基于金磁微粒标记技术的可目视化蛋白质芯片检测方法的建立

目的：应用金磁微粒标记蛋白质技术,建立可目视化蛋白质芯片检测体系,比较金磁微粒和胶体金标记蛋白质技术应用于蛋白质芯片检测效果的优劣。方法：将人IgG点制于环氧基修饰的玻片上,分别与金磁微粒和胶体金标记的羊抗人IgG温育,银染显色,肉眼观察并用普通扫描仪记录结果。结果：基于金磁微粒的蛋白质芯片人IgG最佳点样浓度为0．2mg／ml,37℃温育2h,银染10～15min,检测结果信噪比高;基于胶体金的蛋白质芯片人IgG最佳点样浓度为0．1mg／ml,37℃温育1h,银染15～20min．检测结果信噪比高。结论：金磁微粒标记蛋白质技术应用于蛋白质芯片的检测,具有和胶体金一致的可目视化检测效果。且其标记技术简单,标记的蛋白质可定量。     

O型血孕妇血清IgG抗体效价与新生儿溶血病的关系探讨

目的通过比较母体血型免疫性抗体IgG效价与新生儿溶血病(HDN)发病率的关系,得出产前孕妇血型血清IgG抗体效价检测是优生优育的一个重要方面。方法选取2008年1～12月316对夫妇血型不合的"O"型血妊娠妇女(下称孕妇)及其新生儿为研究对象,用微柱凝胶反应卡分别检测孕妇血清IgG抗体效价及HDN溶血3项指标。结果316例受检孕妇血清IgG抗体效价在1∶64～1∶256之间有185例;在1∶512～1∶1024之间有100例;效价大于1∶1024有31例;HDN总发病患儿66例。结论孕妇血清中血型免疫性抗体IgG是引起HDN的主要原因,且随着IgG抗体效价的增高,HDN的发病率也增高。     

基于磁珠微阵列的多重血型检测技术

目的建立并评价基于磁珠微阵列技术的血型表型的鉴定方法。方法用Tris-HCl(0.01 mol/L,p H 7.4)裂解浓缩红细胞作为待检样本,将探针抗体包被于经醛基修饰的三维基片上,将待测样本与抗体进行反应,最后用C1q包被的磁珠识别抗原抗体复合物,在磁针的磁力吸附下将未结合的磁珠清除。显微摄影每个微阵列检测点,通过Photoshop图像分析判断信号强度。结果针对108个检测样本的5种抗原(A、B、D、M、N)进行分析,以含有40%甘油的PBS做为反应介质能有效避免咖啡环的形成,方法敏感性为86%~93%,特异性为98%~100%。通过C1q包被的磁珠识别抗原抗体复合物能在10~20 min完成检测,所需样本10μL。结论本方法有较高的敏感性与特异性,同一个芯片上可同时检测5种血型抗原,节约样本,且不受样本损伤的影响,为血型鉴定提供了一种新的手段。     

免疫金银染色法在蛋白质微阵列检测TORCH感染的实验研究

目的研制基于免疫金银染色法的TORCH检测蛋白质微阵列。方法将TORCH抗原点样于已经被琼脂糖膜修饰的玻片表面,与待测血清和胶体金标记的抗人IgM(μ链)孵育,用银增强微阵列信号,肉眼观察或普通的平板扫描仪记录结果。以不同浓度的纯化人IgM制备标准曲线。结果蛋白质微阵列的灰度质和IgM的浓度呈线性关系。60份待测血清与ELISA法同时检测,诊断符合率为93.3%。结论该法具有高信息通量、低成本、制备与检测快速简单的优点。     

