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.     

Quantitative nephelometric assay for determining myoglobin evaluated

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of myoglobin (Behringwerke) was evaluated with the BNA Nephelometer. Method precision was good: the intra-assay CV varied between 1.5% and 6.1%; with daily calibration, the interassay CV ranged between 1.5% and 7.5%. For usual sample dilutions, the assay response varied linearly with myoglobin concentrations up to 23.1 nmol/L. After automatic dilution by the instrument, concentrations up to 2310 nmol/L could be measured without high-dose "hook" effect. Further manual dilution allowed measurement of myoglobin concentrations up to 26,000 nmol/L. Calibration was stable for at least seven days. We detected no significant interferences from hemoglobin, haptoglobin, bilirubin, iodine-containing contrast media, and rheumatoid factors. Treating lipemic samples with Lipoclean (Behringwerke) decreased test results. Simultaneously drawn serum and plasma samples from the same subject showed no consistent differences in myoglobin concentrations. The mean reference myoglobin concentration was 1.380 (SD 0.82) nmol/L for men and 0.878 (SD 0.45) nmol/L for women. In patients with renal insufficiency, serum creatinine values were moderately related to serum myoglobin values (r = 0.465). Although a commercial radioimmunoassay (Byk-Sangtec) and the nephelometric assay intercorrelated well (r = 0.929), values obtained by nephelometry were significantly lower (P less than 0.05). By both assays, results for heart and skeletal muscle tissue extracts showed no correlation, a finding that suggests the existence of multiple forms of myoglobin in human tissues. We conclude that immunonephelometry is a rapid, practical, and reliable method for measuring myoglobin in serum.     

Homologous radioimmunoassay of human osteocalcin.

Osteocalcin or bone gamma-glutamic acid-containing protein (GLA protein) was isolated from human bone and used to develop a homologous radioimmunoassay of human osteocalcin. The effect of age on serum osteocalcin was studied in 380 normal children and adolescents and 330 normal adults. The mean (+/- SD) values in adults were higher in men [25 +/- 5 micrograms/L (4.3 +/- 0.8 nmol/L)] than in premenopausal women [20 +/- 6 micrograms/L (3.4 +/- 1.0 nmol/L); P < 0.01], but both were lower than in postmenopausal women [29 +/- 2 micrograms/L (5.0 nmol/L)]. The highest concentrations were seen in girls [ages 10-12 years: 99 +/- 38 micrograms/L (17.0 nmol/L)] and boys [ages 14-16 years: 107 +/- 57 micrograms/L (18.4 nmol/L)]. These mean values were substantially higher than those previously reported for results of heterologous osteocalcin radioimmunoassays but the correlation (r = 0.87, n = 77, P < 0.001) between both sets of results was excellent. In patients with metabolic bone diseases characterized by high or low bone turnover, the increase or decrease in serum osteocalcin observed was as expected. This homologous radioimmunoassay of human osteocalcin thus reflects bone turnover but reports serum concentrations higher than previously suspected.     

Avidin-biotin enzyme immunoassay of osteocalcin in serum or plasma.

We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).     

A method for the determination of sex hormone binding globulin in human serum

A simple and rapid method for serum sex hormone binding globulin (SHBG) was developed. Sera from pregnant women were pooled and the SHBG concentration was measured by Rosner's method. This pool was diluted with charcoal-treated and heated serum to give standards ranging in concentrations from 26.8-0.8 micrograms/L. Two sets of tubes containing dihydrotestosterone (DHT), assay and control tubes, were prepared. Diluted standards and unknowns were added to assay and control tubes along with tritiated DHT. Following incubation, cold saturated ammonium sulfate was added to each tube, mixed, centrifuged and the supernatant counted. Mean SHBG concentration (microgram/L +/- SEM) in normal men was 3.6 +/- 0.4, normal women 11.4 +/- 2.2, pregnant women 58 +/- 2.6, obese women 3.3 +/- 1.0, hirsute women 2.9 +/- 0.2, hypothyroid women 7.3 +/- 1.0, and hyperthyroid women 26.0 +/- 1.6. These results correlate well with previous reports. This method is fast, convenient, and up to 40 samples can be analyzed in one assay.     

