连续监测法测定腺苷脱氨酶

目的 建立连续监测法测定腺苷脱氨酶的方法。方法 用连续监测法对 pH值、腺苷浓度等反应条件进行实验研究，并对所建立的方法进行评价。结果 用双试剂连续监测法；试剂Ⅰ：含α－酮戊二酸6mmol/L,NADH 0.35mmol/L,ADP0.8mmol/L,EDTA-Na2 0.1mmol/L，谷氨酸脱氢酶1000U／L，试剂Ⅱ：腺苷12mmol/L,均用磷酸盐缓冲液（0．1mol/L);pH7.2。线性范围达97U／L，批内CV＝4．90％，批间CV＝6．53％。与波氏显色法有良好的相关性（r=0.9902)。结论 本法操作简便，结果准确，可用于常规分析。     

液体单试剂酶法测定血清碳酸氢根

目的建立一种稳定的单试剂酶法测定血清碳酸氢根（HCO3^-）。方法利用自配单试剂建立了两点法的检测方法，可满足全自动和手工测定，用Bayer 1650生化仪对本方法进行方法学评价。结果本法的线性范围可达50mmol／L，批内变异系数（CV）为2．48％，批间CV为3．24％，平均回收率102．8％，与血气分析仪（X）相关良好，Y=1．019X-0．745，r=0．9903，黄疸、脂血、溶血标本对结果几乎无影响，健康人静脉血HCO3^-浓度为21．5～32．3mmol／L。结论本法操作简便、反应特异、灵敏准确、试剂较稳定，适用临床检测。     

血清葡萄糖及1，5-脱水葡萄糖醇双项同测的建立

目的建立新全酶终点法同时测定血清中1，5-脱水葡萄糖醇（1，5-AG）和葡萄糖（Glu）。方法用葡萄糖激酶（GK）和葡萄糖-6-磷酸脱氢酶（G6PD）偶联系统使血清中Glu彻底转化为不与呋喃糖氧化酶（PROD）反应的6-磷酸葡萄糖酸内酯（6-PGA），以其吸光度变化计算出Glu浓度；以PROD氧化1，5-AG，生成1，5-脱水果糖和H2O2，用Trinders反应比色测定出1，5-AG的浓度。结果1，5-AG在550μmol／L（Y=0．001X＋0．0658，r=0．999）以内，Glu浓度在40mmol／L（Y=0．051X＋0．128，r=0．9989）以内线性良好。Glu和1，5-AG的平均批内变异系数（CV）分别为0．88％和1．05％，批间CV分别为1．40％和1．94％。Glu和1，5-AG的平均回收率分别为100．2％和101．6％。三酰甘油（TG）≤8．9mmol／L、血红蛋白（Hb）≤4g／L、总胆红素≤350μmol／L时不影响Glu和1，5-AG的测定。本法测定l，5-AG与Lana微柱法及GK法与HK法测定Glu相关性均良好（r^2=0．999）。Glu与1，5-AG的95％参考值范围分别为3．7—5．7mmol／L和83．1～240．7μmol／L。结论本法简便快速、灵敏、准确和稳定，可自动化双项同测Glu和1，5-AG，为糖尿病的检测与治疗监控提供了新的方法。     

双试剂酚红比色法全自动测定血清碳酸氢根方法学研究

目的 建立一种能适合各级实验室常规和急诊检测血清HCO3^-的方法。方法 应用自配双试剂建立两步终点测定法，可同时满足全自动和半自动手工测定，用日立7060生化仪对本方法进行方法学评价。结果 比色液吸收蜂为558nm，线性范围可达50mmol／L，批内CV3．16％，批间CV4．55％，平均回收率102．3％，与血气分析仪测定结果相关性良好，Y=1．0059X-0．1038，r=0．9813(n=60)，黄疸、脂血、溶血标本对结果无影响，健康人静脉血HCO3^-浓度21.2～31．2mmol／L．结论 该方法操作简硬、灵敏准确、试剂稳定、成本低廉，可用于血清碳酸氢根的常规分析，更适合自动分析。     

