Development and validation of an automated particle-enhanced nephelometric immunoassay method for the measurement of human plasma c1q

We have developed a sensitive immunoassay based on latex particle agglutination for measuring C1q concentrations in human plasma. In this simple and fast particle-enhanced immunoassay, we used carboxylated latex particles (diameter 210 nm) covalently coated with F(ab)₂ fragments of anti-C1q antibodies. These particles are incubated with diluted sample (400-fold) for 6 min at room temperature, with the resulting agglutination quantified by measuring the change of light-scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hr. This assay generates a standard curve in the range of 24–775 mg/L, showing intraassay and interassay precision of <8% and <10%, respectively. Dilution linearity was validated throughout the dynamic range of the assay. There were no interferences from bilirubin, Intralipid, haemoglobin, and rheumatoid factor. Results obtained in 45 clinical samples correlated well with those obtained by a commercial radial immunodiffusion method (r=0.936), and with those obtained by the Behring immunoprecipitation nephelometric test (r=0.950). The mean concentration in plasma from healthy subjects was 180 mg/L and the reference interval was from 128 to 237 mg/L. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human C1q.     

罗氏 Cobas e601与雅培 Architect i2000检测乙肝表面抗原的比较

目的：比较罗氏电化学发光检测仪Cobas e601与雅培化学发光微粒子免疫分析仪Architect i2000检测乙肝表面抗原的结果。方法选取在雅培 i2000检测仪上检测乙肝表面抗原结果为有反应性,且定量结果小于250 IU/mL 的标本,同时在罗氏 e601进行检测,分析比较检测结果间的差别,并进行直线相关和回归分析。结果共检测46份临床标本,剔除1份两台仪器上检测结果反应性不符的标本外,其余结果具有很好的相关性。其中雅培检测值为0．05～1．00 IU/mL 的15份,两者的直线回归方程为Y ＝17．49X ＋0．843,相关系数 r＝0．979;1．1～10．00 IU/mL 的15份,两者的直线回归方程为Y ＝15．72X ＋21．06,相关系数 r＝0．952;11～250 IU/mL 的15份,两者的直线回归方程为Y ＝29．17X -129,相关系数 r＝1．000。结论两种方法的检测结果具有很好的相关性,并可以通过公式进行相互换算。     

Technicon Immuno 1® PSA assay measures both free and alpha-1-antichymotrypsin-complexed prostate-specific antigen on an equimolar basis

In this study, we investigated the immunoreactivity of the Technicon Immuno 1® PSA assay, a monoclonal-polyclonal sandwich immunoassay, with free and ACT-complexed PSA. Assay calibrators prepared with free PSA (standard immuno 1 calibrators) and calibrators consisting of 90% ACT-complexed and 10% free PSA (90:10 calibrators) yielded virtually identical PSA recoveries at all concentrations tested. Concentrations of total PSA at ∼4 and 10 ng/mL, prepared in varying ratios of free PSA to PSA-ACT complex, recovered from 92–104% at 4 ng/mL and from 98–102% at 10 ng/mL. Additionally, an excellent correlation of serum total PSA values from a panel of 40 prostatic cancer patient samples was obtained using calibration curves generated with the standard Immuno 1 calibrators or the 90:10 calibrators. These results demonstrate that the Technicon Immuno 1® PSA assay measures free and ACT-complexed PSA on an equal molar basis. © 1996 Wiley-Liss, Inc.     

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。     

7种半胱氨酸蛋白酶抑制剂C检测系统的分析性能评价

目的评估7种半胱氨酸蛋白酶抑制剂C（CysC）检测系统的分析性能。方法应用美国临床实验室标准化协会（CLSI）系列文件评估5个颗粒增强免疫透射比浊（PETIA）检测系统和1个均相溶胶颗粒免疫法（SPIA）检测系统的精密度、正确度、空白限（LoB）、抗干扰性以及与SIEMENSBNⅡ颗粒增强免疫散射比浊（PE—NIA）检测系统（简称BNⅡ系统）的相关性和偏差。结果6个检测系统（以A—F表示）和BN1I系统在CysC浓度为0．8～5．0mg／L时的总批内变异系数（CV）均〈3．75％,总批间CV除E系统外均〈5％;正确度验证显示A—E系统和BNⅡ系统测定ERM—DA471的绝对偏倚分别为0．00、0．00、1．01、-0．01、-0．50和-0．35mg／L,相对偏倚分别为0％、0％、18．43％、-0．18％、-9．12％和-6．39％;测定CAP室间质评物显示仅D系统和BN1I系统较为理想。A～F系统及BNⅡ系统的LoB分别为0．00、0．00、0．31、0．01、0．10、0．06和〈0．05mg／L。干扰分析显示Hb≤13g／L、TG≤28mmol／L时,对A、B系统及BNⅡ系统无明显影响（干扰〈±10％）,其他系统均有不同程度干扰。相关分析显示A～F系统与BN1I系统相关性较好[相关系数（r）均〉0．975,P〈0．01]。偏差分析显示,A～F系统与BNII系统的平均偏差分别为－0．02、－0．10、－0．31、0．05、0．04、0．36mg／L。结论CysC各检测系统测定有证参考物质ERM—DA471的最大偏倚可达到lmg／L,最大相对偏倚为18．43％;其中PENIA在测定ERM—DA471和CAP室间质评物时结果低于PETIA;部分检测系统分析性能存在不足。     

