Cloned Enzyme Donor Immunoassay Research Articles

An ImmunoChip prototype for simultaneous detection of antiepileptic drugs using an enhanced one-step homogeneous immunoassay

The development and characterization of a one-step homogeneous immunoassay-based multiwell ImmunoChip is reported for the simultaneous detection and quantitation of antiepileptic drugs (AEDs). The assay platform uses a cloned enzyme donor immunoassay (CEDIA) and a Beta-Glo assay system for generation of bioluminescent signal. Results of the one-step CEDIA for three AEDs (carbamazepine, phenytoin, and valproic acid), in the presence of serum, correlate well with the values determined by fluorescence polarization immunoassay. CEDIA intra- and interassay coefficients of variation are less than 10%. A microfabrication process, xurography, was used to produce the multiwell ImmunoChip. Assay reagents were dispensed and lyophilized in a three-layer pattern. The multiwell ImmunoChip prototype was used to detect and quantify AEDs in serum samples containing all three drugs. Luminescent signals generated from each well were recorded with a charge-coupled device (CCD) camera. The assays performed on an ImmunoChip were fast (5 min), requiring only small volumes of both the reagents (<1 μl/well) and the serum sample. The ImmunoChip assay platform described in this article may be well suited for therapeutic monitoring of drugs and metabolites at the point-of-care setting.

Comparison of Generic Cyclosporine Microemulsion versus Neoral in de Novo Renal Transplant Recipients Managed By 2-Hour Postdose Monitoring

Introduction The bioequivalence of generic formulations is established by measuring pharmacokinetic parameters in healthy volunteers. Cyclosporine (CsA) absorption and exposure is known to differ between healthy volunteers and transplant recipients. Therefore bioequivalence testing may be inadequate to ensure therapeutic equivalence. We sought to compare the efficacy of generic cyclosporine (ArpimuneME, RPG Life Sciences) versus Sandimmune Neoral in de novo renal transplant recipients. Methods A prospective single-center, open-label study enrolled 20 de novo renal transplant patients (group 1: mean age 30.55 ± 9.81 years, M:F 19:1, mean donor age 43.4 ± 10.8). All patients received ArpimuneME along with azathioprine and prednisolone. The results were compared with 17 matched controls (group 2: mean age 28.1 ± 9.5 years, M:F 13:4, mean donor age 47.8 ± 6.8) who received Neoral and were transplanted during the same period. C 2 levels were monitored by the cloned enzyme donor immunoassay (CEDIA). Results Patient and graft survivals were 100% and 100% and 100% and 92.8% in groups 1 and 2, respectively ( P = NS). Six patients (30%) experienced rejection in group 1 as compared eight patients (47.1%) in group 2. Mean CsA levels (ng/mL) during the first month were 1419.1 ± 213.6 and 1460.5 ± 290.7 and at 3 months, 1296.3 ± 227.8 and 1342.4 ± 303.4 in the two groups, respectively ( P = NS). The mean serum creatinine levels (mg%) in group 1 and group 2 were 1.6 ± 0.8 and 2.0 ± 1.4 at discharge and 1.5 ± 0.4 and 1.5 ± 0.8 at 6 months, respectively ( P = NS). Conclusion Use of a generic microemulsion form of CsA provided safe and effective immunosuppression compared with Sandimmune Neoral when drug monitoring was performed by C 2 levels.

Paradoxical Results in Urine Drug Testing for 6-Acetylmorphine and Total Opiates: Implications for Best Analytical Strategy

A major task in urine drug testing is to detect heroin intake. The most common way of doing this is by using morphine as the analytical target in opiate immunoassay screening. However, this strategy sometimes leads to false-positive results because morphine is not a metabolite unique to heroin. The objective of this study was to evaluate the usefulness of the unique heroin metabolite 6-acetylmorphine (6-AM) as the primary analytical target in combination with morphine in the screening assay. A total number of 3521 randomly collected urine samples from 707 patients undergoing heroin substitution treatment were investigated for 6-AM and opiates by CEDIA (cloned enzyme donor immunoassay) and for opiates by DRI immunoassays and by gas chromatography-mass spectrometry (free 6-AM, free morphine, total morphine, and total codeine). The rate of positive outcome in the screening for 6-AM was 9.1% (cutoff 10 microg/L), and for opiates, it was 22.6% (cutoff 300 microg/L), which is in accordance with a known shorter detection time for 6-AM following heroin intake. However, by comparing 6-AM and opiate screening results at different cutoff levels, it was observed that 7-8% of the samples and 12.5% of the patients with detectable 6-AM had an unexpected low content of free and total morphine in the urine. This study confirms earlier observations that certain individuals may escape detection in urine drug testing when morphine is being utilized for the detection of heroin intake. The underlying mechanism for this may be a metabolic defect and/or interaction. It is concluded that 6-AM is a valuable target analyte in the screening of drugs of abuse in urine and may be used in combination with opiate screening in clinical testing.

Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study

Two commercially available drug-screening assays were evaluated: the Roche kinetic interaction of microparticles in solution (KIMS) assay and the Microgenics cloned enzyme donor immunoassay (CEDIA). Urine samples from known drug-abuse patients were analyzed for amphetamines, barbiturates, benzodiazepines, benzoylecgonine, cannabinoids, LSD, methadone and opiates. Samples with discordant findings for the two assays were analyzed by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/electron capture detection (GC/ECD). Amphetamines showed 96.0% concordant results, with two false positive findings by CEDIA, three by KIMS and a further two false negatives by KIMS. Barbiturates showed 99.4% concordant results, with one false negative by KIMS. Benzodiazepines showed 97.4% concordant results, with two false negatives by KIMS (cutoff 100 microg/L, CEDIA cutoff 300 microg/L). Benzoylecgonine showed 17.8% concordant positive and 82.2% concordant negative results and no false finding by either assay. Cannabinoids showed 99.3% concordant results, with one sample negative by KIMS at a cutoff of 50 microg/L and positive by CEDIA (cutoff 25 microg/L). For LSD, 6.7% of findings were not in agreement. Methadone showed 97.5% concordant results, with two false positives by CEDIA, and one false positive and one false negative by KIMS. Opiates showed 96.9% concordant results, with no false KIMS results, but four false positives by CEDIA. The results indicate that the agreement of the CEDIA and KIMS results for the eight drugs is rather good (93.3-100%).

Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors

Application of immunoassay to biosensors for use in the point-of-care setting ideally requires immunoassay without separation steps and with small volumes of both sample and reagents. The suitability of cloned enzyme donor immunoassay (CEDIA), one of a few homogeneous immunoassays available, was investigated for application to biosensors. This method is based on the bacterial enzyme β-galactosidase, which has been genetically engineered by others into two inactive fragments, enzyme donor (ED) and enzyme acceptor (EA). Association of the ED and EA fragments in the assay results in formation of active enzyme, which acts on substrate to generate a detectable signal. Sensitivity of commercially available CEDIA kits were compared, with respect to the sample and reagent volumes, using three different signal generation processes. The CEDIA kit for valproic acid and three substrates, a colorimetric (chlorophenol red-β- d-galactopyranoside), a chemiluminescent (Lumi-Gal 530), and a bioluminescent (Beta-Glo Assay System), were employed in the study. Our results indicate that the high sensitivity of the bioluminogenic substrate, d-luciferin- O-β-galactopyranoside, with short assay time and small volumes of sample and reagents required for the assay, simple handling, and relatively low expense, make this substrate, together with CEDIA, suitable for application to biosensors intended for drug and metabolite monitoring in the point-of-care setting.

Modeling of homogeneous cloned enzyme donor immunoassay

One of the most widely used analytical techniques for sensitive detection of biologically and clinically significant analytes is the immunoassay. In recent years direct immunoprobes allowing label-free detection of the interaction between the antibody and the target analyte have proved their capabilities as fast, simple, and nevertheless highly sensitive methods. Cloned enzyme donor immunoassay (CEDIA) homogeneous assay is based on the bacterial enzyme β-galactosidase, which has been genetically engineered into two inactive fragments, enzyme donor and enzyme acceptor. Reassociation of the fragments in the assay forms active enzyme, which acts on substrate to generate a colored product. A comprehensive kinetic model of CEDIA is developed to aid in understanding this method and to facilitate development of a truly homogeneous version, potentially applicable to a dipstick-type multianalyte point of care analytical device (ChemChip). Although the standard assay involves a two-step process, we also chose to model a single-combined process, which would be simpler to apply in a ChemChip device. From the modeling simulation, we obtain the time courses of the amounts of product and active enzyme, from which the dynamic ranges can be obtained as 10 −6–10 −7 and 10 −5–10 −7 M analyte concentration for two-step and single-combined processes under the conditions of the assumed parameters, respectively. A simple one-step immunoassay has the merit of reducing time and cost and has an improved dynamic range.

