镧系元素在免疫学检测技术中的应用

镧系元素与普通荧光相比其具有荧光衰变时间长、Stokes位移大和发射光谱窄等优点。以镧系元素及其螯合物为标记物应用到免疫分析技术中,成为目前超微量物质分析中最有发展前途的技术之一。自从时间分辨荧光免疫分析技术创立以来,经过30多年的发展,以镧系元素及其螯合物作为标记物与其他技术相结合已应用到不同的免疫检测技术中。本文就镧系元素在免疫检测技术中的应用作一综述。     

单克隆抗体组建黄曲霉毒素酶免疫检测试剂盒的研究

单克隆抗体组建黄曲霉毒素酶免疫检测试剂盒的研究季永镛，林国妹，叶敏（中国科学院上海细胞生物学研究所，上海２０００３１）陆建华，李文广（江苏启东肝癌防治研究所，江苏启东２２６２００）本文报告以高亲和力抗黄曲霉毒素（ＡＦ）单克隆抗体为基础组建的ＡＦＮ免疫...     

免疫学科技市场预测:到1990年免疫检测试剂和设备的销售将成倍增长

<正> 今后各临床实验室用抗原—抗体反应来诊断和治疗疾病将会激增。据一项新的“免疫检测试剂和设备”的调查预测,到1990年,免疫检测方面用品的销售量将超过目前的一倍,达22,600万美元。检测技术的迅速变更将改变市场的结构。酶免疫测定(EIA)在5种主要市场部份中独占鳌头,它可用现有的临床分析仪和分光光度计进行检     

单克隆抗体在食品安全和临床中的应用

单克隆抗体问世后取得了广泛的应用，随后由单克隆抗体衍生出的各种免疫技术如免疫-PCR、免疫传感器等，大大提高了免疫学实验的特异性和敏感性，从而促进了免疫学快速检测的发展。目前免疫检测技术已广泛应用于临床、食品安全以及人类健康保障事业的各个领域，如应用于动物性食品检测，包括对β类兴奋剂、抗生素类、激素类药物及病原微生物等的检测；植物性食品中农药残留、生物毒素及病原微生物等的检测，还有肿瘤、传染和感染性疾病、免疫性疾病、器官移植排斥反应、正常人群的健康检查、进出口岸人群健康检查等。     

上转换荧光材料的化学修饰及在免疫检测的应用

上转换纳米材料是近年来发展起来的新兴稀土荧光材料。在近红外光的激发下可将其转变成可见光这一光学特性,使其具有抗光漂白能力强、宽的反斯托克斯位移、低毒性以及无自体荧光干扰等优点,在免疫检测中可提高灵敏度和信噪比。上转换稀土纳米颗粒水溶性和分散性较差,因此要通过化学修饰成水溶性和分散性较好的上转换稀土颗粒,以进一步与生物分子偶联。本文主要在合成上转换纳米颗粒的基础上,对化学修饰的方法及荧光共振能量传递机制、基于磁分离富集的免疫检测技术、固相微孔板荧光标记技术、免疫传感技术和免疫层析技术的应用研究做一综述。上转换技术的成功应用解决了传统的纳米材料应用中低灵敏度的问题,在医学检测领域具有很大的应用潜力。     

微囊藻毒素亮氨酸精氨酸(MC-LR)损伤睾丸支持细胞免疫反应及细胞生物学行为的机制研究进展

微囊藻毒素亮氨酸精氨酸(MC-LR)是水华爆发时微囊藻属、鱼腥藻属等蓝藻产生的一种对人类有致癌性的藻毒素。MC-LR可对雄性动物生殖系统产生毒性，导致不育，主要表现为诱导细胞凋亡、破坏细胞骨架、干扰DNA损伤修复途径或损伤血睾屏障(BTB)功能等。睾丸支持细胞(Sertoli细胞)是生精小管中与生精细胞直接接触的体细胞，可通过分泌多种细胞因子和免疫抑制因子调节免疫反应以维持睾丸免疫稳态，亦可与邻近生殖细胞形成BTB,为各级生精细胞提供营养、支持和保护作用的微环境。MC-LR可引发睾丸Sertoli细胞炎症、诱导细胞凋亡、破坏BTB完整性，进而造成睾丸生精功能障碍。     

