Enzyme-multiplied Immunoassay Research Articles

Results compared for tricyclic antidepressants as assayed by liquid chromatography and enzyme immunoassay.

The tricyclic antidepressants amitriptyline, nortriptyline, imipramine, and desipramine in serum of patients taking one of the drugs were quantified in two laboratories by high-performance liquid chromatography (HPLC) and enzyme-multiplied immunoassay (EMIT; Syva). Results for split samples were highly correlated, but EMIT gave higher results in most cases, and the slopes of the correlation lines for each analyte were greater than 1. Detection limits for the two procedures were such that 18% of the EMIT results for the drug(s) were considered negative, as compared with 4% of the HPLC results. Additional assay of desmethyl or hydroxy antidepressant metabolites by HPLC did not explain the higher EMIT results. The relatively high detection limit for EMIT greatly limits its use in therapeutic drug monitoring, where low concentrations of tricyclic antidepressants are as important as high ones for dose adjustment or determination of compliance. Other problems with EMIT measurement of tricyclic antidepressants are discussed.

More-sensitive enzyme-multiplied immunoassay technique for procainamide and N-acetylprocainamide in plasma, serum, and urine.

A commercially available (Syva Co.) enzyme-multiplied immunoassay technique (EMIT) for the quantitative determination of procainamide (PA) and N-acetylprocainamide (NAPA) was modified to allow automated quantitative analysis of approximately 100 samples per day, in a working range of 0.1 to 2.0 micrograms/mL. Such a test was needed to evaluate the pharmacokinetic characteristics of controlled-release dosage forms characterized by long half-lives at low plasma concentration. Analytical recovery of PA and NAPA from serum, plasma, and urine was satisfactory, but at extreme ratios for PA:NAPA the accuracy of determining the lower-concentration component became unsatisfactory. In fact, however, we found no such ratios in 5400 clinical samples assayed by this procedure.

Determination of methotrexate and its major metabolite, 7-hydroxymethotrexate, using capillary zone electrophoresis and laser-induced fluorescence detection

High-dosage methotrexate therapy requires careful monitoring of the drug in serum to ensure minimal toxic effects. A simple, rapid and sensitive method for the separation and quantitation of methotrexate and its major metabolite, 7-hydroxymethotrexate, using high-voltage capillary zone electrophoresis combined with laser-induced fluorescence detection is described. The detection limit for methotrexate is as low as 5·10 −10 M (signal-to-noise ratio=3), while that for 7-hydroxymethotrexate is 2·10 −9 M. The linearity of the system extends over nearly four orders of magnitude for both methotrexate and 7-hydroxymethotrexate. The extraction efficiency for the drug and its metabolite from serum is 80–85% using a Sep-Pak C 18 cartridge. Quantitation of methotrexate in serum was possible in the 10 −10 M range, nearly two orders of magnitude lower than that currently obtainable by existing methods. Good correlation ( r=0.99) for serum methotrexate concentrations was obtained with an enzyme-multiplied immunoassay technique. Comparison with an enzyme inhibition assay also provided similar results

Rapid confirmation of enzyme-multiplied immunoassay test (EMIT) for phencyclidine-positive urine samples with capillary gas chromatography—nitrogen—phosphorus detection

Rapid confirmation of enzyme-multiplied immunoassay test (EMIT) for phencyclidine-positive urine samples with capillary gas chromatography—nitrogen—phosphorus detection

Fluorescence polarization immunoassay evaluated for screening for amphetamine and methamphetamine in urine.

We studied the recently developed Abbott fluorescence polarization immunoassay (FPIA) for amphetamine and methamphetamine in urine and compared the results with those of the Syva enzyme-multiplied immunoassay technique (EMIT) and a gas-chromatographic assay. The FPIA method showed a limit of quantification of 0.3 mg/L, comparable with the lower cutoff of the EMIT assay. FPIA demonstrated greater specificity than the EMIT assay: phenylpropanolamine and ephedrine showed extremely limited cross reactivity with the FPIA antibody. Analysis of 249 urine specimens by all three methods clearly demonstrated the FPIA method to be acceptable for screening for amphetamine and methamphetamine in urine.

Concentrations of morphine and codeine in serum and urine after ingestion of poppy seeds.

