用ELISA法和核酸电泳法检测牛轮状病毒

本文用ELISA、核酸电泳法检测腹泻病牛粪便标本。21例中检出轮状病毒抗原有4例为阳性,占19.05%。ELISA法与核酸电泳法结果相符,前者被认为是最值得推广的方法。本研究在江西省首次证明了引起牛腹泻的牛轮状病毒的存在,为防治牛腹泻病采取有效手段提供了病原学依据。     

用ELISA一步法检测白色念珠菌

用白色念珠菌临床分离株免疫家兔并经纯化获得特异性抗体,建立了检测临床标本中白色念珠菌抗原的ＥＬＩＳＡ一步法,此法的敏感性为９９％,特异性为９８３％,与其它念珠菌和临床常见细菌均无交叉反应,整个反应只需１小时即可完成,可望成为一种取代培养法的快速诊断方法     

ELISA技术筛选200种中草药抗HBsAg的实验研究

使用ＥＬＩＳＡ技术对２００种中草药水提取物进行抗ＨＢｓＡｇ的实验研究,共筛出有效药物７种（占总数的３．５％）。若按５种不同剂量（０．３、０．６、１．２、２．５、５．０ｍｇ／１００μｌ）的药物、２种不同浓度的ＨＢｓＡｇ（１０．９２、１４．２６Ｐ／Ｎ值）与３种不同接触时间（立即、１ｈ、２ｈ）的１０项Ｐ／Ｎ值均数来综合评价药效指数时,７种有效药物的次序为青蒿（１．６７Ｐ／Ｎ值）、大蒜（２．１９Ｐ／Ｎ值）、红孩儿（２．３１Ｐ／Ｎ值）、仙鹤草（２．３１）、魔芋（２．３２Ｐ／Ｎ值）、冬瓜皮（２．６３Ｐ／Ｎ值）和猕猴桃（２．８９Ｐ／Ｎ值）。     

斑点ELISA法检测幽门螺旋菌感染的研究

用幽门螺旋菌(HP)的超声粉碎物为抗原,建立了斑点ELISA法检测人血清中抗HP—IgG方法。该法敏感性为94.4％,特异性为87.5％,阳性预测值为97.1％,阴性预测值为77.8％。对271例儿童抗HP抗体调查,发现HP感染与儿童性别无关(P>0.05)。1月以内新生儿抗体检出率59.4％,1～12月婴儿抗体检出率最低(29.1％),1岁后开始逐渐上升,2岁后即可达到50％以上,接近成年人水平。结果表明,我国HP感染年龄早,感染率高。     

双单克隆抗体ELISA间接夹心法检测流行性出血热病毒抗原

建立了检测流行性出血热(EHF)病毒抗原的双单克隆抗体(McAb)ELISA同接夹心法,用本法和间接荧光抗体技术(IFAT)相比较,IFAT检出感染细胞内病毒抗原的高峰在感染后第8天,而ELISA检测感染上清中病毒抗原的高峰在第14天,两方法检测179份人工感染EHF病毒的乳鼠脑和肺组织标本,阳性检出率分别为72.1％和68.2％,实验结果表明,本法特异,敏感,简便,不仅可用于EHF病原学研究,也适用于流行病学调查检测大量鼠肺标本。     

抗HBsAg─抗HRP双特异单克隆抗体的鉴定及初步应用

应用本实验组建立的分泌抗ＨＢｓＡｇ─抗ＨＲＰ双特异单克隆抗体的杂交─杂交瘤细胞株注射Ｂａｌｂ／Ｃ小鼠腹腔,诱生腹水。经用双亲和柱层析,获得较纯双特异单克隆抗体,检测结果表明该抗体具有特异性好、亲和力高等特性。用以建立单克隆抗体与双特异抗体夹心法ＥＬＩＳＡ,初步用于临床血清标本中ＨＢｓＡｇ检测,取得满意效果。并与上海实业科华生化制品有限公司乙肝ＨＢｓＡｇ诊断试剂（卫生部推荐使用试剂）比较,符合率为１００％。此双特异抗体诊断试剂有望广泛用于临床血清标本ＨＢｓＡｇ的检测,为乙型肝炎的诊断提供价廉、快速、特异而敏感的诊断试剂。     

人胎盘型谷胱甘肽S-转移酶的ELISA及用于肝癌的诊断

从人体胎盘提纯GST-π,制备兔抗GST-π血清,纯化其IgG,并降解成Fab′片断,通过SMCC与HRP交联成Fab′-HRP,用于夹心ELISA以测定GST-π,灵敏度可达11pg,超过国外的放射免疫法。用此法测定正常人血清GST-π为1.06±0.94ng/ml,原发性肝癌血清较正常高20倍以上。阳性率达90％左右。     

