Measurement of cyclosporine in plasma of cardiac allograft recipients by fluorescence polarization immunoassay

The Abbott TDx fluorescence polarization immunoassay (FPIA) procedure for measuring cyclosporine A (CsA) was evaluated and compared with the Sandoz polyclonal radioimmunoassay (CsA RIA kit) method. This drug assay was evaluated for precision, calibration, stability, and accuracy. Within-run precision studies utilizing 25 replicate analyses of the three control preparations (containing CsA in the 60-800 ng/ml range) resulted in coefficients of variation (CV) ranging from 1.0 to 9.1%. The CVs of between-run precision determined by assaying the same control drug levels for five consecutive working days ranged from 3.9 to 4.6%. Calibration curve stability was assessed by examining the drift in control values over a 2-week period. Maximum plasma ranged from 82.6 to 108.2%. Four hundred plasma samples were obtained from 30 heart-transplant patients during the first 6 months of CsA therapy and each sample was analyzed simultaneously by TDx and RIA. Linear regression analysis of the results obtained for each patient (x = RIA, y = FPIA) revealed the following mean values: r = 0.87, (CV = 13.7%), slope = 1.47 (CV = 39.2%). Moreover, the concentration of CsA was determined in 35 patient samples both by TDx and high-performance liquid chromatography (HPLC). FPIA results up to 12 times higher than HPLC results have been noted.     

Comparison of a monoclonal antibody fluorescent polarization immunoassay with monoclonal antibody radioimmunoassay for cyclosporin determination in whole blood.

We have compared the whole blood concentrations of parent cyclosporin A (CsA) using monoclonal fluorescence polarization immunoassay (FPIA) and radioimmunoassay (RIA) as well as polyclonal FPIA in kidney, heart, and bone marrow transplant patients (n = 89). A good correlation was found between monoclonal FPIA and monoclonal RIA (r = 0.96) and a slightly worse one between polyclonal and monoclonal FPIA (r = 0.90). The interassay coefficient of variation was satisfactory for all the methods-less than 5% for monoclonal FPIA. The monoclonal FPIA assay with Abbott TDx appears to provide rapid, precise, and accurate measurement of parent CsA. It is therefore useful for therapeutic monitoring of CsA in whole blood in kidney, heart, and bone marrow transplant patients.     

Comparative analyses of cyclosporine in whole blood and plasma by radioimmunoassay, fluorescence polarization immunoassay, and high-performance liquid chromatography

Cyclosporine (CsA) concentrations were prospectively monitored for 6 months after transplantation in 66 consecutive renal transplant recipients. Analysis for CsA was performed with polyclonal radioimmunoassay (RIA), high-performance liquid chromatography (HPLC), and fluorescence polarization immunoassay (FPIA) in whole blood and plasma, and with specific and nonspecific monoclonal RIA in whole blood. Precision within and between runs was best with FPIA, followed by HPLC and RIA. A strong correlation was observed between the results obtained with HPLC and specific monoclonal RIA (r = 0.98). Correlation coefficients between the other assays ranged from 0.56 (plasma HPLC versus blood polyclonal RIA) to 0.91 (blood nonspecific monoclonal RIA versus blood FPIA). The highest values for CsA concentrations in blood were found with nonspecific monoclonal RIA, followed by FPIA and polyclonal RIA (mean ratio versus specific monoclonal RIA was 3.3, 2.9, and 2.4, respectively). Specific monoclonal RIA had 7% higher values than HPLC. There was a more than threefold interindividual variation in the mean ratio between specifically and nonspecifically measured CsA concentrations in whole blood and plasma. The ratio between CsA concentrations, determined with specific versus nonspecific methods in whole blood, decreased significantly during the first month after transplantation (from 0.42 to 0.35, as assayed with specific monoclonal RIA versus polyclonal RIA in whole blood; p less than 0.05). The concentrations in blood were, on the average, three times higher than those in plasma, although there was a more than fourfold interindividual variation in the mean values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the AxSYM monoclonal cyclosporin assay and comparison with radioimmunoassay