Performance of opus immunoassays for thyroxine and beta-human chorionic gonadotrophin in serum

Two representative immunoassays for measuring thyroxine and β-subunit of human chorionic gonadotrophin in serum, using the Opus immunoassay analyzer, were evaluated by comparing them to the reference RIA for T4 and β-HCG enzyme immunoassay. Both assays were superior in accuracy and precision than the reference methods and exhibited good linearity throughout the concentration range needed for discriminating abnormally low and elevated concentrations from the established reference ranges of thyroxine and β-human chorionic gonadotrophin in serum. Correlation between the results of the Opus immunoassays and the reference assays for T4 and β-HCG was very good with correlation coefficients of 0.92 and 0.98, respectively. © 1996 Wiley-Liss, Inc.     

胰岛素化学发光法测定的建立与评价

目的：建立化学发光法测定胰岛素及临床糖尿病患者胰岛素治疗跟踪与稳定的可行性评估。方法：CLISA采用一步免疫分析夹心法与RIA的测定结果进行比较,并将该法运用于临床非胰岛素依赖型糖尿病进行观察。结果：CLISA测胰岛素的批内与批间的变异系数分别为4．8％和6．4％,平均回收率为98．6％,与RIA的相关系数0．8505（P＞0．05）。结论：CLISA法测胰岛素的各技术参数优于RIA法,能及时有效地满足糖尿病患者胰岛素治疗的临床需要。     

Are immunoassays for digoxin reliable?

The specificity of radioimmunoassay and fluorescence polarization immunoassay procedures for the measurement of digoxin has been assessed. Many steroids and lipids have been found to interfere and give false positive results for digoxin under the conditions of the study. Caution is recommended in the interpretation of digoxin measurements made by immunoassay procedures.     

Evaluation of the highly sensitive chemiluminescent enzyme immunoassay “Lumipulse HBsAg‐HQ” for hepatitis B virus screening

Background

Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, “Lumipulse HBsAg‐HQ”, a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the “Lumipulse G1200”.

Methods

Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in‐house.

Results

The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg‐HQ reagent for dilution testing showed good linearity in the 0.005‐150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg‐HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14‐day blood sample was positive. The sensitivity against HBsAg‐HQ “ad” and “ay” subtypes was 0.025 ng/mL. Comparisons among the HBsAg‐HQ, HISCL, and Architect HBsAg reagents were performed using the Bland‐Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg‐HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005‐0.05 HBsAg IU/mL.

Conclusions

According to these results, the Lumipulse HBsAg‐HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients.

     

两种检测高值甲胎蛋白出现钩状效应的方法研究

目的：探讨高值甲胎蛋白出现钩状效应的情况以及钩状效应对实验结果产生的影响。方法取其中79例原发性肝癌患者的高值甲胎蛋白（＞105μg/L）标本进行倍比稀释,用电化学发光法（罗氏）和化学发光法（索灵）检测并观察是否会出现钩状效应,并对其结果进行比较分析。结果通过两种方法检测发现在甲胎蛋白浓度超过4×104μg/L 的标本中有可能出现钩状效应,且出现钩状效应的点均在最大检测范围之上（罗氏的有效线性范围0～1210μg/L;索灵有效线性范围0～1000μg/L）。同时,用两种方法验证在线性范围内的结果线性良好。结论电化学发光法和化学发光法检测高值甲胎蛋白均存在钩状效应,并且因出现钩状效应的点在最大检测范围之上,从而较好的避免了因钩状效应而导致异常低值结果。建议在用这两种方法检测甲胎蛋白时,对于出现超过有效线性范围的结果进行有效稀释;而对于线性范围内的结果均可视为试验的真实结果。     