Unchanged androgen-binding properties of sex hormone-binding globulin in male patients with liver cirrhosis.

BACKGROUND: Men affected by liver cirrhosis frequently show clinical features of hypogonadism due to hormonal changes, in particular in the metabolism of 17beta-estradiol (E2) and testosterone (T). Sex hormone-binding globulin (SHBG), the major binding protein of these steroids in serum, is regularly elevated in such patients, with its androgen-binding properties possibly altered. In the present study, surface plasmon resonance biosensor techniques were used to determine whether the functional binding properties of this transporter are maintained in this pathology. METHODS: We selected 33 male patients with cirrhosis, Child-Pugh grade A or B, and 32 healthy males served as controls. Serum concentrations of T, E2, dehydroepiandrosterone sulfate (DHEAS) and SHBG were measured. In addition, ligand-binding properties of SHBG partially purified from sera of 23 cirrhotic patients and 20 controls were analyzed by a real-time biosensor technique using a surface-coated dihydrotestosterone derivative. RESULTS: The sensorgrams revealed that SHBG was fully bioactive in all samples investigated without any changes in binding kinetics. Moreover, total T concentrations were not significantly different in the cirrhotic patient sera (mean+/-SD 18.0+/-8.6 nmol/L) compared to controls (15.6+/-3.7; n.s.), whereas E2 was higher (152+/-60 vs. 96+/-29 pmol/L; p<0.0001) and DHEAS was lower (1493+/-1410 vs. 5099+/-2844 nmol/L; p<0.0001). CONCLUSIONS: Owing to elevated SHBG levels without changes in the steroid-binding properties in sera of cirrhotic male patients, free or bioavailable T concentrations are lower. This causes a shift of the hormonal balance in favor of E2, which exhibits a lower affinity for SHBG than androgens and accounts for the endocrine symptoms.     

Radioimmunoassay of testosterone not bound to sex-steroid-binding protein in plasma

To measure the concentration of testosterone (T) that is not bound to sex-steroid-binding protein (SBP) in plasma, we quantified by radioimmunoassay the T in the supernates of plasma samples after precipitation with 50%-saturated ammonium sulfate. The concentrations of non-SBP-bound T. directly measured with this assay, correlated significantly (P less than 0.001) with those deduced from measurement of the percentage of non-SBP-bound T determined with [3H]T as tracer or from mathematical models according to the law of mass action. It also correlated significantly with the ratio of T to SBP and with the concentration of nonbound T. As determined with this assay, the mean concentration of non-SBP-bound T in normal men was higher in young (4.67, SD 2.68 nmol/L; n = 30) than in older (greater than 40 years) subjects (2.48, SD 1.61 nmol/L; n = 35; P less than 0.001) and lower than normal in hyperthyroid (1.61, SD 0.91 nmol/L; P less than 0.01) or infertile men (3.28, SD 1.70 nmol/L; P less than 0.01). In women, non-SBP-bound T was higher in hirsute patients (0.24, SD 0.11 nmol/L; P less than 0.01) and was lower during pregnancy (0.09, SD 0.05 nmol/L; P less than 0.05) than in normal women during the follicular phase (0.16, SD 0.07 nmol/L). We conclude that this direct measurement of non-SBP-bound T in plasma is suitable for routine use and represents a reliable index of androgenicity in human pathology, particularly when alterations of the binding capacity of SBP modify the concentrations of total T.     

