NSE时间分辨荧光免疫分析法的建立

采用夹心法建立神经元特异烯醇化酶（NSE）时间分辨荧光免疫分析技术（TRFIA）来测定血清中NSE的含量。以NSE单克隆抗体E1包被板,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu3＋及标记NSE单克隆抗体E7,发光增强系统为以β-二酮体为主的增强液。采用平衡饱和法建立NSE-TRFIA,数据采用双对数函数数据处理程序处理。本方法的连续批内和批间CV分别为2.4%和4.8%,平均回收率为102.6%,灵敏度为0.31ng/mL,可测范围为0.31～320ng/mL,ED20、ED50和ED80分别为20.72ng/mL、57.23ng/mL和157.25ng/mL。本方法与AFP、CEA均无交叉反应。Eu3＋标记抗体-20℃保存8个月免疫反应基本无损失,同批试剂连续8个月应用分析结果稳定。临床应用检测结果与E170所测值高度相关。实验结果表明,本文所建立的NSE-TRFIA的灵敏度、特异性、准确度等均符合临床应用要求。     

HBeAg时间分辨荧光免疫分析法的建立

采用平衡饱和法建立了乙型肝炎病毒e抗原(HBeAg)时间分辨荧光免疫分析法.以针对HBeAg的单克隆抗体G8包被板,双功能螯合剂异氰酸苄基二乙烯三胺四乙酸络合Eu3+及标记C4单抗,发光增强系统为以β-二酮体为主的增强液.数据采用Log-Logit法函数和四参数Logitc函数数据处理程序处理.结果表明方法的批内和批间CV分别为2.39%和5.28%,平均回收率为97.62%,灵敏度为0.58NCU/mL,可测范围为12.01-529.84NCU/mL,ED20、ED50和ED80分别为6.36NCU/mL、26.85NCU/mL和136.7NCU/mL.本方法与HBsAg有13.1%的交叉反应.Eu3+标记抗体-30℃保存6个月免疫反应性基本无损失,同批试剂连续5个月应用分析结果稳定.HBeAg时间分辨荧光免疫分析的质量参数优于EIA和IRMA.     

基于磁性微球AFP时间分辨荧光免疫分析法的建立

目的 建立基于磁性微球(磁珠)AFP时间分辨荧光免疫分析法.方法 用磁珠偶联标记抗AFP单克隆抗体(301M),Eu3标记抗AFP单克隆抗体(304M),采用双抗体夹心法建立基于磁性微球法的AFP-TRFIA,并对106名我院健康体检人员和住院患者进行该方法血清学检测.结果 其检测灵敏度为0.02 IU/mL,线性范围2.12～ 1210 IU/mL;癌胚抗原(CEA)、CA199、CAl25对AFP-TRFIA均无交叉反应.磁珠偶联301M 4℃保存3个月免疫反应性基本无损,而Eu3+标记304M于-20℃下保存6个月免疫反应性也基本无损;同批试剂连续3个月应用分析结果稳定,样品的平均添加回收率为98.75％,低、中、高质控的批内和CV分别为20.46IU/mL和4.75％,50.58 IU/mL和4.25％,120.51 IU/mL和2.89％.批间和CCV分别为19.54IU/mL和9.55％.46.32 IU/mL和7.38％,125.10 IU /mL和3.69％.血清AFP检测结果与传统时间分辨荧光法检测结果对比,结果高度相关,相关系数为0.976.结论 基于磁性微球建立的AFP时间分辨荧光免疫分析法敏感度、特异性、准确度等均符合临床应用要求,可用于临床血清标本中AFP含量的免疫测定.     

乙肝病毒表面抗体TRFIA方法的建立及其应用

建立乙型肝炎病毒表面抗体(抗-HBs)的时间分辨荧光免疫分析法(TRFIA).检测体系由纯化的HBsAg包被的微孔板、Eu3 标记的HBsAg、抗-HBs系列标准品和增强液组成,采用双抗原夹心法定量检测人血清或血浆抗HBs.测定药检所2、5、10 mIU/mL SAb血清盘均可准确检出,阴性血清(20/20)符合率为100%,CV为2.5%.测定范围为5～2000 mIU/mL,正常参考值小于10 mIU/mL.用本法与固相RIA、ELISA和ECLIA对1189份临床血样进行考核,并与参比试剂盒临床符合率一致,具有同等的临床诊断价值.     

