时间分辨荧光免疫分析法测定抗环瓜氨酸肽抗体方法学的建立

目的 建立高灵敏度、宽量程的定量检测患者血清中的抗环瓜氨酸肽(CCP)抗体的时间分辨荧光免疫分析方法.方法 以CCP重组抗原和鼠抗人IgG抗体分别作为固相抗原和Eu3+记抗体,如果样本中存在抗CCP抗体,则形成CCP重组抗原-抗CCP抗体-Eu3+标记鼠抗人IgG抗体复合物,加入解离增强液解离铕离子,检测荧光强度,样本中抗CCP抗体含量与荧光强度成正比;对建立的时间分辨荧光免疫(TRFIA)法检测抗CCP抗体的线性范围、精密度、检测范围进行分析;对32例阳性血清标本分别利用TRFIA及酶联免疫吸附试验(ELISA)法检测抗CCP抗体,分析其相关性;收集50名健康献血者,32例系统性红斑狼疮,26例干燥综合征,10例硬皮病,20例混合性结缔组织病,18例多发性硬化症,利用TRFIA检测其血清中的抗CCP抗体,计算临床特异度.TRFIA与ELISA 2种方法检测结果的一致性检验采用Mc Nemar检验.结果 TRFIA法检测高、中、低3种浓度混合血清的批内(20份)精密度分别为2%、3%和4%,批间(8份)精密度分别为3%、4%和7%;平均回收率为101%;方法的灵敏度为1.0 U/ml;TRFIA法检测健康献血者、系统性红斑狼疮、干燥综合征、硬皮病、混合性结缔组织病及多发性硬化症患者血清中抗CCP抗体,临床特异度分别为98%、97%、96%、100%、95%、100%;2种方法检测的相关系数为0.989;TRFIA比ELISA的检测范围更宽,稳定性能好.结论 首次建立稳定的高灵敏度和宽检测范围的TRFIA法检测人血清中的抗CCP抗体,对早期诊断RA及监测疗效具有重要意义,该方法以其优势有望在各检验科室得以普遍应用.     

免疫-PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体方法的建立

目的　建立高灵敏性和高特异性的免疫 -PCR方法检测肾综合征出血热 (HFRS)患者血清中抗核衣壳蛋白抗体。方法　用汉坦病毒 ( 76 - 118株 )重组核衣壳蛋白为抗原包被聚苯乙烯微滴度板 ,与经ELISA法和临床均已确诊为HFRS患者的血清进行抗原抗体特异性反应 ,以链霉亲和素为桥梁将生物素标记的抗体与生物素标记的DNA指示分子相连 ,PCR扩增DNA指示分子 ,通过检测指示分子DNA来反映肾综合征出血热患者血清中抗核衣壳蛋白抗体的水平 ,并与传统的ELISA法进行比较。结果　免疫 -PCR检测肾综合征出血热患者血清中抗核衣壳蛋白抗体的敏感性是ELISA法的 2 5 0 0 0倍 ;30例HFRS阳性患者血清免疫 -PCR检测均为阳性 ,2 0例甲型肝炎阳性血清和 2 0例正常人血清免疫 -PCR检测均为阴性。结论　免疫 -PCR方法敏感性高 ,特异性强 ,有可能作为一种高敏感性的检测方法用于HFRS的临床辅助诊断     

谷氨酸脱羧酶抗体检测的生物素-链霉亲合素放大ELISA系统构建及其临床应用

目的建立检测谷氨酸脱羧酶抗体(GADA)的新方法,即生物素-链霉亲合素放大ELISA系统(ELISA,BAS ELISA),并进行验证及初步临床应用。方法人脑组织经过破碎、抽提、二次凝胶过滤层析与阴、阳离子交换层析分离蛋白,收集纯化的谷氨酸脱羧酶(GAD);以链霉亲和素(SA)预包被,使GAD生物素化(bio-GAD),探讨检测GADA的最优实验条件,并对BAS ELISA的灵敏度、特异性、准确性及精密性进行验证。然后选择新诊断的成年1型糖尿病患者106例(T1DM组),以健康体检者98例为对照组,抽取空腹外周静脉血3 ml,分离血清冷冻标本,用构建的BASELISA和德国宝灵曼公司Anti-GAD65 ELISA检测血清中的GADA,对比研究并进行统计学分析。结果成功构建了检测GADA的BAS ELISA新方法;在T1DM组中,BAS ELISA方法测得T1DM的GADA阳性率显著高于anti-GAD65 ELISA方法(82.08%vs 52.83%,P<0.01);在对照组中BASELISA方法测得T1DM的GADA阳性率也明显高于anti-GAD65 ELISA方法(10.20%vs 6.12%,P<0.05)。结论利用生物素-链霉亲合素放大系统可以构建检测GADA的ELISA新方法;生物素-链霉亲合素放大ELISA系统在检测GADA方面具有较高的特异性、灵敏度、准确性和精密性,提高了GADA检测的阳性率,降低T1DM的漏检率,为早期检测T1DM提供新方法。     

