用ELISA双抗夹心法检测sHLA-Ⅰ在不同贮存成分血中的浓度

目的　观察贮存血中sHLA I浓度变化并探讨其意义。方法　采用ELISA双抗夹心法定量检测血清中sHLA I ,检测 6 0例正常广东人sHLA I水平 ,2 0名献血者成分血中sHLA I含量。结果　采用ELISA ,技术 ,可溶性HLA I最低检测限为 2 .84ng/ml,批内变异系数为 5 .80 % ,批间变异系数为 9.0 0 % ,回收率为 98.5 7%～ 1 0 0 .1 5 % ,广东人sHLA I参考值为 (6 99.5 4± 36 0 .1 0 )ng/ml。贮存 2 8d的RBC和随机供者血小板的sHLA I的浓度明显高于其它成分血 ,并且与成分血中残存的白细胞数和贮存时间有关。结论　用ELISA法检测sHLA I灵敏、特异、稳定。在临床输血过程中可考虑选择性输注含有不同浓度sHLA I的成分血     

用ELISA双抗夹心法检测sHLA-Ⅰ在不同贮存成分血中的浓度

目的观察贮存血中sHLA-I浓度变化并探讨其意义.方法采用ELISA双抗夹心法定量检测血清中sHLA-I,检测60例正常广东人sHLA-I水平,20名献血者成分血中sHLA-I含量.结果采用ELISA,技术,可溶性HLA-I最低检测限为2.84ng/ml,批内变异系数为5.80%,批间变异系数为9.00%,回收率为98.57%～100.15%,广东人sHLA-I参考值为(699.54±360.10)ng/ml.贮存28d的RBC和随机供者血小板的sHLA-I的浓度明显高于其它成分血,并且与成分血中残存的白细胞数和贮存时间有关.结论用ELISA法检测sHLA-I灵敏、特异、稳定.在临床输血过程中可考虑选择性输注含有不同浓度sHLA-I的成分血.     

ELISA测定血清可溶性HLA—Ⅰ类抗原

目的　建立定量测定可溶性HLA I类抗原的ELISA方法。方法　测定上海地区汉族正常人群血清可溶性HLA I类抗原的含量 ,并进一步研究可能影响这一含量的因素。结果　测得 116例上海地区汉族正常个体血清可溶性HLA I类抗原的含量为 5 2 9 3 2±2 82 88ng/ml。结论　性别和常见HLA A位点抗原型别对血清可溶性HLA I类抗原的含量未造成显著影响。     

可溶性人类白细胞抗原G在选择理想移植胚胎中的意义

目的 通过研究早期胚胎可溶性人类白细胞抗原G(sHLA - G)的表达,探讨sHLA - G在移植时选择理想胚胎的意义.方法 应用酶联免疫吸附实验(ELISA)定量检测体外培养2 d胚胎培养液中sHLA - G浓度.结果 ①60例胚胎培养液中sHLA - G阳性率为36.6%(22/60),其中妊娠组58.3%(14/24),未妊娠组22.2%(8/36);两者相比,差异有统计学意义(P<0.05);②胚胎培养液中平均sHLA - G浓度,妊娠组(2.0±0.8)U/ml,未妊娠组(1.2±1.0)U/ml;两者相比,差异有统计学意义(P<0.05);③双原核受精卵发育形成的胚胎sHLA - G 阳性率为36.6%,与未受精卵子阳性率(6.25%,1/16)相比,P<0.05,差异有统计学意义;与多原核受精卵发育形成的胚胎阳性率(25%,4/16)相比,P>0.05,差异无统计学意义.结论 早期细胞期胚胎有sHLA - G表达,其浓度高低与胚胎发育及种植有关,可作为选择理想移植胚胎的重要依据.     

