Fluorescence Polarization Immunoassay Assay Research Articles

Fluorescence polarization immunoassay for rapid determination of dehydroepiandrosterone in human urine.

In this paper, five fluorescein-labeled dehydroepiandrosterone (DHEA) derivatives (tracers) with different chain lengths between the fluorescein and hapten were synthesized and featured so as to establish a fluorescence polarization immunoassay (FPIA) for DHEA detection in human urine samples with previously prepared polyclonal antibody against DHEA. The outcomes of the structure of tracer on FPIA sensitivity were investigated. Under the optimal condition, the fluorescence polarization value (FP) decreases linearly in DHEA concentration, ranging from 1.6 to 243.3ngmL-1, with the limit of detection of 1.1ngmL-1 and IC50 value of 25.1ngmL-1. Moreover, the developed FPIA was time-saving as it could complete the detection within 3min. FPIA and commercial enzyme-linked immunosorbent assay kit were both applied to analyze the spiked human urine samples with DHEA. Excellent recoveries (92.1-108.0%) and satisfactory correlation coefficient (R2 = 0.98) were acquired with the two methods, indicating that the developed FPIA was a fast and efficient screening immunoassay with accuracy and sensitivity for DHEA detection in human urine samples. Graphical abstract.

Rapid and homologous immunoassay for the detection of herbicide propisochlor in water

ABSTRACT The herbicide propisochlor can be introduced into the water cycle via agricultural activities, thus possibly leading to harmful ecological effects. It is significant for the development of a rapid method for propisochlor analysis in water. In this work, a rapid homogeneous immunoassay based on fluorescence polarization immunoassay (FPIA) was firstly developed for the determination of propisochlor in pond water. The developed FPIA assay can be completed within only 14 min; the 50% inhibition concentration (IC50) was 125.1 ng/mL and the limit of detection was 8.4 ng/mL. The recoveries of propisochlor in pond water ranged from 80.0% to 86.6%. A high performance liquid chromatography confirmation showed a good correlation with the developed FPIA (R 2 = 0.9748). The developed FPIA exhibited satisfactory rapidity, high sensitivity and excellent specificity, meeting the standards of the screening assay of propisochlor in pond water on site. The molecular modeling showed that the alkoxyalkyl of chloroacetamide herbicides exerted important effects on the herbicide–antibody binding.

Unexpected overestimation of methotrexate plasma concentrations: analysis of a single center pediatric population.

At this center, therapeutic drug monitoring of methotrexate (MTX) used to be performed by fluorescence polarization immunoassay (FPIA). We observed an increasing number of unusual high MTX concentrations at 48 and 72 hours during a couple of years. This study aimed to identify the causes of this variation. A retrospective analysis was conducted on 272 patients hospitalized between January 2008 and October 2012. The whole MTX use system was analyzed using Ishikawa's method. The proportion of MTX concentrations ≤0.2 μmole/L at 48 (P48h) and 72 hours (P72h) was recorded and compared between both FPIA and EMITSiemens assays. A χ or a Fisher exact test was used (α = 0.05). Because of an announced withdrawal of the FPIA reagent, the method was switched in 2009 to an immunoenzymatic technique (EMITSiemens). Both P48h and P72h dropped significantly after 2009 (P48h: 45% versus 5% and P72h: 91% versus 47%; P < 0.0001). The replacement of the EMITSiemens reagent by the EMITARK Diagnostics reagent in 2012 led to an increase in both P48h and P72h. No significant difference was found in the proportions of MTX ≤0.2 μmole/L concentrations between FPIA and EMITARK Diagnostics at 48 (45% and 40%; P = 0.556) and 72 hours (91% and 100%; P = 0.231). Both internal and external quality control assessments gave regular satisfactory results during the study period. Furthermore, the interassay comparisons that were performed with internal quality controls and spiked serum samples showed similar results at the time of both shifts. The other changes observed in the MTX circuit were not associated with MTX concentration variations. The overestimation of the plasma concentration of MTX was concluded to be because of the assay reagent. A further study is consequently necessary to assess the impact of this analytical pitfall on the patients' survival.