Recently, a semiautomated fluorescence polarization immunoassay (FPIA) for determination of parent cyclosporin (CsA) has been developed for the Abbott AxSYM system. The new CsA assay measures the drug from an extracted whole blood specimen. The authors report here the evaluation of this new assay and the comparison with a previously validated radioimmunoassay (RIA) method (CYCLO-Trac SP). To assess the imprecision, the authors used tri-level controls supplied by both Abbott and Bio-Rad manufacturers. The within-run CV ranged from 4.4% to 7.3% and the between-day CV ranged from 4.4% to 7.6%. Mean recovery of the drug from clinical specimens spiked with kit calibrators was 108.4%. Fluorescence polarization immunoassay AxSYM (y) was correlated to RIA (x) by using 132 trough blood specimens (44 renal, 44 liver, and 44 heart) from transplant recipients and resulted in the following Passing-Bablok linear regression equation: y = 6.7 + 0.97x, r = 0.989, S(x/y) = 12.9. The percentage of overestimation (mean, range) by FPIA AxSYM versus RIA was (3.8%, range -17.7% to 39.1%). The results observed with this new method from follow-up studies in patients during the early course after transplant were not consistently higher than those obtained by RIA. These findings contrast with previously reported results that compared FPIA TDx assay with RIA. The authors conclude that FPIA AxSYM is a precise method for measuring CsA and offers results similar to those obtained by RIA with a marked reduction in assay time.     

Comparison of nonspecific radioimmunoassay, high-performance liquid chromatography, and fluorescence polarization immunoassay for cyclosporine monitoring in renal transplantation

Three methods, i.e., nonspecific radioimmunoassay (RIA; Incstar), fluorescence polarization immunoassay (FPIA; TDx Abbott), and high-performance liquid chromatography (HPLC), have been used for monitoring cyclosporine blood levels in renal transplantation patients. The levels obtained from 135 samples showed a modest correlation between RIA and HPLC, FPIA and HPLC, RIA and FRIA. The mean ratios of RIA to HPLC, FPIA to HPLC, and RIA to FPIA were 2.96, 4.14, and 0.73. The significant variations in cyclosporine levels result from the cross-reaction of antibody with some cyclosporine metabolites, by which these two methods often overestimate the true blood cyclosporine level. HPLC is a more effective and reliable method for pharmacokinetic studies and blood level monitoring of cyclosporine in clinical practice.     

Cyclosporine blood concentrations determined by different assay methods in heart transplant patients

Cyclosporine (CsA) through blood concentrations were determined by polyclonal nonspecific radioimmunoassay (RIA) (Sandoz), monoclonal specific and nonspecific RIA (Sandimmun, Sandoz), monoclonal specific and polyclonal nonspecific RIA, (Cyclo-Trac, INCSTAR) and fluorescence polarization immunoassay (TDx, Abbott), at multiple points in time, in two patients receiving CsA for immunosuppression after heart transplantation. Results obtained by the different nonspecific methods have a correlation (r) from 0.76 to 0.94. Concentrations determined by Sandimmun nonspecific RIA, Cyclo-Trac nonspecific RIA and TDx were consistently higher than those by Sandoz nonspecific RIA, owing to different cross-reactivity with CsA metabolites, giving ratios of 1:8, 1:4, and 1:6, respectively. In the first patient, the ratios between nonspecific and specific results were higher during days 1-20 than later on, which coincides with the high serum bilirubin level observed (r = 0.72, n = 31). This observation was not made in the second patient, whose liver function was within normal limits. Because CsA and its metabolites are eliminated primarily in the bile, the high values obtained with the nonspecific assay indicate that CsA metabolites accumulate in blood when liver function is impaired.     

An evaluation of the cyclosporine and metabolites whole blood TDx assay

The new Abbott TDx cyclosporine and metabolites fluorescent polarization immunoassay procedure provides a 20-min sample turn-around time, using 50 microliters of sample for the analysis of cyclosporine in whole blood. A precipitation agent and a lysing agent are utilized as a pretreatment step. The range of the whole blood assay is from 0 to 2,000 ng/ml, with a sensitivity of 50 ng/ml. Precision studies at 3 control levels provided coefficients of variation of 2.1-4.8% for both assays. In order to compare this assay with the currently used Sandoz polyclonal radioimmunoassay (RIA) method. 200 whole blood samples were obtained from 20 renal, cardiac, and hepatic transplant recipients. The mean whole blood cyclosporine concentrations for samples above the sensitivity level were as follows: TDx 754 ng/ml (+/- 31) and RIA 619 ng/ml (+/- 22). Blood TDx levels correlated strongly with RIA levels, with a regression coefficient of r = 0.915. This new assay provides reliable blood cyclosporine concentrations that correlate well with RIA measurements. This assay offers rapid sample turn-around times, making same-day results for outpatient drug monitoring possible.     

