Establishment of norvancomycin fluorescence polarization immunoassay for therapeutic drug monitoring

To establish a rapid and simple fluorescence polarization immunoassay method for determination of norvancomycin serum concentration, we collected 300 serum samples from the patients receiving norvancomycin in the hospitals localized in Shanghai, China. The drug concentrations were measured by the established HPLC method and FPIA with vancomycin kit. A FPIA algorithm for the determination of norvancomycin concentration was established according to the correlation between the FPIA and HPLC results. The methods and algorithm were validated in another 70 clinical samples. HPLC determination showed a good linear correlation within the range of 0.5-100?mg?l(-1) of norvancomycin concentrations. The method was validated via extraction recovery, intra- and inter-day methodological recovery and stability of norvancomycin in serum. Correlation analysis between the measurements of HPLC and FPIA in 300 serum samples gave the linear regression equation: (concentration by HPLC)=0.760 × (concentration by FPIA)-0.577 (P<0.001, R(2)=0.982). An algorithm was derived from this correlation for measuring the serum norvancomycin concentrations with FPIA. When it was validated in additional 70 serum samples from patients, 'FPIA algorithm' showed good accuracy versus HPLC: 'FPIA algorithm'=0.93 (HPLC)+0.63, R(2)=0.962, and 94.3% of the results from FPIA algorithm fell within the range of -20%/+20% of HPLC. This algorithm developed in this study can be easily used for determination of norvancomycin using TDx analyzer with vancomycin kit indirectly. It may also be useful for norvancomycin therapeutic drug monitoring.     

Overestimation of vancomycin concentrations utilizing fluorescence polarization immunoassay in patients on peritoneal dialysis

During a study of vancomycin pharmacokinetics in patients undergoing continuous ambulatory peritoneal dialysis (CAPD), a discrepancy was noted when serum concentrations were determined by high performance liquid chromatography (HPLC) in comparison to a fluorescence polarization immunoassay (FPI) technique. Following three weekly intraperitoneal doses (30 mg/kg/2 L), peak serum concentrations (at the end of the 6-h dwell) by FPI were 42.1 +/- 9.1, 43.1 +/- 8.7, and 45.6 +/- 7.4 micrograms/ml. In comparison, the same samples when analyzed by HPLC yielded 36.3 +/- 9.4, 32.2 +/- 8.9, and 31.6 +/- 9.1 micrograms/ml, respectively. A subsequent in vitro study of vancomycin (40 micrograms/ml) in serum indicated a degradation half-life of 693 (FPI) compared with 210 (HPLC) h. These data suggest that vancomycin degradation products accumulate in CAPD patients and lead to an overestimation of vancomycin serum concentrations when measured by FPI.     

高效液相色谱法和荧光偏振免疫法测定万古霉素血清浓度的比较

目的:比较高效液相色谱(HPLC)法与荧光偏振免疫(FPIA)法分别测定血清万古霉素浓度的结果,探讨两者的相关性。方法:收集临床检测万古霉素药物浓度的血清43份,分别用HPLC法和FPIA法进行测定,运用配对t检验,Bland-Altman分析和线性回归分析比较2种方法的测定结果。结果:HPLC法和FPIA法测定的万古霉素血清浓度具有良好的相关性, 回归方程为:Y_FPIA=1.103X_HPLC+0.831 5(R²=0.957 2); Bland-Altman评价分析2种方法一致性较好;配对t检验显示2种方法测定结果之间有显著统计学差异(P<0.000 1)。结论:相较于FPIA法,HPLC法测定不受代谢和降解产物干扰,能准确检测血清万古霉素浓度,适合应用于治疗药物监测。     