可溶性白细胞介素-6受体结合链免疫放射检测方法的建立及应用

目的建立一种可溶性白细胞介素-6受体结合链(IL-6Rα)免疫放射检测方法.方法采用B淋巴细胞杂交瘤技术获取分泌鼠抗人可溶性IL-6Rα单抗的杂交瘤细胞株;经体内诱生腹水法制备单抗;免疫亲合层吸法纯化单抗;用 125I标记单抗后,采用竞争抑制分析法对单抗识别的抗原表位进行鉴定,选择识别不同抗原表位的2株单抗H126和SI10分别作为包被单抗和检测单抗,建立双单抗夹心的可溶性IL-6Rα免疫放射检测方法.分析该检测方法与可溶性IL-6Rα相关的细胞因子白细胞介素-6(IL-6)及可溶性IL-6信号传导链(IL-6Rβ,也称gp130)的交叉反应性;采用加入-回收法对检测方法的准确性进行鉴定,并绘制标准工作曲线,然后测定正常人及多发性骨髓瘤患者血清中可溶性IL-6Rα的含量.结果建立的可溶性IL-6Rα免疫放射检测方法的灵敏度为10 ng/ml,具有良好线性关系的检测范围是10～400 ng/ml,与IL-6及可溶性gp130(sgp130)无交叉反应性,加入重组可溶性IL-6Rα的回收率在92%～108%之间.正常人血清中可溶性IL-6Rα的含量为(81.96±7.23)ng/ml,多发性骨髓瘤患者血清中可溶性IL-6Rα的含量为(237.58±70.96)ng/ml,明显高于正常对照组(P<0.01). 结论该检测方法能够对血液中可溶性IL-6Rα进行准确定量,在可溶性IL-6Rα的基础研究及临床相关性疾病的辅助诊断中具有重要的意义.     

ELISA法测定人血清中apo A Ⅴ浓度

目的 建立测定人血清中apo AⅤ浓度的ELISA双抗体夹心法,并对该法的敏感性、重复性、干扰因素等作评价.方法 测定了该法的apo AⅤ标准曲线,最低检测限,批间、批内变异系数(CV),非特异性脂蛋白的干扰;测定了505例血脂正常人群apo AⅤ的浓度,建立本实验室的参考值.结果 本法的线性范围为(5.86～187.5)ng/ml;最低检测限为2.34 ng/ml;脂蛋白(a)、apo A Ⅰ和apo B用本法检测吸光度值均＜0.3;批内、批间CV均＜10%.结论 该方法灵敏度和特异性均好,为apo AⅤ的临床检测提供了一种可行的方法.     

双抗体夹心ELISA测定人血浆可溶性血管内皮钙粘蛋白方法的建立及其应用研究

目的:建立测定人血浆可溶性血管内皮钙粘蛋白(sVE-cadherin)浓度的双抗体夹心酶联免疫吸附试验(ELISA)方法,并探讨其临床应用价值。方法:应用原核表达的人VE-cadherin重组蛋白作为抗原免疫BALB/c小鼠制备单克隆抗体,采用棋盘格滴定法筛选出最佳双抗体组合,其中检测抗体采用辣根过氧化物酶(HRP)标记,建立检测人血浆sVE-cadherin双抗体夹心ELISA方法,并进行方法学评价。同时采用该法检测28名健康体检者和60例肿瘤患者的血浆sVE-cadherin浓度。结果:经筛选共获得6株单克隆抗体,通过筛查配对,成功建立检测人血浆可溶性VE-cadherin的双抗体夹心ELISA方法。该方法的检测限为24.7 pg/ml,批内变异系数(CV)为4.1%-7.7%,批间CV为8.7%-10.8%,平均回收率为96.7%。用该法检测28名健康体检者血浆VE-cadherin浓度为262.1±11.75 pg/ml,而用该方法检测24例白血病患者血浆VE-cadherin浓度为173.9±17.98 pg/ml,显著低于正常人(P0.01),14例胃癌患者血浆VE-cadherin浓度为311.7±25.24 pg/ml(P0.05),11例肺癌患者血浆VEcadherin浓度为206.8±25.01 pg/ml,均低于正常人(P0.05),11例其他肿瘤(9例乳腺癌,1例胶质瘤,1例肝癌)患者血浆VE-cadherin浓度为310.7±11.82 pg/ml(P0.05)。结论:建立的双抗体夹心ELISA试剂盒有较好的灵敏度及特异性,可用于人血浆中可溶性VE-cadherin含量的检测,并可作为肿瘤诊断的一种新检测指标。     