Evaluation of the QMS® Teicoplanin Immunoassay (ThermoFisher Scientific) on Cobas® 8000 System (Roche Diagnostics) and Comparison to Fluorescence Polarization Immunoassay for the Determination of Teicoplanin Concentrations in Human Plasma

The performances of the QMS(®) Teicoplanin immunoassay recently developed on Cobas(®) 6000/8000 systems were evaluated and compared to a fluorescence polarization immunoassay (FPIA) [Teicoplanin Innofluor(®) Assay (Thermo Fisher Scientific, Indianapolis, IN)] on FLX analyzer (Abbott Laboratories, Abbott Park, IL)]. The validation was performed according to the Cofrac (French Accreditation Committee) document SH GTA 04. For the comparison, 48 plasma samples were analyzed by FPIA and QMS assays. The QMS assay is accurate (intra assay and inter assay inaccuracy ≤ 2.4%) and precise (intra assay and inter assay imprecision ≤ 10.2%). A linear relationship [QMS = 1.0319 × FPIA - 2.8518, r(2) = 0.9246 (P < 0.001)] between FPIA and QMS was found. In the Bland-Altman plots, no systematic bias was found even if QMS results trends to be lower (mean of the ratio QMS concentration/FPIA concentration = 0.91). These results between QMS and FPIA are consistent, which indicates that QMS(®) Teicoplanin immunoassay on Cobas(®) 8000 System is an alternative to FPIA.

Rapid detection of oleander poisoning by Dimension Vista digoxin assay (Flex Reagent Cartridge)

Oleander poisoning can be detected by digoxin immunoassays and for last two decades the fluorescence polarization immunoassay (FPIA) has been used for rapid detection of oleander poisoning in clinical laboratories. Recently, Abbott Laboratories (Abbott Park, IL) discontinued this assay. Therefore, we explored the possibility of using another digoxin assay (Dimension Vista Flex Reagent Cartridge, Tina Quant, EMIT 2000 and old FPIA assay for comparison) for rapid detection of oleander poisoning. When aliquots of drug-free serum pools were supplemented with pure oleandrin or oleander extract, we observed the highest apparent digoxin values using Dimension Vista digoxin assay (Flex Reagent Cartridge). We also observed significant apparent digoxin values in vivo in sera of mice both 1 and 2 hr after feeding with oleander extract. When a serum pool prepared from patients taking digoxin was further supplemented with various amounts of oleander extract, the highest falsely elevated digoxin values were observed with Dimension Vista digoxin assay. Monitoring free digoxin using Dimension Vista digoxin assay (Flex Reagent Cartridge) did not eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract and such effect can be monitored by observing significant reduction in apparent free digoxin levels in the presence of Digibind as measured in the protein-free ultrafiltrate using Dimension Vista digoxin assay (Flex Reagent Cartridge).

Effect of Chinese Medicines Chan Su and Lu-Shen-Wan on Serum Digoxin Measurement by Digoxin III, a New Digoxin Immunoassay

Chan Su and Lu-Shen-Wan are Chinese medicines that crossreact with digoxin immunoassays. Recently, Abbott Laboratories released a new digoxin immunoassay, Digoxin III. We studied potential interference of Chan Su and Lu-Shen-Wan with the Digoxin III assay by comparing results obtained by using Digoxin II and fluorescence polarization immunoassay, also manufactured by Abbott Laboratories. Aliquots of a drug-free serum pool were supplemented with aqueous extract of Chan Su or Lu-Shen-Wan and apparent digoxin concentrations were measured using all three digoxin assays. Significant crossreactivity of Chan Su and Lu-Shen-Wan was observed with the new Digoxin III assay. Moreover, when mice were fed with Chan Su or Lu-Shen-Wan, significant apparent digoxin concentrations were also observed in the sera of mice using the Digoxin III assay indicating that such interferences are also present in vivo. When serum pools prepared from patients receiving digoxin were further supplemented with Chan Su or Lu-Shen-Wan extract, falsely elevated digoxin values were observed with both Digoxin III and fluorescence polarization immunoassay, but digoxin values were falsely lowered using the Digoxin II assay. For example, when one aliquot of Digoxin Serum Pool 1 containing 0.94 ng/mL of digoxin was supplemented with 5.0 microg/mL of Chan Su extract, the digoxin concentration was falsely elevated to 6.60 ng/mL as measured by the Digoxin III assay and 6.99 as measured by the fluorescence polarization immunoassay assay. In contrast, the observed digoxin value was falsely lowered to 0.72 ng/mL using the Digoxin II assay. Interference of Chan Su and Lu-Shen-Wan in the Digoxin III assay cannot be eliminated by monitoring free digoxin concentrations. Digibind neutralizes digoxin-like immunoreactive components of Chan Su and such effect can be monitored by measuring apparent free digoxin concentrations using the Digoxin III assay. We conclude the both Chan Su and Lu-Shen-Wan significantly interfere with serum digoxin measurements by the new Digoxin III assay.