C2 monitoring in maintenance renal transplant recipients: is it worthwhile?

Presently, there is little knowledge regarding cyclosporine (CsA) concentration at 2 hr post-dose (C2) monitoring in maintenance patients. This study evaluates the actual C2 range in stable renal transplant recipients (who underwent transplantation >12 months ago). In addition, we investigated whether underexposure or overexposure to CsA (assessed by C2) affects graft function (as measured by serum [S]-creatinine). All renal transplant recipients in Norway receiving CsA were asked to participate; 1447 fulfilled the criteria. Valid C2 and CsA trough concentration (C0) measurements were performed in 1032 renal transplant recipients (71%) monitored by C0. Target C0 level was 75 to 125 mumol/L. CsA levels were measured using a Cloned Enzyme Donor Immunoassay method, and all analyses were performed in the same laboratory (overall mean [+/-standard deviation] CsA C0=112+/-31 mug/L, CsA C2=697+/-211 mug/L [range 81-1580 mug/L], CsA dose [mg/day]=208+/-61, CsA dose [mg/kg/day]=2.8+/-1.1, and S-creatinine=141+/-58 mumol/L). A univariate analysis of variance showed that patients with C2 levels between 700 and 800 mug/L (n=203, S-creatinine=136+/-49 mumol/L) had significantly lower S-creatinine levels compared with patients with C2 levels greater than 950 mug/L (n=94, S-creatinine=152+/-56 mumol/L) (P<0.02). The same was true for patients with C2 levels less than 450 mug/L (n=95, S-creatinine 141+/-72 mumol/L) (P<0.05) when compared with patients with C2 levels greater than 950 mug/L. There was no significant difference in S-creatinine between patients in the low and intermediate C2 group; 666 patients had C0 levels in the therapeutic range (75-125 mumol/L). A linear regression showed a significant relation between S-creatinine and C2 for these patients (P=0.03). The corresponding relation between S-creatinine and C0 was nonsignificant (P=0.3). Monitoring of C2 in maintenance patients is a valuable tool to detect overexposure to CsA. Until results from prospective studies are available, we recommend C0 in the therapeutic range and reduction in CsA in overexposed patients, aiming at a C2 value between 700 and 800 mug/L.

Multiplex assay of amphetamine, methamphetamine, and ecstasy drug using CEDIA technology.

Using Microgenics unique technology, cloned enzyme donor immunoassay (CEDIA), the multiplex assay (CEDIA Amphetamines/Ecstasy) has been developed for the detection of amphetamine, methamphetamine, and ecstasy drugs at cutoff level either of 500 ng/mL or 1000 ng/mL applicable for either qualitative screening or semiquantitative measurement. The multiplex assay detects the total concentration of amphetamine, methamphetamine, and ecstasy drugs in urine samples. In addition, the assay detects metabolites of parent drugs and structurally related drugs including d,l-amphetamine (67.2%), d,l-methamphetamine (58.4%), MDA (113%), MDMA (199%), MDEA (207%), MBDB (123%), BDB (72%), PMA (24%), and PMMA (100%). The assay is highly specific and exhibits minimal cross-reactivity with the undesirable, structurally related over-the-counter amphetaminelike drugs. Imprecision results (n = 120) demonstrate an intra-assay < 6.3% CV and an interassay < 9.2% CV as performed by a modified NCCLS protocol. Using the SAMHSA mandatory guidelines of a 500-ng/mL cutoff for the immunoassay and a 250-ng/mL cutoff for GC-MS, the assay detects 164 out of 165 GC-MS-positive samples. For drug abuse testing, the multiplex assay presents an excellent method for the detection of multiple drugs with greater sensitivity and broader detection.

Rapid detection of benzoylecgonine in vitreous humor by enzyme immunoassay.