EVALUATION OF Tb-NT-P-BSA ANTIGEN IN THE SERODIAGNOSIS OF TUBERCULOSIS

用人工合成的结核杆菌酚糖酯三糖抗原Tb-NT-P-BSA作抗原,用ELISA法检测了40例结核患者、52个健康对照的血清标本。结果表明本方法的特异性、敏感性分别是在检测抗Tb-NT-P-BSA IgG抗体时为94.2％和20％;在检测抗Tb-NT-P-BSA IgM抗体时为79.6％和45％。该抗原的特异性、敏感性似不理想,有待于进一步改进。     

Der f 1血清特异性IgE ELISA检测方法的建立

目的利用粉尘螨变应原第1组分重组蛋白rDer f 1建立特异性IgE ELISA检测方法,并对哮喘患者血清进行检测。方法以rDer f 1为包被抗原,分别建立间接ELISA法和亲和素-生物素复合ELISA(ABC-ELISA)法,检测Der f 1特异性IgE阳性标准血清和39例哮喘患者血清中的Der f 1特异性IgE,并分析检测结果。结果间接ELISA法和ABC-ELISA法检测Der f 1特异性IgE的最适抗原包被浓度和血清稀释倍数均为15μg/ml和1︰2,间接ELISA法辣根过氧化物酶(HRP)标记抗人IgE抗体适宜稀释倍数为1︰1 000,ABC-ELISA法生物素标记抗人IgE抗体和HRP标记亲和素的稀释倍数分别为1︰1 000和1︰2 000。用间接ELISA和ABC-ELISA检测IgE的灵敏度分别为0.68U/ml和0.73U/ml,检测患者血清中Der f 1特异性IgE的阳性率分别为92.3%和97.4%,差异无统计学意义(P>0.05)。结论基于重组变应原rDer f 1的ELISA法检测粉尘螨特异性IgE具有较高的敏感度,可用于螨过敏性疾病的实验诊断。     

弓形弓形虫抗原检测方法的研究

本研究制备了与多种寄生虫抗原无交叉反应的抗弓形虫单抗B6C3和兔抗弓形虫多抗,多抗为捕获抗体,单抗为检测抗体,建立单-多抗夹心ELISA方法。用亲和素连接分别标记生物素的抗体和酶,建立ABC(avidin-biotin-peroxidase complex)-ELISA[D1]方法。同样用亲和素连接标记生物素的抗体和DNA片段,利用PCR反应对DNA片段巨大的扩增能力,提高检测灵敏度,发展单-多抗夹心ELISA为免疫-PCR(I-PCR)检测弓形虫抗原。单-多抗夹心ELISA检测弓形虫抗原最低浓度为0.6μg/ml,ABC-ELISA检测最低浓度为0.075μg/ml。I-PCR检测最低浓度为0.1ng/ml,检测弓形虫抗原灵敏度高于传统ELISA法6000倍,高于ABC-ELISA法750倍。     

夹心免疫-PCR检测旋毛虫循环抗原的研究(英文)

目的建立以多抗为捕获抗体和单抗为检测抗体的夹心免疫-PCR检测旋毛虫循环抗原。方法利用杂交瘤技术制备抗旋毛虫单抗;旋毛虫抗原免疫家兔,分离纯化兔血清,制备兔抗旋毛虫多抗。多抗作为捕获抗体包被酶标板,单抗为检测抗体,选择适宜多抗和单抗浓度,建立夹心ELISA方法,再利用亲和素和生物素的高结合力连接起标记了生物素的抗体和DNA片段,将抗原抗体特异反应的免疫技术同无限扩增DNA片段的基因检测技术结合,利用PCR反应对DNA片段的扩增能力,提高检测灵敏度。结果制备了兔抗旋毛虫多抗,间接ELISA法筛选出与多种寄生虫抗原无交叉反应的单抗F4C6,夹心ELISA检测旋毛虫抗原最低浓度为0.05μg/ml,免疫PCR方法检测最低浓度为0.1ng/ml。结论首次建立夹心免疫-PCR检测旋毛虫抗原的方法,检测旋毛虫抗原的灵敏度高于传统ELISA方法104倍。     