孕妇产前IgG血型抗体效价预测ABO-HDN的诊断价值

目的探讨孕妇产前IgG血型抗体效价预测新生儿ABO溶血病(ABO-HDN)的诊断价值。方法选择56例O型Rh(D)阳性孕妇实行产前IgG血型抗体效价检测,并对其分娩后的新生儿进行ABOHDN 3项试验的检测。结果入组的孕妇IgG血型抗体以低效价为主,90.7%的孕妇抗体效价1∶256,其中25.9%(14/56)的孕妇抗体效价1∶64,24.1%(13/56)的孕妇抗体效价为1∶64。抗体效价为1∶128的孕妇占18.5%(10/56),抗体效价为1∶256的孕妇占22.2%(12/56)。对入组孕妇的新生儿进行ABO-HDN 3项检测,发现76.8%(43/56)的新生儿可确诊为ABO-HDN,23.2%(13/56)的新生儿未能证实ABO-HDN。当IgG血型抗体效价分别为1∶64、1∶64、1∶128、1∶256、≥1∶512时,相应的ABO-HDN检出率为56.3%、69.2%、90.0%、91.7%、100.0%。A型新生儿ABO-HDN阳性率为72.7%,B型新生儿ABO-HDN阳性率为79.4%,但两组差异无统计学意义(P=0.56)。对43例ABO-HDN患儿的母亲进行产前抗体效价分析,结果显示,在抗体效价1∶64时,尚有20.9%的新生儿发病。结论孕妇产前IgG血型抗体效价越高,提示新生儿发生ABO-HDN的概率越大,但产前IgG血型抗体效价1∶64依然有部分新生儿发生溶血病,提示该实验室所设定的界值需要进一步改进。     

3种方法检测O型血孕妇IgG血型抗体效价分析

目的应用微柱凝胶试剂卡、微柱玻璃珠试剂卡、经典试管法同时检测夫妇ABO血型不合的O型孕妇IgG血型抗体效价,探讨微柱凝胶试剂卡、微柱玻璃珠试剂卡检测孕妇血型抗体效价的界值。方法用微柱凝胶试剂卡、微柱玻璃珠试剂卡、经典试管法平行检测O型血孕妇Ig G血型抗体效价。结果微柱凝胶试剂卡、微柱玻璃珠试剂卡以1∶128为界值与试管法1∶64比较,阳性检出率差异无统计学意义(P0.05)。结论使用微柱凝胶试剂卡、微柱玻璃珠试剂卡检测O型孕妇IgG血型抗体效价的临床参考值应设为≥1∶128。     

量子点标记技术同步检测抗弓形虫IgG和IgM抗体的实验研究

目的建立抗弓形虫(TOX)IgG和IgM抗体同步检测体系。方法分别用不同发射波长的量子点标记羊抗人IgG、羊抗人IgM作为检测抗体,以免疫磁珠作为固相载体,建立抗弓形虫IgG和IgM抗体同步检测体系,并对该检测体系进行了优化;同时检测280份临床血清标本,并与进口酶联免疫吸附测定(ELISA)试剂盒进行方法学比较。结果 TOX抗原的最适固化浓度为50μg/mL,固化效率在35%～55%之间。抗TOX IgG、IgM抗体检测结果与进口ELISA试剂盒相比符合率分别为96.4%,97.1%,本体系同时检测两种抗体的阳性检测率达8.2%。结论成功建立了抗TOX IgG和IgM抗体同步检测体系,该体系操作简便快速,灵敏度高,具有有良好的临床应用价值。     

用免疫磁珠法检测抗白念珠菌烯醇化酶抗体

摘要：目的：建立检测人血清抗白念珠菌烯醇化酶(Eno)IgG类抗体的免疫磁珠法,并评估其在念珠菌血症诊断中的价值。 方法：将Eno重组蛋白耦联至磁性微珠上,以辣根过氧化物酶（HRP）标记的羊抗人IgG为二抗,优化免疫磁珠法的反应条件,考察其精密度和特异性。收集经血培养确诊的念珠菌血症患者（n=113）、念珠菌定植者（n=50）、细菌菌血症患者（n=30）和健康人对照者（n=100）血清,用免疫磁珠法测定抗Eno抗体,并与ELISA法结果进行比较。 结果：免疫磁珠法测定抗Eno抗体的批内、批间变异系数（CV）分别为8.2%和14.2%,重组Eno抗原对相应抗体的阻断率为91.6%。以吸光度（A）值0.29为cut off值时,诊断念珠菌血症的敏感性、特异性、阳性预测值和阴性预测值分别为80.5%、92.3%、90.1%和84.5%。免疫磁珠法的敏感性和特异性与ELISA法比较无统计学差异,但检测流程从37 ℃ 2.5 h缩短至常温1 h。 结论：免疫磁珠法检测抗Eno IgG类抗体简便、快捷、可靠,在侵袭性念珠菌感染研究中具有潜在的应用价值。     