幽门螺旋菌感染血清学诊断

运用建立起来的酶联免疫吸附试验对80名胃镜确诊胃炎及消化性溃疡患者的血清中抗幽门螺旋菌(Helicobacter pylori)IgG、IgA IgM进行了检测。结果表明,抗H.pylori IgG、IgA都可作为H.pylori感染的指标,但前者敏感性较好(92.0%),后者特异性较高(94.4%);抗H.pyloriIgM升高和降低与H.pylori感染无明显关系。抗H.pylori IgG、IgA水平与H.py     

汉坦病毒H8205部分核壳蛋白基因在E.coli中的表达

根据汉坦病毒Ｈ８２０５株ＮＰ基因的序列,设计一对引物,扩增ＮＰ前２９２个氨基酸多肽基因片段,克隆于表达载体ｐＧＥＸ３Ｘ,与载体中２６ｋＤ的谷胱苷肽巯基转移酶（ＧＳＴ）融合表达。ＳＤＳＰＡＧＥ显示表达产物（ＧＳＴＮＰ）主要以包涵体形式存在。Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ表明此融合蛋白有抗原性。包涵体经分离、洗涤、溶解后,Ｓｅｐｈａｒｏｓｅ６Ｂ层析纯化,用此融合蛋白作抗原,进行ＥＬＩＳＡ法检测临床ＨＦＲＳ病人标本的ＩｇＧ和ＩｇＭ,有很好的特异性和敏感性。有生物活性的汉坦病毒Ｈ８２０５ＮＰ的体外表达成功,为汉坦病毒基因工程抗原的大量制备奠定了基础,也为汉坦病毒的临床检测和流行病学调查提供了一种廉价、安全、可靠的抗原。     

肺炎支原体ELISA检测试剂盒的临床应用

目的评价肺炎支原体ELISA检测试剂盒在临床应用的效果。方法用肺炎支原体ELISA检测试剂盒检测呼吸道感染患儿的咽拭子标本,并以肺炎支原体快速检测培养基试剂做同步盲法对照试验,分析该试剂盒的准确性及批内、批间产品的稳定性。结果在100例呼吸道感染患儿咽拭子标本中,肺炎支原体ELISA检测法阳性率为38%,肺炎支原体快速检测培养基法阳性率为37%,两种方法阳性结果符合率为97%。同步盲法试验结果显示,肺炎支原体ELISA检测试剂盒批内、批间产品阳性结果的一致率均为100%。结论该试剂盒具有较好的准确度和特异性,并且操作简便、快速,临床可推广应用。     

人巨细胞病毒IgG酶联免疫检测试剂盒的研制

为研制人巨细胞病毒 (HCMV)IgG酶联检测试剂盒 ,将HCMV接种人二倍体细胞 ,收获的病毒经纯化后用作包被抗原 ,辣根过氧化物酶 (HRP)标记羊抗人IgG为检测抗体 ,采用间接ELISA制备酶联免疫检测试剂盒并进行检定。该试剂盒操作简便、特异性强 ,稳定性、线性及精密性符合体外诊断试剂的要求。     

糖尿病大鼠膀胱和骶髓背根神经节中NGF 的表达与尿流动力学 改变的研究

目的:探讨糖尿病大鼠膀胱与骶髓背根神经节(DRG)中神经生长因子(NGF)的表达与尿流动力学改变。方法:建立糖尿病大鼠模型10只,对照组10只,应用酶联免疫吸附试验(ELISA)法,分别检测大鼠膀胱组织及骶髓DRG中NGF的变化情况,结合代谢笼及尿流动力学改变,探讨糖尿病膀胱病变的可能发病机制。结果:造模12周后,糖尿病大鼠膀胱容量较正常对照组明显增大(1.47±0.28vs0.71±0.12,p<0.05),残余尿量明显增多(0.52±0.18vs0.07±0.08,p<0.01),排尿效率明显下降,膀胱及骶髓DRG中NGF表达水平明显降低。结论:NGF在糖尿病大鼠膀胱和骶髓背根神经节中低表达,在糖尿病膀胱病变中发挥着重要作用。     

Level of the nerve growth factor activity in the submaxillary glands of genetically dystrophic mouse (C57BL/6J)

To examine the relationship between NGF activity and neurologic disorders, the level of NGF activity was studied in the submaxillary glands of both normal and dystrophic mice. The level of NGF activity in the mice was remarkably lower than that observed in normal mice. The data suggest that NGF activity may have some relation to the cause of the disease.     