Development of one-step fluorescence polarization immunoassay for progesterone

A one-step fluorescence polarization immunoassay (FPIA) was developed to measure progesterone level using an immunocomplex single reagent (SR), a preequilibrated mixture of antibody and tracer. Several fluorescence-labeled progesterone tracers were synthesized using the combination of two progesterone derivatives, 11alpha-hemisuccinyloxyprogesterone (P-11HS) and progesterone-3-(O-carboxymethyl)oxime (P-3CMO), and three fluorescence labels, fluoresceinamine isomer I and II, and ethylenediamine fluoresceinthiocarbamyl (EDF). Antiserum was prepared using a progesterone-bovine serum albumin (BSA) imunogen. The influence of the tracer label was significantly different in titer and sensitivity for antibody binding. The best pair of antibody and progesterone tracer was selected for the antigen-antibody reaction. They were the antibody produced from P-11HS-BSA immunogen and P-11HS-EDF tracer. One-step FPIA is a speedy, homogeneous type of immunoassay which needs neither incubation nor separation of free and bound analyte to measure fluorescence polarization. The detection limit of progesterone by SR-FPIA is approximately 2.7 ng/ml with 50 microl samples. The performance characteristics are acceptable for standard curve reproducibility (coefficient of variation (CV): 0.6-6.4%), precision (CV: 3-13%), and mean dilution recovery (95-102%). The total assay time for 10 samples is about 7 min. This immunocomplex SR has proven to be stable compared with the respective solutions of antibody and tracer.     

Comparison of bioassay, high-performance liquid chromatography, and fluorescence polarization immunoassay for quantitative determination of vancomycin in serum

This investigation was designed to compare three assay techniques, the traditional bioassay (agar diffusion), and two more recent techniques, high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA), for the determination of vancomycin concentrations in serum. One hundred clinical samples obtained from patients receiving vancomycin were assayed by each method. The results from each assay were compared using linear regression analysis. The resultant correlation coefficients were as follows: 0.9996 for the HPLC versus FPIA, 0.7773 for the FPIA versus bioassay, and 0.7779 for HPLC versus bioassay. The FPIA technique was the easiest and fastest of the three methods; FPIA and HPLC were the most accurate.     

Evaluation of fluorescence polarization immunoassay for quantitation of serum salicylates

A fluorescence polarization immunoassay (FPIA) for serum salicylates that has been developed for use with the Abbott TDx analyzer is evaluated with regard to precision, accuracy, and stability of the standard curve. The FPIA method is also compared with a well-established high performance liquid chromatography (HPLC) technique in a clinical laboratory environment. The FPIA demonstrates excellent precision, and the standard curve is sufficiently stable to perform reproducible measurements over a 29-day period without recalibration. Superior accuracy of the FPIA method is indicated for salicylate concentrations between 50 and 800 micrograms/ml by recovery studies and by favorable comparison with the reference method. The performance of the FPIA for salicylate concentrations between 0 and 50 micrograms/ml is somewhat less favorable and should be used with caution in this range. The present method is more appropriate than HPLC for the management of patients receiving chronic high doses of salicylates or in cases of acute salicylate overdose and is also more rapid.     

Performance of a fluorescence polarization immunoassay system evaluated by therapeutic monitoring of four drugs.

Fluorescence polarization immunoassays (FPIA) for amikacin, gentamicin, quinidine, and theophylline (supplied by Roche Diagnostic Systems, made using a Cobas Fara centrifugal analyzer) were evaluated and compared with widely used monitoring analysis methods. For each drug, the between-assay imprecision was ascertained by calibration on the day of assay and by a stored calibration curve made at the beginning of the study. The precision of the amikacin and theophylline assays was acceptable [total coefficient of variation (CV) less than 7.5%] at all concentrations tested for each calibration mode. Imprecision of quinidine and gentamicin assays was significant at low concentrations (1.9 mg/L): total CV = 9.0% for quinidine assessed with stored calibration curve and total CV greater than 8.5% for gentamicin measured with the two calibration modes. The calibration curves for all four assays had a good stability (greater than 30 days). Linear regression analysis demonstrated close agreement between the FPIA (y) and the following comparative techniques (x): Abbott TDx assay for amikacin and gentamicin (r = 0.988, r = 0.974, respectively); Stratus fluorometric enzyme immunoassay for quinidine (r = 0.979); and EMIT Syva assay for theophylline (r = 0.993). It is concluded that fluorescence polarization immunoassay is a rapid and reliable method for the therapeutic monitoring of the four drugs tested. Moreover, the use of reagents on an instrument that can be implemented for a wide range of chemistries has significant advantages and cost benefits over dedicated instruments.     