Predictive performance study of two digoxin assays in subjects with various degrees of renal function

This prospective study was conducted to compare the predictive performance of fluorescence polarization immunoassay (FPIA, Abbott TDx Digoxin II) and radioimmunoassay (RIA, Kallestad Labs) with combined low-pressure liquid chromatography/RIA (LPLC/RIA) digoxin assay in measuring 15-17 serum digoxin concentrations (SDC) obtained after a single 10 microg/kg intravenous digoxin dose in patients with various degrees of renal function and at different SDC ranges. Eighteen men and women were stratified into 3 age- and gender-matched groups based upon renal function [N = 6 in each, group I (Cl(cr) < 10 mL/min), group II (Cl(cr) = 10-50 mL/min), and group III (Cl(cr) > 50 mL/min)]. Serum digoxin concentrations were measured at time zero; at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, and 12 hours; and at 2, 3, 4, and 5-7 days after the digoxin dose, using the three different digoxin assays. TDx Digoxin II was unbiased [mean error -0.09 (95% CI -0.19, 0.01)] and RIA biased [mean error -0.29 (95% CI -0.36, -0.21)] to over-predict SDC by 14.2%. In group I patients, the analysis revealed a bias to over-predict SDC by 6.0% for TDx Digoxin II [mean error -0.16 (95% CI -0.29, -0.07)] and an unbiased performance by RIA. In groups II and III, both TDx Digoxin II and RIA showed biased performance, the mean magnitude of bias was low (< 20%). For intermediate SDC range (> 0.5 ng/mL and < or = 3.0 ng/mL), TDx Digoxin II was unbiased in predicting SDC, whereas RIA was biased to under-predict SDC [mean error 0.13 (95% CI 0.10, 0.16)] by 9.9%. The magnitude of bias observed in all cases was less than 20%. Both assays, TDx Digoxin II and RIA, imprecisely measured SDC for all samples combined, different groups and SDC ranges. In all time-paired samples, TDx Digoxin II (FPIA) performed better than the RIA. In conclusion, the magnitude of bias observed with either assay at different groups and SDC ranges was not likely to be clinically relevant. Therefore, either assay may be used to measure SDC in clinical practice.     

Comparison of fluorescence polarization immunoassay and high performance liquid chromatography for the quantitative determination of phenytoin, phenobarbitone and carbamazepine in serum

The Abbott TDx fluorescence polarization immunoassay (FPIA) system has been evaluated and compared with well-established high performance liquid chromatography (HPLC) for the determination of three anticonvulsant drugs: phenytoin, phenobarbitone and carbamazepine. These assays were evaluated for precision, calibration curve stability, specificity and accuracy. Within-run precision studies using control samples (n = 15) in the subtherapeutic, therapeutic, and toxic concentrations, resulted in coefficients of variation in the range of 1.79-3.99% (FPIA) and 1.16-2.52% (HPLC), respectively. Between-run precision ranged from 2.32-6.34% for FPIA and from 2.04-3.38% for HPLC. Comparison of 122 patient samples assayed with both methods indicated an extremely good analytical correlation (r = 0.96) for all three comparisons. The FPIA method offers significant advantages in calibration curve stability while maintaining accuracy and precision comparable with those of established HPLC procedures.     

Digoxin-like immunoreactive substance: monoclonal and polyclonal RIA and FPIA compared

Digoxin-like immunoreactive substance (DLIS) was measured in 34 samples obtained from subjects not receiving digoxin: 10 uremic, 10 third trimester pregnancy, seven cord blood and seven normal. DLIS concentration was measured by four commercial polyclonal radioimmunoassay (RIA) systems: Clinical Assay (CA), Corning Immophase (CI), Diagnostic Products Double Antibody (DP), Kallestad Quantitope (KK), a monoclonal antibody (MA) assay and a Fluorescence Polarization Immunoassay (FPIA). In general, the cord blood samples were richer in DLIS. Digoxin immunoassays, MA and DP showed minimal interference by DLIS in all samples, whereas FPIA and CA exhibited the maximal cross-reactivity with DLIS. In cord blood samples, mean +/- SD DLIS concentration ranged from 0.41 +/- 0.13 (by CA) to 0.034 +/- 0.02 ng ml-1 as measured by MA assay. In uremics, the mean DLIS concentration was below the detection limit of all RIA assays. The FPIA method showed a higher degree of cross-reactivity to DLIS, especially in the cord and pregnancy samples (0.42 +/- 0.13 and 0.4 +/- 0.14 ng ml-1, respectively). DLIS in uremics was below the FPIA detection limit of 0.2 ng ml-1. Overall, the degree of interference by DLIS in decreasing order was FPIA greater than CA greater than CI greater than or equal to KQ greater than DP greater than or equal to MA. The cord blood samples were re-analysed by FPIA (Digoxin-II assay) 4 months later, resulting in 2-4-fold higher DLIS concentrations for these samples. This appears to be due to the substitution of 5-sulphosalicylic acid as a protein precipitating reagent and this effect may have been accentuated by freeze-thaw cycles.     