Electroimmunoassay of sex hormone binding globulin. Enhanced sensitivity by autoradiography using A-ring 125I-17-beta-oestradiol

Four different 125I-iodinated steroids were tested for their binding to human sex hormone binding globulin (SHBG) using an electrophoretic technique. 17-beta-oestradiol iodinated in its A-ring was found to bind with high affinity to SHBG. This radioactive steroid was used to increase the sensitivity of the electroimmunoassay of SHBG by adding the steroid to the samples before electroimmunoassay. The radioactive steroid incorporated into the immunoprecipitates could be observed by autoradiography. The sensitivity of the assay, which employed a rabbit antiserum against purified human SHBG and was standardized with pure SHBG, was about 0.2 mg/l. The precision of the method, calculated as the coefficient of variation within and between assays, was 2.4% and 2.6%, respectively, for values within the normal range. The mean SHBG concentration in healthy regularly menstruating women (n = 50) was 3.50 +/- 0.74 (SD) mg/l when measured in plasma, and 3.78 +/- 0.80 mg/l when measured in serum. The corresponding mean concentrations in healthy men (n = 28) were 2.26 +/- 0.45 mg/l in plasma and 2.44 +/- 0.49 mg/l in serum. The modified electroimmunoassay described in this paper is a simple modification, which increases the sensitivity sufficiently to permit reliable quantification of SHBG over the entire range of concentration which could be relevant in clinical practice.     

Study on the binding of dihydrotestosterone, testosterone and oestradiol with sex hormone binding globulin

Saturation studies on sex hormone binding globulin (SHBG) were performed with increasing levels of the three steroids: dihydrotestosterone (DHT), testosterone (T) and oestradiol (E2) in normal female sera. The curves obtained showed the clearly increasing SHBG affinity for E2, T and DHT, respectively. With T as saturating ligand, the mean values of SHBG binding capacity in normal and pathological sera, expressed as 10(-8) M SHBG were, for normal women, 5.87 +/- 0.2, for normal men, 3.69 +/- 0.1 and for hirsute women, 5.05 +/- 0.2. In hirsutism, SHBG levels were measured together with T and DHT levels. Hyperandrogenia was often better reflected by the T/SHBG ratio than by T levels alone. SHBG binding capacity, measured in pregnancy from the 7th to the 40th week, increased progressively as far as the 30th week. Values up to 5 times those of the mean value of the cycle were observed at the end of pregnancy. Variations in DHT, T and E2 binding as functions of SHBG concentrations, were also studied by adding a constant amount of each steroid to increasing SHBG concentrations in serum. DHT showed the greatest binding capacity, followed by T and then E2. Since the unbound fractions of T and DHT are believed to be biologically active, and since T and DHT are more sensitive than E2 to SHBG variations, the results obtained further support the hypothesis that SHBG plays an important role in the sex steroids balance.     

Particle-enhanced turbidimetric immunoassay of sex-hormone-binding globulin in serum

A particle-enhanced turbidimetric immunoassay (PETIA) for human sex-hormone-binding globulin (SHBG) is described. The method involves use of antibody covalently coupled to latex particles and is almost fully automated, with sample processing being complete in less than 20 min. The working reagents are stable for at least three months, and full calibration of the assay each day is not essential. A particular advantage is that pretreatment of samples is rarely required because the working range of the assay is from 2.0 to 320 nmol/L for nondiluted serum. Intra- and interassay CVs were less than 4.5% and 8.5%, respectively, and mean analytical recovery was 101.5%. SHBG concentrations of 129 serum samples determined by this method and by a commercially available immunoradiometric assay correlated highly.     

Differential immunoadsorption coupled with rate nephelometry for estimation of DNA-binding immunoglobulins

We describe a technique for estimating the mass of anti-DNA antibodies by immunonephelometry of serum immunoglobulins (IgG, IgA, IgM) before and after adsorption onto DNA bound to agarose-polylysine columns. Sixteen patients with systemic lupus erythematosus and 16 age- and sex-matched controls were studied. Precision was determined for high-value (in 10 patients) and low-value (in nine controls) ranges for each of the immunoglobulins. Within-run CVs ranged from 3.0% (IgG, controls) to 11.8% (IgA, patients); between-run CVs ranged from 15.5% (IgG, patients) to 25.2% (IgM, patients). We found anti-DNA antibody concentrations (mean +/- SD) in systemic lupus erythematosus of 1.981 +/- 1.015 g/L for IgG (controls: 0.243 +/- 0.231, p less than 0.001), 0.257 +/- 0.215 g/L for IgA (controls: 0.038 +/- 0.035, p less than 0.001), and 0.282 +/- 0.234 g/L for IgM (controls: 0.191 +/- 0.165, p greater than 0.05). Sensitivity and linearity are such that fivefold dilutions of patients' serum with either a buffered albumin solution or control serum yielded values close to the expected values for IgG. Similarly diluted sera gave inordinately high values in the radiometric binding assay. Neither parametric (linear regression) nor nonparametric correlation methods (Spearman's rank and Kendall's tau) show a significant correlation between patients' data obtained by the present technique and that by a radiometric binding assay (p greater than 0.05), although combined data from patients and controls demonstrate a significant nonparametric correlation (p less than 0.005 for Spearman's and p less than 0.02 for Kendall's).     