弓形虫IgG时间分辨免疫荧光分析法的建立

建立了高灵敏弓形虫-IgG的时间分辨荧光免疫分析(TRFIA)检测方法.样品或参考标准加入到由弓形虫抗原包被的96孔微孔板上,用Eu3+标记羊抗人IgG,建立间接弓形虫-IgG TRFIA检测法.本法检测范围为0.7～200 U/mL, 灵敏度为0.7U/mL;方法的平均批内和批间CV分别为4.5%和5.6%;标准曲线可测范围为0.7～14U/mL. 本文建立的弓形虫-IgG的TRFIA法灵敏度高、稳定性好,具有良好的应用前景.     

双标记AFP及Free-β-HCG时间分辨荧光免疫分析法的建立

目的：研制能同时检测人血清AFP和Free-β-HCG的双标记时间分辨荧光免疫分析（TrFIA）试剂盒。方法：铕（Eu3＋）标记抗AFP单克隆抗体,用钐（Sm3＋）标记抗Free-β-HCG单克隆抗体,采用双抗体夹心法建立AFP/Free-β-HCG双标记TrFIA试剂,对试剂的各项性能指标进行评价。结果：AFP分析灵敏度为0.1U/ml,分析内和分析间的精密度分别为1.2%～5.9%和2.8%～6.3%,检测试剂的测量范围为（0.1～500）U/ml;Free-β-HCG分析灵敏度为0.25ng/ml,分析内和分析间的精密度分别为1.5%～6.2%和3.5%～5.6%,检测试剂的测量范围为（0.25～200）ng/ml。孕妇血样用本试剂盒与进口的同类试剂盒同时检测,AFP和Free-β-HCG的相关系数分别为0.987、0.968,具有较好的一致性。结论：自制AFP/Free-β-HCG双标记TrFIA试剂盒的各项性能指标均能达到临床检测要求,可替代国外同类产品。     

脂肪酸结合蛋白放射免疫分析方法的建立及初步应用

用人工重组的人脂肪酸结合蛋白(FABP)多次免疫大耳白兔,获取高效价的FABP抗体.用氯胺T法制备125I-FABP,再经Sephdex G-25纯化;抗原抗体反应采用平衡一步法,4℃培养24h后,经PR试剂分离结合和游离的标记抗原.该法测定范围5-405ng/mL,最低检出量为9ng/mL,批内和批间CV分别小于6.4%和8.5%.用该法测得人血清FABP含量:健康人(45名)为15.68±2.9ng/mL;重症监护病人(46例)为72.08±32.64ng/mL;心脏病患者(73例)为78.95±24.83ng/mL,明显升高.结果表明,本法稳定,简便特异,其灵敏度适于检出人血清FABP水平的变化.     

血清铁蛋白时间分辨荧光免疫分析试剂盒的研制

目的 血清铁蛋白(sFER)时间分辨荧光免疫(TRFIA)分析试剂盒的研制.方法 采用双抗体夹心法建立sFER-TRFIA试剂盒,并对试剂盒的各项指标进行评价.结果 试剂盒的线性测量范围为2.2～1 070 ng/mL,灵敏度为0.22 ng/mL,批内、批间的精密度分别为4.6%～7.2%、5.4%～8.9%,与癌胚抗原(CEA)、甲胎蛋白(AFP)和人白蛋白无交叉反应.86份血清样本用本试剂盒与国外其他试剂盒同时检测,其相关系数为0.994.结论 试剂盒各项指标均达到临床检测要求,可替代国外同类产品试剂盒.     