ELISA法测定人血清中apo AV浓度

目的建立测定人血清中apoAV浓度的ELISA双抗体夹心法,并对该法的敏感性、重复性、干扰因素等作评价。方法测定了该法的aimAV标准曲线,最低检测限,批间、批内变异系数（CV）,非特异性脂蛋白的干扰;测定了505例血脂正常人群aimAV的浓度,建立本实验室的参考值。结果本法的线性范围为（5．86～187．5）ng／ml;最低检测限为2．34ng／ml;脂蛋白（a）、apoAI和apoB用本法检测吸光度值均〈0．3;批内、批间CV均〈10％。结论该方法灵敏度和特异性均好,为apoAV的临床检测提供了一种可行的方法。     

尿胰蛋白酶原激活肽间接竞争ELISA检测法的建立及方法学评价

目的建立酶联免疫吸附法(ELISA)以检测人尿液中胰蛋白酶原激活肽(TAP)的含量。方法以TAP-BSA交联抗原为包被抗原,TAP为竞争抗原,两者与定量的TAPmAb反应,建立检测TAP的间接竞争ELISA测定法。结果最佳抗原包被浓度为250ng/ml,最佳单抗工作浓度为25μg/ml,HRP-抗小鼠IgG工作浓度为1∶4000。可测量最适范围为0.69~1000ng/ml,灵敏度为0.69ng/ml,批内和批间变异系数分别为9.10%和10.33%,平均回收率为97.70%。结论建立了定量测定尿TAP含量的酶联免疫吸附试验。     

层粘连蛋白酶联免疫分析方法的建立及初步应用

目的　建立酶联免疫吸附试验 ,对层粘连蛋白进行定量检测。方法　将鼠抗人层粘连蛋白单克隆抗体包被于聚苯乙烯微孔板 ,兔抗人层粘连蛋白抗体作第一抗体 ,酶标羊抗兔抗体作为第二抗体 ,在对有关试验条件进行优化后 ,检测 73份献血员标本。结果　本法的标准曲线为 2 5～ 16 0 0ng/ml,但在 5 0～ 80 0ng/ml范围内线性理想 ,其线性回归系数大于 0 .95。批内和批间变异系数分别为 8.6 %和 9.3% ,灵敏度为 2 5ng/ml,平均回收率为 98.1%。 17份样品用本方法与放射免疫法同时检测 ,其相关系数为 0 92 96。 73例献血员血清层粘连蛋白水平为 (115 .7± 17.3)ng/ml。结论　本方法可替代放射免疫法 ,用于人血清和组织液中层粘连蛋白的定量检测     

血清载脂蛋白A5 ELISA的建立及其初步应用

目的建立人血清中载脂蛋白A5(ApoA5)双抗体夹心酶联免疫吸附试验(ELISA),并初步观察其在2型糖尿病(T2DM)中的应用价值。方法用棋盘滴定法确定捕获抗体、检测抗体的最佳工作浓度;绘制标准曲线,确定线性范围、检测限;检测该方法的精密度和特异性,并分别检测T2DM患者及健康人血清中ApoA5水平以及其它生化指标。结果本研究建立的方法测定范围为5-640ng/ml,最低检出量2ng/ml,批内和批间变异系数(CV)分别为5.6%和6.7%。T2DM患者血清中ApoA5水平为(494.2±233.1)ng/ml,显著低于健康对照组(668.2±357.0ng/ml,P=0.001);血清ApoA5浓度与HDL-c呈正相关(r=0.16,P=0.031),与TG呈负相关,但是没有达到统计学差异(r=-0.028,P=0.717)。结论本研究建立的人血清ApoA5双抗体夹心ELISA灵敏度高,特异性好,可应用于临床ApoA5的检测;ApoA5可能是T2DM的独立危险因素,监测ApoA5浓度对于T2DM的预防和干预具有一定临床意义。     

