Evaluation of enzyme-multiplied immunoassay technique (EMIT) for determination of serum digoxin.

Sixty-seven digoxin-containing sera were analyzed by both radioimmunoassay and EMIT. After some important modifications of the EMIT method, agreement between the two methods was very good. Reproducibility of the EMIT assay was excellent; daily variations in values found for control sera were quite small, and recovery of added digoxin was good. Slight hemolysis hadnegligible effects, but highly hemolyzed specimens gave low recoveries of digoxin.     

Trimethoprim alters the disposition of procainamide and N-acetylprocainamide

The steady-state pharmacokinetics and pharmacodynamics of procainamide and its active N-acetyl metabolite (NAPA) were assessed alone and in combination with trimethoprim. Eight healthy men received oral sustained-release procainamide, 500 mg every 6 hours for 3 days, alone and with oral trimethoprim, 200 mg daily for 4 days. Concomitant trimethoprim significantly increased the plasma AUC(0-12) of both procainamide and NAPA (63% and 52%, respectively), with concurrent decreases in their renal clearances (47% and 13%, respectively) and a 39% increase in the mean urinary recovery of NAPA (as percentage of procainamide and NAPA recovery). After trimethoprim coadministration, there was also a trend toward a decrease in the apparent acetylation clearance of procainamide (19%, p = 0.057). The change in procainamide and NAPA renal clearances after trimethoprim coadministration strongly correlated with their baseline renal clearances (r = 0.84 and r = 0.74, respectively, p less than 0.0001). There was small but significant increase in the corrected QT interval with procainamide administration, which increased further with trimethoprim coadministration. We conclude that trimethoprim increases the plasma concentrations of procainamide and NAPA by decreasing their renal clearances and allowing more conversion of procainamide to NAPA.     

Fluoroimmunoassays for procainamide and N-acetylprocainamide compared with a liquid-chromatographic method

We measured procainamide and its active metabolite, N-acetylprocainamide (NAPA), in 80 sera from 37 patients by a new fluorimmunoassay procedure and an established "high-performance" liquid-chromatographic method. Additive and proportional differences between the methods were 0.07 mg/L and 9%, respectively, for procainamide and 0.62 mg/L and 16% for NAPA. Between-day CVs by the chromatographic and immunoassay methods, respectively, were 3.9% and 2.2% for procainamide at a concentration of 6 mg/L, and 5.1% and 1.2% for NAPA (14 mg/L). We applied a modification of the fluoroimmunoassay for determination of procainamide concentrations, using sera obtained during a pharmacokinetic study, and demonstrated excellent agreement with the chromatographic method.     

酶增强免疫分析法与微粒子酶联免疫吸附法检测他克莫司药物浓度的结果对比研究

目的对酶增强免疫分析法（EMIT）与微粒子酶联免疫吸附法（MEIA）进行比对实验,评估SYVAViva-E临床化学分析仪（简称SYVA Viva-E）和Abbott IMX分析仪（简称Abbott IMX）2种检测系统测定他克莫司（FK506）药物浓度的一致性。方法将Tac/Csa CONTROL质控品分别用SYVA Viva-E（EMIT）和Abbott IMX（MEIA）进行批内、批间精密度测定。FK506的靶值（检测范围）：A1水平为4.3（2.1～6.5）ng/mL、A2水平为8.9（5.3～12.5）ng/mL、A3水平为18.0（14.0～22.0）ng/mL。将158份临床患者样本分为极低浓度（0.1～〈2.0 ng/mL）、低浓度（2.0～〈8.0 ng/mL）、中浓度（8.0～〈14.0 ng/mL）、高浓度（14.0～〈20.0 ng/mL）和极高浓度（20.0～24.0 ng/mL）5个组,用SYVA Viva-E和Abbott IMX平行测定FK506浓度,观察2种检测系统测定临床样本的相关性。结果当FK506浓度处于A1水平时,SYVA Viva-E的批内、批间精密度及检测结果均值均高于Abbott IMX（P〈0.05）;处于A2、A3水平时,2种检测系统批内、批间精密度及检测结果均值相近,差异无统计学意义（P〉0.05）。Abbott IMX的检测精密度优于SYVA Viva-E。当临床样本浓度〈8.0 ng/mL时,SYVAViva-E检测结果的均值比Abbott IMX约高1.0 ng/mL,二者比较差异有统计学意义（P〈0.01）,与测定质控品的结果一致;当样本浓度≥8.0 ng/mL时,SYVA Viva-E检测结果的均值与Abbott IMX相近,二者比较差异无统计学意义（P〉0.05）。2种检测系统测定临床样本的总体相关性较好（r=0.977）,但SYVA Viva-E检测结果均略高于Abbott IMX。结论应用EMIT检测FK506时,其检测结果略高于MEIA,尤其在低浓度范围更加明显。2种检测系统在检测FK506浓度时可能存在系统偏差。     