电化学发光免疫法和化学发光微粒免疫分析法在HBsAg检验中的价值比较

目的比较电化学发光免疫法(CI)和化学发光微粒免疫分析法(CMIA)在乙肝表面抗原(HBsAg)检验中的价值。方法纳入我院收治的乙型肝炎疑似病例396例,分别使用CMIA、CI法进行HBsAg水平的测定。以CMIA为金标准,分析CI对HBsAg的诊断灵敏度、特异度和符合率。计算CMIA、CI检测HBsAg的检测批次内和批次间精密度。使用Bland-Altman偏差分析对CI、CMIA的诊断一致性进行分析。结果以CMIA为检测金标准,CI检测的诊断灵敏度为95.03%(287/302),诊断特异度为97.87%(92/94),诊断符合率为95.71%(379/396)。CI、CMIA对HBsAg检测的批次内变异系数和批次间变异系数比较,差异无统计学意义(P>0.05)。CI和CMIA对HBsAg诊断的组内相关系数为0.896,CI、CMIA在对HBsAg的诊断中,一致性较好。经Bland-Altman偏差分析得,两种检测方法的偏倚程度置信区间为(-6.36~23.24),CI、CMIA在0~23 IU/mL的区域内具有良好的一致性,随着机体HBsAg水平的增加,偏差逐渐上升,2.02%(8/396)的点位于偏倚程度95%置信区间以外。结论与CMIA相比,CI在对HBsAg的检验中具有良好的诊断灵敏度、符合率以及精密度。此外,CMIA、CI对HBsAg的诊断一致性良好,然而在高水平HBsAg的检测中仍然存在一定偏差。     

不同方法学检测乙型肝炎血清标志物结果的评价分析

目的对酶联免疫吸附试验（ELISA）、时间分辨免疫荧光法（TRFIA）、化学发光法（CLIA）、电化学发光法（ECLIA）检测乙型肝炎血清标志物[乙型肝炎表面抗原（HBsAg）、乙型肝炎表面抗体（抗HBs）、乙型肝炎e抗原（HBeAg）、乙型肝炎e抗体（抗HBe）、乙型肝炎核心抗体（抗HBc）]的结果进行分析,评价不同方法学检测结果之间是否具有一致性。方法用ELISA、TRFIA、CLIA、ECLIA分别检测100例乙型肝炎病毒（HBV）感染者和50名正常体检者的乙型肝炎血清标志物。以ECLIA作为参考方法,通过Kappa检验评价不同方法学检测结果之间的一致程度。结果 4种方法检测乙型肝炎血清标志物的结果具有高度以上一致性,CLIA和ECLIA在检测敏感性上更有优势。结论目前广泛使用的检测乙型肝炎血清标志物的方法具有较好的一致性,为实验室结果互认提供了依据。其中ELISA适用于人群初筛,CLIA和ECLIA对于肝炎患者疗效判断更有价值。     

Effect of substrate size on immunoinhibition of amylase activity

Immunoinhibition assays are hypothesized to work by antibodies blocking substrate access to enzyme active sites. To test this hypothesis, the inhibition of amylase isoenzymes by monoclonal and polyclonal antisera was assessed using substrates of varying sizes: chromogenic sustrates 3, 5, or 7 glucose units in length, novel synthetic macromolecular substrates, and starch. The synthetic macromolecular substrates consisted of small oligosaccharide substrates linked to an inert polymer that conferred a large size to substrate molecules as determined by gel filtration chromatography. When substrate size increased, amylase activity could be inhibited equivalently by antibody concentrations that are 10-fold lower. Progressively less polyclonal serum was required to inhibit amylase activity as substrate length increased from 3 to 5 to 7 glucose units and as size was increased by linkage to a polymer. Different effects of substrate size were observed with two monoclonal antibodies. One monoclonal antibody blocked amylase activity independent of substrate size, while another monoclonal antibody had little inhibitory effect except using starch as substrate. We conclude that use of larger substrates can expand the repertoire of inhibitory epitopes on enzymes and convert a noninhibitory antibody into an inhibitory one.     