Measurement of urinary annexin V by ELISA and its significance as a new urinary-marker of kidney disease

To confirm the significance of excretion of annexin V into the urine and the change of urinary annexin V concentration in kidney disease, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two monoclonal antibodies. Urinary annexin V concentration was measured in healthy individuals and patients with kidney and other diseases. Urinary annexin V did not change over a range of pH between 5.0 and 8.0, and was stable during the course of the study for 24 h at room temperature and for 8 days at 4 degrees C. The mean urinary annexin V concentration in 105 normal healthy individuals was 1.5+/-1.5 ng/ml, while that in patients with nephrotic syndrome and systemic lupus erythematosis (SLE) nephritis was 9.3+/-9.1 and 6.6+/-6.7 ng/ml, respectively, and that in IgA nephropathy and chronic renal failure was 2.6+/-2.1 and 1.3+/-0.7 ng/ml, respectively. Annexin level correlated with urinary protein concentration (r=0. 717), but not the serum creatinine concentration, blood urea nitrogen (BUN) and 24-h creatinine clearance. Mean urinary annexin V concentration in patients with ischemic heart disease, hypertension, and diabetes mellitus was 1.4+/-1.0, 1.4+/-1.1, and 1.7+/-1.3 ng/ml, respectively. In one case of relapsing nephrotic syndrome, the urinary annexin V concentration was markedly increased in the early phase after admission and then decreased. This patient later required hemodialysis. These results suggest that a high urinary annexin V concentration may be an indicator of acute renal injury related to the urinary protein level.     

Enzyme linked immunosorbent assays (ELISA) for the quantitative determination of human leukocyte collagenase and gelatinase

A competitive and a sandwich enzyme linked immunosorbent assay (ELISA) were developed for human leukocyte collagenase and gelatinase. The competitive assay could detect 0.5 ng collagenase and 0.05 ng gelatinase. The detection limit of the sandwich ELISA was 0.05 ng for collagenase and 0.02 ng for gelatinase. No cross reactivity between human leukocyte collagenase and gelatinase was detected. The sandwich ELISA was used to determine plasma levels of these enzymes. The 90% range for collagenase was between 0 and 50 micrograms/l; the 90% range for gelatinase was between 27 and 94 micrograms/l.     

人组织激肽释放酶6双抗夹心ELISA方法的建立及临床初步应用

目的通过制备的人组织型激肽释放酶6(hK6)单克隆抗体(mAb),建立双抗夹心酶联免疫吸附测定(ELISA)检测方法,并用于胃癌诊断。方法采用该实验室保存的hK6重组蛋白,免疫BALB/c小鼠后通过杂交瘤技术制备特异性的mAb,经鉴定纯化、酶标记后,建立双抗夹心ELISA方法,检测胃癌患者血清中hK6水平,并联合检测血清中的癌胚抗原(CEA)水平,探讨hK6作为胃癌生物标记的可行性。结果成功建立了检测血清中hK6的双抗夹心ELISA方法,并确定该方法包被抗体的最适浓度为5μg/mL,酶标记抗体的最佳稀释比例为1∶2 000。该方法检测各组血清中的hK6水平,与胃溃疡组hK6水平[(3.59±1.02)ng/mL]和健康体检组hK6水平[(3.35±0.67)ng/mL]比较,胃癌组hK6水平[(5.78±1.66)ng/mL]明显高于其他两组,差异有统计学意义(P0.05);而胃溃疡组与健康体检组hK6水平比较,差异无统计学意义(P0.05)。胃癌患者hK6和CEA检测的阳性率分别为69.70%和45.46%,两者联合检测的阳性率为78.79%。结论成功建立了hK6双抗夹心ELISA检测方法;hK6是较好的胃癌血清肿瘤标记物,同时检测hK6与CEA可提高胃癌的检出率,减少漏诊的发生,有助于胃癌的诊断。     