We measured morphine and codeine in commercially available poppy seeds and in serum and urine samples from healthy adults who had ingested these poppy seeds. Four brands of black poppy seeds, examined by gas chromatography-mass spectroscopy (GC-MS) with deuterated internal standards, contained from 17 to 294 micrograms of morphine and 3 to 14 micrograms of codeine per gram of seeds. Morphine was detected by GC-MS in hydrolysates of serum as late as 24 h after ingestion, with a maximum mean concentration of 100 ng/mL (range 82-131) measured 2 h after the subjects ingested 25 g of seeds. Opiates were detectable (greater than 300 micrograms/L) in urine by enzyme-multiplied immunoassay (EMIT; Syva Co.) and by radioimmunoassay screening procedures for as long as 48 h after ingestion. The identity and quantities of morphine and codeine in poppy seed extracts and in hydrolysates of serum and urine were confirmed by GC-MS. Therefore a positive finding of morphine or codeine in blood and urine may sometimes be due to ingestion of poppy seeds.

Enzyme immunoassay method for comprehensive drug screening in micro-samples of urine.

We adapted the reagents from 11 different enzyme-multiplied immunoassay technique (EMIT; Syva Co., Palo Alto, CA 94304) drug-detection kits for use in a centrifugal analyzer. The antibody reagents were mixed into a single dilute solution, and the enzyme-labeled drug derivatives were combined similarly (Mixed EMIT reagents), for use in testing urine samples for the presence of multiple drugs. The assay, a rapid comprehensive drug-screening technique, requires 100 microL of sample and is capable of testing seven samples, in duplicate, simultaneously, in less than 10 min. Clinical evaluation (n = 325) by comparison with thin-layer chromatography (TLC) had the following results: 230 samples were negative by TLC and Mixed EMIT, 77 samples were positive by TLC and Mixed EMIT, 16 samples were negative by TLC and positive by Mixed EMIT, and two samples were positive by TLC and negative by Mixed EMIT.

Urinary pregnandiol-3-glucuronide and estrone conjugates to creatinine ratios in early pregnancies complicated by vaginal bleeding

There is no simple and rapid test available to predict the outcome of an early pregnancy complicated by vaginal bleeding. In this prospective study, 15 women with normal pregnancies collected a weekly urine sample between 6 and 13 weeks' gestation. A single random urine sample was obtained from 15 women with bleeding who continued to carry their child and 50 women who proceeded to have a spontaneous abortion (SAB). Pregnandiol-3-glucuronide (PDG) was determined with the use of enzyme-multiplied immunoassay technique (EMIT) and estrone conjugates (E1C) were measured by radioimmunoassay (RIA). The ratios of these metabolites to creatinine (C) were calculated. PDG/C ratios in normal women rose gradually from 6 weeks on. All women with bleeding during a normal pregnancy had ratios in the normal range, but 94% of women with a SAB had ratios below the normal range. The E1C/C ratio remained unchanged from 6 to 11 weeks and then rose rapidly. Until 11 weeks, there was no clear separation between the E1C/C ratios of the women with a SAB and the women with bleeding who continued their pregnancies. The prognosis of threatened abortion can be made by a urinary PDG/C ratio but not by an E1C/C ratio. EMIT is simple and quick and uses technology present in many laboratories.

Comparison of the Seralyzer and EMIT systems for determination of theophylline concentrations

A reflectance photometry assay for measurement of serum theophylline concentration was evaluated by comparison with an enzyme-multiplied immunoassay technique (EMIT). The concentration of theophylline in blood samples obtained from patients receiving intravenous theophylline therapy in the pediatric intensive-care unit was measured by both the Seralyzer and the EMIT systems. The interday and intraday variability of each method were also determined by means of calibrators of known concentration (5 to 40 micrograms/mL). There was significant correlation between the serum theophylline concentrations determined by the Seralyzer system and the EMIT system. The overall standard error of the estimate was 2.3 micrograms/mL, but with operator experience this improved to 1.3 micrograms/mL. Intraday variability was 1.2 micrograms/mL for the Seralyzer and 0.7 micrograms/mL for EMIT; the respective values for interday variability were 1.4 micrograms/mL and 0.8 micrograms/mL. The Seralyzer method measures theophylline concentrations with reliability, convenience, and speed. It could be potentially useful in a satellite, clinic, or acute-care pharmacy area.

