铝残留含量检测电感耦合等离子体质谱仪法建立及优化

目的建立及优化电感耦合等离子体质谱仪(ICP-MS)法的铝(Al)残留含量检测方法。方法采用硝酸溶液作为消解试剂,对供试品及标准品进行处理,优化相应消解浓度及设备参数,并考察优化后检测方法的专属性、线性、重复性、准确性、检测限、定量限、中间精密度等。确认该方法是否适用于人血白蛋白中Al残留含量的检测。结果硝酸溶液浓度:5%;消解时间:4 h。ICP-MS设备条件:射频功率:1 600 W,采样深度:10 mm,雾化器/载气流速:1.0 L/min,载气补偿流速:0.5 L/min,实验模式:标准模式,积分时间:0.2 s,数据采集:3次。专属性:Al高/低浓度加样回收率分别为92%,98%;RSD为3.5%,4.9%;线性:在0～40μg/L范围内,标准品/样品的Al浓度与光能量信号值(cps)相关系数r均>0.999 0;准确性/重复性:高中低浓度样品Al加样回收率分别为:108%,110%,110%;RSD(%)分别为4.7%,4.9%,2.8%;检测限/定量限分别为:0.006μg/L,0.019μg/L;中间精密度:人员因素P>0.05,日期因素P>0.05,RSD_(12次)=2%,无显著差异。ICP MS法与原子吸收法(AAS法)检测结果比对:ICP-MS法与AAS法检测结果偏差为8%,加标样品结果偏差为3%,2种检测方法检测结果无显著差异。结论经优化后,Al残留量ICP-MS检测方法具有良好的专属性、线性、重复性、准确性、检测限、定量限、中间精密度,适用于本公司人血白蛋白制品的Al残留含量检测。     

LC-MS/MS检测血清25-羟基维生素D替代校准品基质的选择与评价

目的探讨如何选择和评价液相色谱-串联质谱(LC-MS/MS)检测血清25-羟基维生素D[25(OH)D]替代校准品基质。方法分别用4%牛血清白蛋白(BSA)溶液和30%乙醇水溶液2种替代基质,配制25(OH)D的标准溶液并绘制标准曲线。通过比较内标在不同基质中的响应值差异及基质效应混合实验、准确度验证,对替代基质进行全面的评价。结果内标在4%BSA和血清样本中的响应值差异无统计学意义(P>0.05),在30%乙醇水溶液和血清样本中的响应值差异有统计学意义(P<0.05)。4%BSA溶液通过了基质效应混合实验验证,30%乙醇水溶液未通过;使用4%BSA基质的标准曲线对室间质量评价样本的定量结果满足准确度要求;30%乙醇水基质未满足要求,有2个样本的定量结果偏移>25%。结论4%BSA可作为LCMS/MS检测人血清25(OH)D的替代校准品基质。     

对石油醚萃取法检测人血白蛋白制品中辛酸钠含量可靠性的验证

目的对石油醚萃取法检测人血白蛋白制品中辛酸钠含量的可靠性进行学验证。方法采用石油醚作为萃取剂,利用0.01 mol/L氢氧化钠滴定液确定辛酸钠的含量。结果回收率为0.98～1.01,变异系数(CV)(%)为0.60～1.53。结论利用石油醚萃取法检测人血白蛋白制品中辛酸钠含量的方法可靠,与现行中国生物制品规程所要求的气相色谱结果一致。     

高效液相色谱法同时测定血清色氨酸和犬尿氨酸浓度的方法建立及应用

目的建立一种高效液相色谱-紫外检测法同时测定血清中色氨酸(Trp)和犬尿氨酸(Kyn)浓度的方法。方法色谱柱采用Agilent Hypersil ODS(125.0mm×4.0mm,5μm),流动相为15mmol/L乙酸钠缓冲液(pH4.0)和乙腈95∶5(V/V),流速为0.8ml/min,柱温25℃,紫外检测λKyn=360nm,λTrp=278nm。结果Kyn和Trp的保留时间分别为3.3min和5.2min,线性范围分别为0.083~21.000μmol/L和2.650~678.000μmol/L,检测限分别为0.028μmol/L和0.053μmol/L,日内和日间相对标准偏差分别低于5%和10%,回收率分别为91.99%~113.89%和95.81%~118.82%。结论该方法简便、快速、特异,适合于临床检测。     

