Direct spectrophotometric determination of alpha-amylase activity in salive, with p-nitrophenyl alpha-maltoside as substrate.

We describe a simple, direct kinetic method for determination of salivary alpha-amylase (1,4, alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1). The assay makes use of a well-defined substrate, p-nitrophenyl alpha-maltoside, which is hydrolyzed by alpha-amylase to a chromogenic product, p-nitrophenol. Activity is determined by directly monitoring the increase in absorbance of the reaction mixture. Amylase activity can be defined in international (IUB) units of micromoles of product/min per liter of saliva. For 22 healthy subjects, the mean +/- SD of amylase activity in mixed saliva was 2.77 +/- 1.12 U/liter. Activity and instrumental response were linearly related over the entire range tested (0.224 to 11.90 U/liter). The within-run precision (CV) over this range was better than 3% for all but the lowest activities. Values obtained with this assay correlate well with those obtained with a modified Nelson-Somogyi saccharogenic method (r = 0.979). The precision and simplicity of this assay suggest that it is the method choice for determining amylase activity in human saliva.     

Alterations in serum pancreatic elastase 1 content in acute and chronic pancreatitis: comparison with alpha-amylase activity

Serum pancreatic elastase 1 content in 45.9% of 203 samples from 32 patients with acute pancreatitis was found to be greatly elevated to greater than 1000 ng/dl, much higher than the control values (90 to 270 ng/dl), whereas serum alpha-amylase activity, in 62.3% of the same samples, was within the control range (100 to 460 IU/L). The increased pancreatic elastase 1 content persisted for several days after serum alpha-amylase activity returned to normal, suggesting that, compared with serum alpha-amylase activity, pancreatic elastase 1 content truly reflects the clinical course of acute pancreatitis. The measurement of pancreatic elastase 1 content appears to be more valuable in the diagnosis and prognosis of acute pancreatitis than that of serum alpha-amylase activity. In 41 patients with chronic pancreatitis, on the other hand, pancreatic elastase 1 content and serum alpha-amylase activity were within the normal range in 38.3% and 56.2% respectively, of 336 samples.     

An automated method for determination of serum and urine alpha-amylase.

An automated method for the determination of alpha-amylase activities of serum and urine using the Phadebas Amylase Test is described. The procedure has been adapted for the System Olli 3000 analyser using amylase tablets whose weight is half of that of normal tablets. The results are calculated by the computer with the aid of a standard curve calculated theoretically. This automated procedure is fast, as many as 120 analyses per hour can be carried out. Intra-assay precision of 2.0 and 2.2% (CV) is obtained from serum samples containing 301 and 181 u/l of alpha-amylase, respectively (n = 20), and inter-assay precision is 4.4 and 3.3% with mean values of 337 and 196 u/l, respectively (n = 20). When the automated procedure is compared with the manual procedure on 85 sera with alpha-amylase activities below 1000 u/l, a good correlation r = 0.992, and a regression equation y = 1.01x-6 are found. In the case of serum and urine samples, which contain high enzyme activities, the automated method gives slightly higher results than the manual method.     

Studies on determination of alpha-amylase with p-nitrophenyl-alpha-D-maltotetraoside

Nitrophenylmaltodextrins are alpha-amylase substrates which allow a continuous determination with a zero order kinetics over a period of at least 10 min, without deviations from linearity. Only one auxiliary enzyme is necessary. Practicability and clinical evidence of alpha-amylase determinations by means of p-nitrophenyl-alpha-D-maltotetraoside are demonstrated. The interserial precision of 0.84% cannot conceal an only moderate correlation with previous methods. This fact, however, does not negate the advantages.     

An enzymatic method for the alpha-amylase assay which comprises a new procedure for eliminating glucose and maltose in biological fluids

An alpha-amylase assay in biological fluids, characterized by a new procedure for eliminating glucose and/or maltose, was developed. The reagent includes thermostable glucokinase, glucosephosphate isomerase and phosphofructokinase obtained from a thermophile Bacillus stearothermophilus. Up to 4,000 mg/dl glucose or 600 mg/dl maltose had no effect on the measured value of alpha-amylase activity when measured at 37 degrees C, even at a serum volume fraction in the reagent of 1/50. Alpha-Amylase activity was monitored by the absorbance increase at 340 nm due to NADPH production. The assay has a high degree of precision, with the within-run and day-to-day coefficients of variation being 2.17% at 75.0 U/l and 2.49% at 112 U/l, respectively, and is linear up to about 2,500 U/l. Regarding interferences, bilirubin, urate, ascorbate, pyruvate, EDTA, sodium fluoride and others were found to have no effect on the assay, and the reagent in solution is stable for about 2 wk at low temperatures (6-8 degrees C).     