妊娠妇女产前不规则抗体检测分析

目的 检测妊娠妇女血清中IgG抗A(B)及Rh血型抗D抗体效价,探讨妊娠妇女不规则抗体筛查在新生儿溶血病预防中的意义.方法 常规检测夫妇ABO及RhD血型,对夫妇ABO及Rh血型不合的妊娠妇女血清进行不规则抗体筛选及特异性鉴定.结果 在421例夫妇ABO血型不合的妊娠妇女血清中,IgG抗A(B)效价大于或等于1∶64者168例(39.9%),在2例RhD阴性妊娠妇女中检出抗D抗体1例(50.0%).结论 产前对夫妇进行ABO、RhD血型及IgG抗A(B)和抗D效价检测,发现异常及时进行治疗,可减少因母婴血型不合而产生的新生儿溶血病的发生率;血型不完全抗体检测卡检测IgG抗A(B)及抗D抗体效价,操作简便,结果准确.     

自建微阵列化学发光免疫分析法定量检测心肌肌钙蛋白I自身抗体及其初步临床应用

目的基于微阵列蛋白质芯片技术建立定量检测心肌肌钙蛋白I自身抗体(cTnIAAb)的微阵列化学发光免疫分析法,并评价该方法的检测性能。方法以心肌肌钙蛋白I(cTnI)-C融合蛋白为检测抗原,对抗原点样浓度、封闭液、封闭时间和二抗进行筛选,以检测结果信噪比最大为标准优化条件。以100名健康体检者(正常对照组)cTnIAAb检测灰度值的第95百分位数为临界值,建立cTnIAAb检测的标准曲线,并评价微阵列化学发光免疫分析法的检测性能(稳定性、精密度、最低检测限、线性范围等)。采用微阵列化学发光免疫分析法检测500例血清cTnI>0.1 ng/mL的急性冠状动脉综合征(ACS)患者(ACS组)的血清cTnIAAb,采用免疫印迹法验证其特异性。结果最优实验条件:抗原点样浓度为50μg/mL,封闭液为2%人乳,最佳封闭时间为12 h,二抗为辣根过氧化物酶(HRP)-羊抗人IgG抗体。微阵列化学发光免疫分析法检测高、低值cTnIAAb的精密度良好,变异系数(CV)均<15%;空白限(Lo B)、检测限(LoD)分别为0.12、0.23 AU/mL;线性范围为0.23~815.00 AU/mL。免疫印迹法结果显示,cTnIAAb阳性样本和阳性对照在相对分子质量40000~55000处均出现特异性条带。ACS组血清cTnIAAb阳性率为8.4%(42/500),明显高于正常对照组[2%(2/100)](χ²=5.023,P<0.05);但血清cTnIAAb浓度2个组之间差异无统计学意义(P>0.05)。Spearman相关性分析结果显示,ACS组cTnIAAb与cTnI无相关性(r²=0.1,P>0.05)。结论自建的定量检测cTnIAAb的微阵列化学发光免疫分析法具有良好的检测性能,可为cTnIAAb的临床应用提供较好的方法学基础。     