肺癌患者血清中游离DNA含量与肿瘤标志物相关性分析

通过对52例肺癌患者、8例肺部良性疾病患者和10名健康人血清游离DNA含量与肿瘤标志物(TM)CA19-9、CA125、CYFRA21-1、CEA和NSE的联合检测,对两种检测结果作相关性分析。将表皮生长因子受体(EGFR)作为肿瘤基因标志物,运用荧光定量RT-PCR方法检测血清游离DNA含量。结果显示,10名健康人血清游离DNA含量平均为18.81ng/mL(范围:0.17~54.64ng/mL)。若以19ng/mL作为血清游离DNA含量的阳性临界值,则87.5%的健康人低于此水平。8例肺部良性疾病患者游离DNA含量均值为76.86ng/mL(范围:5.33~189.7ng/mL),其中4例(50%)高于阳性临界值。52例肺癌患者游离DNA均值为107.6ng/mL(范围:6.39~1617ng/mL),37例(71.2%)的肺癌患者血清游离DNA含量高于正常值。血清游离DNA含量诊断肺癌的价值等同于肿瘤标志物五项指标联合检测。其灵敏度、特异性、准确性分别为:71.15%,50%,68.3%,提示血清游离DNA含量作为一项新颖肿瘤标志物具有潜在的临床诊断价值。     

β2微球蛋白时间分辨荧光免疫检测法的建立及初步应用

为了建立β2微球蛋白(β2-m)时间分辨荧光免疫分析。以羊抗兔抗体包被板条,双功能螯合剂异氰酸苄基二乙烯三胺五乙酸络合Eu3+及标记β2-m,发光增强系统为以β二酮体为主的增强液。采用竞争法建立β2-m时间分辨荧光免疫分析,数据采用双对数法函数和四参数logistic函数数据处理程序处理。结果此法的批内和批间CV分别为2.25%和2.91%,平均回收率为101.06%,灵敏度为0.06mg/L,可测范围为0.06～12mg/L。本方法与白蛋白、肌红蛋白均无交叉反应。溶血对本法无影响。临床初步应用所检测结果与放免法吻合。试验结果表明,β2-m-TRFIA法的敏感度、特异性、准确度等指标符合临床诊断要求。     

癌胚抗原时间分辨荧光免疫测定法

本文报告了癌胚抗原时间分辨荧光免疫测定法。采用McAb包被微量滴定条,以双功能基团螯合剂标记Eu~(3+)为示踪物。本法最小检出值为0.7ng/ml,标准曲线范围为2.5～500ng/ml,批内和批间CV平均为7.09％和10.56％,回收率为106.00％。测定方法简便快速,每天一个技术人员可完成300份样品。     

Tissue engineering of flexor tendons: optimization of tenocyte proliferation using growth factor supplementation

A significant problem in flexor tendon repair is the lack of suitable graft material for reconstruction. The ex vivo production of flexor tendon graft constructs requires the expansion of primary cells. Growth factors, such as platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor-1 (IGF-1), and basic fibroblast growth factor (bFGF), are known to promote tendon healing and tendon cell proliferation. The purpose of these experiments was to optimize tenocyte proliferation in 3 tendon cell populations using growth factor supplementation. Cells of the synovial sheath, epitenon, and endotenon were isolated from rabbit flexor digitorum profundus tendons and maintained in culture. Cell cultures were supplemented with IGF-1, PDGF-BB, and bFGF alone and in combination. The conditions used for individual growth factor supplementation were IGF-1 (10, 50, and 100 ng/mL), PDGF-BB (1, 10, and 50 ng/mL), and bFGF (0.5, 1, and 5 ng/mL). The conditions used for combinations of growth factors were IGF-1 + PDGF-BB (50 + 10 and 100 + 50 ng/mL, respectively) and IGF-1 + PDGF-BB+ bFGF (50 + 10 + 1; 50 + 10 + 5; 100 + 50 + 1; and 100 + 50 + 5 ng/mL, respectively). For all 3 tendon cell populations, proliferation at 72 h was greater in the presence of individual growth factors as compared to controls. With PDGF-BB (50 ng/mL) supplementation, mean absorbance values increased 97% (0.57 to 1.13) in S cells, 37% (0.51 to 0.70) in E cells, and 33% (0.33 to 0.44) in T cells ( p < 0.001). In addition, a synergistic effect was observed. The combination of growth factors resulted in greater proliferation as compared to maximal doses of individual growth factors. In cultures supplemented with IGF-1 (100 ng/mL) +PDGF-BB (50 ng/mL), mean absorbance increased 114% (0.57 to 1.22) in S cells, 63% (0.51 to 0.831) in E cells, and 47% (0.33 to 0.48) in T cells ( p < 0.001). IGF-1 (100 ng/mL) + PDGF-BB (50 ng/mL) + bFGF (5 ng/mL) resulted in the greatest amount of cell proliferation for all 3 tendon cell populations. The mean absorbances increased 251% in S cells, 98% in E cells, and 106% in T cells ( p < 0.001). In summary, IGF-1, PDGF-BB, and bFGF can be used in combination to maximize tenocyte proliferation. Synergism among growth factors may provide a means to facilitate tendon engineering.     