Effect of alpha-glucosidase inhibitor on human pancreatic and salivary alpha-amylase

The mode of inhibition of a new complex oligosaccharide that inhibits the alpha-glucoside hydrolase activity of pancreatic and salivary alpha-amylase was studied. Kinetic analysis revealed a non-competitive type of inhibition with a Ki of 1.47 +/- 0.03 micrograms when tested against human pancreatic alpha-amylase and 3.89 +/- 0.08 micrograms against human salivary alpha-amylase. The inhibitory action of alpha-glucoside hydrolase inhibitor (alpha-GHI) on pancreatic amylase was observed over a wide range of pH (6.0--7.9), whereas the inhibition of salivary amylase was optimal at pH 6.5. Column chromatographic investigations suggested the possible formation of an enzyme-inhibitor complex because the mixture of alpha-GHI and pancreatic alpha-amylase was eluted as a single component through a Sephadex G200 column. However, this enzyme-inhibitor complex was easily separated into each component and the enzyme activity was fully recovered after electrophoresis.     

Coupled reactions of immobilized enzymes and immobilized substrates: clinical application as exemplified by amylase assay.

We described a partitioned enzyme-sensor system, which incorporates an immoblized substrate and three or more discrete immobilized enzymes. This instrument measures alpha-amylase activity by passing the solution containing alpha-amylase over a column packed with immobilized starch. The resulting oligosaccharides are successively exposed to a column or columns containing immobolized glucose oxidase, catalase, glucoamylase or maltase, and glucose oxidase. The resulting hydrogen peroxide is detected by a three-electrode amperometric cell. All immobilized reagents were immobilized on a particulate, porous alumina to allow rapid and constant flow rate. With use of less than optimum immobilized reagents, alpha-amylase activity has been measured from about 5 to 200 kU/liter with a 50 microliter sample size. Lack of sensitivity is predominantly attributable to the low activity and low stability of immobilized maltase and glucoamylase. We believe that a clinical test using this system is feasible and desirable because the immobilized reagent system should allow for testing of alpha-amylase with excellent precision, convenience to the operator, and low cost.     

Determination of alpha-amylase activity in serum and dialysate from patients using icodextrin-based peritoneal dialysis fluid.

OBJECTIVE: Low serum activity of alpha-amylase has been reported in peritoneal dialysis (PD) patients following treatment with icodextrin-based peritoneal dialysis fluid (IPDF). However, these results have been questioned because icodextrin interferes with the polysaccharide reagent included in the assay as a substrate for alpha-amylase in the sample. DESIGN: We adapted a routine method using p-nitrophenol maltoheptaoside as substrate for the analysis of total alpha-amylase in serum and dialysate from 27 patients using IPDF. Serum from 12 healthy volunteers and serum and dialysate from 19 PD patients using glucose-based peritoneal dialysis fluid (GPDF) were used as controls. For the PD patients, time on dialysis ranged from 1 to 24 months (mean 5.7 months) and time of exposure to IPDF ranged from 1 to 52 weeks. RESULTS: To test for interference and recovery, and thus to validate the alpha-amylase assay, samples were spiked with IPDF and synthetic alpha-amylase. This revealed that addition of up to 75% IPDF did not interfere with the assay. Furthermore, alpha-amylase was fully recovered when spiked in serum from patients treated with IPDF. We show that total alpha-amylase activity is considerably lower in the serum of IPDF patients (20.3 +/- 16.5 U/L, p < 0.001) than GPDF patients (85.5 +/- 51.7 U/L) and healthy persons (55.1 +/- 13.6 U/L). CONCLUSIONS: We have shown that the IL method (ILTest; Instrumentation Laboratory, Lexington, MA, USA) measures alpha-amylase activity in samples containing icodextrin metabolites. The clinical significance of reduced plasma alpha-amylase activity, as well as the relative importance of pancreatic versus salivary and tissue-bound alpha-amylase, in PD patients using IPDF is not known.     