混合型固相萃取技术及毛细管电泳检测血浆和尿液中4种喹诺酮类药物

目的建立固相萃取及毛细管电泳法快速检测血浆和尿液中喹诺酮类药物洛美沙星、加替沙星、环丙沙星和氧氟沙星的方法。方法毛细管为熔融石英毛细管(75/365μm,40/47cm),运行缓冲液为40mmol/L硼砂缓冲液(pH 9.0),电压13kV,柱温为20℃,检测波长280nm。样品经固相萃取后进样分析。结果 4种喹诺酮类药物在6min内完全分离。在1~40μg/mL浓度范围内,洛美沙星、加替沙星、环丙沙星和氧氟沙星峰面积和浓度的线性关系良好,相关系数r分别为0.998 7、0.997 6、0.998 3和0.994 2。尿液和血浆中4种喹诺酮类药物加标回收率为80.1%~107.6%,精密度相对标准差(RSD)为2.1%~6.2%。结论该方法具有快速、简便、准确度和精密度高等特点,适用于血浆和尿液中洛美沙星、加替沙星、环丙沙星和氧氟沙星水平的快速检测。     

人凝血因子Ⅷ抗原含量ELISA检测方法的建立、验证及应用

目的双抗体夹心法测定标本中人凝血因子Ⅷ(human coagulation factor Ⅷ, FⅧ)抗原水平并进行验证和应用。方法建立检测人FⅧ抗原(FⅧ∶Ag)含量的双抗体夹心ELISA法;用人FⅧ/VWF标准血浆(NIBSC 07/316)标定内控标准品(S.A.R.P);根据方法学验证的要求对此方法进行线性、准确度、精密度、样品稳定性的研究分析。将验证后的该方法用于FⅧ纯化工艺的监控及产品质量分析。结果以NIBSC 07/316标定分装冻存的S.A.R.P的FⅧ∶Ag为1.12 Ag/mL,标准曲线的线性范围为(0.003—0.112)Ag/mL,各浓度回收率在97.62%—102.19%,CV<5%,R~2≥0.99;不同浓度样品检测结果的批内准确度在90.70%—95.23%,批间准确度在91.50%—94.20%;批内精密度CV值在1.69%—4.98%,批间精密度CV值在2.63%—4.83%。样品添加实验的回收率在87.50%—89.50%,特异性回收率在94.64%—101.78%。具有生物学活性的样品应-80℃冻存,复融1次后立即检测。武汉血制公司FⅧ纯化工艺中聚乙二醇4000(PEG 4000)沉淀和离子交换层析去除了大约60%的FⅧ抗原,工艺稳定性较好,生产规模可线性放大,不同来源产品FⅧ∶C/FⅧ∶Ag的比值接近。结论本方法具有良好的线性、准确度和精密度。用该方法进行FⅧ抗原含量检测可作为FⅧ纯化工艺内部质量控制的一个指标。     

毛细管气相色谱法测定消毒产品中邻苯二甲醛

为准确测定消毒产品中邻苯二甲醛的含量,采用毛细管气相色谱法,对以50%乙醇溶液稀释后的邻苯二甲醛样品进行了测定观察。结果,毛细管气相色谱方法的线性范围宽,线性相关系数r>0.9999。测定结果的精密度高,测得标准样品的相对标准偏差为0.7%~2.2%,实际样品的标准偏差为0.5%~4.0%。对3个消毒产品进行加标回收率实验,其平均回收率为97.6%~99.2%。方法检出限为2.0mg/L。结论,本方法操作简便、本实验条件下未发现干扰现象,方法的灵敏度、精密度与准确度高,完全适宜于测定消毒产品中邻苯二甲醛。     

血液制品密封性检查方法的建立与确认

目的建立和验证真空衰减法用于血液制品密封性检查。方法建立血液制品包装密封性检查方法,根据方法学验证的要求进行检测限、线性、范围、准确度、精密度、耐用性的确认;验证后的方法用于血液制品生产过程包装密封性检查。结果该方法检测限为2.5μm,线性相关系数r=1,阳性样品压差值在准确度允许范围之内,耐用性符合要求,重复性试验6次检测结果与中间精密度试验12次结果RSD均<10%,所有验证项目均符合可接受标准。结论真空衰减法可以用于检查血液制品包装密封性。     

分光光度法测定碘酊中的甲醇含量

目的建立分光光度法测定碘酊中甲醇含量的方法。方法经对样品特殊预处理,采用分光光度计对碘酊中的甲醇含量进行了实验室检测。结果用分光光度计,能检出甲醇含量在0.1~1.5 mg/m l范围内,甲醇浓度与吸光度呈线性相关;相关系数r=0.9998,回归方程为Y=0.2551X-0.0068。样品的平均加标回收率为93.17%~96.42%,精密度高,误差率(RSD)2.0%。结论分光光度法适用于测定碘酊中甲醇的含量,检测准确度高。     