免疫检测信号放大技术研究进展

随着疾病早期诊断及生物标志物开发、功能界定等需求的出现,免疫检测技术的灵敏度不足这一问题日益显著,如何提高检测灵敏度去突破瓶颈成为现在生物分析领域的一个重大挑战。对检测信号进行放大是提高检测灵敏度最直接的一种方法。生物素-亲和素系统(biotin-avidin system,BAS)、酪胺信号放大技术(tyramide signal amplification,TSA)以及免疫-聚合酶链反应(immuno-polymerase chain reaction,Im-PCR)是较为经典的信号放大技术,可显著提高免疫检测的灵敏度。近年来,研究发现,基于催化信号分子沉淀的信号放大技术、基于纳米颗粒的信号放大技术及基于杂交链式反应的信号放大技术等信号放大手段的出现,使得免疫检测的灵敏度可以进一步提高3个数量级。本文将针对近年来研究较多的几种用于免疫检测信号放大的技术进行总结,并对其优缺点进行比较分析,以期为已开发的信号放大技术向临床转化及进一步开发超高灵敏度的免疫学检测技术提供参考。     

广州市水源水、出厂水及珠江广州河段微囊藻毒素污染现状调查

目的了解广州市自来水厂水源水、出厂水和珠江广州河段微囊藻毒素（MCs）污染情况。方法采集广州市自来水厂水源水、出厂水和珠江广州河段水样超声破碎微囊藻毒素并过滤后采用ELISA试剂盒进行检测。结果本次调查所采水源水和出厂水各6个样品中,有2个水源水样本MCs浓度超过国家标准（1μg/L）。出厂水亦可检出MCs,但与相应的水源水相比,浓度均明显下降且没有超出标准规定的限制。珠江广州河段的16个样品检测结果提示珠江水中也存在有MCs污染,个别河段MCs浓度大于1μg/L。结论本调查结果提示广州市水源水、出厂水及珠江广州河段均存在MCs污染且个别样品浓度较高,应引起充分重视。     

艰难梭菌A毒素对人甲状腺乳头癌细胞系的毒性作用研究

艰难梭菌 (Ｃｌｏｓｔｒｉｄｉｕｍｄｉｆｆｉｃｉｌｅ)Ａ毒素是艰难梭菌引起疾病的主要致病因子 ,具有肠道毒性、细胞毒性、小鼠致死活性、促血球凝集活性、致血管通透性亢进等生物学活性〔1,2〕 。艰难梭菌Ａ毒素对组织和细胞有毒性作用 ,但其作用机制未完全阐明。目前国内对艰难梭菌Ａ毒素的细胞毒性的研究刚刚起步 ,我们曾研究过艰难梭菌Ａ毒素对ＨＥＰ2 等 6种细胞的毒性作用 ,其中人甲状腺乳头癌细胞 (ＴＰＣ 1)对Ａ毒素的敏感性较高〔3〕。我们进一步观察Ａ毒素对ＴＰＣ 1细胞生长、细胞膜损伤、细胞形态和生化特征等方面的影响。…     

电化学免疫检测技术

一、概述电化学免疫检测是近十几年发展起来的一种新的微量检测技术,主要用于生物活性物质:抗体、抗原以及其它生物体内分泌物质的检测。免疫检测是基于抗原抗体特异结合,从而进行抗原、抗体浓度测量的。电化学免疫检测大致可以分为直接测量和间接测量两大类。直接测量是利用抗原、抗体反应产生抗原抗体复合物的膜电位变化,用电化学传感元件测量电位变化,从而测出抗原或抗体浓度。间接测量是利用抗     

Mitotic aberrations induced by carbaryl reflect tyrosine kinase inhibition with coincident up-regulation of serine/threonine protein phosphatase activity: implications for coordination of karyokinesis and cytokinesis.