A rapid and specific method for the estimation of glucose using and oxygen electrode and simple differentiating circuit

A simple differentiating circuit is described which, together with an oxygen electrode assembly, provides a rapid (20 sec) and cheap system for the estimation of glucose in body fluids using a sample volume of 20 microliter. Response was linear over the range 0-22 mmol/l. The complete system has been evaluated using plasma and whole blood. "Within-day" precision on control plasmas over the concentration range 2.73-8.33 mmol/l lies between 2.0 and 3.3% (C.V., n = 23); "day-to-day" precision was 4.6% (C.V., n = 23). On whole blood, to which nitrate had been added, precision was 4.5% (C.V., n = 15); precision using standard glucose solutions was never greater than 3.3% (C.V., n = 15) over the concentration range 1.1-22.2 mmol/l. Recoveries of added glucose to plasma and whole blood were greater than 90%. Good correlation between glucose values obtained by the oxygen electrode system described here and an automated system were obtained for plasma (r = 0.9902, n = 35) and whole blood (r = 0.9610, n = 35), although whole blood gave values on average 27% lower than plasma. The use of the system for the measurement of plasma cholesterol is also demonstrated.     

罗氏尿微量清蛋白检测试剂盒性能评价

目的评价罗氏第2代尿微量清蛋白(ALBU2)在Cobas 8000C702全自动生化分析仪上的检测性能。方法 (1)精密度评价:以CLIA′88规定的允许误差TEa为依据,要求重复精密度1/4TEa,中间精密度1/3TEa;(2)线性范围及可报告范围评价:采用EP6-A方案,并延伸计算平均稀释回收率,以平均稀释回收率在90%~110%,评价临床可报告范围;(3)携带污染评估:评估血清清蛋白对尿量微量清蛋白检测的携带污染,以携带污染率≤0.5%标准判定;(4)方法比对分析:以Siemens BNⅡ为参考系统,将罗氏Cobas 8000C702与BN2结果进行相关性比对分析。结果重复精密度:低浓度CV为1.98%,高浓度CV为1.64%;中间精密度:低浓度CV为4.35%,高浓度CV为1.20%。线性范围验证:测量范围为5.6~413.55mg/L;临床可报告范围:最大稀释倍数为30倍,临床可报告范围为5.6~12 406.5mg/L。携带污染率:血清清蛋白(42.6g/L)对尿液微量清蛋白(6.9mg/L)的携带污染为0.28%。室内比对:标本浓度在200mg/L时,回归直线为Y=0.896 X+5.049,r2=0.994 4,医学决定水平处系统偏移通过;当标本浓度在201~413.55mg/L时,回归直线为Y=0.848 X-10.44,r2=0.917,医学决定水平处系统偏移未通过。结论罗氏ALBU2在Cobas 8000C702平台上检测能满足临床的需求,不同仪器间的比对,在不同浓度范围内有差异,应根据各自仪器的系统建立独立的参考范围。     

One-step sandwich enzyme immunoassay for insulin using monoclonal antibodies

An enzyme-linked immunosorbent assay for the measurement of insulin in human serum has been developed. The test is based on the sandwich technique with two monoclonal antibodies directed against two different epitopes of insulin using coated plastic tubes as the solid phase and horse radish peroxidase as the label. The immunoreactions are completed in one step within 2 h. The horse radish peroxidase activity bound to the tube wall is measured photometrically after an additional 1-h incubation with the substrate. The standards used cover the range from 0 to 260 mU insulin/L. Employing the Enzymun-Test System ES 22 modular batch analyzer, the detection limit was found to be 3.7 mU insulin/L. Coefficients of variation (CV's) between 1.4-7.8% for intraassay precision and 5.6-10% for interassay precision were obtained over the concentration range of 17-107 mU Insulin/L. The correlation between the procedure described here (y) and a commercially available double antibody radioimmunoassay (x) is expressed by the following equation: y = 1.07x + 1.14 mU insulin/L.     

COMPARISON OF FLUORESCENCE POLARIZATION IMMUNOASSAY AND HPLC FOR THE DETERMINATION OF THEOPHYLLINE IN SERUM

Theophylline in serum was measured by fluorescence polarization immunoassay (FPIA) and by high-performance liquid chromatography (HPLC). Within-run precision studies using control samples in the subtherapeutic, therapeutic and toxic concentrations, resulted in coefficients of variation in the range of 2.86-3.12% (FPIA) and 2.1-3.66% (HPLC), respectively. Between-run precision ranged from 2.76-6.2% for FPIA and from 2.51-6.0% for HPLC. The mean recovery for three spiked controls was 98.9% for FPIA and 98.8% for HPLC. Comparison of 60 patients' samples, assayed with both methods, indicated an extremely good analytical correlation (r = 0.990). The FPIA method displayed a slight but consistent positive bias in relation to the concentration of theophylline present in patients sera. Caffeine was found to exhibit a positive bias to 13%, over a caffeine concentration range of 10-40 micrograms/ml. The HPLC method offers an advantage for measurements of both caffeine and theophylline simultaneously. The FPIA offers significant advantages in speed of analysis and turnover-time, while maintaining accuracy and precision compared with those of established HPLC procedures.     

