Immunofluorescence assay (IFA)

免疫荧光分析 (IFA)
  • 文章类型: Journal Article
    莱姆病的争议之一是螺旋体螺旋体在指南指导的抗菌治疗后可能持续存在。螺旋体的直接检测对于探索这种现象至关重要,鉴于感染通常是隐匿性的,并且很少在血液和其他体液中观察到。此外,通过在受影响的组织中检测,已经检查了螺旋体感染在神经退行性疾病病因中的作用。在这一章中,我们描述了专门鉴定疏螺旋体DNA的方法,RNA,和组织中完整的生物体(通过蛋白质)用于莱姆病的研究。
    Among the controversies in Lyme disease is the potential for Borrelia spirochetes to persist after guideline-directed antimicrobial therapy. Direct detection of the spirochetes has been essential to explore this phenomenon, given that the infection is often occult and infrequently observed in blood and other body fluids. In addition, the role of spirochetal infection has been examined in the etiology of neurodegenerative diseases through detection in affected tissues. In this chapter, we describe methodology to specifically identify Borrelia DNA, RNA, and intact organism (via protein) in tissue for studies of Lyme Borreliosis.
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  • 文章类型: Journal Article
    细胞病变效应包括由病毒感染产生的一组细胞改变。它具有很大的相关性,因为它构成了感染的直接标记。同样,这些改变通常是病毒特异性的,这使它们成为许多病毒物种的表型标记。通过对感染产生的损伤进展的动力学研究,所有这些特征已用于补充对病毒-细胞相互作用动力学的研究。各种方法已被用于监测细胞病变效应,从光学显微镜,免疫荧光测定,用荧光染料直接标记,随着时间的推移,用于表征感染的斑块测定。在这里,我们讨论了细胞病变效应研究的相关性,并描述了其应用的不同实验替代方案。
    The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.
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  • 文章类型: Journal Article
    疱疹是一种众所周知的传染性感染,同样影响两性。在用于治疗的许多抗病毒药物中,阿昔洛韦(ACY)是首选药物。目前可用的ACY疗法存在局限性,例如口服生物利用度差(10-15%)和高剂量要求。本科学研究旨在探索pluronic卵磷脂有机凝胶(PLO)作为ACY的新型药物递送平台,以通过局部途径改善其递送。有机凝胶的性质如生物相容性和两亲性质(其促进具有不同溶解度特性的各种药物的溶解以及增强活性分子的渗透潜力)使其成为用于治疗局部疾病的有利的药物递送平台。所开发的PLO配方具有微聚特性,viz.,zeta电位,药物含量百分比,有机凝胶形态,皮肤渗透,保留,和稳定性研究。通过诱导皮肤单纯疱疹病毒1型感染,然后通过免疫荧光和PCR分析确认病毒载量,将选定的局部制剂与市售制剂的治疗功效进一步比较。如在损伤评分指数和PCR分析中所反映的,开发的制剂显示出相对于市售产品的显著改善。Further,与对照组相比,在t.i.d.治疗方案中,它被证明对市售制剂更好。导致提高的生物利用度和安全性的总体性能的改善归因于赋形剂性质和制剂特征之间的协同作用。这种微环境中的药物ACY不仅发现了改进的递送载体,而且还提供了增强的药物-靶标相互作用。
    Herpes is a well-known contagious infection equally affecting both sexes. Among many antiviral drugs employed for its treatment, acyclovir (ACY) is the drug of choice. The currently available therapies of ACY suffer from limitations like poor oral bioavailability (10-15%) and high-dose requirement. The present scientific study aims to explore pluronic lecithin organogel (PLO) as a novel drug delivery platform for ACY to bring an improvement in its delivery through topical route. The properties of organogel like biocompatibility and amphiphilic nature which facilitates dissolution of various drugs of different solubility characteristics along with enhancing the permeation potential of active molecules make it a favorable drug delivery platform for the management of topical diseases. The developed PLO formulations were characterized for micromeritic characteristics, viz., zeta potential, percentage drug content, organogel morphology, skin permeation, retention, and stability studies. The selected topical formulation was further compared with the marketed one for its therapeutic efficacy by inducing cutaneous Herpes simplex virus type 1 infection followed by confirmation of viral load by immunofluorescence and PCR analyses. The developed formulation showed significant improvement over the marketed product as reflected in lesion scoring index and PCR analysis. Further, it proved better to the marketed formulation in t.i.d. treatment regimen in comparison to control. The improvement in overall performance leading to enhanced bioavailability and safety is attributed to the synergism between excipient properties and formulation characteristics. The drug ACY in this micro environment not only finds an improved delivery vehicle but it also offers enhanced drug-target interactions.