化学发光免疫分析检测丙肝病毒抗体在血液筛查中的应用评价

目的:评价化学发光免疫分析(CLIA)测定丙肝病毒(HCV)抗体的方法学性能在血液筛查中的应用。方法:以辣根过氧化物酶(HRP)为酶标记物,以鲁米诺为发光底物,采用双抗原夹心法检测血样中的抗-HCV。建立抗-HCV化学发光酶免疫分析方法,与常规使用的抗-HCV酶联免疫分析方法(ELISA)同时检测20例发光值(RLU)大于14 000的抗-HCV血清阳性标本,再用荧光定量RT-PCR检测;每天用卫生部临床检验中心指定生产的HCV的质控血清对CLIA进行检测,建立自己实验室的该项目临界值浓度,连续6个月对HCV质控血清检测,计算S/CO值,统计分析精密度、CV;用EQA的质控物检测HCV抗体,进行标本符合率计算。结果:CLIA方法的灵敏度为0.02ng/mL,阳性符合率为100%,ELISA的阳性符合率为75.2%,荧光定量RT-PCR检测的阳性符合率为100%。结论:HCV抗体CLIA检测抗-HCV试验方法的敏感性、准确性、稳定性较好,能满足临床输血早期筛查,以排除假阳性,杜绝漏检的风险。     

特异性神经元抗Hu抗体对小细胞肺癌早期诊断价值的研究

目的 探讨特异性神经元抗Hu抗体对小细胞肺癌（SCLC）早期诊断的价值。方法 采用新合免疫细胞组织化学卵白素－生物素过氧化物酶复合物法（ABC法），蛋白免疫印迹法（Western blot)对98例SCLC患者的血清进行检测，分析其临床意义。结果 （1）亲合免疫细胞组织化学ABC法在约40％不伴神经系统副肿瘤综合征（PNSNS）的SCLC患者血清中检测出特异性神经元抗体，滴度为1：1000－1：8000；在约89％伴PNSNS的SCLC患者血清中检测出特异性神经元抗体，滴度为1：1000－1：64000；（2）Western blot在不伴PNSNS的SCLC患者血清中检测出2例可识别正常人大脑神经元细胞核提取物中分子量为35000－40000蛋白和40000的HuD克隆纯化蛋白抗原；在伴PNSNS的SCLC患者血清中检测出16例可识别上述蛋白抗原；（3）正常人血清中未检测出特异性神经元抗体。结论 特异性神经元抗Hu抗体对SCLC的早期诊断具有重要意义，可以作为早期筛选SCLC的一面指标。     

旋毛虫病患者血清中循环抗原及抗体检测的研究

111例旋毛虫病患者用斑点ELISA 试验检测循环抗原(CAg),薄板ELISA试验检测特异性抗体。其中未经治疗的患者36例,两法检出率均为100％;75例经治疗后3个月的患者血清中CAg 检出率为78.67％,抗体 IgG为 97.47％、IgM为 84.82％,IgE为 83.54％,两组间的 CAg、特异性 IgM、IgE抗体都有明显差异。健康人血清、日本血吸虫病患者血清试验,均有一定的假阳性与交叉反应。斑点ELISA检测旋毛虫病患者血清中CAg,ELISA检测IgM、IgE仍属敏感的和特异的,并且重现性好。特别是斑点ELISA采用第一和第二抗体标记酶进行两次放大的方法比之选用生物素亲和素放大法价廉,易于制备,从而可以取代传统的活检法,具有实用价值。     

胰岛素瘤的解剖分布特点分析

目的胰岛素瘤是最常见的胰腺神经内分泌肿瘤，因其临床表现多样，导致诊断困难。影像学诊断尤其是超声内镜(EUS)在胰岛素瘤的诊断中起着重要作用，拥有较高的敏感性和特异性。本研究拟通过明确胰岛素瘤的解剖分布特点，以期有助于提高影像学的诊断准确率和降低漏诊率，尤其是在教育和培训实践中对于EUS的学习者更具有指导价值。 方法回顾性分析解放军总医院第一医学中心病案资料数据库1993年1月至2019年11月经外科手术、病理确诊为胰岛素瘤的患者的临床资料，检索方法采取搜索术后病理诊断为"胰岛素瘤"的病例，通过查阅病例的方法，提取出胰岛素瘤的大小和解剖分布等数据，进一步分析其特点。 结果共检索到确诊为胰岛素瘤的患者116例，其中，男45例、女71例，年龄13～76岁，平均年龄(44.4±14.85)岁。胰岛素瘤单发110例(94.8%)、多发6例(5.2%)。位置分布：头颈部46例(39.7%)，单发45例、多发1例；体尾部68例(58.6%)，单发65例、多发3例；全胰腺多发2例(1.7%)。病变大小特点：最大径0.4～3.4 cm，平均大小(1.53±0.58)cm。≤1 cm 29例、>1 cm而≤1.5 cm41例、>1.5 cm而≤2.0 cm28例，≤3 cm 15例，>3 cm 3例。年龄与肿瘤的大小相关，≤44岁患者肿瘤平均大小为(1.36±0.51)cm、>44岁患者肿瘤平均大小为(1.70±0.60)cm，P<0.05。头颈部的肿瘤大于体尾部的肿瘤，头颈部肿瘤平均大小(1.66±0.63)cm，体尾部(1.42±0.52)cm，P<0.05。 结论胰岛素瘤在胰腺体尾部较头颈部更好发；绝大多数单发，但可以全胰腺多发；多数小于1.5 cm，肿瘤的大小与患者年龄和肿瘤的解剖分布相关。     