A method for measuring testosterone-estradiol-binding globulin--new analysis of experimental data and comparison with two commercially available kits

Some binding assays have been suggested to measure serum testosterone--estradiol-binding globulin (TeBG) concentration. They have led to discrepant results. This study brought to surface the lack of these methods and the interest of a method using an accurate analysis of the binding isotherm. These observations led us to propose an absolute reference method especially suitable for the radioimmunological assays standardization. Our method used Con A Sepharose as a solid phase and DHT as a radioligand. We succeeded in solving the difficulties related to binding methods by evaluating the non-specific binding of the ligand and ligand--albumin binding. A careful analysis of experimental data, by setting equations specific to this DHT-TeBG system permitted the systematic errors to be corrected and allowed a precise determination of the serum TeBG concentration (27.3 +/- 6.0 nmol/l for 30 healthy men; 40 +/- 13 nmol/l for 15 healthy women). Moreover, the affinity constant of the ligand for the protein was accurately evaluated KDHT = (0.7 +/- 0.1)10(9) (mol/l-1) at 20 degrees C, which was not permitted by other methods. Two lines of evidence supported our assumptions and results. On the one hand, using estradiol as a radioligand instead of dihydrotestosterone, we found the same value for serum TeBG concentration; on the other hand, our assay favourably compared with two different kits commercially available. The correlation (r = 0.89) with 3H-SBP kit Merieux was good though this kit was not suitable for correctly assaying serum TeBG concentrations between 30 nmol/l and 60 nmol/l. An excellent correlation (r = 0.97) with SHBG IRMA Kit Farmos was found. This antibody-based assay and our binding assay yielded quite similar values that mutually validated both methods.     

Radioimmunoassay of human sex hormone binding globulin: improved radioiodination procedure

Sex hormone binding globulin (SHBG), purified by affinity chromatography from retroplacental blood plasma, was reacted with 3-(p-hydroxyphenyl) propionic acid N-hydroxysuccinimidyl ester (PHPPS, Bolton-Hunter reagent). The derivative of SHBG obtained (parahydroxyphenylpropionyl-SHBG; PHPP-SHBG) was stable and could, in contrast to underived SHBG, be efficiently 125I-iodinated with a lactoperoxidase technique. The PHPP-SHBG labelled with 125I had good antiserum binding and stability properties and was used for radioimmunoassay (RIA) of SHBG in serum. The RIA requires a total incubation time of 3 h. It has been standardized with purified SHBG and has a sensitivity of 5 micrograms/l, giving a lowest detectable concentration in the routine procedure (samples diluted 1:40) of about 0.2 mg/l. Variation within and between assay was 4.1% and 7.2%, respectively, for samples with values within the normal range. Values obtained by this RIA procedure correlate well with those obtained by a dihydrotestosterone binding method and by an electroimmunoassay technique. The mean serum concentration of SHBG in healthy, regularly menstruating women (n = 42) was 3.7 +/- 1.0 (SD, standard deviation) mg/l and in healthy men (n = 100) 2.0 +/- 0.9 mg/l.     