The insecticide carbaryl and its metabolite 1-naphthol cause partial uncoupling of karyokinesis and cytokinesis in V79 Chinese hamster fibroblasts; karyokinesis is blocked in metaphase, the microtubules of the spindle depolymerize and the chromosomes and spindle remnants become displaced to the periphery of the cell. A high frequency of these disturbed cells elongate and a smaller fraction initiate a cleavage furrow. Here, we attempt to determine the potential targets for carbaryl and 1-naphthol in cytokinesis-specific signalling, led by the fact that the potential protein phosphatase inhibitor 1-naphthyl phosphate was previously identified in treated cells. We found that the typical cytological pattern induced by carbaryl and 1-naphthol could be obtained with tyrphostins, specific tyrosine kinase inhibitors, indicating that the carbaryl-induced effects could be due to tyrosine kinase inhibition. This was confirmed by tyrosine kinase assays showing that carbaryl, 1-naphthol and 2-naphthol were equally efficient at inhibiting tyrosine kinase activity as tyrphostin B44(-). As tyrosine kinases can act as regulatory factors in determining dephosphorylation rates, the activities of type-1 (PP1) and type-2A (PP2A) serine/threonine protein phosphatases were also determined. There was a clear up-regulation of the overall PP1/PP2A activities in cells treated with carbaryl, 1-naphthol or tyrphostin B44(-). This stimulation was shown to be indirect because these compounds had no effect on the activity of purified human PP1 in the test tube. 2-Naphthol, which has been found to be less efficient with regard to displacement of chromatin, did not cause up-regulation, but a significant decrease in PP1/PP2A activity. We suggest that a net decrease in tyrosine kinase activity in combination with a net increase in PP1/PP2A activity is a precondition for cell elongation and cytokinesis in mammalian cells and that the corresponding enzymes are targets in the network of activities serving to coordinate karyokinesis and cytokinesis.     

A role of PP1/PP2A in mesenteric lymph node T cell suppression in a two-hit rodent model of alcohol intoxication and injury

This study examined the role of protein phosphatase type-1 (PP1), type-2A (PP2A), and mitogen-activated protein kinase phosphatase-1 (MKP-1) in altered mesenteric lymph node (MLN) T cell function in a two-hit model of alcohol (EtOH) intoxication and burn injury. Male rats (250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dL prior to burn or sham injury (25% total body surface area). MLN T cells harvested 24 h after injury show a significant decrease in p38 and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation in T cells from rats receiving a combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. Treatment of cells with inhibitors of PP1/PP2A [calyculin A (CA) and okadaic acid (OA)] prevented the suppression in T cells p38 and ERK-1/2 activation. In addition, the suppression in interleukin-2 and interferon-gamma production was attenuated in T cells cultured in the presence of CA and OA. MKP-1 inhibitor triptolide did not prevent the suppression in T cells p38/ERK-1/2 and cytokine production. Furthermore, there was a significant decrease in PP1alpha phosphorylation (Thr320) and an increase in PP2A (Tyr307) phosphorylation in T cells following a combined insult of EtOH intoxication and burn injury. As phosphorylation of PP1 at Thr320 and PP2A at Tyr307 led to an inhibition of their enzymatic activities, the decrease in the PP1alpha phosphorylation correlates with an increase in its enzyme activity. Thus, these results suggest that activation of PP1 is likely to play a predominant role in T cell suppression following a combined insult of EtOH intoxication and burn injury.     

桩蛋白在两型星形胶质细胞及神经元中的差异表达

目的:观察桩蛋白在大鼠两型星形胶质细胞及神经元中的差异表达情况。方法:通过细胞培养并结合免疫荧光和RT-PCR方法,观察了桩蛋白的mRNA及蛋白在大鼠两型星形胶质细胞T1A和T2A以及神经元中的表达。结果:RT-PCR显示体外培养的T1A表达桩蛋白mRNA,而T2A和神经元未表达桩蛋白mRNA;免疫荧光染色显示桩蛋白主要分布于T1A的胞浆及突起中。结论:桩蛋白在T1A中表达,但未表达于T2A和神经元中;本实验结果为进一步研究桩蛋白在T1A中的生物学作用奠定了基础。     