Detection of hepatitis B virus PreS1 antigen using a time-resolved fluoroimmunoassay

The hepatitis B virus (HBV) PreS1 antigen is expressed at the distal most region of the envelope protein and contains the hepatocyte receptor-binding site. The presence of the HBV PreS1 antigen in serum and liver of HBsAg-positive patients is a new marker used for diagnosing HBV infection, and is indicative of viral replication. Our objective is to establish a method of time-resolved fluoroimmunoassay (TRFIA) with higher sensitivity and broader detection range for detecting serum HBV PreS1 antigen. Eu(3+) labeling of antibodies was performed with respective labeling kits, and Eu(3+) fluorescence intensity was measured with an auto DELFIA1235 TRFIA analyzer. The established method was evaluated for its performance. Serum specimens (574 in total) from Wuxi People's Hospital were analyzed for PreS1 antigen using the TRFIA and ELISA. The precision, specificity, and sensitivity of the TRFIA were clearly better than ELISA. The detection limit was 0.01?ng/mL. The average recovery rate for PreS1 antigens was 103.3%. There was significant correlation between the PreS1 antigen results obtained by TRFIA and ELISA in 374 serum samples with HBV >10(3)?IU/mL (χ(2)?=?25.04, p?相似文献   

Two immunoassays for antimullerian hormone measurement: analytical and clinical performances

Today, serum antimullerian hormone (AMH) is considered as an interesting marker of fertility potential in women to determine follicular status and in men to evaluate testicular function. We study analytical and clinical characteristics of two AMH commercialized immunoassays: Immunotech and DSL methods. The detection limits (close to 0.13 ng/mL), functional sensitivities (close to 0.30 ng/mL) are equivalent, and imprecision results are acceptable for entirely manual assays. The Immunotech method is linear within the calibration range (up to 21 ng/mL) and the DSL method presents a lack of linearity making it accurate only up to 11 ng/mL (and not up to 14 ng/mL as it is indicated by the manufacturer). The two methods allow to measure human AMH, don't cross react with TGF-beta superfamily proteins and the DSL immunoassay recognize mouse (25%), rat (68%) and calf (100%) AMH. The comparison between the two methods (from 0.3 to 200 ng/mL) shows a good correlation (r = 0.979) with not statistically different results (p = 0.31). From a clinical point of view, the two methods allow the evaluation of follicular status in normo-ovulatory women and in women with polycystic ovary syndrome. Results are in agreement with studies showing that AMH serum concentration is strongly correlated with the number of antral follicles. In conclusion, the Immunotech method seems to be more efficient than the DSL method even if the two methods are suitable for clinical applications needing AMH measurements.     

Importance of prostate-specific antigen (PSA) as a predictive factor for concordance between the Gleason scores of prostate biopsies and RADICAL prostatectomy specimens

OBJECTIVE:

To evaluate the concordance between the Gleason scores of prostate biopsies and radical prostatectomy specimens, thereby highlighting the importance of the prostate-specific antigen (PSA) level as a predictive factor of concordance.