血清骨钙素N端中分子片段的测定及临床应用

目的　研究各年龄组血清骨钙素N端中分子 (N MID)片段的含量 ,了解其稳定性 ,并探讨与各种骨代谢疾病的关系。方法　用电化学发光免疫测定法 (ECLIA)测定血清骨钙素N MID片段 ,其中对照组 183例 ,各种骨代谢疾病患者 99例。将样品放置于室温和 4℃中 ,于不同时间分别测定血清骨钙素N MID片段和全段骨钙素。结果　女性绝经后含量显著升高 ,男性各年龄组差异无统计学意义 ,女性高于男性 ,各类疾病组与对照组比较差异均有显著性。样品在室温和 4℃冷藏 ,骨钙素N MID片段很稳定 ,而全段骨钙素逐渐下降。结论　用ECLIA法测定血清骨钙素N MID片段 ,灵敏度高 ,稳定性 ,能很好地反映骨转换的变化 ,是骨代谢疾病研究非常有价值的敏感指标。     

化学发光免疫法测定血清前列腺特异性抗原

目的调查前列腺癌、前列腺增生患者、前列腺炎患者和正常人血清中前列腺特异性抗原(PSA)含量。方法采用化学发光免疫法测定33例前列腺癌患者,89例前列腺增生患者,25例前列腺炎患者和20例正常人血清样本中的前列腺特异性抗原(PSA)。结果正常人血清PSA含量为1.46±0.84x±s,与报道相近;前列腺癌患者血清PSA含量明显高于正常人、前列腺增生患者和前列腺炎患者血清PSA含量(P<0.001);前列腺癌患者血清fPSA含量明显高于前列腺增生患者和前列腺炎患者(P<0.001);前列腺癌患者fPSA/PSA比值明显低于前列腺增生病人和前列腺炎患者,两者差异非常显著(P<0.001)。结论在前列腺癌患者发病后的不同时期进行血清PSA含量测定,对病情的监测和预后有一定意义。     

根据NCCLS-EP9-A2评价2种发光免疫分析法的一致性

目的根据NCCLS-EP9-A2对2种发光免疫分析法进行比对试验,研究2种方法检测结果的一致性。方法收集40例临床血清标本,分别使用Access分析仪和Elecsys2010分析仪同时测定血清标本IgE水平,分析2组结果的相关性和2种方法检测结果的一致性。结果2种方法所得的结果相关性良好(r=0．994),2组结果预期偏差在允许偏差范围内,可以接受。结论在严格按操作规程进行校准和质控在控的前提下,使用这2种发光免疫分析方法测定血清中IgE水平的结果基本一致,在同一实验室可以同时使用这2种方法检测相同项目。     

健康人群胰岛素测定稳定性及参考范围调查

目的 建立本实验室人血清胰岛素(Ins)参考范围并分析测定稳定性.方法 对Architect I2000化学发光免疫分析系统进行方法学评价;对413例健康成年人进行血清Ins检测,分析生理和实验室指标对Ins的影响,建立Ins参考范围;对Ins在不同温度下的稳定性进行探讨.结果 Ins检测批内和天间不精密度分别为1.67%和2.60%.各年龄组Ins水平无统计学差异(P>0.05).女性Ins水平(中位数5.7 mU/L)高于男性(中位数5.0 mU/L)(z＝3.696,P＜0.05).以第97.5%位数为参考值上限,第2.5%位数为参考值下限,女性Ins参考范围为2.6～11.8 mU/L,男性为2.3～11.6 mU/L.胰岛素与体质量指数、三酰甘油呈正相关,与高密度脂蛋白呈负相关.Ins水平在25、4、-20 ℃至少可稳定4 h、24 h、7 d.结论 性别是影响人血清Ins水平的重要因素,应建立不同性别Ins参考范围;胰岛素水平在各种温度条件下并非很稳定.     

荧光偏振免疫法检测血浆同型半胱氨酸水平的临床评价

目的对荧光偏振免疫法(FPIA)检测血浆同型半胱氨酸(HCY)进行方法学评价，并初步探讨其临床应用价值。方法系统研究了FPIA法测定HCY的精密度、灵敏度、稀释线性、校准曲线的稳定性及干扰因素，并分析了其与高效液相色谱法(HPLC)检测结果的相关性。同时应用FPIA法测定了北京地区77名正常人及61例冠心病患者血浆HCY水平。结果FPIA法检测HCY具有较高的精密度(总CV为1.93%～3.60%)，分析灵敏度为0.21μmol/L，校准曲线至少可稳定3周，稀释标本回收率为92.28%～102.41%，黄疸、脂血和溶血现象基本不影响测定结果。FPIA法(X)与HPLC法(Y)具有良好相关性(Y=0.977X+0.690，r=0.992)。冠心病患者血浆HCY［(16.83±6.70)μmol/L］明显高于正常人［(9.67±3.00)μmol/L］(P<0.05)。结论FPIA法操作简单、结果准确可靠、自动化程度高，且检测速度快，具有较高的精密度，非常适合普通实验室应用。