Automated fluorescence polarization immunoassay for monitoring vancomycin

We have extended fluorescence polarization immunoassay (FPIA) technology for the measurement of drugs to include the complex amphoteric glycopeptide antibiotic vancomycin (molecular weight, 1,449). Fluorescein-labeled vancomycin was employed as a tracer, and antisera specific for vancomycin were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum were combined, and the polarization of tracer fluorescence is determined in a specially designed fluorometer (Abbott TDx). Because of instrument design, the possibility of fluorescent interferences is minimized. The assay can measure as little as 0.6 mg/L of vancomycin and is free of interferences from hemolysis, lipemia, bilirubin, and changes in protein concentration. The coefficient of variation within assay was 3% (n = 5) and between assays was 5% (n = 5). The FPIA assay (TDx Vancomycin) was compared to a liquid chromatographic (LC) assay for vancomycin and to a commercially available radioimmunoassay (RIA) for 98 clinical specimens. A linear least-squares regression analysis gave a correlation coefficient for LC of 0.980 from the equation FPIA = 1.09 LC + 3.04, and a correlation coefficient for RIA of 0.957 from the equation FPIA = 1.036 RIA + 1.66.     

Comparison of whole-blood cyclosporine levels measured by radioimmunoassay and fluorescence polarization in patients post orthotopic liver transplant

This study compared the analysis of whole blood cyclosporine concentrations measured by fluorescence polarization immunoassay (FPIA) and radioimmunoassay (RIA) polyclonal and monoclonal procedures. Fifteen orthotopic liver transplant patients with a mean age of 39 +/- 11.06 years were included in the study. One hundred thirteen levels were analyzed using FPIA, RIA polyclonal, and RIA monoclonal procedures. There was no difference statistically in comparing FPIA and RIA polyclonal results (p greater than 0.05). There was a statistical difference between FPIA and RIA monoclonal results p = 0.0001). With use of least squares simple linear regression analysis, FPIA results showed good correlation with RIA polyclonal results (R2 = 0.87). Poor correlation was shown between FPIA and RIA monoclonal results (R2 = 0.51). In this study population, FPIA produced results 2.5% higher than the RIA polyclonal procedure.     

Changing characteristics of the TDx digoxin II assay in detecting bufadienolides in a traditional Chinese medicine: for better or worse?

In 1992, apparent digoxin concentrations determined by the Abbott TDx II assay 5 hours after the ingestion of 10 pills of traditional Chinese medicine containing toad secretions (chan su) by 7 volunteers, yielded results that were equimolar to bufalin measured by 2 in-house bufalin radioimmunoassays (RIAs). Recently, a 17-year-old Chinese female unintentionally took 100 (instead of 10) of these pills for a sore throat but suffered no ill effects. The blood bufalin concentration at 3 hours by 1 of the 2 RIAs was 10.93 nmol/L, which was commensurate with the dose. However, the apparent digoxin measured by a TDx II assay produced in 2004 was only 3.08 nmol/L, which probably reflects the change in the specificity of the polyclonal digoxin antisera used in the assay over the years. In 1989, the TDx assay was commended for its ability to detect poisoning from plant and animal cardenolides, a property that seems to be waning and, thus, bad news for those wishing to use the assay to detect alternative cardenolides. But, on the other hand, it possibly eliminates the "specter" of digoxin-like immunoreactive substance (DLIS) that has afflicted some digoxin assays, which can only be good news.     