Recovery of phenytoin from an enteral nutrient formula

The recovery of phenytoin from phenytoin oral suspension dispersed in an enteral nutrient formula was determined. The study was conducted in two phases. In phase 1, diluted phenytoin oral suspension was added to 10 1-mL samples of full-strength Osmolite and 10 1-mL samples of a distilled water control solution to produce a theoretical concentration of 10 micrograms/mL. The samples were filtered through an ultrafiltration membrane and assayed for phenytoin concentration by a homogeneous enzyme-multiplied immunoassay technique. In phase 2, varying amounts of diluted phenytoin oral suspension were added to 30-mL quantities of half-strength Osmolite or control solution to determine the effect of phenytoin concentration on recovery of phenytoin; also, a constant amount of diluted phenytoin oral suspension was added to 30-, 60-, and 90-mL quantities of half-strength Osmolite or control solution to determine the effect of solvent volume on recovery of phenytoin. Duplicate samples of each phase 2 mixture were filtered and assayed in the same manner as phase 1 samples. The mean concentration of phenytoin in phase 1 samples was 3.70 +/- 0.28 microgram/mL for Osmolite and 9.87 +/- 0.27 microgram/mL for control solution; this difference was significant. The percentage of phenytoin recovered from phase 2 samples of Osmolite increased with increasing phenytoin concentration and decreased with increasing volumes of Osmolite. The decreased recovery of phenytoin from the enteral nutrient formula used in this study has potential clinical importance, but further research in humans is needed to substantiate these in vitro observations.

Evaluation of the Ames Seralyzer for therapeutic drug monitoring of theophylline.

Dry reagent technology and reflectance photometry are combined in the Ames Seralyzer to offer a solid-phase plastic strip assay methodology. Light reflected from serum placed on the antibody-impregnated strip is quantitated with a two-point standard curve to display a theophylline result in 90 seconds. The accuracy and precision of the Seralyzer for serum theophylline are compared to the enzyme-multiplied immunoassay technique (Emit) which is commonly used to determine drug concentrations. Liquid calibrators (5 and 25 micrograms/ml) and liquid-spiked sera (10, 15, and 20 micrograms/ml) were measured six times daily simultaneously by both methods for twenty days. Eighty patient samples (theophylline range: 0-33 micrograms/ml) were measured twice each during the twenty days. Acceptable assay limits were established and maintained by measuring 15 micrograms/ml spiked sera prior to and during the evaluation period. All within-run and between-run means for the Seralyzer and Emit were within +/- 1 microgram/ml of the spiked value. All ranges of within-run and between-run means were within +/- 2 micrograms/ml of the spiked value. Mean between-run coefficients of variation for the Seralyzer at the 5, 10, 15, 20, and 25 micrograms/ml values were 11.5, 6.7, 4.9, 4.6, and 4.9 percent, respectively. Linear regression on the 80 patient samples gave a slope of 1.02, intercept of -0.28, and correlation of 0.984. The Seralyzer's accuracy and precision compare favorably with Emit for theophylline determinations greater than 7.5 micrograms/ml.

Evaluation of an extension set for intermittent intravenous drug delivery to infants

An intravenous administration set designed for delivery of drug doses to pediatric patients was tested in vitro for the effect of fluid density and flow rate on drug delivery, and delivery of drug by this extension set was compared in vivo with delivery by other methods. Gentamicin (as the sulfate salt) and penicillin G potassium were used to represent low-density and high-density drugs, respectively; a 1-mL solution of each drug, labeled with carbon 14, was tested with each of two primary infusion solutions: 0.45% sodium chloride injection and 10% dextrose injection. The drug dose was injected via a port into a piston-containing chamber from which an equivalent amount of the primary fluid was displaced. Serial samples collected from the end of the filter-containing extension set were analyzed for drug concentration using a liquid scintillation technique. In 12 infants receiving i.v. gentamicin, this delivery method was compared in a randomized crossover trial with delivery by a syringe pump and by i.v. push. Each delivery system was used on one of three consecutive days, and serum gentamicin concentrations were measured by enzyme-multiplied immunoassay. The time required for in vitro delivery of the dose was dependent on flow rate. Density of the drug solution or the primary i.v. fluid did not significantly affect drug delivery. Serum gentamicin concentrations were not significantly different for the three delivery methods, but variability of drug delivery was greatest with the pediatric extension set. This pediatric extension set provides accurate and reliable drug delivery at primary infusion flow rates slower than 10 mL/hr when the drug dosage volume is 2-3 mL or less.