电感耦合等离子体质谱检测人全血中的汞

目的研究电感耦合等离子体质谱法(ICP-MS)检测全血中汞的可行性。方法采用盐酸作为样本基质,用微波消解仪对全血进行消解,然后用ICP-MS检测全血汞,验证ICP-MS的检测下限、功能灵敏度、分析测量范围、回收率、准确度和精密度。结果 ICP-MS的检出限为4.525×10~(-2)ng/mL,功能灵敏度为1.495 ng/mL,分析测量范围为0.18~13.83 ng/mL,回收率为92%~103%。低值[(20±5)ng/mL]和高值[(50±8)ng/mL]质控品20 d检测均值分别为22.22和51.39 ng/mL,批间精密度分别为6%和5%;1 d内检测临床随机混合全血、低值和高值全血质控品各20份,批内精密度分别为4.8%、4.8%、4.4%。结论 ICP-MS检测全血汞可以满足临床要求。     

Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302

The amidolytic activity of activated kallikrein in plasma can be measured by use of the chromogenic substrate, S-2302 (H-D-Pro-Phe-Arg-pNA). Plasma prekallikrein was activated to kallikrein by exposure to 50 mg/L dextran sulfate in acetone/water (35/65 by vol) at 0 degrees C for 15 min. The acetone slows anti-kallikrein activity and increases the kallikrein activity by 30%. The 37 degrees C reaction mixture contained 0.54 mmol of S-2302 substrate per liter of Tris buffer (pH 7.5 at 37 degrees C). We monitored the change in absorbance at 405 nm for 60 s. The specificity of the substrate for kallikrein was demonstrated by using plasma deficient in prekallikrein (Fletcher trait) diluted with pooled normal human plasma. We recommend collecting blood specimens with sodium citrate as the anticoagulant and with use of a double-syringe technique and all-plastic containers. Plasma kallikrein activity with Chromozym-PK (Bz-Pro-Phe-Arg-pNA) as substrate (y-axis) compared with S-2302 as substrate (x-axis) gave the relation: y = 0.28x + 0.82 (r = 0.94). Day-to-day analytical variation was 2.4% for a pooled plasma with a mean value of 85.9 mukat/L. The mean (and 2 SD) for 50 healthy adults was 86.4 (32.4) mukat/L.     

血清碳酸氢根测定方法的改良

目的　改良血清碳酸氢根测定方法 ,提高准确性及精密度。方法　改良终点法为两点速率法 ,进行线性、准确性、精密度、回收试验、临床应用等研究。结果　速率法和终点法的变异系数分别为 3 .69%和 7.66%。临床应用结果显示两法具有一定的相关性 ( r=0 .93 8) ,检测结果的差异具有显著性 ( P<0 .0 5 )。速率法和终点法平均回收率分别为 96.5 %和91 .2 %。结论　改良后的速率法 ,简便易行 ,准确度及精密度高 ,值得推广应用。     

Plasma prekallikrein: quantitative determination by direct activation with Hageman factor fragment (beta-XIIa)

This report describes a plasma prekallikrein assay which, unlike methods that employ contact activation, is not affected by the factor XII or HMW kininogen content of the plasma analyzed. In this assay beta-XIIa, a potent fluid-phase activator of prekallikrein, is added to diluted plasma in the presence of 20% acetone (to inactivate kallikrein inhibitors) at 30 degrees C and the kallikrein generated is measured with the chromogenic substrate S-2302. Prekallikrein is fully activated under these conditions and the activity remains stable for at least 30 hr. The mean prekallikrein concentration in plasma samples from 24 healthy individuals was 1.50 +/- 0.35 (S.D.) S-2302 U/ml, corresponding to 20.3 +/- 4.7 micrograms/ml prekallikrein (the specific activity of highly purified human prekallikrein was determined to be 74 S-2302 U/mg). In contrast, the mean concentration in five plasma samples from patients deficient in HMW kininogen was 0.38 +/- 0.02 S-2302 U/ml. No activity was generated in prekallikrein-deficient plasma, and essentially normal levels (1.35 +/- 0.18 S-2302 U/ml) were measured in plasmas from three patients with factor XII deficiency. Plasma prekallikrein was also quantitated by radial immunodiffusion, which gave results similar to those obtained by functional assay with beta-XIIa. The determination of plasma prekallikrein by direct activation with beta-XIIa in the presence of acetone offers several advantages over the use of contact activators such as dextran sulfate. These advantages include complete inactivation of kallikrein inhibitors and total activation of prekallikrein (even in plasmas deficient in other contact factors) without simultaneous generation of plasmin.     