Evaluation of fluorescence polarization immunoassay for determination of cyclosporin in plasma

The fluorescence polarization immunoassay (FPIA) method for determination of cyclosporin in plasma was evaluated and compared with the high-performance liquid chromatography (HPLC) and the radioimmunoassay (RIA) methods. The coefficients of variation for the within-run and between-run precision were less than 5 and less than 8%, respectively, for samples ranging in concentration from 50 to 600 ng/ml. Recoveries were determined by adding cyclosporin at concentrations from 25 to 1,000 ng/ml to patient plasma; they were, on average, 98.5%. The calibration curve was stable throughout a 10-week study period. There was no clinically significant interference due to hemolysis, icterus, lipemia, or other commonly used drugs. There was considerable variation of the ratio of the FPIA result to the HPLC result, whereas there was a good correlation between the FPIA and the RIA results (r = 0.975, n = 25, y = 1.2x - 36.4), when evaluated using specimens from renal transplant patients receiving cyclosporin orally. It was concluded that the FPIA is an appropriate, rapid method for patient cyclosporin analysis in plasma and serves as a practical alternative to the RIA.     

Evaluation of a fluorescence polarization immunoassay procedure for quantitation of methotrexate

A fluorescence polarization immunoassay (FPIA) procedure for measuring methotrexate was evaluated. The dynamic range of the assay is from 0.05 to 810 microM, and the calibration curve can be stored for at least 2 weeks. The FPIA procedure is automated and rapid; one result can be obtained in 18 min and five results in 25 min. There was no interference from hemoglobin (800 mg/dl), triglycerides (500 mg/dl), bilirubin (20 mg/dl), and protein (12.1 g/dl). Cross-reactivity with 7-hydroxy methotrexate and 2,4-diamino-N-methylpteroic acid was 0.6 and 44%, respectively. The coefficient of variation for the within-run and between-run precision was less than 5.0%. For the comparison studies, the samples were divided into four groups. The methotrexate concentrations in group 1 were 0.05-2.1 microM; in group 2, 2.2-9.3 microM; in group 3, 10-80 microM; and in group 4, greater than 80 microM. Linear regression analysis of the results obtained with the FPIA procedure and the enzyme multiplied immunoassay gave a correlation coefficient of at least 0.95 for all groups.     

Comparison of gas—liquid chromatography and fluorescence polarization immunoassay for therapeutic drug monitoring of flecainide acetate

A gas chromatographic (GC) method for quantitation of flecainide acetate in human plasma is described and compared with a fluorescence polarization immunoassay (FPIA) for therapeutic drug monitoring. The GC method includes a solid-phase extraction procedure and electron capture detection (ECD) without the need of derivatization. Within-day and between-day coefficients of variation were <7% for GC and FPIA. Recovery was between 89–101% for the GC method. Plasma from 36 patients were analysed by both GC and FPIA and the results showed a good correlation (slope = 0.96; INTERCEPT = 0.009 μg ml⁻¹; r = 0.987).     

Comparison of particle-enhanced turbidimetric inhibition immunoassay and fluorescence polarization immunoassay using monoclonal antibodies for theophylline

The DuPont theophylline assay reagent kit, a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) method adapted for use on a centrifugal fast analyzer, was evaluated. It was compared with fluorescence polarization immunoassay (FPIA). Day-to-day precision was 4.7% at 6.8 micrograms/ml, and 3.3% at 26.6 micrograms/ml. The assay is linear to a concentration of 40 micrograms/ml. Good correlation was found between the two methods (PETINIA/FPIA: y = 1.04x + 0.15, r = 0.988, Syx = 1.19) in the evaluation of 176 patients receiving theophylline. This method offers a precise and accurate alternative to FPIA.     

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.     