Theophylline

This study was undertaken to evaluate a method of prospectively estimating appropriate aminophylline infusion rates in acutely ill, hospitalized patients with bronchospasm. Steady-state serum theophylline concentrations (Css), clearances (Cl), and half-lives (t1/2) were estimated by the Chiou method using serum concetrantions obtained 1 and 6 h after the start of a constant-rate intravenous aminophylline infusion in 10 male patients averaging 57 years of age. Using an enzyme-multiplied immunoassay (EMIT) system for theophylline analysis, pharmacokinetic estimations were excellent for Css (r = 0.9103, p less than 0.01) and Cl (r = 0.9750, p less than 0.01). The mean estimation errors were 9.4% (range 0.8-21.5) for Css and 12.3% (range 1.3-28.0) for Cl. There was no correlation between patient age and Cl. This method is useful for rapidly individualizing aminophylline therapy in patients with acute bronchospasm.

Comparison of enzyme immunoassay, radioimmunoassay, and microbiologic assay for amikacin in plasma

An enzyme-multiplied immunoassay technique (EMIT), radioimmunoassay (RIA), and microbiologic assay (MBA) were compared as methods of measuring amikacin in human plasma. Accuracy of the three methods was assessed in plasma with amikacin added in concentrations of 2.5-50 micrograms/ml. Correlations between the assay methods were compared over a range of 0.5-50 micrograms/ml. Amikacin was also assayed in plasma to which had been added other drugs, including 16 antibiotics and 3 antineoplastic agents; also tested were samples that had been stored at 5 degrees C or -20 degrees C. Within the amikacin concentration range of 2.5-50 micrograms/ml, the coefficients of variation of all methods were within 10%. Correlation was good between EMIT and RIA as well as between EMIT and MBA. Of the other drugs tested, only tobramycin, dibekacin, and kanamycin affected amikacin EMIT determinations, while only kanamycin affected amikacin RIA determinations. No effect of cold and freezing was observed on amikacin determinations. EMIT assay is an acceptable method for routine analysis of amikacin plasma samples. The amikacin assay results for the three methods were highly correlated.

Determination of aminoglycosides in rat renal tissue by enzyme immunoassay.

The enzyme-multiplied immunoassay technique for the determination of gentamicin or amikacin in serum was evaluated for use in rat renal tissue. After the addition of either gentamicin or amikacin to tissue homogenate, the assay of the supernatant showed a reduced recovery, depending on the amount of renal tissue present per milliliter of homogenate. The recovery was independent of the amount of aminoglycoside added. This reduction could be due to either protein binding or interference with the enzyme-multiplied immunoassay technique by substances released from the renal tissue. When the enzyme-multiplied immunoassay technique is used for aminoglycoside assay in renal tissue, care should be taken to minimize the amount of tissue present in the homogenate or to correct for the decreased recovery.

Reliability of Substrate-labeled Fluorescent Immunoassay Method for Determination of Amikacin Concentration in Serum and Comparison with Other Methods

The reliability of substrate-labeled fluorescent immunoassay (SLFIA) method was examined using amikacin sulfate (AMK) samples of 5 known concentrations in spiked serum. The result of this SLFIA method were compared with those of enzyme-multiplied immunoassay technique (EMIT), radioimmunoassay (RIA) and bioassay methods. The correlation coefficient of the SLFIA method was 0.9999 and the regression line was Y=1.116X + 0.069. The correlation coefficients of the EMIT, RIA and bioassay methods were more than 0.999. The within-day and between-day coefficients of variation of the SLFIA assay were 2.82-9.13% and 3.04-8.14%, respectively. In AMK-spiked serum samples, the correlation coefficient between the SLFIA and EMIT methods was 0.9996, and the regression line was Y=1.137X-0.181. The value of SLFIA was slightly higher than that of EMIT. Compared with SLFIA and RIA or bioassay, the high correlation was also obtained in AMK-spiked serum samples. In serum of patients undergone AMK therapy, furthermore, the results obtained by SLFIA were compared with those obtained by EMIT, RIA and bioassay. These showed high correlation with each other, though, the value of SLFIA was slightly lower than those of other methods. These results suggest that the SLFIA method may be used for the routine clinical monitoring of serum AMK.

Variability of phenytoin protein binding in epileptic patients.