对吡喃糖氧化酶法测定血清中葡萄糖的评价

目的对毗喃糖氧化酶法测定血清中的葡萄糖进行评价。方法进行方法学评价试验和方法间比较试验。结果检测限为0．039mmol／L,反应进程检测低值血清498s时吸光度趋于稳定,反应吸光度为0．35A;检测高值血清10min时吸光度趋于稳定,反应吸光度为2．0A,批内CV低、中、高值分别为0．70％、0．58％、0．70％,日间CV低、高值分别为2．32％、0．63％,线性范围36．61mmol／L,平均回收率102．7％,血红蛋白14．2g／L以下、总胆红素546．65μmol／L以下、甘油三酯25．78mmol／L以下、维生素C（Vc）100mg／dL以下无显著性干扰,与己糖激酶法相关,Y=0．9906X-0．0412,r=0．9998;与葡萄糖氧化酶法相关,Y=0．9980X-0．0379,r=0．9998,相关良好。结论该法简单、稳定,与己糖激酶法和葡萄糖氧化酶法相关性良好,能较好地抗溶血、黄疸、乳糜、Vc的干扰。     

己糖激酶法与葡萄糖氧化酶电极膜法测定血糖的比较

目的了解己糖激酶（HK）法和葡萄糖氧化酶电极膜法（GOD-膜法）测定血糖的性能。方法用上述两种测定血糖的方法．分别进行了精密度实验、灵敏度实验、可报告范围检测以及方法对比实验。结果①精密度：HK法低值定值血清总CV为1．95％．高值定值血清总CV为1．67％：GOD-膜法低值定值血清总CV为1．37％,高值定值血清总CV为1．68％;②HK法的灵敏度为1mmol／L．GOD-膜法的灵敏度为1．8mmol／L。③HK法的报告上限为55mmol／L,GOD-膜法的报告上限为45mmol／L。④方法对比实验：以HK法为比较方法（Y）。GOD-膜法为实验方法（X）,血糖的回归方程式为Y=1．0029X,R^2=0．9948。结论两种方法检测血糖的精密度、灵敏度、可报告范围及方法比对实验均符合临床要求,适用于血糖的常规和自动化分析。     

低密度脂蛋白胆固醇的直接测定法研究

目的用甾类糖苷化合物与胆固醇酯酶和胆固醇氧化酶制备的高亲和性酶化舍物,结合特殊表面活性剂,通过对测定条件的优化,建立了低密度脂蛋白胆固醇直接法方法。方法本方法线性范围达12mmol／L,与friedewal公式法（以下简称F法）计算结果及日本第一化学株式会社（以下简称日本一化）试剂测定相关性良好,分别为相关系数（r）：0．9955,Y—1．044X1-0．1535;r：0．9989,Y—1．0058X2＋0．0571。批内变异系数（CV）〈1．5％,日间CV〈2．0％,回收率（100土5）％。TG浓度达20mmol／L,抗坏血酸小于4．5mmol／L、血红蛋白小于4．5g／L和胆红素小于540μmol／L时无显著干扰。当用纯的不同浓度的高密度脂蛋白胆固醇加入准确定值的新鲜血清中,观察脂蛋白在血清中的反应。结果纯高密度脂蛋白胆固醇浓度在6．4mmol／L以内对本法无显著干扰。结论本文建立的低密度脂蛋白胆固醇直接测定法,其性能指标符合临床使用要求。标本无需预处理,精密度好,准确性高,适用于各种自动生化分析仪。     