False-positive Serum Tricyclic Antidepressant Concentrations Using Fluorescence Polarization Immunoassay Due to the Presence of Hydroxyzine and Cetirizine

A recent report indicates that hydroxyzine and its active metabolite cetirizine interfere with the PENTINA carbamazepine assay. The potential interference of hydroxyzine and cetirizine with the fluorescence polarization immunoassay (FPIA) and CEDIA assay of carbamazepine as well as with the fluorescence polarization immunoassay of tricyclic antidepressants (TCA) was studied. Aliquots of drug-free serum pools were supplemented with various concentrations of hydroxyzine and cetirizine representing therapeutic, mild to moderate toxic as well as very toxic concentrations. Then apparent carbamazepine and TCA concentrations were measured by immunoassays. Although no interference of hydroxyzine and cetirizine was observed with carbamazepine assays (FPIA and CEDIA), significant apparent TCA concentrations were observed when aliquots of drug-free serum were supplemented with hydroxyzine or cetirizine. Mathematical formula was devised to predict hydroxyzine and/or cetirizine concentration in serum based on observed apparent TCA levels. Hydroxyzine and cetirizine also falsely increased total TCA values when aliquots of serum pool prepared from patients receiving TCA were further supplemented with these drugs. In conclusion, hydroxyzine and cetirizine do not interfere with the FPIA and CEDIA carbamazepine assays but interfere with the measurement of total TCA using the FPIA.

New Enzyme-Linked Chemiluminescent Immunosorbent Digoxin Assay Is Free From Interference of Chinese Medicine DanShen

DanShen is a traditional Chinese medicine indicated for cardiovascular diseases. The potential interference of DanShen with serum digoxin measurement was investigated using a new enzyme-linked chemiluminescent immunosorbent (ECLIA) digoxin assay. Aliquots of drug-free serum were supplemented with ethyl acetate extract of DanShen (4 different brands studied), and apparent digoxin concentrations were measured by the ECLIA as well as fluorescence polarization immunoassay (FPIA) and a turbidimetric assay for comparison. Mice were also fed 4 DanShen preparations and apparent digoxin concentrations were subsequently measured. In another experiment, serum pools containing digoxin were further supplemented with DanShen extracts and digoxin concentrations were measured again by all 3 assays. No apparent digoxin concentration was observed when aliquots of drug-free serum pools were supplemented with DanShen and digoxin concentrations were measured by the ECLIA or the turbidimetric assay. In contrast, significant apparent digoxin concentrations were observed using FPIA, and the highest apparent digoxin concentration was observed with brand 4 of DanShen extract. Similarly, when mice were fed with this herb, significant apparent digoxin concentrations were also observed using FPIA, but neither ECLIA nor turbidimetric assay showed any apparent digoxin concentration. When aliquots of digoxin pool were further supplemented with various DanShen extract, the apparent digoxin concentrations were significantly increased when FPIA was used. In contrast, digoxin concentrations in the presence of DanShen extract compared well with the digoxin concentration of the original pool when ECLIA or turbidimetric assay was used. We conclude that DanShen does not interfere with serum digoxin measurement using a more recently released ECLIA digoxin assay.