新生大鼠大脑皮质O-2A祖细胞的体外诱导分化

目的　在获取高纯度O 2A祖细胞的基础上 ,探讨O 2A祖细胞在体外分化的形态学特点。方法　采用“两次恒温摇床振荡法”和差速贴壁法 ,结合使用神经营养因子 (bFGF、PDGF AA) ,进行O 2A祖细胞体外纯化和扩增培养 ,并观察O 2A祖细胞在有血清和无血清培养条件下的分化情况 ,用免疫细胞化学鉴定分化成熟的 2型星型胶质细胞和少突胶质细胞。结果　培养的O 2A祖细胞能形成克隆球 ,具有增殖能力 ,经免疫细胞化学鉴定细胞纯度达 90 %以上。在含血清的培养基中能定向分化为 2型星形胶质细胞 ,其胞体较大 ,从胞体上伸出细长突起 ,由突起上再发出更为细小的分支 ,GFAP和A2B5抗体标记均为阳性 ;在无血清的化学条件培养基中能定向分化为少突胶质细胞 ,其胞体较小 ,突起短而细 ,相互交织呈“蜘蛛网”样 ,CNPase抗体标记阳性。结论　 (1)培养的O 2A祖细胞保持定向干细胞的特性 ,具有增殖和双向分化的潜能 ;(2 )O 2A祖细胞定向分化为 2型星形胶质细胞过程中经历较长的增殖期 ,而分化为少突胶质细胞过程中则增殖期较短 ;(3)分化的 2型星形胶质细胞和少突胶质细胞在形态学和抗原表达上存在差异。     

Oligodendrocyte progenitor cells derived from mouse embryonic stem cells give rise to type-1 and type-2 astrocytes in vitro

Oligodendrocyte progenitor cells (OPCs) in primary culture can give rise to mature oligodendrocytes and type-2, but not type-1 astrocytes depending on the culture conditions. The OPCs thus are called oligodendrocyte-type-2 astrocyte (O2-A) progenitor cells. Mouse embryonic stem cells (mESCs) have been efficiently differentiated into OPCs; however, the fate plasticity of mESC-derived OPCs is not well characterized. In the present study, using GFP-Olig2 mESC line, we showed that the Olig2(+)/GFP(+)/A2B5(+)/NG2(+) OPCs derived from GFP-Olig2 mESCs can mature into oligodendrocytes when co-cultured with mESC-derived neurons. Interestingly, when induced to astrocytic differentiation by bone morphogenetic protein-4, these mESC-derived OPCs can not only generate type-2 astrocytes, but also type-1 astrocytes. These results challenge the dogma that OPCs in culture can only generate type-2, but not type-1 astrocytes, and support the in vivo finding that during perinatal development, OPCs can give rise to a subset of type-1 astrocytes.     

蛋白脂蛋白在两型星形胶质细胞和少突胶质细胞中的表达

目的:探讨蛋白脂蛋白(PLP)在分化成熟的两型星形胶质细胞和少突胶质细胞中的表达。方法:用激光共聚焦双重免疫荧光标记技术检测PLP在分化成熟的两型星形胶质细胞和少突胶质细胞中的表达情况。结果:在O-2A谱系来源的成熟2型星形胶质细胞和少突胶质细胞中PLP抗体标记为阳性,而在T1A谱系来源的成熟1型星形胶质细胞中则未检测到PLP的表达,从而在蛋白质水平验证本室研究两型星形胶质细胞基因表达谱差异基因芯片结果中PLP mRNA在T2A中高表达,而在T1A中低表达的现象。结论:PLP等脂代谢相关基因可能在O-2A谱系发生和分化过程中起重要作用,O-2A谱系与神经髓鞘发生以及脑内脂质代谢内在机制密切相关。     

Immunoglobulin M and G antibody response to type- and subtype-specific antigens after primary and secondary exposures of mice to influenza A viruses.

A mouse model of influenza infection was studied to help define parameters that may affect serodiagnosis of human infections by immunoassays. Antibodies to both type- and subtype-specific influenza A antigens were measured by a solid-phase immunofluorometric assay. Dilute mouse sera were added to purified influenza virus that had been covalently bound to polyaminostyrene microbeads, and the bound antibody was detected by fluorescein isothiocyanate-labeled isotype-specific antisera. Results were consistent in that upon exposure of mice by either infection alone or by vaccination after infection, both immunoglobulin M (IgM) and IgG antibodies reactive with newly encountered subtype specific viral antigens were measured. IgG antibody was usually detectable by the solid-phase immunofluorometric assay several days before it could be detected by a hemagglutination inhibition test. Increased levels of antibody of the IgG1, IgGa, IgG2b, and IgG3 subclasses were also measured during influenza infection. Surprisingly, response to type-specific viral antigens was of the IgG class in primary as well as in secondary exposure. The results suggest that for serodiagnosis of influenza infections by detection of specific IgM antibody, the assay should use subtype-specific antigens.     