Thermosensitive heparin‐poloxamer hydrogel encapsulated bFGF and NGF to treat spinal cord injury

The application of growth factors (GFs) for treating chronic spinal cord injury (SCI) has been shown to promote axonal regeneration and functional recovery. However, direct administration of GFs is limited by their rapid degradation and dilution at the injured sites. Moreover, SCI recovery is a multifactorial process that requires multiple GFs to participate in tissue regeneration. Based on these facts, controlled delivery of multiple growth factors (GFs) to lesion areas is becoming an attractive strategy for repairing SCI. Presently, we developed a GFs‐based delivery system (called GFs‐HP) that consisted of basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and heparin‐poloxamer (HP) hydrogel through self‐assembly mode. This GFs‐HP was a kind of thermosensitive hydrogel that was suitable for orthotopic administration in vivo. Meanwhile, a 3D porous structure of this hydrogel is commonly used to load large amounts of GFs. After single injection of GFs‐HP into the lesioned spinal cord, the sustained release of NGF and bFGF from HP could significantly improve neuronal survival, axon regeneration, reactive astrogliosis suppression and locomotor recovery, when compared with the treatment of free GFs or HP. Moreover, we also revealed that these neuroprotective and neuroregenerative effects of GFs‐HP were likely through activating the phosphatidylinositol 3 kinase and protein kinase B (PI3K/Akt) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) signalling pathways. Overall, our work will provide an effective therapeutic strategy for SCI repair.     

A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.     

HCMV infection depress NGF expression in human glioma cells

Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.     

神经生长因子生物活性检定方法的建立

采用鸡胚背根神经节体外培养法,建立了神经生长因子生物活性的检定方法,对判定神经生长因子的生物活性单位制定了明确统一的判定标准     

狂犬病毒抗体ELISA检测试剂盒的改进研究

为了提高狂犬病毒抗体检测的灵敏度和特异性,采用狂犬病毒单克隆抗体包被酶标板,再分别加入重组的狂犬病毒糖蛋白或细胞培养抗原做固相层的方法(抗体捕捉法),用传统的间接ELISA法做对照,按常规方法检测抗狂犬病毒抗体。结果显示,抗体捕捉法的非特异性反应低于间接法,而灵敏度达到0．51U水平,高于间接法。在800份临床标本检测中,检出率明显高于间接法。用15份阳性血清作小鼠中和试验,并和抗体捕捉ELISA法比较具有高度的一致性。试验结果充分表明,该方法优于传统的ELISA间接法。因此可作为临床注射狂犬疫苗后检测血清中狂犬病毒抗体的常规方法。     

糖尿病大鼠膀胱和骶髓背根神经节中NGF的表达与尿流动力学改变的研究

目的：探讨糖尿病大鼠膀胱与骶髓背根神经节（DRG）中神经生长因子（NGF）的表达与尿流动力学改变。方法：建立糖尿病大鼠模型10只,对照组10只,应用酶联免疫吸附试验（ELISA）法,分别检测大鼠膀胱组织及骶髓DRG中NGF的变化情况,结合代谢笼及尿流动力学改变,探讨糖尿病膀胱病变的可能发病机制。结果：造模12周后,糖尿病大鼠膀胱容量较正常对照组明显增大（1.47±0.28vs0.71±0.12,p〈0.05）,残余尿量明显增多（0.52±0.18vs0.07±0.08,p〈0.01）,排尿效率明显下降,膀胱及骶髓DRG中NGF表达水平明显降低。结论：NGF在糖尿病大鼠膀胱和骶髓背根神经节中低表达,在糖尿病膀胱病变中发挥着重要作用。     

神经生长因子通过抑制内质网应激促进外周神经损伤后髓鞘碎片的清除

神经生长因子 (never growth factor,NGF）可促进损伤外周神经的修复并加速轴突和髓鞘的再生,但对外周神经损伤前期作用的研究报道较少。本研究主要探究在损伤外周神经前期,NGF能否加速施旺细胞 (Schwann cells, SCs) 对髓鞘碎片的清除及其调控机制。将坐骨神经损伤的Wistar雄性大鼠连续5 d给予NGF治疗,并运用分子生物学检测手段分析损伤坐骨神经内部髓鞘碎片的清除,细胞的凋亡及内质网应激 (endoplasmic reticulum stress, ERS) 的表达和变化。免疫荧光分析结果显示,与模型组相比,NGF给药组显著加速髓鞘碎片的清除,并促进SCs的增殖 (46.33 ± 5.68 vs. 66.69 ± 8.76, P＜ 0.05 for MPZ; 47.58 ± 4.52 vs. 37.69 ± 2.50, P＜ 0.01 for GFAP)。TUNEL免疫组化证实,NGF可有效抑制SCs的凋亡(25 ± 4 vs. 37 ± 6, P＜ 0.05),Western印迹结果显示,模型组坐骨神经内部内质网应激水平被过度激活,给予NGF治疗后,相关蛋白质表达被逆转 (1.03 ± 0.03 vs. 1.24 ± 0.07, P＜ 0.01 for PDI; 1.16 ± 0.16 vs. 1.48 ± 0.10, P＜ 0.05 for GRP-78; 1.33 ± 0.11 vs. 1.76 ± 0.17, P＜ 0.01 for Caspase-12; 1.01 ± 0.05 vs. 1.39 ± 0.16, P＜ 0.01 for CHOP)。上述结果证实,NGF可通过抑制内质网应激减少神经组织内细胞的凋亡,并加速髓鞘碎片的清除,促进外周神经损伤的修复。