Analytic Performance Evaluation of a New Turbidimetric Immunoassay for Phenytoin on the ADVIA 1650® Analyzer

Phenytoin is an anticonvulsant that requires therapeutic drug monitoring. Recently, Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of phenytoin on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained in 52 patients receiving Phenytoin using this new assay with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA). The new turbidimetric immunoassay for phenytoin showed the following imprecision with the low, medium, and high controls: total CV of 5.2% (mean 4.81 microg/mL), 3.7% (mean 16.24 microg/mL), and 4.1% (mean 22.65 microg/mL), respectively. The detection limit of the assay was 0.79 microg/mL, and the assay was linear up to a phenytoin concentration of 46.1 microg/mL. The assay showed excellent dilution recovery and recovery of spiked samples (mean recovery 101.4% and 94.4%, respectively). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y=1.06 x-0.61, r=0.98, n=52). We also determined the cross-reactivity of 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, and of oxaprozine, an analogue with a similar chemical structure to phenytoin, in both phenytoin assays. Both assays showed almost no cross-reactivity to oxaprozine and only small (5%-8%) cross-reactivity to HPPH. We also found that the turbidimetric assay was free from interference at least up to 1200 mg/dL of hemolysis, 30 mg/dL of free bilirubin, 34.5 mg/dL of conjugated bilirubin, and 750 mg/dL of triglyceride (Intralipid). When a drug-free serum was followed by a serum sample containing 38.5 microg/mL of phenytoin, no sample probe carryover effect was observed. We conclude that the new turbidimetric assay can be used for routine monitoring of phenytoin in clinical laboratories.

Analytic Performance Evaluation of a New Turbidimetric Immunoassay for Carbamazepine on the ADVIA 1650 Analyzer

Carbamazepine, an anticonvulsant, requires therapeutic drug monitoring. Recently Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of carbamazepine on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained with this new assay in sera of 54 patients receiving carbamazepine with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA) and a chemiluminescent immunoassay (CLIA). The new turbidimetric immunoassay for carbamazepine showed excellent precision. The low control showed a total CV of 4.9% (mean 2.86, SD 0.14 microg/mL), the medium control demonstrated a total CV of 3.5% (mean 7.79, SD 0.27 microg/mL), and the high control showed a total CV of 4.8% (mean 16.15, SD 0.78 microg/mL). The assay was linear up to a carbamazepine concentration of 20 microg/mL. The assay showed excellent dilution recovery and recovery of samples supplemented with carbamazepine (mean recovery 102.2%). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y = 0.96 x - 0.46, r = 0.99, n = 54). We also observed excellent correlation between the values obtained by the CLIA (x-axis) and the turbidimetric (y-axis) assay (y = 1.10 x -0.32, r = 0.99, n = 54). However, the slope of 1.10 was higher than the slope of 0.96 observed with the regression equation obtained by using values obtained by the FPIA and the turbidimetric assay. The positive bias obtained with the new turbidimetric assay compared with the CLIA assay resulted from lower cross reactivity of carbamazepine 10,11-epoxide, the active metabolite of carbamazepine, with CLIA. On the other hand, the cross reactivity of the metabolite is similar between the new turbidimetric assay and the FPIA assay. We conclude that the new turbidimetric assay can be used for routine monitoring of carbamazepine in clinical laboratories.