Simultaneous quantitation of quinidine, procainamide, and N-acetylprocainamide in serum by gas-liquid chromatography with a nitrogen-phosphorus selective detector

We describe a single-run method for quantitating quinidine, procainamide, and N-acetylprocainamide, involving gas-liquid chromatography with a nitrogen-phosphorus selective detector. Within-run precision (CV) was 3% (x = 2 mg/L, n = 20), 6.9% (x = 4 mg/L, n = 10), and 1.5% (x = 8 mg/L, n = 8) for quinidine; 7.7% (x = 4 mg/L, n = 14), 1.6% (x = 8 mg/L, n = 16), and 2.3% (x = 12 mg/L, n = 12) for procainamide; and 6.3% (x = 5 mg/L, n = 6), 3.6% (x = 10 mg/L, n = 20), and 4.0% (x = 20 mg/L, n = 10) for N-acetylprocainamide.l Between-run precision was 3.0%(x = 2 mg/L, n = 20), 7.0% (x = 4 mg/L, n = 9), and 2.8% (x = 8 mg/L, n = 9) for quinidine; 4.7% (x = 4 mg/L, n = 10). 3.3% (x = 8 mg/L, n = 20), and 1.9% (x = 12 mg/L, n = 10) for procainamide; and 9.3% (x = 5 mg/L, n = 6), 4.3% (x = 10 mg/L, n = 20), and 3.8% (x = 20 mg/L, n = 10) for N-acetylprocainamide. Tube stoppers that contain a rubber plasticizer interfere with the technique. Clinical application and correlation with drug concentrations by this technique are discussed.     

Levofloxacin and ciprofloxacin decrease procainamide and N-acetylprocainamide renal clearances

Ten healthy adults participated in a randomized, crossover drug interaction study testing procainamide only, procainamide plus levofloxacin, and procainamide plus ciprofloxacin. During levofloxacin therapy, most procainamide and N-acetylprocainamide (NAPA) pharmacokinetic parameters, including decreased renal clearances and renal clearance/creatinine clearance ratios, changed (P < 0.05). During ciprofloxacin treatment, only procainamide and NAPA renal clearances decreased significantly.     

The quantitative disposition of procainamide and N-acetylprocainamide in the rat

The objectives of this study were to investigate: 1) the rat acetylator phenotype, 2) the systemic availability of oral procainamide (PA), 3) the kinetic disposition of PA and its N-acetyl metabolite (NAPA) and 4) the relationship between PA dose and steady-state blood PA and NAPA levels. The rat acetylator phenotype seems to be monomorphic in type. The systemic availability of PA was estimated to be 78%. The half-life (T 1/2) of PA elimination was 55 minutes and that of NAPA was 51 minutes. PA clearance was 64 ml/kg/min and NAPA clearance 22.4 ml/kg/min. The apparent distribution volume for PA was 4.92 liters/kg and for NAPA 1.64 liters/kg. Acetylation accounted for 38% of PA disposition, urinary excretion 34% and other metabolism 28%. Urinary excretion of NAPA accounted for 72% of administered drug. Steady-state blood PA levels showed a linear increase with dose whereas NAPA did not. The latter observation suggests saturation of PA acetylation at higher PA doses.     

基质效应对荧光偏振免疫测定技术和酶放大免疫测定方法检测环孢素A结果的影响

目的 通过荧光偏振免疫测定(fluorescence polarization immunoassay,FPIA)和酶放大免疫测定技术(enzyme-multiplied immunoassay technique,EMIT)测定环孢素A(cyciosporine,CsA),了解基质效应对检测结果的影响,解释在CsA室问质评结果中两方法的检测结果的差别,并找到纠正方法.方法 选择不同浓度的临床全血标本100份,用FPIA和EMIT技术进行检测,对比检测结果;通过添加Cu2+,zn2+评估离子残留对免疫反应的影响;用添加等体积血红蛋白富集液后再次抽提的方法,评价非全血基质对质控品和CsA标准品抽提率的影响.结果 检测临床全血标本FPIA(X)和EMIT(Y)检测结果具有良好的相关性(Y=0.926 8X-8.115,R2=0.996 9);金属离子残留对FPIA法和EMIT法均不产生下扰(P>0.05);非全血基质样本会降低两方法的抽提率;样本中血红蛋白含量≥30 g/L就可纠正非全血基质对抽提率的影响.结论 下发质控品的非全血基质是导致CsA室间质评结果中两系统的检测结果不一致的主要原因,可通过添加血红蛋白富集液进行纠正;遇器官移植受者有极低血红蛋白(<30 g/L)时,要允分考虑低血红蛋白对CsA检测结果的影响.     

