Importance of using molar concentrations to express cross-reactivity in immunoassays.
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Digoxin↗
Biomedical subjects
R Valdes
Publications and source records attributed to R Valdes.
Digoxin immunoassay with cross-reactivity of digoxin metabolites proportional to their biological activity.
Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.
Animals↗
Mechanism and elimination of aspirin-induced interference in Emit II d.a.u. assays.
The presence of salicylates in urine reduces the signal in Emit assays (Syva), potentially yielding false-negative drugs-of-abuse screening results. We demonstrate that the principal urinary metabolite of salicylate, salicyluric acid (SUA; 2-hydroxybenzoylaminoacetic acid), interferes with the measurement of NADH formed in the assay by reducing the molar absorptivity of NADH at 340 nm. Thus, for a given concentration of d.a.u. analyte the change in absorbance over the assay time interval is less in the presence of SUA. With the Emit cocaine assay on the Hitachi 704 analyzer, the rate of absorbance change (delta AR) monitored at 340 nm for a specimen containing approximately 270 micrograms/L benzoylecgonine (BE) was 57 +/- 1.9 mA/min without SUA and 29 +/- 2.7 mA/min with 5 g/L SUA (n = 20). In contrast, delta AR determined at 376 nm was 18.6 +/- 0.5 mA/min with and 17.9 +/- 0.8 mA/min without 5 g/L SUA (n = 20). Measuring the Emit assay signal at wavelengths where SUA has no absorbance (376 nm) eliminates the interference due to SUA while maintaining the precision of the assay near the cutoff concentration for BE (300 micrograms/L).
Aspirin↗
Understanding the sodium pump and its relevance to disease.
Na,K-ATPase (sodium pump; EC 3.6.1.37) is present in the membrane of most eukaryotic cells and controls directly or indirectly many essential cellular functions. Regulation of this enzyme (ion transporter) and its individual isoforms is believed to play a key role in the etiology of some pathological processes. The sodium pump is the only known receptor for the cardiac glycosides. However, endogenous ligands structurally similar to digoxin or ouabain may control the activity of this important molecular complex. Here we review the structure and function of Na,K-ATPase, its expression and distribution in tissues, and its interaction with known ligands such as the cardiac glycosides and other suspected endogenous regulators. Also reviewed are various disorders, including cardiovascular, neurological, renal, and metabolic diseases, purported to involve dysfunction of Na,K-ATPase activity. The escalation in knowledge at the molecular level concerning sodium pump function foreshadows application of this knowledge in the clinical laboratory to identify individuals at risk for Na,K-ATPase-associated diseases.
Cardiac Glycosides↗
Cardiac troponin-T immunoassay for diagnosis of acute myocardial infarction.
We evaluated the analytical and clinical performance of an immunoassay for cardiac troponin T (cTnT). Within-run and total imprecision ranged from 1.6 to 11.3%. The sensitivity and linear range was 0.015 and 13 micrograms/L, respectively. Frozen samples were stable for at least 8 weeks. No interferences were seen with lipids or bilirubin (total and conjugated). Hemoglobin caused a negative bias at concentrations > 4 g/L. Heparinized plasma showed a 6% negative bias compared with serum. The clinical utility of cTnT was compared with that of creatine kinase (CK)-MB (mass assay). The sensitivity of cTnT measurements from 63 patients with acute myocardial infarction (AMI) (cTnT cutoff 0.1 microgram/L) was 60% at 0-3 h, 59% at 3-6 h, 94% at 6-9 h, 90% at 9-12 h, 99% at 12-24 h, 92% at 24-48 h, 83% at 48-72 h, and 100% at 72-96 h. Corresponding results for CK-MB (cutoff 5.0 micrograms/L and 2.5% relative index) were 45%, 64%, 82%, 97%, 87%, 81%, 54%, and 59%. The specificity of the markers from 49 non-AMI patients was 46% and 79% for cTnT and CK-MB, respectively. We show that CK-MB is more specific for diagnosis of AMI, and propose that cTnT is more sensitive to myocardial injury.
Bilirubin↗
Twenty-five years' experience with renal transplantation in children of low socioeconomic class.
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Adolescent↗
Decreased signal in Emit assays of drugs of abuse in urine after ingestion of aspirin: potential for false-negative results.