Protein phosphatases and nucleolin in osteoblastic cells: cleavage of nucleolin in apoptotic cells

Protein phosphorylation and dephosphorylation has been recognized as a key mechanism in cell proliferation, differentiation, and apoptosis in various tissues. Okadaic acid is a potent inhibitor of protein phosphatase type 1 (PP1) and type 2A and induces apoptosis in human osteoblastic Saos-2 and MG63 cells. Nucleolin is an abundantly expressed nucleolar phosphoprotein and is located mainly in the nucleolus. The staining pattern of nucleolin in Saos-2 and MG63 cells is similar to that of PP1 delta. Nucleolin was demonstrated to bind to PP1 delta in nucleolus by using immunocytochemical and immunoprecipitation methods. AgNORs and nucleolin, visible as dots in the nucleus of the control cells, disappeared from the apoptotic nuclei. A major band, 110 kDa, was detected in the proteins obtained from the control cells. The level of the 110 kDa protein decreased in the apoptotic cells, whereas an additional band, 80 kDa, appeared and the level of this protein increased in the proteins prepared from okadaic acid-induced apoptotic cells. Our results indicate that PP1 delta directly binds to nucleolin in the nucleolus and that nucleolin is cleaved during apoptosis.     

Type-1 interferon signaling mediates neuro-inflammatory events in models of Alzheimer's disease

A neuro-inflammatory response has been implicated in human patients and animal models of Alzheimer's disease (AD). Type-1 interferons are pleiotropic cytokines involved in the initiation and regulation of the pro-inflammatory response; however, their role in AD is unknown. This study investigated the contribution of type-1 IFN signaling in the neuro-inflammatory response to amyloid-beta (Aβ) in vitro and in the APP/PS1 transgenic mouse model of AD. Enzyme-linked immunosorbent assay confirmed a 2-fold increase in IFNα in APP/PS1 brains compared with control brains. Quantitative polymerase chain reaction also identified increased IFNα and IFNβ expression in human pre-frontal cortex from AD patients. In vitro studies in primary neurons demonstrated Aβ-induced type-1 IFN expression preceded that of other classical pro-inflammatory cytokines, IL1-β, and IL-6. Significantly, ablation of type-1 interferon-α receptor 1 expression in BE(2)M17 neuroblastoma cells and primary neurons afforded protection against Aβ-induced toxicity. This study supports a role for type-1 interferons in the pro-inflammatory response and neuronal cell death in AD and suggests that blocking type-1 interferon-α receptor 1 maybe a therapeutic target to limit the disease progression.     

Phosphatases regulate histamine synthesis in rat brain

Cyclic AMP-dependent protein kinase (PKA) and Ca(2+)-calmodulin dependent protein kinase II (CaMKII)-mediated phosphorylation activate histamine synthesis in nerve endings, but the phosphatases deactivating it had not been studied. In this work we show that the protein phosphatase 2A (PP2A)/protein phosphatase 1 (PP1) inhibitor okadaic acid increases histamine synthesis up to twofold in rat cortical miniprisms containing histaminergic nerve endings. This effect was mimicked by the PP2A/PP1 inhibitor calyculin, but not by the inactive analog 1-norokadaone. Other phosphatase inhibitors like endothall (PP2A), cypermethrin and cyclosporin A (protein phosphatase 2B, PP2B) had much lower effects. The effects of okadaic acid appeared to be mediated by an activation of the histamine synthesizing enzyme, histidine decarboxylase. PKA-mediated activation of histamine synthesis decreased the EC(50) and maximal effects of okadaic acid. On the other hand, CaMKII-mediated activation of histamine synthesis decreased okadaic acid maximal effects, but it increased its EC(50). In conclusion, our results indicate that brain histamine synthesis is subjected to regulation by phosphatases PP2A and PP1, and perhaps also PP2B as well as by protein kinases.