METHODS:

We retrospectively analyzed 253 radical prostatectomy cases performed between 2006 and 2011. The patients were divided into 4 groups for the data analysis and dichotomized according to the preoperative PSA, <10 ng/mL and ≥10 ng/mL. A p-score <0.05 was considered significant.

RESULTS:

The average patient age was 63.3±7.8 years. The median PSA level was 9.3±4.9 ng/mL. The overall concordance between the Gleason scores was 52%. Patients presented preoperative PSA levels <10 ng/mL in 153 of 235 cases (65%) and ≥10 ng/mL in 82 of 235 cases (35%). The Gleason scores were identical in 86 of 153 cases (56%) in the <10 ng/mL group and 36 of 82 (44%) cases in the ≥10 ng/mL group (p = 0.017). The biopsy underestimated the Gleason score in 45 (30%) patients in the <10 ng/mL group and 38 (46%) patients in the ≥10 ng/mL (p = 0.243). Specifically, the patients with Gleason 3 + 3 scores according to the biopsies demonstrated global concordance in 56 of 110 cases (51%). In this group, the patients with preoperative PSA levels <10 ng/dL had higher concordance than those with preoperative PSA levels ≥10 ng/dL (61% x 23%, p = 0.023), which resulted in 77% upgrading after surgery in those patients with PSA levels ≥10 ng/dl.

CONCLUSION:

The Gleason scores of needle prostate biopsies and those of the surgical specimens were concordant in approximately half of the global sample. The preoperative PSA level was a strong predictor of discrepancy and might improve the identification of those patients who tended to be upgraded after surgery, particularly in patients with Gleason scores of 3 + 3 in the prostate biopsy and preoperative PSA levels ≥10 ng/mL.     

Ultrasensitive indirect competitive ELISA and strip sensor for detection of furazolidone metabolite in animal tissues

Nitrofurans are a class of drugs, which are typically used as antibiotics or antimicrobials illegally in aquiculture. In this paper, indirect competitive enzyme-linked immunosorbent assay and strip sensor were developed for detection of furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ). These two methods could detect AOZ with high sensitivity and specificity. The immunosorbent assay with the half-inhibitory concentration of 0.074?ng/mL has a good recovery from 79.9% to 119.8% for detection of AOZ in pork, chicken, fish, shrimp, pork liver and chicken liver. Based on the developed strip sensor, the detection limit was 0.05?ng/g by the naked eyes and the cut-off value was 0.3?ng/g. The cross-reactivity with other nitrofurans, metabolites and some other compounds was all less than 0.1%. The sample recoveries ranged from 97.3% to 115.5%. Both these methods could be used for on-site detection of furazolidone metabolite at trace level in animal tissues.     

Potential use of procalcitonin as a diagnostic criterion in febrile neutropenia: experience from a multicentre study

In order to assess the diagnostic value of procalcitonin, 158 patients with febrile neutropenia from centres across Europe were studied. Patients with fever were diagnosed on the basis of either: (1) clinical, radiological and microbiological criteria; or (2) the procalcitonin value. In the latter case, concentrations of 0.5-1.0 ng/mL were considered diagnostic of localised infection, concentrations of 1.0-5.0 ng/mL of bacteraemia, and concentrations of > 5.0 ng/mL of severe sepsis. Procalcitonin and C-reactive protein were estimated daily in serum by immunochemiluminescence and nephelometry, respectively. Overall, the sensitivity (specificity) of procalcitonin for bacteraemia was 44.2% (64.3%) at concentrations of 1.0-5.0 ng/mL, and 83.3% (100%) for severe sepsis at concentrations of > 5.0 ng/mL. It was concluded that procalcitonin is a marker strongly suggestive of severe sepsis at concentrations of > 5.0 ng/mL. Estimated concentrations of < 0.5 ng/mL indicate that infection is unlikely, but it was observed that bacteraemia associated with coagulase-negative staphylococci may fail to elevate serum procalcitonin levels.     