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  • 文章类型: Journal Article
    严重发热伴血小板减少综合征(SFTS)是由SFTS病毒(SFTSV)引起的。我们调查了SFTS患者血清学反应的详细动力学。2015年7月至2018年10月纳入28例年龄≥18岁的患者。通过使用逆转录聚合酶链反应检测其血浆中的SFTSVRNA来确认SFTS。使用免疫荧光测定(IFA)和酶联免疫吸附测定(ELISA)测量SFTSV特异性IgG和IgM。我们发现在症状发作后第5-9天检测到SFTSV特异性IgG,在疾病过程中,它的滴度在上升。SFTSV特异性IgM滴度在症状发作的第2-3周左右达到峰值。使用IFA和ELISA,症状发作后第5-9、10-14、15-19和20-24天的SFTSV特异性血清阳性率为63%,76%,90%,100%,58%,86%,100%,100%,分别,对于IgG,而他们是32%,62%,80%,100%,53%,62%,70%,100%,分别,IgM。延迟的IgM应答可归因于SFTSV特异性IgMIFA或ELISA的低敏感性和/或受损的免疫应答。我们在这项研究中使用的使用IFA或ELISA的IgM测试是,因此,不足以早期诊断SFTS。
    Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV). We investigated the detailed kinetics of serologic response in patients with SFTS. Twenty-eight patients aged ≥18 years were enrolled between July 2015 and October 2018. SFTS was confirmed by detecting SFTSV RNA in their plasma using reverse transcription polymerase chain reaction. SFTSV-specific IgG and IgM were measured using immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). We found that SFTSV-specific IgG was detected at days 5-9 after symptom onset, and its titer was rising during the course of disease. SFTSV-specific IgM titer peaked at around week 2-3 from symptom onset. The SFTSV-specific seropositive rates for days 5-9, 10-14, 15-19, and 20-24 from symptom onset using IFA and ELISA were 63%, 76%, 90%, and 100%, and 58%, 86%, 100%, and 100%, respectively, for IgG, whereas they were 32%, 62%, 80%, and 100%, and 53%, 62%, 70%, and 100%, respectively, for IgM. The delayed IgM response could be attributed to the low sensitivity of SFTSV-specific IgM IFA or ELISA and/or impaired immune responses. The IgM test using IFA or ELISA that we used in this study is, therefore, insufficient for the early diagnosis of SFTS.
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  • 文章类型: Evaluation Study
    鼠斑疹伤寒是由伤寒立克次体引起的热带病,在东南亚国家等资源有限的环境中流行。伤寒杆菌感染的早期诊断有助于适当的管理并降低严重疾病的风险。然而,由于立克次体低,血液中伤寒的分子检测不敏感。此外,免疫荧光测定(IFA)血清诊断的黄金标准是繁琐的,主观,不切实际,在许多流行地区都没有。为了确定一种可以在印度尼西亚应用的实用诊断方法,我们使用先前研究的伤寒杆菌PCR阳性病例和其他已知感染的对照配对血浆,评估了商业伤寒杆菌IgM和IgG酶联免疫吸附测定(ELISA)和IFA的性能.IgM和IgG抗体联合ELISA的敏感性和特异性。使用配对标本的伤寒表现优异(95.0%和98.3%,分别),与结合的IFAIgM和IgG(97.5%和100%,分别);仅来自急性标本的ELISAIgM敏感性较差(45.0%),但特异性优异(98.3%)。IFAIgM较敏感(77.5%),但单个标本的特异性较低(89.7%)。
    Murine typhus is a tropical disease caused by Rickettsia typhi and is endemic in resource-limited settings such as Southeast Asian countries. Early diagnosis of R. typhi infection facilitates appropriate management and reduces the risk of severe disease. However, molecular detection of R. typhi in blood is insensitive due to low rickettsemia. Furthermore, the gold standard of sero-diagnosis by immunofluorescence assay (IFA) is cumbersome, subjective, impractical, and unavailable in many endemic areas. In an attempt to identify a practical diagnostic approach that can be applied in Indonesia, we evaluated the performance of commercial R. typhi IgM and IgG enzyme-linked immunosorbent assay (ELISA) and IFA using paired plasma from previously studied R. typhi PCR-positive cases and controls with other known infections. Sensitivity and specificity of combined ELISA IgM and IgG anti-R. typhi using paired specimens were excellent (95.0% and 98.3%, respectively), comparable to combined IFA IgM and IgG (97.5% and 100%, respectively); sensitivity of ELISA IgM from acute specimens only was poor (45.0%), but specificity was excellent (98.3%). IFA IgM was more sensitive (77.5%), but less specific (89.7%) for single specimens.