低密度脂蛋白胆固醇(LDL-C)表面活性剂法检测试剂的研制

目的 研制低密度脂蛋白胆固醇（LDL-C）表面活性剂直接消除法测定试剂。方法低密度脂蛋白（LDL）与试剂Ⅰ中的表面活性剂Ⅰ形成稳定复合物，而非LDL中胆固醇被胆固醇酶、过氧化物酶消除，反应5min后，加入试剂Ⅰ，与表面活性剂Ⅱ和色原反应，LDL释放胆固醇并显色，LDL的浓度与呈色的深浅成正比。结果LDL-C检测试剂：在方法性能测定中，在2min的测定时间内，LDL-C浓度5．0mmol／L-27．0 mmol／L范围内，线性关系良好（r=0．9912），特异性良好，平均回收率为99．52％，LDL-C低、中、高值血清标本的批内和批间变异系数（CV）分别为2．6％-3．3％和3．5％-4．2％，乳糜微粒、胆红素和血红蛋白等于扰不明显；方法对比实验表明，其与PVS沉淀法、Friedewald公式法及其日本第一化学试剂相关良好，r值分别为0．967，0．9913，0．9779。结论 LDL-C检测试剂各项指标符合临床要求。     

血清乙醇脱氢酶活性测定及其临床意义

本文建立一种用自动生化分析仪双试剂法检测血清乙醇脱氢酶（ADH）活性的方法。同时对100例肝功正常者与9例原发性肝癌、10例肝硬化、40例肝炎患者测定血清该酶活性。在反应体系中，NAD 和乙醇浓度分别为10．0mmol／L和25．0mmol／L，缓冲液为pH9．00的0．1mol／L甘氨酸-NaOH溶液。ADH对NAD 和乙醇的Km值分别为1．67mmol／L和3．03mmol／L。批内CV为0．7％～5.4％。肝功正常者血清ADH活性为1．4±1．2U／L。原发性肝癌为11．4±2．5U／L，肝硬化为4．6±1．5U／L和肝炎为5．5±1．6U／L。这些患者血清ADH活性显著高于肝功正常者。原发性肝癌患者血清ADH活性显著高于肝硬化和肝炎患者。这种简便、准确、灵敏的测定ADH活性方法可用于临床常规分析。     

改良双缩脲双试剂二点终点法测定血清总蛋白的研究与应用

目的研究建立改良双缩脲双试剂二点终点法测定血清总蛋白的方法，以消除标本溶血、黄疸、脂浊、含葡聚糖的干扰。方法采用改良试剂，测定方法的精密度、回收率、检测限、线性范围、干扰试验，以确定所建立方法的基本性能；与Doumas法比较以确定方法的可靠性与消除干扰的效果。结果样品与试剂比例为5：220μl，线性范围达140g／L。回归方程：Y=484．0＋48．06X，r=0．9993。批内CV（20次）0．24％，日间CV（20d）1．07％。回收率95．6％～103％，平均99．5％。检测限2．36g／L。血红蛋白（Hb）2．1g／L以下，胆红素（Bil）148μmol／L以下，三酰甘油（TG）aS．2mmol／L以下，葡聚糖30g／L以下干扰不显著。改良法与Doumas法测定外观正常标本结果高度相关，回归方程：Y=-2．4354＋1．0374X，r=0．9882。结果间差异无统计学意义，t=0．156，P=0．877。测定溶血、高脂、黄疸、含葡聚糖标本二种方法间差异有统计学意义。结论采用改良双缩脲双试剂二点终点法测定血清总蛋白，能有效消除溶血、黄疸、高脂和葡聚糖的干扰，提高结果准确性。     

Automatic bioluminescent glucose determination using commercially available reagent kits coupled to the bacterial NAD(P)H-linked luciferase system