Pathogenesis of carcinoma of the papilla of Vater

Most adenomas and carcinomas of the small intestine and extrahepatic bile ducts arise in the region of the papilla of Vater. In familial adenomatous polyposis (FAP) it is the main location for carcinomas after proctocolectomy. In many cases symptoms due to stenosis lead to diagnosis at an early tumor stage. In about 80%, curative intended resection is possible. Operability is the most relevant prognostic factor. Most ampullary carcinomas resp. carcinomas of the papilla of Vater develop from adenomatous or flat dysplastic precursor lesions. They can be sited in the ampulloduodenal part of the papilla of Vater, which is lined by intestinal mucosa. They also can develop in deeper parts of the ampulla, which are lined by pancreaticobiliary duct mucosa. Intestinal-type adenocarcinoma and pancreaticobiliary-type adenocarcinoma represent the main histological types of ampullary carcinoma. Furthermore, there exist unusual types and undifferentiated carcinomas. Many carcinomas of intestinal type express the immunohistochemical marker profile of intestinal mucosa (keratin 7?, keratin 20+, MUC2+). Carcinomas of pancreaticobiliary type usually show the immunohistochemical profile of pancreaticobiliary duct mucosa (keratin 7+, keratin 20?, MUC2?). Even poorly differentiated carcinomas, as well as unusual histological types, may conserve the marker profile of the mucosa they developed from. These findings underline the concept of histogenetically different carcinomas of the papilla of Vater which develop either from intestinal- or from pancreaticobiliary-type mucosa of the papilla of Vater. Molecular alterations in ampullary carcinomas are similar to those of colorectal as well as pancreatic carcinomas, although they appear at different frequencies. In future studies, molecular alterations in ampullary carcinomas should be correlated closely with the different histologic tumor types. Consequently, the histologic classification should reflect the histogenesis of ampullary tumors from the two different types of papillary mucosa.     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.     

Demonstration of the mechanism of action of biguanides

Summary Palmitic acid oxidation in rat diaphragm homogenate is depressed by biguanide concentrations that are still incapable of inhibiting oxidative phosphorylation. Glucose oxidation is not directly effected by the same biguanide concentrations: however, the inhibitory effect of palmitic acid on glucose oxidation is partly removed by biguanides. Inhibition of fatty acid oxidation, which accounts for most of the metabolic effects caused by these drugs, can be regarded as the fundamental mechanism of action of biguanides. There is some evidence suggesting that these drugs might interact with carnitine, thus preventing long-chain fatty acids from being transported across the mitochondrial membrane to the site of oxidation. Traduzione a cura degli AA.     

血吸虫童虫生长发育及其机制的研究进展

血吸虫童虫是宿主免疫系统攻击的重要靶标,包括皮肤型、肺型和肝门型童虫。宿主分子对童虫生长发育具有重要作用。童虫生长发育机制包括免疫调节、信号转导、性别发育及凋亡等。肌动蛋白、组织蛋白酶、烯醇化酶和葡萄糖基转移酶等分子为血吸虫童虫生长发育的重要分子。本文对血吸虫童虫生长发育及其机制的研究进展做一综述。     

124例耐多药结核分枝杆菌基因突变特征分析

目的对临床分离的耐多药结核分枝杆菌相关基因的突变特征进行分析。方法对124例耐多药结核分枝杆菌以及50株敏感株的耐药相关基因(包括异烟肼inh A、kat G、oxyR-ahp C间隔区以及利福平rpo B)进行序列测定,分析其基因突变情况。结果异烟肼耐药inh A基因突变率为14.5%;kat G基因突变率为70.2%(87/124),主要位于315位;oxyR-ahp C间隔区突变率为15.3%;inh A、kat G两种基因同时突变率75.0%,三种基因同时突变率为89.5%。利福平rpo B基因突变的检出率高达95.2%,突变主要发生在531、526、516位点。结论我省耐多药菌异烟肼耐药相关基因最常见突变为kat G 315、inh A C-T(-15)、axyR-ahp C间隔区(-10)C-T,利福平为rpo B531、526、516。结合MDR-TB耐药相关基因的特征分析,可以建立一种快速、准确、特异的适合于我省的检测结核菌耐多药性的新方法。     