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  • 文章类型: Journal Article
    Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disorder affecting multiple systems of the body. Clinical features show wide variations in patients with the different ethnic background. Renal involvement is a predictor of poor prognosis. Immunological workup is an integral part of SLE diagnostic criteria. Anti-ribosomal P Protein (anti-P) antibodies are highly specific for SLE. They may be present in Antinuclear Antibodies (ANA) negative SLE patients. Their role in Lupus Nephritis (LN) is under debate, some researchers found them associated with poor prognosis whereas others found favorable effect of these antibodies on renal disease.
    In this study, we investigated frequency of anti-P antibodies and the effect of these antibodies on renal functions in the LN patients.
    A total of 133 SLE patients were enrolled in this study. All patients had ANA in their sera. Anti-P antibodies along with other autoantibodies against extractable nuclear antigens (anti-Sm, anti- SS-A, anti-SS-B, anti-histones and anti-RNP) were detected by Immunoblot assay. Anti-dsDNA antibodies were detected by indirect Immunofluorescence Assay (IFA).
    We found anti-P antibodies in 10.5% LN patients. Interestingly their presence in association with anti-dsDNA was associated with improved renal functions in comparison to those who had antidsDNA antibodies in isolation (serum creatinine: 1.3 ± 0.8 mg/dl vs. 3.0 ± 3.0; P= 0.091).
    Anti-dsDNA antibodies are directly involved in renal pathology in SLE patients. As these antibodies are nephrotoxic, concomitant occurrence of anti-P antibodies seems to offer a shielding effect on renal functions, which was evident by normal serum creatinine levels. Therefore, anti-P antibodies may be considered as a good prognostic marker in these patients.
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  • 文章类型: Journal Article
    2016年,以色列报告了骆驼皮肤病变的疾病爆发。为了确定这种疾病的病因,我们采用了多学科诊断方法。病变物质的透射电子显微镜(TEM)分析显示存在类正痘病毒,根据其特征砖的形状。来自皮肤损伤的病毒成功感染绒毛尿囊膜,并在Vero细胞中诱导细胞病变效应,随后被正痘特异性抗体阳性染色。通过两个独立的qPCR对病毒进行了明确的鉴定,其中之一是在这项研究中开发的,然后对病毒基因组的几个区域进行测序。qPCR和测序结果证实了骆驼痘病毒(CMLV)的存在,并表明它不同于先前注释的可从GenBank获得的CMLV序列。这是以色列首例CMLV病例,和隔离的CMLV亚型的第一个描述。
    An outbreak of a disease in camels with skin lesions was reported in Israel during 2016. To identify the etiological agent of this illness, we employed a multidisciplinary diagnostic approach. Transmission electron microscopy (TEM) analysis of lesion material revealed the presence of an orthopox-like virus, based on its characteristic brick shape. The virus from the skin lesions successfully infected chorioallantoic membranes and induced cytopathic effect in Vero cells, which were subsequently positively stained by an orthopox-specific antibody. The definite identification of the virus was accomplished by two independent qPCR, one of which was developed in this study, followed by sequencing of several regions of the viral genome. The qPCR and sequencing results confirmed the presence of camelpox virus (CMLV), and indicated that it is different from the previously annotated CMLV sequence available from GenBank. This is the first reported case of CMLV in Israel, and the first description of the isolated CMLV subtype.
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