不同免疫分析仪在环孢素A血药浓度监测中的差异

目的:考察不同分析方法对环孢素A(CsA)血药浓度监测中的影响.方法:71例肝肾移植术后CsA样品分别用AxSYM和TDx测定.结果:TDx监测结果大于AxSYM监测结果.结论:AxSYM和TDx在环孢素A血药浓度监测中存在差异.     

Specific enzyme-multiplied immunoassay and fluorescence polarization immunoassay for cyclosporin compared with Cyclotrac [125I]radioimmunoassay.

The analysis of cyclosporin-A (CsA) has proved a valuable adjunct to clinical care of patients who have received organ grafts. The measurement of CsA in whole blood by specific methods has recently taken a new direction with the introduction of a range of rapid methods, including a homogeneous enzyme immunoassay technique (EMIT) and a monoclonal fluorescence polarization immunoassay (FPIA). The present paper compares these two methods with the established Cyclotrac specific [125I]RIA (radioimmunoassay) using both commercial CsA-spiked control material as well as a group of 60 patient specimens (predominantly renal transplants). While each of the new methods showed acceptable precision and accuracy with the commercial quality control material, significant differences were demonstrated with patient specimens, such that FPIA was 12.5% greater than [125I]RIA (p less than 0.0001), which was in turn 5.9% greater than EMIT (p = 0.007). These data suggested that the FPIA may have residual CsA-metabolite interference and that the EMIT method was the most "specific" for parent CsA of the three tested, potentially therefore more comparable to high-performance liquid chromatography (HPLC).     

Measuring endogenous digoxin-like substance and exogenous digoxin in the serum of low-birth-weight infants

The interference with three serum digoxin assay methods of endogenous digoxin-like substance (EDLS) in the serum of low-birth-weight (LBW) infants was assessed. The serum from 5-mL blood samples obtained from each of 19 LBW infants was divided into four 0.5-mL portions. Each portion was spiked with 10 microL of a distilled water-ethanol solution with or without digoxin to produce final digoxin concentrations of 0 (control), 0.49, 0.98, or 1.96 ng/mL. Each portion in each patient was then analyzed by radioimmunoassay (RIA), fluorescence polarization immunoassay (FPIA), and radial partition immunoassay (RPIA) using the control portions to measure EDLS. Serum digoxin concentrations measured by each assay method were calculated by subtracting the EDLS concentrations in the control portions from the measured digoxin concentrations in the spiked samples. The mean +/- S.D. concentrations of EDLS measured by RIA and FPIA were 0.26 +/- 0.13 ng/mL and 0.33 +/- 0.16 ng/mL, respectively. Of the 19 control samples assayed by RPIA, 18 had EDLS concentrations less than 0.1 ng/mL; one sample reflected an apparent concentration of 0.11 ng/mL. Mean recovered digoxin concentrations by RIA at each spiked digoxin concentration were significantly different from those obtained by FPIA and RPIA. A low but significant correlation was noted between EDLS concentrations in serum samples assayed by RIA and FPIA. The RPIA method appears to be preferred over the RIA and FPIA methods used in this study for serum digoxin analysis in LBW infants because of acceptable accuracy and minimal interference by EDLS.     

Evaluation of the immunotech cyclosporine direct radioimmunoassay for the determination of whole-blood cyclosporine in renal transplant patients

Therapeutic drug monitoring (TDM) of cyclosporine (CsA) has been an accepted as an essential tool in the management of solid organ transplant recipients. The authors evaluated a new CsA method, Immunotech cyclosporine direct radioimmunoassay (Beckman Coulter, Prague, Czech Republic), for the measurement of whole-blood CsA concentrations. The performance was compared with CEDIA Plus method as well as group mean data for HPLC and other immunoassays available from the International CsA Proficiency Testing Program (www.bioanalytics.co.uk). Regression analysis of patient samples gave a relationship of RIA = 1.0822 CEDIA(+) + 69.84 (r(2) = 0.933). External CsA-spiked proficiency-testing (PT) samples gave a regression equation of RIA = 0.9672 CEDIA(+) + 4.99 (r(2) = 0.996). The correlation with the CEDIA Plus method using patient specimens (hence, including CsA metabolites) suggested that the test RIA method possibly had slightly inferior specificity for parent CsA. The results suggest that the Immunotech cyclosporine direct RIA kit is suitable for the measurement of whole-blood CsA concentrations and maintained clinically acceptable analytic precision and accuracy, displaying CVs of less than 15% and biases of less than 10%. The PT program CsA-metabolite-free samples showed that calibration between methods was comparable with the possible exception of mFPIA/TDx.     