PK7300全自动血型分析仪测定ABO血型抗体效价的初步研究

目的：探索使用PK7300全自动血型分析仪测定ABO血型抗体效价的方法,探讨献血者血型检测中与ABO血型抗体效价相关的影响因素及注意事项。方法仪器微板法测定抽样标本的ABO血型抗体效价;仪器微板法及手工试管法测定各血型混合血浆的ABO血型抗体效价;统计分析该中心2013年5～7月检测出的ABO血型抗体效价降低的情况。结果A型献血者与B型献血者的ABO血型抗体效价比较,差异无统计学意义（P＞0．05）,O型献血者的ABO血型抗体效价明显高于A型或B型献血者（P＜0．05）;手工试管法的灵敏度明显优于仪器微板法,而手工试管法二的灵敏度又明显优于手工试管法一（P＜0．05）;ABO血型抗体效价降低的A型献血者明显多于B型或O型（均P＜0．01）,ABO血型抗体效价降低的男性献血者明显多于女性（P＜0．01）。结论仪器微板法测定ABO血型抗体效价操作简便、结果判读客观、重复性好,1∶1～1∶80的稀释比例范围能满足绝大多数标本的测定需要,采用梯度稀释有助于减小测定误差;血型、性别、年龄、亚型等因素可能与献血者ABO血型抗体效价降低相关。     

新生儿脐血AB0血型鉴定结果质量控制方法探讨

本研究探讨一种新生儿脐带血ABO血型鉴定的质量控制方法。采用血型血清学试验对脐血标本作ABO血型鉴定,对不能定出结果的疑难血型采用聚合酶链反应-序列特异性引物（Sequence Specific Primer PCR,PCR—SSP）作进一步确认。结果表明：在76120例脐血AB0血型正定型鉴定中,检出78例ABO血型弱抗原反应和难以判定血型结果的疑难标本（1％。）,经复查排除了弱凝集反应30例。检测260例脐血ABO血型反定型,相符血型148例（56．92％）,不相符血型112例（43．08％）。PCR—SSP基因分型检测58例中45例血清学表型结果与基因分型结果一致,3例表型结果与基因定型结果不相符,10例正反定型不相符标本,基因分型结果与正定型完全一致。结论：采用红细胞抗原（正定型）方法结合DNA基因分型技术检测新生儿脐带血抗原反应弱、抗体效价低的ABO血型质控效果好,是一种高效、灵敏的质量控制方法,适用于新生儿脐血AB0疑难血型的鉴定。     

Antigen microarrays for serodiagnosis of infectious diseases.

BACKGROUND: Progress in robotic printing technology has allowed the development of high-density nucleic acid and protein arrays that have increased the throughput of a variety of assays. We generated protein microarrays by printing microbial antigens to simultaneously determine in human sera antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2 (ToRCH antigens). METHODS: The antigens were printed on activated glass slides with high-speed robotics. The slides were incubated first with serum samples and subsequently with fluorescently labeled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. Both microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. RESULTS: The detection limit (mean + 2 SD) of the microarray assay was 0.5 pg of IgG or IgM bound to the slides. Within-slide, between-slide, and between-batch precision profiles showed CVs of 1.7-18% for all antigens. Overall, >80% concordance was obtained between microarray assays and ELISAs in the classification of sera; for T. gondii, CMV, and HSV1, concordance exceeded 90%. CONCLUSIONS: The microarray is a suitable assay format for the serodiagnosis of infectious diseases and can be easily optimized for clinical use. The ToRCH assay performs equivalently to ELISA and may have potentially important advantages in throughput, convenience, and cost.     

Validation of a hospital-laboratory workstation for immunohematologic methods

BACKGROUND: The FREELYS Nano system (Diagast) is a manual workstation for ABO/D grouping, Rh phenotyping, K typing, and antibody screening (ABS) for immunoglobulin G (IgG) antibodies only and works with the erythrocyte-magnetized technology (EMT). The principle of EMT is based on magnetization of red blood cells and avoids centrifugation and washing steps.
STUDY DESIGN AND METHODS: A total of 304 samples were tested with our routine blood bank methods, 100 samples for ABO/D grouping, 196 samples (100 at first evaluation, 96 at second evaluation) for Rh phenotyping and K typing (PK7200, Olympus), and 108 samples for ABS (DiaMed). All samples were tested in parallel with the FREELYS Nano.
RESULTS: We found a 100% concordance between the observed (FREELYS Nano) and the expected (Olympus PK7200) results for ABO/D grouping in all 100 samples. For Rh phenotyping and K tests, in 24 of 100 samples false-positive reactions were observed in the first evaluation by the FREELYS Nano. After changing the test kit batch for Rh phenotyping by the manufacturer, a complete concordance in Rh phenotyping and K tests was observed in a second evaluation. For ABS, the FREELYS Nano showed in 4 of 108 samples (3.7%) false-negative reactions for IgG antibodies (two anti-K, one anti-E, one anti-C^w), and one (0.9%) false-positive reaction.
CONCLUSIONS: The FREELYS Nano is reliably suited to ABO/D grouping, Rh phenotyping, and K testing. The rate of false-negative reactions for IgG antibodies should be reduced.     