During routine drug analysis with the Syva d.a.u. Emit immunoassays we observed a high frequency of urines with lower rates of changes in absorbance (delta A R) than the rate for a drug-free urine calibrator. Many of these urines contained salicylates. Among 40 urines with apparent salicylate concentrations between 15 and 420 mg/dL tested for benzoylecgonine (BE), 20 had delta A R < -4 (range +2 to -28 mA/min). The rates decreased with increasing salicylate: delta A R = -0.057 x (salicylate, mg/dL) -0.22 mA/min (r = 0.85, n = 40, P < 0.01). Urines from 100 control subjects (no salicylate) had mean +/- SD delta A R values of -1.05 +/- 2.2 mA/min (range +3 to -7; only two were < -4 mA/min). Although direct addition of salicylic acid (200 mg/dL) to urine specimens did not reproduce the negative bias, ingestion of aspirin (acetylsalicylic acid) did by -0.09 mA/min per 1 mg/dL (72.4 mumol/L) salicylate. Negative biases observed for other Emit d.a.u. assays after salicylate ingestion lead us to conclude that ingestion of therapeutic doses of aspirin may cause false-negative results for drug screens in urines by this technology.
Aspirin↗
Demonstrating instrument-reagent flexibility: a carbamazepine enzyme immunoassay reagent system.
Versatility of immunoassay reagents is beneficial to laboratories seeking cost-effective combinations of tests and automated instrumentation. In such cases, both immunoassay analytical performance and instrument independence must be assessed. Considering this, we determined the compatibility of a new carbamazepine EMIT 2000 reagent system with two fully automated but different kinetic rate analyzers (Hitachi 704 and Cobas MIRA), comparing results to a reagent-dedicated fluorescent polarization automated device (TDx) as reference. In order to more stringently assess reagent antibody specificity, we tested recovery of purified carbamazepine spiked into sera pooled from different hospital groups (normal, renal failure, hepatic failure, term pregnancy, cord blood). Cross-reactivity was additionally tested using patient sera containing various amounts of tricyclic antidepressants, compounds structurally but not functionally related to carbamazepine. Despite distinct operational differences between analyzers, precision (< 5.5% CV) and accuracy (> 95% recovery) compared well to the TDx method. However, when carbamazepine was spiked into sera from patients with hepatic failure or at term pregnancy, all three methods measured a negative bias in recovery of 16-20%. No significant cross-reactivity was observed at normal therapeutic concentration of certain tricyclic compounds, though measurable cross-reactivity was detected when present at toxic serum concentrations. We conclude that the EMIT carbamazepine immunoassay is adaptable to the different kinetic rate analyzers studied. Analytical specificity should, furthermore, be assessed in the context of interferences likely to be clinically encountered.
Antibody Specificity↗
The dosage alone determines whether a substance is toxic.
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Dose-Response Relationship, Drug↗
Improving the specificity of digoxin immunoassays.
The overall reliability of measuring digoxin in serum improved significantly with the discovery and application of immunoassays. However, because of the low concentration of digoxin being measured, its narrow therapeutic range in serum, and the presence of endogenous digoxin-like immunoreactive factors (DLIF), developing assays for measuring digoxin still pose formidable challenges. In this presentation, recent developments in the characterization of DLIF from bovine adrenal cortex and human serum are described. Data accumulated to date suggest there is one principal endogenous molecular factor (DLIF) in humans that cross-reacts with anti-digoxin antibodies. This factor exists at sufficiently high concentrations in some patients to interfere with measurements of digoxin by most digoxin immunoassays. All digoxin immunoassays should be tested to interference from this endogenous factor. Various techniques for reducing DLIF cross-reactivity are reviewed. The isolation and purification of DLIF now provides new approaches for selecting specific anti-digoxin antibodies used in developing more accurate digoxin immunoassays.
Blood Proteins↗
A multicenter evaluation of lipid profiling with a compact analyzer (Miles Clinistat).