Potential problem with fluorescence polarization immunoassay cross-reactivity to vancomycin degradation product CDP-1: its detection in sera of renally impaired patients

During the development of a homogeneous immunoassay for the antibiotic vancomycin, we observed in certain patient samples a quantitation difference between the enzyme multiplied immunoassay technique (EMIT) method and the comparison method, fluorescence polarization immunoassay (FPIA). This prompted us to evaluate the integrity of vancomycin in samples from renally impaired patients. Since it has been reported in the scientific literature that vancomycin degrades into an antibiotically inactive crystalline degradation product (CDP-1) in vitro, we developed high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) methods to determine whether CDP-1 is present in patient sera. HPLC and LC/MS analysis on samples from renally impaired patients positively identified CDP-1 in fresh samples. Next, we tested the cross-reactivity of three currently available vancomycin immunoassays, radioimmunoassay (RIA) FPIA, and EMIT, to CDP-1 prepared in our laboratory. Our data suggest that CDP-1 is recognized by FPIA and RIA, both polyclonal antibody-based methods, but not by EMIT, which uses a monoclonal antibody.     

A modified fluorescence polarization immunoassay method incorporating fat emulsion (FE-FPIA) to determine cyclosporin A concentrations in rat skin.

We have developed a simple, sensitive and reliable assay procedure for cyclosporin A (CyA), a modified fluorescence polarization immunoassay method incorporating fat emulsion (FE-FPIA), to determine the CyA content in rat skin. The conventional fluorescence polarization immunoassay (FPIA) method for CyA using a commercially available FPIA kit, TDX cyclosporine monoclonal whole blood, was modified. A fat emulsion for intravenous infusion, Intralipos, was incorporated for dissolving the CyA extracted from the skin tissue, and a mixture of MeOH/purified water was used as the sample pretreatment medium instead of the precipitation reagent in the conventional FPIA kit intended for whole blood samples. These modifications enabled us to produce a reliable and the sensitive assay of CyA in skin tissue. The reproducibility (coefficient of variation), detection limit, and assay time for FE-FPIA were below 2%, 25 ng/ml, and about 24 min/24 samples, respectively, and were comparable with those for the whole blood samples determined by the conventional FPIA. Pre-purification of samples required by the HPLC assay is not needed in the FE-FPIA method. The usefulness of the FE-FPIA method in evaluating the topical pharmacokinetics of CyA in skin is discussed.     

False-positive serum digoxin concentrations determined by three digoxin assays in patients with liver disease

The incidence and magnitude of false-positive serum digoxin concentrations (SDCs) determined by three digoxin assays in patients with liver disease were studied. Patients with biochemical evidence of liver disease were enrolled in the study if they had never received a cardiac glycoside, were not pregnant, were not receiving spironolactone, did not have moderate to severe renal impairment, and did not have transient elevations in liver function test results. Blood specimens from each patient were assayed for apparent SDCs in triplicate using a fluorescence polarization immunoassay (FPIA, TDx Digoxin II, Abbott) and a digoxin radioimmunoassay (RIA, GammaCoat I125, Clinical Assays) and in duplicate using a fluorometric enzyme immunoassay (Dade Stratus, American Dade). Forty-two patients met the study criteria. The percentage of patients exhibiting detectable apparent SDCs (greater than or equal to 0.2 ng/mL) was 57% with RIA, 55% with FPIA, and 28% with the fluorometric enzyme immunoassay. Apparent SDCs ranged from 0.2 to 0.6 ng/mL (RIA), 0.2 to 1.56 ng/mL (FPIA), and 0.2 to 0.38 ng/mL (fluorometric enzyme immunoassay). Values obtained using the fluorometric enzyme immunoassay were significantly different from the apparent SDCs determined using RIA and FPIA; however, no significant difference was found between the values obtained using RIA and FPIA. Significant correlations were found between the apparent SDCs determined using RIA and serum bilirubin values and between the apparent SDCs determined using the fluorometric enzyme immunoassay and alkaline phosphatase values. Of the three assay methods tested, the fluorometric enzyme immunoassay showed the least cross-sensitivity to digoxin-like immunoreactive substance (DLIS).(ABSTRACT TRUNCATED AT 250 WORDS)