Theophylline, dyphylline, caffeine, acetaminophen, salicylate, acetylsalicylate, procainamide, and N-acetylprocainamide determined in serum with a single liquid-chromatographic assay

We describe a single set of liquid-chromatographic conditions for assay of theophylline, dyphylline, caffeine, acetaminophen, salicylate, acetylsalicylate, procainamide, and N-acetylprocainamide in serum. The chromatographic system includes a Waters Associates mu-Bondapak C18 column and acetonitrile in 0.1 mol/L potassium phosphate buffer, pH 4.0 (9.75/90.25 by vol), as the mobile phase. Only 50 microL of serum is required, and drug concentrations as low as 0.5 mg/L can be detected. Absolute and relative analytical recoveries range from 95 to 101%. Day-to-day variation of the method is less than 6% for each drug. Linearity extends to 1 g/L for all drugs. Recycling of the mobile phase under pressure eliminates the need to prepare and de-gas solvents. The use of the single stationary and mobile phase provides a practical and economical approach to routine and urgent therapeutic drug monitoring.     

Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine

BACKGROUND: Bence Jones proteins or monoclonal immunoglobulin kappa and lambda free light chains (FLCs) are important markers for identifying and monitoring many patients with B-cell tumors. Automated immunoassays that measure FLCs in urine and serum have considerable clinical potential. METHODS: Sheep antibodies, specific for FLCs, were prepared by immunization with pure kappa and lambda molecules and then adsorbed extensively against whole immunoglobulins. The antibodies were conjugated onto latex particles and used to assay kappa and lambda FLCs on the Beckman IMMAGE protein analyzer. RESULTS: The unconjugated antibodies showed minimal cross-reactivity with intact immunoglobulins or other proteins. With latex-conjugated antibodies, kappa and lambda FLCs could be measured in normal sera and most normal urine samples. Patients with multiple myeloma had increased concentrations of the relevant serum FLC, whereas both FLCs were increased in the sera of patients with systemic lupus erythematosus. CONCLUSIONS: We developed sensitive, automated immunoassays for kappa and lambda FLC measurements in serum and urine that should facilitate the assessment of patients with light chain abnormalities.     

Comparison of the autonomic effects of procainamide and N-acetylprocainamide in the dog

The autonomic effects of procainamide (PA) and N-acetylprocainamide (NAPA) were studied in anesthetized dogs. High plasma concentrations of PA (31 +/- 1.3--64 +/- 3.4 micrograms/ml) and NAPA (64 +/- 3.4--127 +/- 8.3 micrograms/ml)) reduced base-line mean arterial pressure and heart rate and attenuated the pressor and positive chronotropic responses to bilateral carotid occlusion and the negative chronotropic response to vagal stimulation. Neither drug reduced the pressor or positive chronotropic responses to catecholamines (epinephrine, phenylphrine, isoproterenol), however. In fact, at some doses PA and NAPA accentuated the pressor and positive chronotropic effects of epinephrine. Similarly, the depressor response to acetylcholine was not reduced by these drugs; it was significantly increased at some doses. In the isolated hindlimb (constant flow) PA and NAPA reduced the pressor response to preganglionic (sympathetic chain) stimulation but not to postganglionic (femoral and sciatic nerves) stimulation. We conclude that NAPA, like PA, at high plasma levels is vagolytic and attenuates baroreceptor-mediated reflexes associated with reduced arterial pressure. These effects appear to be due to ganglionic blockade.     