The variability of protein binding of phenytoin in epileptic patients was evaluated using a rapid ultrafiltration system. Thirty-one pairs of total and free (unbound) phenytoin plasma concentrations were reviewed. Filtered plasma for free phenytoin determination was obtained using an ultrafiltration membrane system. All samples were analyzed by an enzyme-multiplied immunoassay technique. A good correlation was obtained for total phenytoin concentration v free phenytoin concentration. Comparison of obtained and predicted free phenytoin values differed significantly. Of nine patients demonstrating toxic effects, six had total phenytoin concentrations within the accepted therapeutic range with free concentrations above the therapeutic range. The results of this study indicate a wide variation of phenytoin protein binding in chronic epileptic patients, implying that total phenytoin concentration must be interpreted cautiously, and use of free phenytoin concentration appears to be a more appropriate guide to therapy. The availability of a rapid method of ultrafiltration makes free phenytoin plasma levels readily available to the clinician.

Metoprolol or propranolol does not alter the kinetics of procainamide.

Eight healthy volunteers received a single 500 mg intravenous dose of procainamide hydrochloride by 30-min infusion on three occasions in random sequence. The three modes of administration were (a) control, without concurrent drugs; (b) during coadministration of propranolol, 80 mg three times daily; and (c) during coadministration of metoprolol, 100 mg two times daily. Procainamide kinetics were determined from multiple serum concentrations measured by enzyme-multiplied immunoassay (EMIT) during 10 h after each dose. A metaproterenol infusion study verified a high degree of beta-blockade during trials 2 and 3. Mean procainamide half-life during the three trials (1.9, 2.2, and 2.3 h, respectively) tended to show prolongation during beta-blocker treatment, but the overall difference was of borderline significance (0.05 less than p less than 0.1). Total procainamide clearance (16.2, 14.1, and 13.7 ml/min/kg) did not differ significantly between the three trials, nor was there a significant change in area under the serum concentration curve for N-acetylprocainamide, the major metabolite. Thus the kinetics of procainamide in healthy persons are not importantly altered by typical therapeutic doses of two beta-adrenergic blockers.

The General Toxicology Unknown II. A Case Report: Doxylamine and Pyrilamine Intoxication

A general toxicology unknown case is presented to demonstrate our systematic approach. A 20-year-old male was found dead with multiple suicide notes. Overdose was suspected but substances were not known. Blood alcohol was negative. Urine was analyzed by enzyme-multiplied immunoassay technique and was negative for all drugs assayed. Urine was then extracted with ethyl acetate:hexane (1:1) at pH 10 and back-extracted into 1.0N sulfuric acid. The acidic layer was adjusted to pH 10, and re-extracted with ethyl acetate:hexane (1:1). The residue was analyzed by gas chromatography (GC) on a 3% OV-101 column. It was found to be negative for all commonly screened substances. However, several unknown peaks were observed. Electron impact mass spectra of these unknown peaks were obtained and searched for in our computer library of more than 25000 mass spectra. These unknown peaks were identified as doxylamine and pyrilamine by gas chromatography/mass spectrometry. The base peak and molecular ion for pyrilamine were at m/z 121 and 285, respectively. The base peak for doxylamine was at m/z 58. No molecular ion was observed for doxylamine. Both doxylamine and pyrilamine are antihistamines, but are promoted and used in the management of insomnia. Quantitation was performed on a GC using dexbrompheniramine as an internal standard. Blood concentrations for doxylamine and pyrilamine were 0.7 and 7.0 mg/L, respectively. Concentrations in other tissues were determined. Death was caused by combined doxylamine and pyrilamine intoxication; the manner of death was suicide.

Logit-log calibration curves for EMIT assays

The logit-log and four-parameter logistic procedures when appropriate for calculation of enzyme-multiplied immunoassay (EMIT) data have the advantage that they can be applied regardless of the kinetic analyzer or reaction conditions. To use these procedures correctly one must determine the change in absorbance at an infinite drug concentration (delta A infinity). The marked variation of delta A infinity with equipment and reaction conditions and the difficulty in determining this value have hindered broad use of these otherwise universally applicable procedures. We have evaluated two simple methods for determining delta A infinity, both based on its equivalence to delta A in the absence of specific antibody: (1) cross-kit reaction using antibody/substrate and enzyme-drug reagents from kits for different drugs, and (2) substitution of an antibody-free substrate reagent with composition based on direct analysis. The cross-kit procedure was tested with EMIT assays for phenobarbital, primidone, phenytoin, carbamazepine, ethosuximide, and theophylline. In some cases an unexpected type of cross-reaction occurred, giving an erroneously low value for delta A infinity. The antibody-free substrate reagent always permitted accurate determination of delta A infinity.