We evaluated the Clinistat Analyzer (Miles Inc., Diagnostics Division, Elkhart, IN) for measuring cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol at three medical centers. The system, based on multilayer film technology, uses precalibrated, dry film reagent disks. Ten microliters of serum is applied to the dry film reagent disk in the test procedure. For HDL-cholesterol measurement, serum is pretreated by precipitation with phosphotungstic acid and magnesium chloride. Total precision (CVs) of each of the three assays was less than or equal to 5%. The assay ranges were linear and satisfactory for clinical use. Patients' results compared well with established methods. No significant interferences were found with hemolysis, icterus, and lipemia.
Chemistry, Clinical↗
Isolation of digoxin-like immunoreactive factors from mammalian adrenal cortex.
Endogenous digoxin-like immunoreactive factors (DLIF) are present in serum and tissues of humans and animals. To date, a tissue source for these factors has not been rigorously defined nor have these factors been isolated to identifiable homogeneity. In this study, we define the distribution of DLIF in mammalian tissues, demonstrate the adrenal cortex to be the principal source of this factor in bovine, and isolate DLIF to chromatographic homogeneity using high performance liquid chromatography (HPLC). DLIF concentrations in tissue extracts from rats measured as follows: adrenal glands, 44.3; serum, 6.3; liver, 5.2; kidney, 1.2; heart, brain, or lungs, less than 1.4 ng of digoxin-equivalent per g of protein. Human tissues showed similar results. In dogs, the ratio of the DLIF concentration in lumbar vein serum to that in infrarenal inferior vena cava serum was 3.3 +/- 0.4 (mean +/- S.E., n = 4). Bovine adrenal cortex contained 7 times more DLIF per g of tissue than the adrenal medulla. 70 +/- 4% (n = 7) of the total bovine cortical DLIF activity (6,159 pg of digoxin-equivalent) applied to a reverse phase HPLC column eluted as one definitive fraction. 60% of the digoxin-like immunoreactivity extracted from bovine serum also co-eluted with DLIF from adrenal. None of the 14 steroid molecules or 7 cardiac glycoside congeners co-eluted with the major DLIF activity. Our data indicate that 947 pmol of DLIF is equivalent to 1 pmol of digoxin-equivalent immunoreactivity. Preliminary mass spectral analysis suggests that purified DLIF has a molecular mass of 780 daltons comprised of one 390-dalton aglycone component plus several sugar moieties. This study establishes a definitive link between DLIF in serum and the adrenal cortex as a source tissue. We also demonstrate a method for purifying DLIF to chromatographic homogeneity with an extraction capacity of 1.2 nmol of DLIF per g of adrenal cortex.
Adrenal Cortex↗
Approaches to minimizing interference by cross-reacting molecules in immunoassays.
Here we review techniques useful in eliminating or reducing interferences caused by molecules that cross-react in immunoassays. The biochemical rationale for using these techniques is discussed. Examples are taken from recent studies aimed at reducing interferences caused by endogenous molecules such as digoxin-like immunoreactive factors or steroid hormones. In this context the role of protein-binding of cross-reacting molecules is also considered. Immunoassay ligand selectivity can be inherently limited by the heterogeneity of the antigenic response or by the structural similarity of epitopes on multiple ligands. Certain empirical approaches have proved useful in maximizing the analytical specificity of immunoassays. These approaches include isolating the relevant ligands before immunoassay, adjusting the kinetic or equilibrium conditions used during the assays, and developing more specific antisera. The physicochemical properties of the cross-reacting molecule best dictate which technique(s) to use. The approaches discussed here are general and apply to minimizing interference caused by a wide variety of both endogenous and exogenous cross-reacting molecules.
Antibody Specificity↗
Radial partition fluorescent immunoassay of thyrotropin. Analytic evaluation and clinical correlation.
The authors evaluated the analytic and clinical performance of a sensitive radial partition fluorescent enzyme immunoassay for thyrotropin (TSH) performed on Stratus and compared it with a nonsensitive radioimmunoassay (RIA) method. Sensitivity of 0.15 mIU/L was obtained, and precision, specificity, and linearity were acceptable. A good correlation was observed between the two assays in samples from 311 hospitalized patients (r = 0.976). Stratus TSH results were outside the reference range for 20% of clinically euthyroid patients (n = 126), and 2.4% had undetectable levels. The clinically hyperthyroid group (n = 11) with the exception of one patient had TSH values below 0.2 mIU/L. Only 39% of hypothyroid patients on thyroid hormone replacement (n = 74) had TSH values in the reference range, with 38% and 23% exhibiting low and high values, respectively. All untreated primary hypothyroid patients (n = 8) had elevated TSH concentrations. The authors conclude that this sensitive TSH assay is useful for diagnosing hyperthyroidism when there is a clinical suspicion but cannot be recommended for thyroid screening in hospitalized patients.