Rapid Detection of Oleander Poisoning Using Digoxin Immunoassays

Oleander is an ornamental shrub that grows in the United States, Australia, India, Sri Lanka, China, and other parts of the world. All parts of the plant are poisonous because the presence of cardiac glycoside oleandrin. Despite its toxicity, oleander extract is used in folk medicines. Because of its structural similarity, oleandrin cross-reacts with the fluorescence polarization immunoassay (FPIA) for digoxin. We studied the potential of detecting oleandrin in serum using 5 common digoxin immunoassays (FPIA, MEIA, both from Abbott; Beckman digoxin assay on Synchron LX, Chemiluminescent assay, CLIA from Bayer Diagnostics) and a recently FDA-approved turbidimetric assay on the ADVIA 1650 analyzer (Bayer). Aliquots of drug-free and digoxin-like immunoreactive substances (DLIS)-free serum pools were supplemented with ethanol extract of oleander leaves or oleandrin (Sigma Chemicals) in amounts expected in vivo after severe overdose. We observed significant apparent digoxin concentration with FPIA, Beckman, and the new turbidimetric assay (1 mL drug-free serum supplemented with 5.0 microL of oleander extract: apparent digoxin 2.36 ng/mL by the FPIA, 0.32 ng/mL by the MEIA, 0.93 ng/mL by the Beckman, 0.82 ng/mL by the new turbidimetric assay). The CLIA showed no cross-reactivity. Similar observations were made when serum pools were supplemented with oleandrin. Because cross reactivity should be tested in the presence of the primary analyte, we supplemented serum pools prepared from patients receiving digoxin with oleander extract or oleandrin. The measured digoxin concentrations were falsely elevated with the FPIA, Beckman, and turbidimetric assays, the highest false elevation being observed with the FPIA. Surprisingly, apparent digoxin concentrations were falsely lowered when MEIA was used. Digibind neutralizes free apparent digoxin concentration in vitro in serum pools supplemented with oleander extract, and this effect can be measured by the FPIA. We conclude that FPIA is most sensitive to detect the presence of oleander in serum. In contrast, the CLIA (no cross-reactivity) should be used for monitoring digoxin in a patient receiving digoxin and self-medicated with a herbal remedy containing oleander.

Comparison of fluorescence polarization immunoassay and high-performance liquid chromatography methods for assay of teicoplanin: can correlation be improved?

Teicoplanin is a glycopeptide indicated in serious infections due to Staphylococcus aureus and requires monitoring and dose adjustment based on measurement of trough concentration. Measurement of teicoplanin can be carried out by the fluorescence polarization immunoassay (FPIA) method or by high-performance liquid chromatography (HPLC) methods. We aimed to compare HPLC method using the sum of the peaks of A2-2, A2-3, A2-4 and A2-5 with FPIA method. Thirty-six serum samples obtained over a 6-month period from patients treated by teicoplanin were first analyzed by a HPLC method by determining both the A2-2 peak and the sum of areas of A2-2 to A2-5 and were then stored at –20 °C for FPIA assay. Correlation between FPIA and HPLC A2-2 methods was as: HPLC A2-2 = 0.694FPIA – 0.892 with r 2 = 0.93. A paired t-test revealed significant differences between the results ( P < 0.001). To improve the FPIA and HPLC correlation, we performed a linear regression between FPIA and the sum of A2-2, A2-3, A2-4 and A2-5 obtained in the HPLC method: HPLC A2-2 to A2-5 = 0.953FPIA – 0.915 with r 2 = 0.96. A paired t-test did not show any significant difference between the results of the two methods ( P = 0.94). These results show a better correlation between FPIA and HPLC when the sum of A2-2 to A2-5 was used for calculations. In conclusion, HPLC A2-2 to A2-5 must be preferred to HPLC A2-2 for the assay of teicoplanin, even if the HPLC A2-2 to A2-5 runtime is higher than in the HPLC A2-2 method.

Monitoring urinary excretion of cannabinoids by fluorescence-polarization immunoassay: a cannabinoid-to-creatinine ratio study.