The usual specimens submitted by a medical examiner for toxicological analysis include blood, urine, bile, vitreous humor, stomach contents, and solid-organ tissue. The detection of drugs in these specimens typically involves a combination of techniques including colorimetry, immunoassay, and gas chromatography. Although many laboratories rely principally on urine for the detection of drugs of abuse by immunoassay, these assays may be applied to other specimen types. An evaluation of Microgenics Corporation's cloned enzyme donor immunoassay (CEDIA) was conducted in order to evaluate its use in the detection of cocaine/cocaine metabolites in vitreous humor specimens. During a 14-month period, 392 vitreous humor specimens were analyzed by the CEDIA DAU Cocaine assay. Instrument parameters were set according to published manufacturer's guidelines. All presumptive positive immunoassay results prompted confirmatory testing and quantitation by gas chromatography-mass spectrometry (GC-MS) of other specimens including blood. Vitreous humor specimens were not tested by GC-MS. Using a approximately 100-ng/mL cutoff, the CEDIA assay produced 23 presumptive positive results, 22 of which were confirmed by GC-MS. The only specimen which could not be confirmed, elicited an immunoassay screen value near the cutoff limit. Routine analysis of blood, urine, bile, and/or bladder wash specimens by gas chromatography-nitrogen phosphorus detection revealed the presence of cocaine/cocaine metabolites in only 7 (31.8%) of the 22 confirmed cases. The concentration ranges of cocaine and benzoylecgonine in the blood specimens were none detected to 337 ng/mL and 17 to 8598 ng/mL, respectively. Cocaethylene was not detected in these cases. Analysis of vitreous humor specimens by CEDIA improved the detection rate of cocaine/cocaine metabolites by 0.7% in the cases submitted to our laboratory during the 14-month period.

Improved cross-reactivity to alpha OH triazolam in the BMC CEDIA DAU urine benzodiazepine assay.

Immunoassays designed to detect use of older benzodiazepines such as oxazepam or diazepam often cannot detect triazolam use because of the low doses of triazolam administered, rapid biotransformation to metabolites with poor cross-reactivities, and the small amount of alpha OH triazolam glucuronide excreted in the urine. Previous studies have demonstrated that certain immunoassays have high cross-reactivity to alpha OH triazolam but are unable to detect therapeutic triazolam use in urine. The objectives of this study were to characterize the immunoreactivity toward alpha OH triazolam in the reformulated cloned enzyme donor immunoassay (CEDIA) drug abuse urine benzodiazepine assay and to measure the immunoreactivity of urine specimens from subjects who were administered single oral doses of triazolam. Alpha OH triazolam standards were prepared in drug-free urine and the new CEDIA assay gave a positive result at concentrations from 100 to 200 ng/ml, which indicates an eight-fold improvement in CEDIA cross-reactivity to alpha OH triazolam standards in the reformulated CEDIA assay. With a 200 ng/ml cut-off, 4/30 of the urine specimens screened positive for benzodiazepines without enzymatic hydrolysis and 6/30 after enzymatic hydrolysis. When using an in-house 100 ng/ml nitrazepam cut-off calibrator, 10/30 urine specimens were positive in the reformulated CEDIA assay without hydrolysis and 22/30 were positive with enzymatic hydrolysis before screening.

Evaluation of nonisotopic binding assays for measuring vitamin B 12 and folate in serum

We report the evaluation of the microparticle enzyme immunoassay (MEIA) for estimating vitamin B 12 and the ion capture (IC) assay for estimating folate in serum. The essays were performed with the Abbott IMx analyzer. Intra-assay coefficients of variation were from 3.4% to 8.0% for vitamin B 12 and from 1.5% to 4.2% for folate, respectively. Interassay coefficients of variation ranged from 6.3% to 8.8% for vitamin B 12 and from 3.9% to 11.3% for folate, respectively. Linearity was satisfactory, with an analytical recovery of 119 ± 4% for vitamin B 12 and 95 ± 8% for folate. The detection limit was 56.2 pmol/l for vitamin B 12 and 1.9 nmol/l for folate. Results of these assays correlated well with those from the cloned enzyme donor immunoassay (CEDIA), our current routine method: r = 0.97 for vitamin B 12 ( n = 47) and r = 0.97 for folate ( n = 50). The MEIA and the IC assay were easy to perform with the IMx analyzer.