人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的评价与应用

目的对研制的人血清微量sCR1蛋白双抗体夹心ELISA试剂盒的重复性、稳定性进行评价,并了解其实际应用效果。方法选取中、晚期肝硬化患者50例和健康体检者50例分别作为肝病组和正常对照组,以鼠抗人CD35单克隆抗体、兔抗人sCR1多克隆抗体和辣根过氧化物酶标记的山羊抗兔IgG为包被抗体、夹心抗体和检测抗体,以纯化后的重组人sCR1蛋白为标准品,建立人血清微量sCR1蛋白双抗体夹心ELISA试剂盒,进行试剂盒重复性和稳定性检验,并应用该试剂盒检测两组血清sCR1蛋白水平。结果双抗体夹心ELISA法检测人血清微量sCR1蛋白的线性范围是15.60~250.00ng/mL;血清sCR1蛋白水平对吸光度值的回归方程为Y=112.10 X2+18.21 X+1.694(r2=0.998);重复性检测中,高、低水平标准品检测值的批内相对标准偏差(RSD)分别为6.20%、7.40%,批间RSD分别为6.70%和7.90%;试剂盒稳定性检测中,RSD均不大于0.01;肝病组血清sCR1表达水平显著高于正常对照组(P0.01)。结论人血清微量sCR1蛋白双抗体夹心ELISA试剂盒线性范围广、重复性好、易于保存,适合于临床和科研检测工作。     

高迁移率族蛋白B1单克隆抗体制备与竞争ELISA测定法的建立

目的制备高迁移率族蛋白B1(HMGB1)单克隆抗体和建立HMGB1的直接竞争ELISA测定方法。方法采用PEG细胞融合技术、有限稀释法和间接ELISA法制备和筛选杂交瘤细胞。直接竞争ELISA法测定大鼠血清HMGB1水平。结果共获得7株HMGB1特异性杂交瘤细胞株。建立的直接竞争ELISA方法的测定范围为1~320 ng/ml,回收率为97%~106%,变异系数为2.6%~7.3%。用该方法测定大鼠血清HMGB1水平发现,糖尿病大鼠模型HMGB1水平高于正常大鼠(P<0.001)。结论直接竞争ELISA可用于HMGB1的测定,该方法较双抗体夹心ELISA法更简便、快速。     

癌症患者血浆PAI-1浓度变化

目的测定多种恶性肿瘤患者血浆中纤溶酶原激活物抑制剂1(Plasminogen activator inhibitor-1,PAI-1)浓度变化情况,探讨PAI-1血浆含量变化作为恶性肿瘤诊断中的作用。方法收集乳腺癌、肺癌、喉癌血液标本各40例,并以正常人血液标本作对照,用ELISA法检测肿瘤患者及正常对照组血浆中PAI-1的含量,并比较其差异。结果与正常对照组(9.84±4.66 ng/ml)相比,乳腺癌(21.49±10.54 ng/ml),肺癌(15.10±8.55 ng/ml),喉癌(14.03±7.14 ng/ml)患者血浆中PAI-1含量均明显升高,其中乳腺癌组与正常组相比差异极为显著(P0.01)。结论血浆中PAI-1的浓度升高可作为肿瘤诊断辅助指标,PAI-1可作为肿瘤治疗潜在靶位。     