A fully automated blood typing system for hospital transfusion services. ABS2000 Study Group

BACKGROUND: The results of routine blood bank testing by a fully automated blood typing system (ABS2000) were compared with those obtained by standard manual methods in six hospital transfusion services. STUDY DESIGN AND METHODS: The ABS2000 system uses microtiter plates for determining ABO and D types, solid-phase red cell adherence (SPRCA) assays for antibody detection, and modified SPRCA plates for IgG crossmatches. The transfusion services used their standard manual test tube methods. RESULTS: Of 3779 donors' samples tested for ABO types (red cell typings only), 3.0 percent could not be interpreted by the ABS2000 system's neural network, because of clots, hemolysis, or lipemic samples. The results for ABO types were concordant for 99.8 percent of the remaining samples. Of 3779 donors' samples tested for D types, the results were concordant for 98.7 percent. Of 7580 patients' samples tested for ABO types (red cell and plasma typings), 5.8 percent could not be interpreted by the ABS2000 system. There was 100-percent concordance of ABO typing results for the remaining 7140 samples. There was 99. 7-percent concordance of results for patients' D types. The results of 96.7 percent of antibody detection tests and 98.8 percent of crossmatches were concordant. Neither method failed to detect a serologically incompatible crossmatch that was associated with a specific, clinically significant alloantibody. The ABS2000 system performed 45 confirmatory donor ABO and D types in 115 minutes, 22 antibody detection tests in 116 minutes, 16 patients' ABO/D types in 149 minutes, and 40 crossmatches in 140 minutes. CONCLUSION: The ABS2000 blood typing system automates routine blood bank tests with accuracy comparable to that of hospital transfusion services' standard manual methods.     

Evaluation of polyethylene terephthalate for ABO and Rh typing and alloantibody screening

BACKGROUND: For many years, hospitals and laboratories have used evacuated glass tubes for blood collection. To improve the safety of blood collection, plastic polyethylene terephthalate (PET) tubes (Vacutainer PLUS, Becton Dickinson) have been developed. The objectives of this study were to compare the accuracy of ABO grouping, Rh typing, and antibody screening of blood samples collected in plastic tubes with that in glass tubes and to determine if refrigerated blood samples collected in plastic tubes remained stable over a 28-day period. STUDY DESIGN AND METHODS: Samples were collected from 121 volunteers, at least 30 from each of the A, B, O, and AB blood groups, in four types of Vacutainer tubes: silica-coated plastic, K(2) EDTA plastic, uncoated glass, and K(2) EDTA glass. Samples from each tube were tested for ABO group and Rh type by use of the microtyping gel identification card system and the tube method. A three-cell antibody screen was performed by the microtyping gel card technique with a monospecific IgG reagent. Initial samples were tested within 3 hours of collection. Refrigerated samples were retested for ABO and Rh type and antibody screening 1, 2, 21, and 28 days later. Agreement between test results was determined by using Cohen's Kappa statistic. RESULTS: Complete agreement was observed between the ABO and Rh typing results in samples drawn into glass and plastic tubes of both the EDTA and nonanticoagulated type (kappa = 1.0). In retesting, there were no examples of a change in ABO or Rh type over the 28-day study period. Only two alloantibodies (1.7%) were identified in the 121 samples, and no difference was observed in alloantibody expression in either plastic or glass Vacutainer tubes over the 28-day study period. CONCLUSION: Samples collected into the PET serum or EDTA tubes provided accurate ABO and Rh typing results that remained consistent over a 28-day period. Samples collected in these tubes also appeared to enable accurate alloantibody identification. However, the number of alloantibodies identified in this study was small, and this result should be confirmed in a larger series.     