Cloned enzyme donor immunoassay (CEDIA) for drugs-of-abuse screening

Large numbers of specimens (5000-18,000) were screened for amphetamines, barbiturates, cocaine, marijuana, opiates, and phencyclidine by RIA (Roche), Emit II (Syva), and a new immunoassay, CEDIA (cloned enzyme donor immunoassay, Microgenics). All immunoassays performed equivalently for cocaine, opiates, and phencyclidine. All immunoassays detected the same amphetamine/methamphetamine-positive specimens, but all also detected numerous specimens containing cross-reacting sympathomimetic amines. CEDIA detected 100%, Emit II 93%, and RIA 82% of the barbiturate-positive specimens. Emit II and CEDIA detected 86-88% of the specimens found by RIA to be marijuana positive. A subset of specimens was additionally screened by OnLine (Roche) and TDx (Abbott) for amphetamines, cocaine, and marijuana. OnLine and TDx also detected all of the amphetamine-positive specimens and numerous specimens containing cross-reacting sympathomimetic amines. All immunoassays performed equivalently for cocaine, and the four nonisotopic tests detected 86-89% of the marijuana positives found by RIA. Interfering sympathomimetic amine drug compounds can be eliminated by using an oxidizing agent, thus decreasing the number of unconfirmable amphetamine presumptive positives. The CEDIAs for all of the major drugs of abuse are reliable and effective for large-volume urine screening programs.

Nonisotopic Binding Assay for Measuring Vitamin B12 and Folate in Serum

We evaluated a nonisotopic method (CEDIA; cloned enzyme donor immunoassay) for estimating vitamin B12 and folate in serum. The assays were performed with a Cobas-Mira analyzer. Intra-assay CVs were from 3.7% to 11.0% for vitamin B12 and from 1.2% to 10.7% for folate. Interassay CVs ranged from 9.5% to 11.9% for vitamin B12 and from 6.1% to 18.5% for folate. Linearity was satisfactory, with analytical recovery of 94% at 8.7 and 25.4 nmol/L for folate and 280 and 554 pmol/L for vitamin B12. The detection limit was 3.6 nmol/L for folate and 12.3 pmol/L for vitamin B12. Results of this assay correlated well with those of a conventional radioassay: r = 0.98 for vitamin B12 (n = 51) and r = 0.97 for folate (n = 57). The CEDIA was easy to perform but appeared to be unreliable for use with samples from myeloma patients.

新しいＥｎｚｙｍｅ Ｉｍｍｕｎｏａｓｓａｙ Ｓｙｓｔｅｍによる血清中ジゴキシン測度測定の評価

A concentration of digoxin and apparent one of methyldigoxin in serum were measured by using the cloned enzyme donor immunoassay (CEDIA) method and the digoxin-NXT (NXT) method, and compared these new enzyme immunoassay systems with the fluorescence polarization immunoassay (FPIA) method.There was a significant correlation between FPIA and NXT methods in the serum concentration of digoxin, and a significant correlation between FPIA and CEDIA methods in the serum concentration of digoxin or apparent one of methyldigoxin was also found, respectively.However, in the serum concentration of digoxin, the mean value of the NXT method was significantly higher than that of the FPIA method.In contrast, the level determined by the CEDIA method was significantly smaller than that by the FPIA method.At the subtherapeutic range measured with the FPIA method (<0.8ng/ml), the value by NXT method shows 1.7-fold higher than that by the CEDIA method.Cross-reactivities among these three methods were compared by using the serum samples of 15 patients, not administered digitalis.It was found that the determinations of NXT and CEDIA methods were interfered with substances other than digitalis, such as the FPIA method.These results indicate that the specificity of reaction differs from one another in these three methods.

血清中ジゴキシン濃度測定におけるＣＥＤＩＡ法の評価

Serum levels of digoxin and methyldigoxin were measured with the cloned enzyme donor immunoassay (CEDIA) method and the fluorescence polarization immunoassay (FPIA) method.In other samples, both of those cardiac glycosides were measured with FPIA method and radioimmunoassay (RIA) method.There was a significant correlation (r=0.835, n=85) between CEDIA and FPIA methods in serum levels of digoxin and an apparent that of methyldigoxin. But in some patients, especially in infants, the values of the CEDIA method were lower than those of FPIA method.In contrast, there was a higher significant correlation (r=0.939, n=63) between FPIA and RIA methods in serum levels of both cardiac glycosides.These results may indicate that sensitivities of these methods to the interfering substances (ex. endogenous digoxin-like substance) differ each other.