A sensitive and specific automatic bioluminescent method is described for glucose determination in serum samples using commercially available reagent-kits. The Boehringer Gluco-quant kit, originally designed for spectrophotometric measurement, was successfully coupled to the bacterial luciferase NAD(P)H-linked system. The method's validity was proven by comparison with a spectrophotometric method. Correlation was excellent (r = 0.98, n = 50). Precision attained by 30 assays was good (CV 1.19%). The assay was verified by determining the glucose concentrations of more than 1000 serum samples. Using a microcomputer-controlled automatic luminescence analyser (Berthold LB 950 T) and reagent kits for luminometry (Boehringer Mannheim, LKB Wallac, Lumac/3M), the complete assay can be performed fully automatically with commercially available reagent kits. More than 200 samples can be assayed by one individual per day. The bioluminescence method is at least 100 times more sensitive than spectrophotometric measurements. Other reagent kits tested (Behring, Merck, Sigma) are also suitable for coupling to the NAD(P)H-linked luciferase system.     

New chromogen for assay of glucose in serum.

We describe a colorimetric assay for glucose determination in human serum, with use of the chromogen 2-amino-4-hydroxybenzenesulfonic acid (AHBS), glucose oxidase, and peroxidase. With this assay, glucose concentrations less than or equal to 27.8 mmol/L can be measured in serum, with a sample/reagent volume ratio as low as 0.0025. The chromogen itself is easily soluble in water and does not require other components for the color change, making the reagent composition less complex. A single working reagent is used, and the reaction is completed within 10 min at 37 degrees C. The absorbance of the yellow reaction product is measured at 415 nm, and a blank sample measurement is not needed. The average analytical recovery of glucose in different human sera was 97.6%, with no significant interference of reducing compounds in serum. The results of the recommended procedure correlated well with those of the phenol/4-aminoantipyrine method of Trinder.     

Development and validation of an automated, enzyme-mediated colorimetric assay of salicylate in serum

This salicylate-specific assay can be adapted for use with most discrete analyzers, for rapid emergency or routine testing with small serum or plasma sample volumes and a single calibration. The basis of this method is as follows: salicylate monooxygenase (EC 1.14.13.1) converts salicylate to catechol in the presence of NADH; the catechol then reacts with 4-aminophenazone under alkaline conditions, catalyzed by manganese ions, to produce a red dye. Incorporation of an NADH-regenerating system, involving glucose and glucose dehydrogenase, into the enzyme reagent ensures that the working reagent is stable for more than two weeks. The standard curve is linear over the drug concentration range 0 to 5 mmol/L. The CV was less than 4% over 20 days. Results correlated well with those by the Trinder colorimetric method and an HPLC method. We saw no interference by any of 80 drugs we tested at therapeutic concentrations or by endogenous compounds in serum.     

Quantitative gas-chromatographic flame-ionization method for p-chlorophenoxyisobutyric acid in human serum and saliva.

We describe a gas-chromatographic method for p-chlorophenoxyisobutyric acid (I) the active metabolite of clofibrate. The drug and internal standards are separated from either serum or saliva by a double extraction procedure and converted to the corresponding butyl esters by reaction with iodobutane in a mixture of methanol and N,N-di-methylacetamide containing tetramethylammonium hydroxide. Within-run CV of this assay at a serum I concentration of 79.2 mg/liter was 2.3% and at a salivary I concentration of 2.5 mg/liter was 2.1%. Precision during four months of the serum and salivary assays at these concentrations was 4.1% and 6.2%, respectively. The mean serum concentration of I (12 h after dose) in patients receiving the drug at an average dose of 28.0 mg/kg per day was 109.6 mg/liter. Serum and salivary concentrations of I as determined by our procedure were used to calculate the unbound fraction of drug in human serum. Such measurements can be used to monitor therapy in patients with renal disease, where drug toxicity may arise from high concentrations of unbound I.     