Time-resolved fluorometric sandwich immunoassay for human growth hormone in serum and urine

A specific and sensitive time-resolved fluorometric sandwich immunoassay for human growth hormone (hGH) in serum and urine is described. A monoclonal anti-hGH IgG1 (5802)-coated polystyrene ball was incubated with serum or dialyzed urine and subsequently with europium ion-labeled monoclonal anti-hGH IgG1 (5801). Europium ion bound to the polystyrene ball was measured by time-resolved fluorometry. The detection limit of hGH was 0.3 pg/tube, which was 15-fold higher than that by sandwich enzyme immunoassay using horseradish peroxidase as label. The assay range of serum hGH was 15-15,000 ng/liter using 20 microliters of serum. The assay range of urinary hGH was 2-2,000 ng/liter using 150 microliters of dialyzed urine. The detection limits of hGH in serum and urine by this immunoassay were satisfactory for measuring hGH levels in serum and urine of healthy children and in serum of healthy adults and higher levels but not in urine of healthy adults and in serum and urine of patients with hGH deficiency.     

Determination of lysozyme in serum, urine, cerebrospinal fluid and feces by enzyme immunoassay

Conjugates of human lysozyme and horseradish peroxidase (HRP) were prepared by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate. A conjugate containing 2 mol HRP/mol lysozyme was isolated by gel filtration and used as a labeled antigen in competitive enzyme immunoassays, in which anti-lysozyme rabbit IgG had been bound to wells of microtiter plates. The assay can detect as little as 1 microgram lysozyme/l. The following reference intervals have been established: 950-2450 micrograms/l for serum, 1.7-123 micrograms/l for urine, 17.6-118 micrograms/l for cerebrospinal fluid and 0.04-1.5 microgram/g for feces.     

A new enzyme immunoassay for prolactin in serum or plasma

This enzyme immunoassay (EIA) of human prolactin (hPRL) involves incubation of sample and anti-hPRL antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) in tubes coated with a second antibody to hPRL. The test can be performed within 60 min. No reaction of the antibodies with human placental lactogen and human somatotropin is detectable. The presence of detergent allows assay of both serum and plasma. Precision was improved by including polyethylene glycol in the reaction mixture. To optimize analytical recovery, we added protease inhibitor. Assay of the EIA standards shows good correlation with results for World Health Organization reference preparations. The measurable range is 1 to 400 micrograms/L. Intra- and interassay CVs are about 5%. Comparisons with two RIAs and two other EIAs show reasonably good correlations. The components of our EIA are stable for 18 months.     

A sensitive enzyme immunoassay system for the measurement of beta-thromboglobulin in plasma and urine

A sensitive enzyme immunoassay system for the measurement of beta-thromboglobulin (beta-TG) in plasma and urine was developed using purified antibodies to beta-TG linked to beta-D-galactosidase from Escherichia coli as label. The assay system consisted of polystyrene beads with immobilized antibody F(ab')2 fragments and the antibody Fab' fragments labeled with beta-D-galactosidase. The assay had a sensitivity of 10 pg/assay tube. beta-TG levels in plasma and urine could be determined with 1-2.5 microliters and 100 microliters samples, respectively. The results obtained with the enzyme immunoassay correlated well with those of a radioimmunoassay (r = 0.946, slope = 0.846, y-intercept = 4.71 ng/ml; n = 60). Plasma beta-TG levels in healthy subjects determined with the present assay system were 38.4 +/- 12.2 ng/ml (+/- 1 SD). Levels were markedly increased in 19 out of 22 patients with myeloproliferative syndrome. Urinary beta-TG levels in healthy subjects were estimated to be 0.17 +/- 0.05 ng/ml (+/- 1 SD). Urine levels were increased in 16 out of the 19 patients with myeloproliferative syndrome whose plasma levels were high. These findings indicate that urinary beta-TG levels may be reflection of the beta-TG in plasma and can be an indicator of platelet activation.     

Substrate-labeled fluorescent immunoassay for measuring dibekacin concentrations in serum and plasma.

We have developed a homogeneous substrate-labeled fluorescent immunoassay for the measurement of dibekacin concentrations in serum and plasma. The fluorogenic enzyme substrate beta-galactosyl-umbelliferone was covalently attached to tobramycin, an aminoglycoside structurally similar to dibekacin, to prepare a fluorogenic drug reagent (FDR). The FDR is nonfluorescent under assay conditions, but fluoresces upon hydrolysis by beta-galactosidase. However, binding of the FDR by antiserum to the cross-reactive drug kanamycin prevents enzyme hydrolysis. The fixed level of FDR competes with dibekacin within the sample for the limiting number of antibody-binding sites in the reaction mixture. Unbound FDR is hydrolyzed by beta-galactosidase to release a fluorescent product that is proportional to the dibekacin concentration in the sample. The assay exhibits good precision, standard curve reproducibility, recovery, sensitivity, and correlation with a comparative method. Additionally, the substrate-labeled fluorescent immunoassay is rapid and easy to perform.