Characterization of theophylline binding to serum proteins in pregnant and nonpregnant women

Sera from 10 subjects in the third trimester of pregnancy and from 10 nonpregnant women were studied to elucidate the mechanism underlying decreased theophylline protein binding during pregnancy. Consistent with the physiologic hypoalbuminemia of pregnancy, serum albumin concentrations averaged only 3.2 +/- 0.3 gm/dl (+/- SD) in pregnant subjects, compared with 4.4 +/- 0.3 gm/dl in control subjects (p less than 1 x 10(-6], and this was the main cause of decreased theophylline binding. Saturation binding studies indicated a single class of theophylline binding sites. Theophylline binding capacity (N) was greater in pregnant (N = 4.3 +/- 1.0) than in nonpregnant (N = 3.3 +/- 0.4) subjects, but binding affinity (ka) averaged only 227 +/- 69 (mol/L)-1 in pregnant subjects, compared with 303 +/- 44 (mol/L)-1 in control subjects (F2,17 = 4.26; p = 0.032). At a theophylline plasma concentration of 10 micrograms/ml, the combined effects of hypoalbuminemia and lowered ka would reduce theophylline binding to 31% +/- 3% in pregnant women, compared to 39% +/- 3% in nonpregnant control subjects (p less than 1 x 10(-5]. Nonesterified fatty acid concentrations were similar in both subject groups and did not contribute to the pregnancy-associated decrease in theophylline binding.     

Dialyzable free cortisol after stimulation with Synacthen

OBJECTIVES: To compare the changes in free vs. total serum cortisol concentrations after acute stimulation of the adrenal cortex. DESIGN AND METHODS: Paired serum samples of ten individuals taken immediately before and 1 h after stimulation with 250 microg ACTH (1-24) (Synacthen) given i.v. were analyzed. Total cortisol was quantified using liquid chromatography tandem-mass spectrometry with an online sample extraction system and tri-deuterated cortisol as the internal standard. Free cortisol was measured with the same method after equilibrium dialysis. Concentrations of the corticosteroid-binding globulin (CBG) were determined by radioimmuno assay. RESULTS: Total cortisol increased by a mean of 106% (mean basal cortisol 312 nmol/L (SD 140 nmol/L), stimulated 686 nmol/L (SD 163 nmol/L); p < 0.001, paired t-test for differences); no significant change of CBG concentrations was found (874 nmol/L (SD 179 nmol/L) before stimulation, 869 nmol/L (SD 225 nmol/L) after stimulation). The mean increase of free cortisol was 263% (mean basal free cortisol 20.3 nmol/L (SD 13.2 nmol/L), stimulated 73.8 nmol/L (SD 26.7 nmol/L); p < 0.001) and thus substantially more pronounced compared to the increase of total cortisol (p < 0.01). The ratio of free to total serum cortisol was significantly increased after stimulation (6.1% (SD 1.7%) before stimulation, 10.6% (SD 1.9%) after stimulation; p < 0.001). CONCLUSIONS: After acute neuroendocrine stimulation of the adrenal cortex the relative increase of free bioactive cortisol concentrations is substantially more pronounced than the increase of total cortisol concentrations.     

Importance of blood-collection tubes in plasma lidocaine determinations.

In 25 clinical samples serum lidocaine concentrations fell from a mean of 6.5 +/- 2.1 mg/L (mean +/- SD) to 4.9 +/- 1.8 mg/L (p less than 0.001) when the blood sample was allowed to make contact with the stopper of the Vacutainer collection tube. In vitro experiments showed that this effect of the stopper occurred only with whole blood and was dependent on sample concentration. The plasma binding of lidocaine decreased from a normal value of 56% +/- 2.2 (mean +/- SD) to 28% +/- 2.2 (p less than 0.001) when exposed to the Vacutainer stopper. We conclude that a chemical leached from such stoppers displaces lidocaine from its plasma-binding sites and that the drug is then redistributed into the erythrocytes, producing spuriously low lidocaine concentrations in plasma or serum. Such artifacts are important in therapeutic drug monitoring and can lead to erroneous clinical decisions.     

Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay

BACKGROUND: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum. METHODS: We quantitatively isolated folates from 275 microL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C(8) analytical column. (13)C-labeled folates were used as internal standards. RESULTS: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were approximately 9% lower than results obtained with LC/MS/MS. The microbiologic assay gave approximately 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5-75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF. CONCLUSIONS: This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.     

Immunoassay for sex hormone-binding globulin in undiluted serum is influenced by high-molecular-mass aggregates

BACKGROUND: The new Elecsys chemiluminescence assay for measurement of homodimeric sex hormone-binding globulin (SHBG) was designed for use with undiluted serum, in contrast to other methods that require predilution. During assay development, unexpected calibration difficulties were observed that were attributable to particular biochemical properties of the highly concentrated SHBG in solution. METHODS: We used a surface plasmon resonance (SPR) biosensor, which enables biomolecular interaction analysis of SHBG, and size-exclusion chromatography for this investigation. The immunoassay was evaluated for imprecision, linearity, and suitability of the dilution medium, and the method was compared with an IRMA for SHBG. RESULTS: The SPR biosensor characterized the special protein properties of SHBG in various concentrations. Above 200 nmol/L there was a strong tendency toward formation of high-molecular-mass aggregates. This was also detectable by size-exclusion chromatography and could be reversed by simple dilution of the sample. On the basis of these results, the dynamic measuring range of the SHBG assay is restricted to 0.350-200 nmol/L. Assay evaluation on a 2010 analyzer revealed excellent precision (CV 相似文献   

Specific binding of cardiac glycoside drugs and endogenous digitalis-like substances to particulate membrane fractions from human placenta

We studied the characteristics of binding of cardiac glycosides to particulate membrane fractions from human placenta, to demonstrate that placental tissue is a suitable source of receptors for digitalis drugs. Moreover, we performed preliminary experiments with 125I-labeled digoxin and placental particulates to develop a radioreceptor assay for measurement of endogenous substances with activity similar to cardiac glycoside drugs (EDLS). Placental membrane fractions were incubated with [3H]ouabain (10 nmol/L) or 125I-labeled digoxin (50 pmol/L). With both ligands, binding followed a pseudo-first-order reaction kinetics and was saturable. Scatchard analysis revealed a single class of sites [for ouabain, KD = 20.2 +/- 5.8 nmol/L (mean +/- SEM), Bmax = 3.1 +/- 0.9 nmol per gram of protein; for digoxin, KD = 29.7 +/- 1.9 nmol/L, Bmax = 24.3 +/- 1.1 nmol per gram of protein]. As expected, digoxin was less potent than ouabain in displacing both tracers from digitalis drugs receptors; progesterone, cortisone, digitoxose, furosemide, bumetanide, and propranolol had no or little effect. Specific 125I-labeled digoxin binding was competitively inhibited by plasma and (or) urine extracts from newborns, adults, pregnant women, and patients with renal insufficiency. Inhibition of binding and volume of plasma and urine assayed were linearly related. These findings support the hypothesis that cardiac glycosides and EDLS can interact with the human placenta and suggest placental tissue to be a suitable source of receptors for cardiac glycosides.     

Liquid chromatography and fluorescence polarization immunoassay methods compared for measuring flecainide acetate in serum

We analyzed 99 patients' serum samples for concentrations of a new antiarrhythmic agent, flecainide acetate, by fluorescence polarization immunoassay (FPIA) and "high-performance" liquid chromatography (HPLC). Within-day and between-day coefficients of variation at concentrations in the low and high ends of the therapeutic range were less than 7% for HPLC and less than 9% for FPIA. There was no statistical difference in the mean (+/- SD) concentrations of the clinical serum samples measured by the two methods (607 +/- 334 micrograms/L by HPLC, 602 +/- 344 micrograms/L by FPIA), but results by each differed by a mean of 0.13%. FPIA and HPLC measurements correlated significantly (r = 0.98, P less than 0.05), and were linearly related (slope = 0.970, intercept = 13 micrograms/L) as assessed by orthogonal regression. Both assay methods produced similar concentration measurements and were sufficiently accurate and precise to be used in therapeutic drug monitoring.