Assessment of quality of life of parents of children with juvenile chronic arthritis

The aim of the study was to assess the quality of life (QOL) and the psychological status of parents of children with juvenile chronic arthritis (JCA). The QOL, anxiety and depression of the parents of 28 children with JCA were evaluated and compared to those of the parents of 28 healthy children. Mothers of JCA children and mothers of healthy children reported similar QOL. The reported anxiety and depression levels were similar for mothers and fathers in both groups. The parents of children with pauciarticular-type JCA reported lower QOL and higher levels of anxiety and depression than the parents of children with other types, namely polyarticular and systemic JCA. These findings may be explained by the fact that the pauciarticular patients had shorter disease duration and were less frequently seen in the outpatient clinic. The QOL of mothers of children with JCA was found to be slightly impaired in the group of children with pauciarticular JCA. Future larger studies are needed to confirm these results, as the number of subjects in the three groups was rather low. Received: 26 September 2001 / Accepted: 8 February 2002     

氯硝柳胺悬浮剂的毒性评价

目的评价氯硝柳胺悬浮剂的毒性，为现场大规模应用灭螺提供依据。方法按照中华人民共和国国家标准GB 15670-1995《农药登记毒理学试验方法》和鱼类毒性试验方法进行。结果经口、经皮肤的LDso雌、雄性大鼠均>5 000 mg/kg，经呼吸道的LCso雌、雄性大鼠均>5 000mg/m3，该药经口、经皮肤、经呼吸道毒性均属微毒类药物；兔眼用药后，观察期内无不良反应，对眼无刺激性；皮肤用药后对皮肤无刺激性。与氯硝柳胺原药、氯硝柳胺乙醇胺盐原药和氯硝柳胺乙醇胺盐可湿性粉剂相比，氯硝柳胺悬浮剂对鱼急性毒性最低。结论氯硝柳胺悬浮剂属微毒类药物，对鱼的毒性低于其乙醇胺盐可湿性粉剂，适合于现场应用。     

Numerical Simulation of the Effect of Rate of Change of Glucose on Measurement Error of Continuous Glucose Monitors

Background

A 5-day in-patient study designed to assess the accuracy of the FreeStyle Navigator® Continuous Glucose Monitoring System revealed that the level of accuracy of the continuous sensor measurements was dependent on the rate of glucose change. When the absolute rate of change was less than 1 mg•dl⁻¹•min⁻¹ (75% of the time), the median absolute relative difference (ARD) was 8.5%, with 85% of all points falling within the A zone of the Clarke error grid. When the absolute rate of change was greater than 2 mg•dl⁻¹•min⁻¹ (8% of the time), the median ARD was 17.5%, with 59% of all points falling within the Clarke A zone.

Method

Numerical simulations were performed to investigate effects of the rate of change of glucose on sensor measurement error. This approach enabled physiologically relevant distributions of glucose values to be reordered to explore the effect of different glucose rate-of-change distributions on apparent sensor accuracy.

Results

The physiological lag between blood and interstitial fluid glucose levels is sufficient to account for the observed difference in sensor accuracy between periods of stable glucose and periods of rapidly changing glucose.

Conclusions

The role of physiological lag on the apparent decrease in sensor accuracy at high glucose rates of change has implications for clinical study design, regulatory review of continuous glucose sensors, and development of performance standards for this new technology. This work demonstrates the difficulty in comparing accuracy measures between different clinical studies and highlights the need for studies to include both relevant glucose distributions and relevant glucose rate-of-change distributions.     

治疗高血压药物的经济学评价

重视高血压治疗中的经济学评价,对利用我国有限的卫生资源来遏制高血压对人民群众的危害有着重要的现实意义。药物经济学对于药物治疗的成本和治疗的结果给予同样的关注。因为治疗高血压的费用,不仅涉及药物价格,还包括患者的危险水平,降压疗效和对临床终点事件的影响,以及治疗的依从性和安全性。因此药物经济学更强调整体成本和价-效比。低危病人,若非药价低廉,治疗的价-效比不够理想。而在高危的患者,价-效比越小越经济而不是药费越便宜越好。