alpha-Amylase determination using maltopentaose as substrate

The rationale of choosing a NADP-coupled continuous method, with the substrate maltopentaose, as a method for the determination of alpha-amylase (EC 3.2.1.1) activity is investigated. The method presented is investigated with respect to all reaction parameters, including possible influence of protein, and shows zero order reaction kinetics after a 5-6 minute lag phase. The blank reaction from maltopentaose substrate is constant and is 13% of the upper limit of the reference interval for serum. The course of the blank reaction can be used to check that the maltopentaose is of adequate purity for use in the assay. Km for maltopentaose is 0.48 mmol/l. There is no interference from endogenous glucose when the total NADP turnover is less than 0.25 mmol/l. Data for sensitivity, linearity and long term precision over an eighteen month period are given, together with reference intervals for serum and for urine. The method is recommended for consideration as a reference method.     

Reference interval for serum alpha-amylase determined with p-nitrophenyl-alpha-D-maltoheptaoside as a substrate

The reference interval for the catalytic concentration of alpha-amylase (EC 3.2.1.1) in serum was determined in 326 blood donors. The analytic method used p-nitrophenyl-alpha-D-maltoheptaoside as substrate, which allows a continuous colorimetric measurement at 37 degrees C of the catalytic concentration of alpha-amylase in serum. The limits obtained were 0.7-3.8 mukat/l (42-228 U/l), which were in agreement with the only previous report found.     

Improved assay for alcohol dehydrogenase activity in serum by centrifugal analysis

We describe an improved method for determination of alcohol dehydrogenase (EC 1.1.1.1) activity in 60 microL of human serum, based on conversion of ethanol to acetaldehyde with simultaneous reduction of NAD+ in glycine NaOH buffer (pH 9.0) at 37 degrees C in a centrifugal analyzer. The final concentration of NAD+ was 10 mmol/L and ethanol was 20 mmol/L. The dilution curve was linear with enzyme activity up to 200 U/L, and results by this method correlated well with those by a manual method (N Engl J Med 279: 241-248, 1968). Within-run precision (CV) was 0.9 to 8.2% over the range of 4.5 to 88.1 U/L, and day-to-day precision was 5.4 to 5.6%. In sera from 198 healthy individuals, mean alcohol dehydrogenase activity was 1.6 (SD 1.2, range 0-5) U/L. To evaluate the clinical utility of determining alcohol dehydrogenase, we measured the activity of alanine aminotransferase and alcohol dehydrogenase in sera from 470 patients with various diseases in our hospital, and found that results for the two enzymes did not correlate well.     

Comparison of precision for the kodak ektachem 400 and technicon autoanalyzer II creatinine methods

We have evaluated the precision of the Ektachem 400 thin film enzymic creatinine method and compared it to that obtained for an AutoAnalyzer II (AAII) picric acid procedure. The results of the study show that the Ektachem method yields imprecise creatinine values over the low concentration range (40-240 mumol/L). Run-to-run precision estimates at a creatinine concentration of 225 mumol/L (by AAII) yielded CV's of 9.3 and 2.6% for the Ektachem 400 and AutoAnalyzer II methods respectively. The Ektachem 400 yielded imprecise creatinine determinations even when sample ammonia concentrations were less than 150 mumol/L. Patient samples analyzed by the AAII and Ektachem 400 over the creatinine range 40-900 mumol/L were highly correlated (r = 0.994). The correlation (r = 0.779) was low for patient data over the creatinine range 40-160 mumol/L. Creatinine results determined by the Ektachem 400 showed improved precision when samples were evaluated in triplicate using Kodak's latest (No. 8.5) software revision.     