Human kallikrein 10 ELISA development and validation in breast cancer sera

BACKGROUND: Human kallikrein 10 (hK10) is a putative secreted serine protease belonging to the same gene family as prostate specific antigen (hK3; PSA). There is significant interest in measuring hK10 which may act as a tumor suppressor in some cancers. We have developed an ELISA for hK10 and determined its analytical and clinical performance in normal and breast cancer sera. METHODS: The assay used a previously described pair of monoclonal anti-hK10 antibodies and recombinant hK10 protein. Serum hK10 was detected quantitatively in a 2-step sandwich ELISA with colorimetric detection. The assay was analytically validated and used to determine serum levels of hK10 in a set of breast cancer, benign breast disease and normal samples. RESULTS: The assay covered a linear range of 0.2 to 15 ng/mL and had a detection limit of 0.08 ng/mL. The within-run and between-run imprecision was <9%. The average spike and dilution linearity recoveries were 96 and 103% respectively. Mean hK10 concentration in normal female sera was 0.79+/-0.26 ng/mL. Concentrations were not age related and were not significantly different from benign fibrocystic disease or breast cancer. However, in a subset of breast cancer patients with both early and late stage disease, serum hK10 levels were elevated, at >1.55 ng/mL, above all normal female and benign disease samples. CONCLUSIONS: We report in detail the analytical performance of a colorimetric hK10 ELISA validated in human serum and report for the first time the hK10 serum concentration in benign and breast cancer samples.     

Sensitive enzyme-linked immunosorbent assay for adult T-cell leukemia-derived factor and normal value measurement

Four different monoclonal antibodies against recombinant adult T-cell leukemia-derived factor (ADF), identical to thioredoxin, were established and used for the determination of ADF concentration in serum. Using two of the monoclonal antibodies, we developed a two-step enzyme-linked immunosorbent assay (ELISA) for ADF. This ELISA showed a highly specific reactivity on ADF with no cross-reactivity to several proteins with homologue sequence on the active center. The detection limit of the assay was 2.0 ng/ml (mean ± 2 SD). The intra- and interassay coefficients of variation (CV) were 0.81–3.74% (n = 8) and 4.78–6.97% (n = 7), respectively. The normal value of ADF mean concentration from 145 healthy donors was 40.8 ng/ml. © 1996 Wiley-Liss, Inc.     

悬浮红细胞上清中与肿瘤相关非细胞成分蓄积规律研究

本研究探讨悬浮红细胞（pRBC）上清中与肿瘤相关的非细胞成分在整个保存期中的蓄积规律。在采集当日对悬浮红细胞进行五联袋分装,于保存第0、7、14、21、28、和35d各取30ml红细胞悬液,以1006×g离心10min后取上清,采用ELISA方法测定正常T细胞表达和分泌因子（RANTES／CCL5）、肿瘤坏死因子α（TNF-α）、血小板衍生生长因子（PDGF）、血管内皮生长因子（VEGF）和单核细胞趋化蛋白-1（MCP-1）蓄积浓度。结果表明：RAN—TES／CCL5和TNF-α在新鲜pRBC上清中保持有较高的浓度,随着保存期的延长,其蓄积浓度未见有明显增高。VEGF在保存当天的pRBC上清中浓度为229．9pg／ml,保存7d末浓度迅速上升至749．08pg／ml,之后较稳定地维持在高水平,保存35d末其浓度为760．67pg／ml,其浓度曲线显示了保存7d后的-个明显上升,但配对t检验未见有统计学差异。PDGF的蓄积规律表现为：PDGF在保存当天的pRBC上清中浓度为13．54ng／L,保存期内稳定上升,28d末略有下降,保存35d末其浓度迅速上升为22．13ng／L（P〈0．05）,显示浓度增长有统计学差异。MCP-1在保存当天的浓度为39．98ng／L,保存期内缓慢上升,35d末浓度为49．83ng／L。结论：随着保存期的延长,pRBC上清中生长因子类物质似乎呈现明显的增长趋势,而RANTES／CCL5和TNF-α在新鲜pRBC上清中即保持有较高的浓度,且随着保存时间的延长蓄积浓度未见有明显增高,看来同保存期的延长无明显相关。