Steady-State Amprenavir,Tenofovir, and Emtricitabine Pharmacokinetics Before and After Reducing Ritonavir Boosting of a Fosamprenavir/Tenofovir/Emtricitabine Regimen from 200 mg to 100 mg Once Daily (TELEX II)

Abstract

Purpose: Evaluate how reducing ritonavir (RTV) boosting from 200 mg to 100 mg once daily (QD) affects steady-state pharmacokinetics of components of a fosamprenavir (FPV)-based regimen. Methods: Prospective, open-label, pharmacokinetic study in 12 HIV-infected patients stabilized on FPV/RTV 1400 mg/200 mg + tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) 300 mg/200 mg QD (TELEX II). Pharmacokinetics were assessed by noncompartmental analysis at baseline and 4 weeks after RTV reduction to 100 mg QD. Results: Baseline median minimum plasma concentration (C_min) and area under the plasma concentration-time curve over 24 hours post dose (AUC_24h) were as follows: APV: 1,708 ng/mL, 84,260 h ? ng/mL; tenofovir: 53 ng/mL, 2,420 h ? ng/mL; FTC: 58 ng/mL, 9,190 h ? ng/mL; RTV: 80 ng/mL, 10,230 h ? ng/mL. Four weeks after reducing RTV, changes in C_min and AUC_24h were: APV: +26%, +0.6%; tenofovir: +77%, +30%; FTC: +188%, +13%; RTV –64%, –79%. Component plasma concentration ranges were consistent with historical values. Median APV C_min was 14.7-fold above the protein-binding-adjusted 50% inhibitory concentration of wild-type HIV. Four weeks after RTV reduction, HIV-1 RNA levels remained <50 copies/mL in all patients, median CD4+ count increased from 465 to 495 cells/mm³, and favorable lipid changes and no adverse events were observed. Conclusion: Reducing RTV boosting from 200 to 100 mg QD of FPV/TDF/FTC QD conferred no detrimental effect on APV, tenofovir, FTC, or RTV pharmacokinetics and maintained virologic suppression.     

血清YKL-40和CA125联合检测在卵巢癌诊断中的应用

目的探讨血清YKL-40和CA125联合检测在卵巢癌诊断中的应用价值。方法分别用酶免分析法（EIA）和微粒子酶免分析法（MEIA）测定39例卵巢癌、27例卵巢良性肿瘤及40例健康对照妇女血清YKL-40和CA125水平,YKL-40以健康对照组95％可信区间的上限值为阳性,比较YKL-40和CA125在三组间、卵巢癌病人不同临床分期和手术治疗前后水平和阳性率的差异。结果正常对照组血清YKL-40水平的95％可信区间的上限值为71．9ng／mL;卵巢癌患者血清YKL-40水平及阳性率显著高于卵巢良性肿瘤组和对照组（t〉4．83,P〈0．01）,而卵巢良性肿瘤组和对照组之间差异无统计学意义（t=0．96,P〉0．05）。Ⅲ／Ⅳ期卵巢癌患者血清YKL-40水平显著高于Ⅰ／Ⅱ期患者（167．9ng／mL vs 87．7ng／mL,t=1．86,P〈0．01）。卵巢癌患者术后第3天和第7天血清YKL-40水平显著低于术前水平（t〉2．92,P〈0．01）;未能手术切除的患者血清YKL-40水平显著高于手术切除者（t=4．06,P〈0．01）。YKL-40联合CA125诊断卵巢癌的灵敏度为87．2％,特异性为91．0％,阳性预测值为85．0％,阴性预测值为92.4％;YKL-40与CA125联合诊断早期卵巢癌的灵敏度从CA125单指标的33．3％提高到66．7％。结论YKL-40是一种新的诊断卵巢癌的肿瘤标志物,联合YKL-40与CA125检测可提高对早期卵巢癌诊断的灵敏度。