Adult↗
More on the new immunometric digoxin assay on the Ames Seralyzer.
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Biological Assay↗
Criteria for identifying endogenous compounds as digoxin-like immunoreactive factors in humans.
Endogenous digoxin-like immunoreactive factors (DLIF) are factors in plasma that interact with anti-digoxin antibodies. In this report we propose specific empirical criteria that must be satisfied by any group of endogenous compounds purported to account for DLIF activity in human plasma. These criteria include immunoreactive potency relative to existing physiologic concentrations as well as the biochemical and protein binding properties of these compounds. Recent studies have identified several congeners of fatty acids and phospholipids, hydrocortisone, and dehydroepiandrosterone-sulfate as compounds likely to account for DLIF activity in plasma. Using the above criteria we demonstrate that the highest reported plasma concentrations of these compounds combined account for less than 25% of DLIF reported in healthy adult subjects, less than 11% in newborns, less than 27% in pregnant women, and less than 39% in patients with renal failure. Human serum albumin at a concentration of 40 g/l completely abolished any detectable interaction of these compounds with both anti-digoxin antibodies or canine kidney Na/K-ATPase. The immunoreactive and physical properties of these compounds are also not consistent with those reported for DLIF. We conclude that these compounds do not account for the plasma DLIF concentrations measured in human subjects nor are they likely to play a role as specific endogenous regulators of Na/K-ATPase.
Antibodies↗
Endogenous digoxin-like immunoreactivity in blood is increased during prolonged strenuous exercise.
Digoxin-like immunoreactive factors (DLIFs) in serum may represent endogenous cardiotropic agents. We determined if blood levels of these endogenous factors changed during prolonged strenuous exercise. Total and loosely protein-bound (LPB) DLIF were measured by radioimmunoassay in the serum of nine healthy subjects during prolonged exercise to exhaustion. Mean total and LPB serum levels of DLIF increased by 72% (580 to 945 pg/mL) and 63% (53 to 91 pg/mL) over baseline values in digoxin equivalents (p less than 0.01), respectively, after three hours of exercise at 70% of VO2max. The prevalent serum nonesterified fatty acids (arachidonic, linoleic, oleic, palmitic, and stearic acids) as well as hydrocortisone did not account for the observed elevations in DLIF. Percent left ventricular fractional shortening (%FS) and mean velocity of left ventricular circumferential fiber shortening (mVCF) measured echocardiographically were lower (-18.0% and -16.4%, respectively, p less than 0.05) after exercise as compared to prior to exercise. Cardiac left ventricular dysfunction as measured by %FS did correlate with blood levels of DLIF (r = -0.680, p less than 0.02). These observations may suggest a relationship between serum levels of DLIF and cardiac fatigue.
Adult↗
Excretion of endogenous digoxin-like immunoreactive factors in human urine is a function of urine flow rate.
We studied the effect of varying water and salt intake on the renal excretion of endogenous digoxin-like immunoreactive factors (DLIF). DLIF were measured in human urine and serum by competitive displacement of 125I-labeled digoxin from anti-digoxin antibodies. Diuresis was selectively induced in normal healthy humans by acute water ingestion, and natriuresis was preferentially induced by acute saline ingestion. We found the amount of endogenous immunoreactivity excreted in urine to be correlated with urine flow rate but not with urinary sodium excretion. Urinary excretion of DLIF, normalized to creatinine, was 3.6-fold greater at a urine flow rate of 5.5 mL/min than at 0.5 mL/min. On the other hand, saline intake increased urine flow rate 1.9-fold and increased sodium excretion threefold, but did not affect urinary excretion of DLIF. Fractional excretion of DLIF was linearly related to fractional excretion of water. This study demonstrates that normalization of DLIF values to urinary creatinine does not make DLIF excretion independent of urine flow rate and underscores the need for information on urine flow rate when DLIF measurements in urine are being interpreted.
Blood Proteins↗