Drug testing in substance abuse treatment programs is focused on urine analysis of parent drugs and major metabolites. Huestis reported that serial monitoring of the major urinary cannabinoid metabolite (delta9-THC-COOH)-to-creatinine ratios in paired urine specimens (collected at least 24 hours apart) could differentiate new marijuana or hashish use from residual cannabinoid metabolite excretion in urine after previous drug use. Subjects with a history of chronic marijuana use were screened for cannabinoids in urine over several months by an enzyme immunoassay (EMIT) with a cut-off value of 50 ng/mL. Presumptive positive specimens were confirmed by gas chromatography-mass spectrometry (GC-MS) for delta9-THC-COOH with a cut-off value of 15 ng/mL. The objective of this study was to determine whether a semiquantitative cannabinoids immunoassay (corrected for creatinine concentration) could differentiate new marijuana use from residual cannabinoid excretion in chronic users of marijuana or hashish compared with GC-MS. The criterion for new marijuana use was a cannabinoid-to-creatinine ratio > or =0.5 (dividing the immunoassay quantitative result to creatinine ratio of specimen 2 by the specimen 1 ratio, specimen 3 by the specimen 2 ratio, etc.). Urine specimens were analyzed by fluorescence-polarization immunoassay (FPIA) on an Abbott TDxFLx analyzer after analysis by GC-MS. In 90 urine specimens (group A) with delta9-THC-COOH values determined by GC-MS, the mean delta9-THC-COOH concentration was 44.4 ng/mL (range, 16-100), and the mean FPIA total cannabinoids value was 91.7 ng/mL (range, 21-204 ng/mL) with a correlation coefficient of 0.993 (group A). In 111 specimens (group B), the mean delta9-THC-COOH concentration was 361 ng/mL (range, 101-960 ng/mL). The mean FPIA value was 657 ng/mL (range, 211-1,270 ng/mL), and the correlation coefficient of the B series was 0.975. Percent cross-reactivity for delta9-THC-COOH standards prepared in drug-free urine by FPIA was 82% at 25 ng/mL, 45% at 50 ng/mL, and 50% at 100 ng/mL. Overall, there was 89% agreement (132 of 148 specimens) between FPIA and GC-MS. In 16 of 148 specimens, however, the FPIA and GC-MS paired urine data did not agree. The sensitivity of the FPIA assay was 95.3%, and the specificity was 44.4%. The authors conclude that FPIA cannabinoid analysis should be further evaluated as an alternative to GC-MS quantitation to help distinguish new marijuana use from residual marijuana metabolite excretion in clinical drug treatment programs.

Bidirectional (positive/negative) interference of spironolactone, canrenone, and potassium canrenoate on serum digoxin measurement: elimination of interference by measuring free digoxin or using a chemiluminescent assay for digoxin

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. Spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with the fluorescence polarization immunoassay (FPIA) for digoxin, and can falsely elevate serum digoxin concentrations. Serum digoxin concentrations were falsely lowered when the microparticle enzyme immunoassay (MEIA) was used. Aliquots of drug-free serum were supplemented with therapeutic and above-therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin activities were measured. We observed digoxin-like activities in the FPIA, but observed no activity with the MEIA or the chemiluminescent assay (CLIA). However, when serum digoxin pools prepared from patients receiving digoxin were supplemented with these compounds, we observed suppression of total digoxin levels with the MEIA. In contrast, no interference was observed in the presence of these compounds when CLIA was used for digoxin measurement. These compounds are strongly protein-bound, and no apparent digoxin activity was observed in the protein-free ultrafiltrate when drug-free sera were spiked with high levels of these compounds. Taking advantage of strong protein binding of these compounds and weak protein binding of digoxin (25%), interference of spironolactone, canrenone, and potassium canrenoate in FPIA and MEIA digoxin assays can be mostly eliminated by monitoring free digoxin concentration. Another approach to avoid this interference is to use the CLIA digoxin assay.