Effect of various storage conditions on a fluorescence polarization immunoassay for tobramycin

The effects of various storage conditions on the results of a fluorescence polarization immunoassay for tobramycin were studied. Two venous blood samples (150 mL each) were drawn one hour and six hours after a single intramuscular dose of tobramycin. From each of these samples, which represented peak (6 micrograms/mL) and trough (1 microgram/mL) concentrations, aliquots of whole blood and of serum were prepared and stored in both glass and polypropylene containers. Serum samples were stored at -20 degrees C and assayed for tobramycin at intervals of 1-372 days. Samples of serum and whole blood were stored at 4 and 25 degrees C and assayed on days 1, 3, and 7. Mean tobramycin concentrations over time and between-run coefficients of variation were calculated for each set of samples. There was no substantial variation in tobramycin concentrations over time. Significant differences between tobramycin concentrations were noted only for peak serum samples in glass versus plastic containers at -20 degrees C and for trough serum samples stored in glass at -20 degrees C versus 25 degrees C. However, these differences were small and are unlikely to be clinically important. Under the conditions tested, the results of a fluorescence polarization immunoassay for tobramycin do not appear to be affected by storage time, storage temperature, container material, or storage medium (whole blood versus serum).     

荧光偏振免疫法测定血中依替米星的浓度

目的:探索应用荧光偏振免疫法测定依替米星血药浓度的实验方法,考察该方法测定依替米星的可行性.方法:使用庆大霉素荧光偏振免疫试剂盒,用TDx对依替米星进行测定.结果:本方法能够检测依替米星的血药浓度,回归曲线为Y=0.439X+0.1772,r=0.9981,在0.5～20μg·mL-1,浓度范围内线性关系良好.日内、日间标准偏差均小于10%,低、中、高不同浓度的加样回收率分别为(93.12～9,74)%,(102.41～2.73)%,(89.50～1.45)%.结论:本方法灵敏度高,操作简便,检测时间短,可以用于临床快速检测依替米星的血药浓度.     

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.     

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).     

荧光偏振免疫分析法测定丙戊酸钠缓释片及药物动力学研究

在6名健康志愿者中,用荧光偏振免疫分析法测定丙戊酸钠血药浓度,并探讨了丙戊酸钠缓释片的药物动力学。结果表明,它的滞后时间是0.22±0.11h,半衰期是21.66±4.25h,达峰时间是9.64±2.31h,峰浓度是31.82±5.51μg/ml,曲线下面积是1391.90±224.26μg·h/ml。     

Evaluation of instrumental, nonisotopic immunoassays (fluorescence polarization immunoassay and enzyme-multiplied immunoassay technique) for cyclosporine monitoring in whole blood after kidney and liver transplantation.

Two new instrumental methods, an enzyme-multiplied immunoassay technique (EMIT) and a fluorescence polarization immunoassay (FPIA), were evaluated for monitoring of cyclosporine (CyA) in whole blood samples of renal and liver transplant patients. They are considered as being specific to the parent drug and they were compared with a specific radioimmunoassay (RIA) and a nonspecific FPIA. The data reveal that the novel procedures provide slightly overestimated CyA levels compared with specific RIA (6-12% for EMIT, 20-25% for FPIA). For both assays, intrarun and interrun reproducibilities were found to be in the 2-8% range. The ease of performance and the possibility of performing approximately 40 assays/h make the two methodologies very attractive for both routine and emergency analyses. These approaches are viewed to be complementary to the only previously available instrumental method, the nonspecific FPIA, which provides three- to fourfold higher CyA levels than those obtained with specific methods. Specific and nonspecific monitoring of CyA levels allowed variations in proportions of metabolites to total CyA and metabolites to be distinguished. A higher percentage and variability of cross-reacting metabolites were found in whole blood samples after liver transplantation compared with those in blood of kidney transplant recipients.     

高效液相色谱法与荧光偏振免疫法测定全血环孢菌素A浓度比较

目的探讨两种方法测定环孢菌素A浓度结果的可靠性。方法分别用高效液相法和荧光偏振免疫法测定全血中环孢菌素A浓度,比较2种方法测定结果的差异。结果2种方法的测定结果有显著性差异,FPIA法所测结果高于HPLC法。结论对环孢菌素A进行治疗药物监测时应考虑测定方法的影响。