Immunoturbidimetry of urinary albumin: prevention of adsorption of albumin; influence of other urinary constituents

I describe a simple, rapid immunoturbidimetric assay for low concentrations of albumin in urine (2 to 260 mg/L). However, in this assay, the human serum albumin (HSA) in the standards binds nonspecifically to the polystyrene or glass tubes. This nonspecific binding cannot be prevented by adding bovine serum albumin (BSA) to standards, because the anti-HSA antibody cross reacts with BSA. Adding Triton X-100 (1 mL/L) to standards effectively prevents this nonspecific binding of HSA from standards to both polystyrene and glass tubes. High concentrations of compounds found in urine from normal and diabetic subjects do not interfere with this assay if pH extremes can be avoided. The between-day CV is 4.8% at means = 18.8 mg/L and 2.0% at means = 183.1 mg/L. Measurements by this immunoturbidimetric method (y) correlate well with those obtained by a radioimmunoassay (x): y = 1.078x - 0.141 mg/L (n = 98; r = 0.984) and with those obtained by a radial immunodiffusion method (x'): y = 1.026x' - 0.117 mg/L (n = 98; r = 0.983). Urinary excretion of albumin by 25 healthy, nondiabetic subjects was less than 8 micrograms/min.     

Development of a rapid and sensitive immunofluorometric assay for glutathione S-transferase A

BACKGROUND: The short half-life of the alpha-class glutathione S-transferases (GSTAs) in plasma combined with their even distribution throughout the liver lobule suggests that they may be useful complements to the more traditionally used liver markers. However, the currently available assays for measuring GSTAs in biological fluids have a poor dynamic range and are cumbersome, requiring multiple steps and prolonged incubation times. METHODS: Hybridomas that secrete monoclonal antibodies to human GSTAs were produced and used to develop a rapid one-step immunometric assay for the determination of GSTA in serum. The assay uses a time-resolved immunofluorometric assay (TR-IFMA) format and requires 35 min of incubation. The reference interval was determined using 208 serum samples from healthy blood donors. We also compared our TR-IFMA with a commercially available enzyme immunoassay (EIA) for GSTAS: RESULTS: The assay had a detection limit of 0.07 microg/L with a measuring range up to 625 microg/L. Within-run imprecision (CV) was 1.8-2.6% over the concentrations of GSTA tested (2.5-311 microg/L), with a between-run CV of <5%. In healthy blood donors, the median values and reference intervals were 2.0 microg/L and 0.6-7.2 microg/L for females and 2.6 microg/L and 0.7-9.8 microg/L for males, respectively. GSTA concentrations determined with the TR-IFMA correlated well with those obtained using a commercially available EIA. CONCLUSIONS: This report describes a new assay for monitoring the concentrations of GSTAs in human serum. The method may be useful in further evaluating the potential of monitoring serum GSTAs in the routine clinical setting.     

Interlaboratory standardization of enzyme results: the Richmond project

Three concentrations of proficiency monitoring material and two concentrations of secondary standard calibrating material were prepared and stored frozen. The materials were prepared in buffer containing amylase from human saliva, aspartate aminotransferase from human liver, creatine kinase from human muscle, human serum albumin (20 g/L), and cofactors. The proficiency monitoring material was assayed by 10 methods in nine laboratories for 15 days to establish baseline performance. Each laboratory then used the secondary standard calibrating material to calibrate their instruments' responses to that of a standardization method, and repeated the assay of the proficiency monitoring material for 15 days. For amylase before calibration, between-laboratory mean values for the three concentrations of proficiency monitoring material were 29% lower than the standardization method, and the between-laboratory CV was 28%. After calibration the mean amylase values were 4% lower and the CV was 6%. For aspartate aminotransferase, the pre-calibration between-laboratory mean values were 24% higher than the standardization method (CV 14%) but only 3% higher (CV 6%) after calibration. CK activity deteriorated at storage temperatures above -70 degrees C. This study demonstrates that, by using a common secondary standard, laboratories can improve calibration of enzyme results.     