Stabilization of blood homocysteine in an epidemiological setting

The major problem in the determination of homocysteine (Hcy), which is thought to be a risk factor in colorectal cancer, is the rise in its concentration if blood is not centrifuged immediately after collection. We assess the interference of 3-deazaadenosine (which inhibits conversion of S-adenosylhomocysteine into Hcy within the erythrocyte), using the fluorescence polarization immunoassay (FPIA) assay, the stabilizing effect of 3-deazaadenosine and the impact of temperature on Hcy stabilization. To assess interference of 3-deazaadenosine, 12 blood samples were extracted; two aliquots were obtained from each and one of them was added 3-deazaadenosine (50 micromol/l). To assess the stabilizing value of 3-deazaadenosine, as well as the effect of temperature, two blood samples were extracted from 24 volunteers. One of the tubes was immediately placed on ice and centrifuged (reference concentration). To the second tube was immediately added 3-deazaadenosine (50 micromol/l), producing six aliquots, three of which were kept at room temperature (25 degrees C) for 1, 4 and 6 hours, the other three kept at 37 degrees C. The mean values (standard deviation) obtained for methodological interference were: 7.32 (3.58) micromol/l without stabilizer, and 7.11 (3.61) micromol/l with stabilizer. There were no statistically significant differences (P = 0.104) and intraclass correlation coefficient was 0.989, suggesting no methodological interference. We did not find any significant differences regarding our reference value in the samples kept at room temperature during the interval studied. A high Pearson correlation coefficient was obtained. Nevertheless, in those samples kept at 37 degrees C, a slight increase was observed in the 4-hour period (P = 0.009). The addition of 3-deazaadenosine may avoid problems in the critical pre-analytical phase in the Hcy measurement. There is no interference with the FPIA assay, nor any dilution effect, and new reference values are not necessary.

The Detection of Cocaine in Hair Specimens Using Micro-Plate Enzyme Immunoassay

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.

Effects of digoxinlike immunoreactive substances and digoxin FAB antibodies on the new digoxin microparticle enzyme immunoassay.

Digoxin-like immunoreactive substance (DLIS) is known to interfere with fluorescence polarization immunoassay (FPIA) (Digoxin II, Abbott Laboratories) and falsely elevates the total digoxin concentrations. Digoxin FAB antibody (Digibind) is also known to affect digoxin results by FPIA assay. The authors studied the effects of DLIS and Digibind on a new microparticle enzyme immunoassay (MEIA) for digoxin recently introduced by Abbott Laboratories, compared with the standard FPIA method and chemiluminescence assay (ACS-digoxin, Ciba-Corning). They studied 30 volume-expanded patients (term pregnancy, liver and renal disease) for the presence of DLIS. None of these patients received digoxin. They observed measurable DLIS concentrations in 12 of 30 patients by the FPIA assay and in only 1 patient by both MEIA and ACS assays. The concentration of DLIS in that patient was 0.31 ng/ml of digoxin equivalent by the MEIA assay, 0.36 ng/ml by the ACS assay, and 1.15 ng/ml by the FPIA assay. When they supplemented serum containing digoxin with low to high concentrations of digibind (0.5, 1.0, 2.0 and 4.0, 10, and 20 micrograms/ml), and measured digoxin concentrations by FPIA, MEIA, and ACS assays, they observed lower than expected values of total digoxin. However, when they supplemented serum containing no digoxin with high concentration of digibind (5.0, 10.0 and 20.0 micrograms/ml) and supplemented protein-free ultrafiltrates with digoxin, they observed expected digoxin concentrations in the ultrafiltrates by all three assays, indicating that the ultrafiltrates are essentially free of digibind.

Pharmacokinetics of two cyclosporine formulations using FPIA and HPLC assay in volunteers

The analytical methods for the analysis of cycloporine (CsA), a fluorescence polarization immunoassay (FPIA) and HPLC method, were compared in a pharmacokinetic study of two CsA soft capsule formulations (Sandimmun®; Sandoz, Implanta®; Hanmi). Sixteen healthy volunteers completed the study and each subject ingested single doses (4×100 mg) of the test and the reference formulations in a two-way crossover design with a one-week drug-free interval between doses. Following each administration, whole blood concentrations of CsA were monitored over a period of 24 hour by both FPIA and HPLC methods. Blood concentrations and pharmacokinetic parameters determined by either analytical method showed large intersubject variation, with the FPIA data showing relatively higher magnitude of intersubject variation than the HPLC data. The blood concentrations determined by FPIA were 1.1–1.3 times higher than those determined by HPLC. There were strong and significant correlations between the two methods (r>0.83: p 0.05. tmax was earlier and Cmax was slightly lower for the test formulation. AUC24h, Cmax, tmax and MRT determined separately from the data obtained by the two methods for the two formulations were examined by analyses of variance (ANOVA) for the bioequivalency evaluation. Results of ANOVA and confidence limits of test/reference ratios of AUC24h, Cmax, tmax and MRT, and statistical tests indicated the bioequivalence of the two formulations (i.e., test/reference ratio was within 100×20%) except for Cmax and tmax. The mean of Cmax showed only 7.9% and 11.6% differences but the detection limit were 26.6% and 21.4% as determined by FPIA and HPLC respectively which is slightly over the 20% criteria. The mean of tmax also showed 11.1% and 9.3% differences but the detection limit were 29.2% and 29.6% as determined by FPIA and HPLC respectively. This experiments suggest that the data yielded for the two formulations demonstrated that they were bioequivalent.