Methods and clinical evaluation of a new enzyme immunologic method for determination of thyroxin binding globulin (TBG) and T4/TBG quotient: a multicenter study

A heterogeneous enzyme immunoassay for the determination of thyroxine binding globulin (TBG) was developed and assessed in clinical trials in 12 laboratories. The assay is based on the competition principle and employs plastic tubes coated with goat anti-TBG. CV's between 1.4-8.9% for intra-assay precision and 2.9-8.6% for inter-assay precision were found over the concentration range of 4-40 mg/l TBG. In comparative studies using highly purified TBG as standard, values with Enzymun-Test TBG were found to be on average 30% lower than those obtained by various TBG-RIAs. A broad-base study, to determine reference values, was carried out on a group of control persons 18 to 50 years old without previous history of thyroid disease. This study revealed a median of 14.33 mg/l TBG, with 95% of all values between 9.6 and 18.5 mg/l TBG. The median in women of 14.5 mg/l TBG was significantly higher than in men (13.4 mg/l TBG). TBG values in hyperthyroid patients were within the reference range while those in hypothyroid individuals were elevated. Highly elevated TBG values were seen in women receiving oestrogen (median: 22.2 mg/l TBG) and in pregnant women (median: 28.5 mg/l TBG). The T4/TBG ratios made it possible to distinguish between euthyroid, hyperthyroid and hypothyroic subjects (median: 4.9, 11.3 and 1.0, respectively). These ratios were significantly lower in pregnant women (median: 3.1) than in the control persons.     

Development and evaluation of a simple, direct, solid-phase radioimmunoassay of serum cortisol from readily available reagents

A simple, rapid solid-phase radioimmunoassay for serum cortisol was developed using cortisol antibody distributed by the Scottish Antibody Production Unit and commercially available radioiodinated cortisol ligand. The assay involves a 1-h incubation at ambient temperature, using the antibody covalently linked by the easily performed carbonyldiimidazole method, to microcrystalline cellulose. A detailed comparison of the accepted 0.125 mol/l citrate, pH 4.0, and an alternative 0.1 mol/l phosphate/8-anilinonaphthalene sulphonic acid, pH 7.4, diluent demonstrated similar precision (within-assay 10% to 5%; between-assay 11% to 6% over the range 100-1000 nmol/l cortisol) and recovery (median recoveries: phosphate 98.6%, citrate 99.5%). Linear regression analysis of human serum cortisol concentrations in comparison with the 'Amerlex' cortisol RIA kit gave the following results: citrate = 1.029. 'Amerlex' - 3.5 nmol/l (n = 80, r = 0.967); phosphate = 1.017, 'Amerlex' + 26.2 nmol/l (n = 80, r = 0.961). Phosphate, pH 7.4 diluent was adopted as the diluent of choice, since it was economical of antibody and maintained good precision over a wider working range of cortisol concentration.     

The influence of the label on the quality of a solid-phase immunoassay: evaluation of a commercial ELISA kit for serum ferritin

A new Enzyme Linked Immuno Sorbent Assay (ELISA) kit for the determination of serum ferritin has been compared with another ferritin kit based on the Immuno Radio-Metric Assay (IRMA) approach, both assays containing similar antibodies. Based on these studies, we found the within-run precision of the ELISA (and IRMA) to have coefficients of variation of 4-10% and 2-6% respectively, over a concentration range of 12-600 micrograms/l. The between-run precision for the same concentration range exhibited a CV range of 9-13% and 7-11% respectively. The sensitivities were found to be 1.4 micrograms/l and 0.9 microgram/l. The mean recovery was 103% for the ELISA procedure. It was found that, using the serum dilution technique, the linearity reached to 1000 micrograms/l. In the ELISA procedure no influence from the so-called "high dose hook effect" was observed. While EDTA-plasma produced 6% lower values than serum in the ELISA technique, no interference from albumin, gamma-globulins and mild haemolysis was observed. Stability problems with the ELISA kit were not encountered. A comparative analysis of multiple specimens demonstrated nearly identical values with r = 0.994 and y = 0.87 x1.01. The quality and ease of operation of the ELISA approach compared with other techniques are discussed. In conclusion it is possible to replace a radio-label in an immunoassay with an enzyme-label with the same degree of reliability and other parameters of quality control exhibited by radioimmunoassays.     

Kinetic measurement of total amylase and isoamylase activities with a centrifugal analyzer

We evaluated a new kinetic assay for alpha-amylase (Phadebas IsoAmylase Test), using modified starch as the substrate and a CentrifiChem 400 centrifugal analyzer system. We determined isoamylase activities by using a selective inhibitor. Results were compared with those obtained with the chromogenic Phadebas dyed-starch technique. The method for total amylase appeared to be rapid and precise (CV = 4%) and results correlated well with the chromogenic method. Samples with activities up to 3000 arb. units/L can be analyzed without dilution. Glucose and pyruvate interfere with the assay, but hemoglobin and bilirubin do not. Pancreatic (P) and salivary (S) isoamylase activity can be determined with acceptable precision (CVP = 8%, CVS = 10%) by an automated routine procedure with commercially available reagents.