An enzyme linked immunosorbent assay (ELISA) for measurement of human serum thyroglobulin. Evaluation of the influence of thyroglobulin auto-antibodies

The aim of the study was to develop an enzyme linked immunosorbent assay (ELISA) for measuring thyroglobulin (Tg) in human serum and to evaluate the influence of serum thyroglobulin auto-antibodies (TgAb) on the ELISA. The sensitivity of the ELISA was 2.1 micrograms/l. Serum Tg levels in healthy controls were from less than 2.1 to 55.5 micrograms/l (n = 46) (95% reference range). With serum Tg concentrations between 19.6 to 90 micrograms/l the within-assay coefficient of variation (CV) was from 4.5 to 6.6% (n = 12) and the between-assay CV from 8.5 to 10.5% (n = 6). The recovery from 20 to 89 micrograms Tg/l serum was from 93 to 101%. There was significant correlation between serum Tg concentrations measured by the ELISA and a RIA method in healthy controls (r = 0.85, n = 46, p less than 0.001) and in patients with differentiated thyroid carcinoma (r = 0.97, n = 28, p less than 0.001). The TgAb interfered with the serum Tg determination both in the ELISA and in the RIA method. The assay is simple and easy to perform, and the equipment is inexpensive and useful for large-scale serum Tg measurements as an alternative to RIA.     

Measurement of glycated protein by a rapid and specific method for absolute quantification of lysine-bound glucose

We modified the liquid-chromatographic assay of Schleicher and Wieland (J Clin Chem Clin Biochem 1981; 19:81-7) for measuring lysine-bound glucose in serum proteins, increasing its performance and practicality. After precipitating serum proteins from 10- to 50-microL samples with ethanol (700 mL/L) and hydrolyzing these in 6 mol/L HCl, we inject 20 microL of the diluted hydrolysate directly into the chromatograph, which consists of an acid-resistant C18 precolumn combined with a high-resolution C18 main column. The eluent is 3.5 mmol/L H3PO4 solution containing 30 mL of acetonitrile per liter. These modifications increase sensitivity, provide excellent resolution and longevity of stationary phases, shorten assay times to 15 to 20 min, and are suited for automation. The assay is highly sensitive and highly specific, quantifying nanomoles of lysine-bound glucose per milligram of protein. A precision (CV) of 5.1% is achievable at physiological and supra-physiological glucose concentrations, and analytical recovery is 99%. This inexpensive method has been applied to serum albumin, bulk serum proteins, and preparations of low-density lipoproteins and immunoglobulins.     

Quantitative nephelometric assay for determining myoglobin evaluated

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of myoglobin (Behringwerke) was evaluated with the BNA Nephelometer. Method precision was good: the intra-assay CV varied between 1.5% and 6.1%; with daily calibration, the interassay CV ranged between 1.5% and 7.5%. For usual sample dilutions, the assay response varied linearly with myoglobin concentrations up to 23.1 nmol/L. After automatic dilution by the instrument, concentrations up to 2310 nmol/L could be measured without high-dose "hook" effect. Further manual dilution allowed measurement of myoglobin concentrations up to 26,000 nmol/L. Calibration was stable for at least seven days. We detected no significant interferences from hemoglobin, haptoglobin, bilirubin, iodine-containing contrast media, and rheumatoid factors. Treating lipemic samples with Lipoclean (Behringwerke) decreased test results. Simultaneously drawn serum and plasma samples from the same subject showed no consistent differences in myoglobin concentrations. The mean reference myoglobin concentration was 1.380 (SD 0.82) nmol/L for men and 0.878 (SD 0.45) nmol/L for women. In patients with renal insufficiency, serum creatinine values were moderately related to serum myoglobin values (r = 0.465). Although a commercial radioimmunoassay (Byk-Sangtec) and the nephelometric assay intercorrelated well (r = 0.929), values obtained by nephelometry were significantly lower (P less than 0.05). By both assays, results for heart and skeletal muscle tissue extracts showed no correlation, a finding that suggests the existence of multiple forms of myoglobin in human tissues. We conclude that immunonephelometry is a rapid, practical, and reliable method for measuring myoglobin in serum.