游离血红蛋白测定:三波长法和邻联甲苯胺法的比较研究

目的对三波长法和邻联甲苯胺法测定血浆游离血红蛋白(FHb)的准确性、线性、精密度、回收率、稳定性和操作简便性进行比较研究。方法分别用2种方法检测FHb标准品和全血样品中的FHb含量,依次进行准确性、线性、精密度、回收率、稳定性各方面的实验,比较并分析结果。结果三波长法检测得到的FHb标准品含量结果与真实值基本相符,并且直接计算即可得出结果,精密度和回收率都符合方法学要求,检测时呈色稳定;邻联甲苯胺法检测的结果需要外加参照才可获得,吸光度值A与FHb浓度在一定范围内(600 mg/L)才成线性,精密度不如三波长法,且呈色反应极不稳定。结论三波长法测定FHb结果真实可信,稳定性高,操作简便;邻联甲苯胺法测定FHb含量步骤相对较繁琐,线性范围较窄,注意事项多,稳定性差,比较而言,三波长法的应用前景更为广阔。     

Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction.Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at : 70°C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.     

PKA活性测定——微量滴定板方法的探讨

本文应用微量滴定板方法测定血液制品中残留的PKA活性.结果表明,用0.05M Tris/0.15M NaCl,pH8.0缓冲液,PKA和PK在室温下培育30分钟,然后加入激肽释放酶显色底物,在室温下再继续培育15分钟是适宜的.该方法重复性好,所测得IVIG中PKA的含量与其生产工艺、大白鼠降压实验相符,并与用芬兰红十字血液中心方法测得的结果基本一致.     

PLASMA PREKALLIKREIN: ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTIVATION

The precursor of the kinin-forming enzyme, prekallikrein, was isolated from rabbit plasma protected from activation during preparatory procedures. Prekallikrein was shown to be a 4.5S γ₁-glycoprotein with an isoelectric point of 5.9 and a mol wt of 99,900. The proenzyme was activated at neutral pH by an enzyme from rabbit or human plasma we have termed prekallikrein activator (PKA) or by trypsin. Prekallikrein was activated by PKA by a process of enzymatic scission. This resulted in the appearance of two fragments; the larger of these possessed kallikrein activity.     

Part of purified human plasma kallikrein removed together with a remaining IgG fraction--immunoblot experiments and functional tests

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved.     

Evaluation of a microassay for human plasma prekallikrein

Current methods for determining plasma prekallikrein, one of three zymogens of the contact phase of plasma proteolysis, are laborious and impractical for general use in a clinical laboratory. Therefore, we have developed a simple, reliable assay using commercially available reagents. By use of the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCI (S-2302), a functional assay, performed in a 96-well microplate, was designed to measure prekallikrein in plasma. Measures were taken to destroy the naturally occurring plasma protease inhibitors of kallikrein without affecting the integrity of the plasma prekallikrein, which allowed complete activation of the zymogen to virtually 100% of predicted activity when compared with that of purified kallikrein. Besides permitting full activation, the use of low pH to destroy critical plasma protease inhibitors allowed the conversion of prekallikrein to kallikrein in as many as 44 plasma samples at one time without the tedious individual timing step usually required to activate each sample. An excellent correlation was found (r = 0.92) when this functional microassay was compared with a functional spectrophotometric assay performed in three subject populations: normal individuals, women receiving oral contraceptives (who frequently exhibit high plasma prekallikrein concentrations), and patients with liver disease (who manifest low plasma prekallikrein levels). This plasma prekallikrein microassay should facilitate the increased determination of plasma prekallikrein in pathophysiologic conditions as well as the monitoring of the progression of various diseases in which contact activation occurs.     

火箭免疫电泳标化蛋白质标准品中的白蛋白

本文报道了用火箭免疫电泳标化蛋白质标准品中白蛋白的方法。白蛋白制品经95×1.4cm Sephadex G—200柱层析,取K_(AV)=0.45的白蛋白单体洗脱部分,用PAGE证实呈单一区带,浓缩后定值,以此作为标准进行白蛋白标化.探讨了该法的最适反应条件并进行了方法学的评价.对平均浓度为18.9、36.6、47.3g/L 的白蛋白样品进行重复性试验,批内变异系数分别为1.55%、1.45%和1.21%;批间变异为3.06%、2.5%和2.12%。回收率范围为97.6～101.8%。胆红素、血红蛋白、球蛋白及加入的防腐剂NaN_3不干扰标化.本文特异性强、准确性高、精密度好、有望成为蛋白质标准品中白蛋白标化的参考方法。