Evaluation of the Abbott TDx serum benzodiazepine immunoassay for the analysis of lorazepam, adinazolam, and N-desmethyladinazolam.

This study involved the evaluation of the Abbott TDx serum benzodiazepine assay, a fluorescence polarization immunoassay (FPIA), for the detection of lorazepam, adinazolam, and N-desmethyladinazolam in serum. Precision of the assay was determined by using three control serums containing 75, 300, and 700 ng/mL nordiazepam. Between-run precision studies (N = 22) gave mean values of 76, 306, and 690 ng/mL with coefficients of variation of 6.5, 3.3, and 5.7%, respectively. Percent cross-reactivity of serum lorazepam standards (35-500 ng/mL) ranged from 29 to 69%. The cross-reactivity of serum adinazolam ranged from 40 to 47% between 50 and 150 ng/mL and from 38 to 55% for N-desmethyladinazolam between 50 and 250 ng/mL. Serum specimens (48) collected from individuals known to be receiving lorazepam were analyzed. Twenty-two specimens were positive for benzodiazepines. Serum specimens were collected from 0.25 to 24 h after administering a 15-mg oral dose of adinazolam to six volunteers. The FPIA results were compared with combined high-performance liquid chromatographic (HPLC) results for adinazolam and N-desmethyladinazolam. The FPIA method did not detect benzodiazepines at 0.25 h after administration of adinazolam but did detect benzodiazepines from 0.5 to 24 h after administration. The correlation between HPLC (N-desmethyladinazolam) and FPIA results by regression analysis gave the following: y = 0.937x + 4.449, r = 0.98, n = 15. It was concluded that the Abbott FPIA assay for benzodiazepines can detect lorazepam when prescribed in therapeutic doses and when present at greater than 25 ng/mL and can semiquantitatively detect adinazolam or N-desmethyladinazolam or both when present at concentrations greater than 50 ng/mL.

Cross-reactivities of cyclosporin G (NVa2 cyclosporin) and metabolites in cyclosporin A immunoassays

Immunoassays of cyclosporin A (CsA) have been routinely used to measure CsG. We investigated the cross-reactivities of CsG and its metabolites, as well as the proportion CsG constitutes in relation to total drug measured, for six CsG metabolites (GM1, GM9, GM4N, GM1c, GM1c9, GM19) in the following CsA assays: Sandimmune selective RIA (SS), Sandimmune nonselective RIA (NS), Cyclotrac SP-RIA (CT), fluorescence polarization immunoassay (FPIA), and enzyme immunoassay (EMIT). The cross-reactivity of CsG in these assays was as follows: SS, FPIA, CT, approximately 100%; NS, approximately 40%; EMIT, < 2%. The cross-reactivities of CsG metabolites were investigated in all assays except EMIT and varied among metabolites and assays. The most significant variance was found with the NS assay, where most of the metabolites exhibited cross-reactivities of > 40%. In contrast, in the SS, FPIA, and CT assays, cross-reactivities of < 5% were observed for most of the metabolites. The ranking of cross-reactivities of CsG metabolites in the assays is SS = CT < FPIA < NS. The degree of cross-reactivity did not change significantly when the SS, CT, and FPIA assays were calibrated with CsG instead of CsA--whether parent CsG was present or not. The data suggest that the SS, CT, and FPIA methods would be suitable for the routine monitoring of CsG.