 2.5-min analysis. RESULTS: Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S(y|x) =1.595 micromol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S(y|x) =1.146 micromol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/L). CONCLUSIONS: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.     

Simple microplate method for determination of urinary iodine

BACKGROUND: Urinary iodine is a good biochemical marker for control of iodine deficiency disorders. Our aim was to develop and validate a simple, rapid, and quantitative method based on the Sandell-Kolthoff reaction, incorporating both the reaction and the digestion process into a microplate format. METHODS: Using a specially designed sealing cassette to prevent loss of vapor and cross-contamination among wells, ammonium persulfate digestion was performed in a microplate in an oven at 110 degrees C for 60 min. After the digestion mixture was transferred to a transparent microplate and the Sandell-Kolthoff reaction was performed at 25 degrees C for 30 min, urinary iodine was measured by a microplate reader at 405 nm. RESULTS: The mean recovery of iodine added to urine was 98% (range, 89-109%). The theoretical detection limit, defined as 2 SD from the zero calibrator, was 0.11 micromol/L (14 microg/L iodine). The mean intra- and interassay CVs for samples with iodine concentrations of 0.30-3.15 micromol/L were < or = 10%. The new method agreed well with the conventional chloric acid digestion method (n = 70; r = 0.991; y = 0.944x + 0.04; S(y|x) = 0.10) and with the inductively coupled plasma mass spectrometry method (n = 61; r = 0.979; y = 0.962x + 0.03; S(y|x) = 0.20). The agreement was confirmed by difference plots. The distributions of iodine concentrations for samples from endemic areas of iodine deficiency diseases showed similar patterns among the above three methods. CONCLUSIONS: Our new method, incorporating the whole process into a microplate format, is readily applicable and allows rapid monitoring of urinary iodine.     

Intact parathyrin in postmenopausal women

To establish a reference range, we measured intact parathyrin (parathyroid hormone, PTH) in 245 healthy postmenopausal women, ages 42-75 years, with use of the Allegro Intact PTH Kit from Nichols Institute Diagnostics. We also assayed serum from a subset of 120 of the women with kits specific for mid-molecule PTH. The mean intact PTH concentration for the 245 women was 32 ng/L (95% confidence interval 14-60 ng/L). Intact PTH values in these subjects were not normally distributed, although calcium concentrations in the same samples were. There was positive, but not significant (r = 0.12, P = 0.06), correlation between intact PTH and age, and a significant negative correlation between serum calcium and intact PTH that was not observed between calcium and mid-molecule PTH. The improved sensitivity of the intact PTH assay makes it useful in studies of calcium homeostasis in the normal population.     

Abbott TDx monoclonal antibody assay evaluated for measuring cyclosporine in whole blood

We report here the evaluation of the Abbott TDx assay with a monoclonal antibody for selectively quantifying cyclosporine (CsA) in whole blood. Over the clinically relevant concentration ranges, results with this assay demonstrated within- and between-run CVs of less than 2.5% and 5%, respectively; sensitivity of 25 micrograms/L; good analytical recovery (100.3%); and linearity with whole-blood specimens. The percentage cross-reactivity of the major CsA metabolites varied from 15.3% for AM9 (M-1), 8.2% for AM1 (M-17), and 3.7% for AM4N (M-21), to less than 3% for the other metabolites tested. Results with the TDx assay (y) correlated well with those by the Sandimmune selective RIA (x; Sandoz) with blood specimens from 44 renal-transplant recipients (n = 44, x= 187.3, y = 198.9, y = 5.49 + 1.03x, r = 0.987). The TDx values were on average 24% higher than those by HPLC (x') with the same patients' specimens (n = 44, x' = 159.9, y = 198.9, y = 15.9 + 1.14x', r = 0.967). We conclude that the Abbott TDx monoclonal antibody assay provides a rapid, precise, and accurate means for quantifying CsA in whole blood.     

Ultrasensitive, specific, two-antibody immunoradiometric assay that detects free alpha subunits of glycoprotein hormones in blood of nonpregnant humans.

This noncompetitive, sensitive, immunoradiometric assay of the free alpha subunit of human pituitary glycoprotein hormones is based on two monoclonal antibodies and an avidin-biotin separation system. The affinity of the first antibody, mouse anti-alpha subunit covalently conjugated to biotin, is 3.8 x 10(11) L/mol. The second antibody, radiolabeled with 125I, has an affinity of 5.4 x 10(11) L/mol. A polystyrene ball coated with avidin serves as the separation system. Tests of "purified" immunochemical-grade intact human glycoprotein hormones yielded cross-reactions of approximately 2% in the assay. Sephadex G-100 column chromatography showed that this "cross-reaction" was caused by contamination of the various hormone preparations with free alpha subunit. When the intact glycoprotein hormones were further purified with specific anti-alpha monoclonal antibody, their reaction in the alpha subunit assay was undetectable (less than 0.01%). Interassay CV averaged 3.5%, and intra-assay CV averaged 7.5% at low concentrations of subunit. The detection limit of the assay (0.01 micrograms/L) is adequate to detect free alpha subunit in the blood of normal humans. Mean (SD) concentrations of free alpha subunit in normal humans were as follows: eugonadal men = 437 (35) ng/L; postmenopausal women = 1231 (40) ng/L; eugonadal women, follicular phase = 1061 (40) ng/L; eugonadal luteal phase = 780 (45) ng/L.     

Evaluation of an automated commercial immunoluminometric assay for alpha-foetoprotein.

We evaluated a two-site immunoluminometric assay (AFP LIA-mat Byk Sangtec) for the determination of alpha-foetoprotein (AFP). The assay, involving two monoclonal antibodies which recognize two different alpha-foetoprotein epitopes, is rapid (4 h) with a wide working range (0-600 x 10(3) IU/l), a good lower limit of detection and good reproducibility (CV less than 10%). The regression equation for the AFP LIA-mat (y) and the immunoradiometric assay AFP Bridge Serono (x) was y = 1.175x - 2.27 (n = 95, r = 0.996) for serum and y = 1.16x + 479.2 for amniotic fluid. It did not display a hook effect, and when we assessed the linearity by assaying a high concentration of alpha-foetoprotein, it gave a linear response down to 3.1 x 10(3) IU/l. We also evaluated the clinical response of AFP LIA-mat in 278 patients with different diseases. Eighteen of the 19 patients with hepatocellular carcinoma had alpha-foetoprotein levels greater than 100 x 10(3) IU/l. In contrast, only 5 of the 47 patients with cirrhosis showed values above 50 x 10(3) IU/l, demonstrating that this assay discriminates fairly well between hepatocellular carcinoma and cirrhosis. Data in agreement with the literature were obtained for testicular tumours; all of seven seminomatous tumours presented values below 5 x 10(3) IU/l, whereas 50% of the non-seminomatous tumours (n = 8) presented values above 20 x 10(3) IU/l.