ABO新生儿溶血病与O型孕妇血清中IgG及其亚类含量的相关分析

目的探讨O型孕妇血清IgG抗体及其亚型含量与新生儿溶血病(HDN)的关系。方法采用血型血清学方法,对317名夫妇血型不合的O型孕妇作IgG抗体效价检测,并对其中有妊娠史的287名孕妇作IgG抗体水平比较;采用ELISA法对71名HDN患儿及其母亲、65名健康O型孕妇和51名健康新生儿的IgG亚类作定量分析。结果1)317名新生儿中发生ABOHDN71例(22.4%),其中抗-A46例、抗-B25例;2)随着妊娠次数的增加,IgG抗体效价≥64者的比例和ABO-HDN发病率升高,>2次妊娠与第2次妊娠间有统计学差异;3)患儿及其母亲体内IgG抗体的含量显著高于正常对照组,且以IgG1抗体为主,患儿体内的IgG1比例(61.9%)高于母体(52.8%)。结论新生儿ABOHDN的发病率随其母亲体内IgG抗体效价的升高而升高,且与母亲体内IgG1呈正相关。夫妇血型不合的O型孕妇应定期检测IgG抗体及其亚类含量。     

自身免疫性溶血性贫血患者的血型检定研究

为了观察自身抗体对ABO和RhD血型检定的干扰，对38例自身免疫性溶血性贫血(autoimmune hemolytic anemia，AIHA)患者血液标本进行ABO和RhD常规定型，氯奎放散试验后定型及基因定型。结果表明：38例AIHA患者中，ABO血型难定者11例(31.6％)，均为间接抗球蛋白试验阳性及正反定型不合。RhD(-)误定RhD( )1例，并含抗-D。采用氯奎放散试验后血型皆正确检定。结论：AIHA患者的自身抗体干扰血型检定，必须采用多种技术才能正确检定血型，确保安全输血。     

肿瘤患者疑难血型鉴定和基因分型的相关探讨

血液病和恶性肿瘤的部分患者,尤其是白血病和多发性骨髓瘤患者,常因病情及治疗导致ABO血型正反定型不符,表现为ABO血型的抗原或抗体减弱。本研究分析正反定型不一致的原因,进行正确的红细胞分型和基因分型,对12份肿瘤患者正反定型不一致血液样本进行的血型血清学试验和吸收放散试验。对存在疑问的标本进行PCR扩增第6、7外显子和5-7内含子,对外显子序列进行基因测序。结果表明,9例标本通过血型血清学、吸收放散定型,6例为A型,2例为O型,1例为B型;3例血型血清学和吸收放散无法鉴定其血型的标本,经测序分别定型为O46,B108和A102型,其中有2例因为PCR扩增效果差或者测序结果矛盾,未能给出ABO基因型。结论：对ABO血型正反定型不一致无法定型的肿瘤患者的血液样本,可以采用血型血清血方法、基因分型及吸收放散的方法正确鉴定ABO血型,以保证输血的安全性和有效性。     

全自动血型分析仪与微板法在ABO血型筛查中的比较分析

目的探讨Beckman Coulter PK7300全自动血型分析仪与微板法在ABO血型筛查中的应用效果。方法选取2016年1-5月无偿献血者EDTA抗凝全血标本12 000例,采用Beckman Coulter Pk7300全自动血型分析仪和STAR加样微板手工比色法分别进行检测分析。结果两种方法检测ABO血型的准确率差异无统计学意义(P0.05)。在ABO血型筛查中,Beckman Coulter Pk7300全自动血型分析仪ABO亚型和抗体减弱的检出率均高于微板法。3例ABO血型正反定型在Beckman Coulter Pk7300全自动血型分析仪检测中一致而在微板法中不一致,4例ABO血型正反定型在Beckman Coulter Pk7300全自动血型分析仪检测中不一致而在微板法中一致。结论 Beckman Coulter Pk7300全自动血型分析仪能够安全有效对献血者进行ABO血型筛查,检测结果可疑的标本仍需结合试管法进行人工判读。