人IA-2基因的部分区段克隆表达及其在1型糖尿病诊断中的应用价值评估

目的:构建人IA-2基因不同区段原核表达载体,诱导表达获得重组蛋白,并初步验证其在1型糖尿病蛋白酪氨酸磷酸酶自身抗体检测中的价值。方法:用RT-PCR方法调取目的基因,构建相应的原核表达质粒,转化大肠杆菌HB101,诱导表达获得纯化重组蛋白;以重组蛋白为包被抗原,初步建立检测蛋白酪氨酸磷酸酶自身抗体的ELISA方法,评价各片段在1型糖尿病诊断中的价值。结果:获得了2种可被1型糖尿病患者血清识别的重组人蛋白酪氨酸磷酸酶抗原区段IA-2(601～979)和IA-2(683～979),检测敏感性和特异性相当,但IA-2(683～979)检测的阳性D450nm值明显高于IA-2(601～979),成为首选的抗原区段。结论:所选重组人IA-2(683～979)抗原区段具有良好的抗原性,可作为1型糖尿病患者辅助诊断试剂的候选抗原。     

量子点免疫层析快速定量检测HCG

近年来,量子点以其独特的光学性质被广泛应用到医学检测上.血清中人绒毛膜促性腺激素(HCG)的含量是诊断早期妊娠的常用指标,也可用于异常妊娠性疾病的早期发现和鉴别诊断.本文采用超声乳化法制备了高质量的亲水性量子点,并将其与免疫层析试纸条技术相结合,在此基础上自主研发了用于试纸条检测的量子点免疫荧光检测仪,对血清中HCG的含量进行了高灵敏度的快速定量检测.结果表明,对于血清中的HCG含量检测,最优检测条件为加样体积50μl,反应时间15 min,检测的灵敏度达到0.85 U/L,高于商品化的胶体金试纸条.这种检测技术简单、快速、灵敏,有望在其他蛋白质类标志物的检测中得到广泛应用.     

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.     

Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay

Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 10⁴ genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.     

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.     

抗心型脂肪酸结合蛋白单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗心型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),并建立侧向免疫层析方法检测血浆中H-FABP。方法:用H-FABP蛋白免疫纯系Balb/c小鼠,采用杂交瘤技术建立能稳定分泌抗人H-FABP的单克隆杂交瘤细胞株。常规制备腹水,纯化后得到特异性抗H-FABP单克隆抗体,进行效价、特异性、亲和力的鉴定分析,并在ELISA平台进行抗体配对,用所筛选到的抗体对初步建立了检测H-FABP的侧向免疫层析方法。结果:成功获得12株稳定分泌抗体的阳性细胞株,并筛选出能相互配对,并应用于侧向免疫层析平台的抗体3D1和5F4,检测临床样品与对照试剂比较总符合率为100%。结论:筛选能稳定分泌抗体的细胞株,配对抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏的检测出临床样品中H-FABP,为临床应用快速检测H-FABP指标提供了方法和关键材料。     

Evaluation of a New Cryptococcal Antigen Lateral Flow Immunoassay in Serum,Cerebrospinal Fluid and Urine for the Diagnosis of Cryptococcosis: A Meta-Analysis and Systematic Review

BackgroundA new lateral flow immunoassay (LFA) for the detection of cryptococcal antigen was developed.ObjectiveWe aimed to systematically review all relevant studies to evaluate the diagnostic accuracy of the cryptococcal antigen LFA on serum, CSF and urine specimens.MethodsWe searched public databases including PubMed, Web of Science, Elsevier Science Direct and Cochrane Library for the English-language literature published up to September 2014. We conducted meta-analyses of sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratios (DOR) and SROC of LFA in serum and CSF, respectively. The sensitivity of LFA in urine was also analyzed. Subgroup analyses were carried out to analyze the potential heterogeneity.Results12 studies were included in this study. The pooled sensitivity and specificity values of LFA in serum were 97.6% (95% CI, 95.6% to 98.9%) and 98.1% (95% CI, 97.4% to 98.6%), respectively. The average PLR of LFA in serum was 43.787 (95% CI, 22.60–84.81) and the NLR was 0.03 (95% CI, 0.01–0.09). The pooled DOR was 2180.30 (95% CI, 868.92–5471.00) and the AUC was 0.9968. The pooled sensitivity and specificity values of LFA in CSF were 98.9% (95% CI, 97.9% to 99.5%) and 98.9% (95% CI, 98.0% to 99.5%), respectively. The average PLR of LFA in serum was 48.83 (95% CI, 21.59–110.40) and the NLR was 0.02 (95% CI, 0.01–0.04). The pooled DOR was 2931.10 (95% CI, 1149.20–7475.90) and the AUC was 0.9974. The pooled sensitivity value of LFA in urine was 85.0% (95% CI, 78.7% to 90.1%)ConclusionsThe study demonstrates a very high accuracy of LFA in serum and CSF for the diagnosis of cryptococcosis in patients at risk. LFA in urine can be a promising sample screening tool for early diagnosis of cryptococcosis.     

A Rapid Latex Agglutination Assay for the Detection of Penicillin-Binding Protein 2′

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2′ (PBP2′) from isolates of staphylococi. PBP2′ present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2′ could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2′ positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2′ negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.     

番茄环斑病毒纳米荧光颗粒试纸条的研制

目的:研发一种快速、便捷检测番茄环斑病毒(ToRSV)的方法。方法:以荧光纳米颗粒为标记物,采用免疫层析试验方法制备ToRSV荧光纳米颗粒试纸条,在紫外灯下观察试纸条上的荧光信号,作为结果判定依据。结果:用制备的荧光纳米颗粒试纸条检测包括ToRSV在内的9种病毒,仅ToRSV有阳性反应,其余待测样品均呈阴性,表明该试纸条具有较好的特异性;用该荧光试纸条与传统胶体金试纸条进行灵敏度测试时,其灵敏性高于胶体金试纸条1倍以上,且稳定性试验结果良好。结论:ToRSV荧光纳米颗粒试纸条的研制,为快速检测ToRSV提供了有效手段,该方法可用于现场检验,具有广阔的应用前景。     

A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA

Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with nucleic acid sequence based amplification (NASBA), this assay can detect concentrations of HIV RNA that match the clinically relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample preparation technologies to serve as an HIV viral load test for low-resource settings.     

A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

Ca²⁺ activated Cl⁻ channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl⁻ secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca²⁺, which not only activates epithelial CaCCs, but also inhibits epithelial Na⁺ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl⁻ secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca²⁺ and Cl⁻ secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.     

人源蛋白酪氨酸磷酸酶SHP-2基因克隆与原核表达

目的:构建蛋白酪氨酸磷酸酶SHP-2的原核表达载体并在大肠杆菌中表达。方法:以人脑组织mRNA为模板,通过RT-PCR扩增出目标cDNA,构建蛋白酪氨酸磷酸酶SHP-2-pEASY-E1重组质粒。将重组质粒转化进E.coli TOP10感受态细胞中,通过菌落PCR和测序进行阳性克隆的筛选和验证,将正确的质粒转化E.coli Transetta感受态细胞中,通过SDS-PAGE和western-blot进行蛋白检测和验证,酶促动力学分析SHP-2可溶性蛋白的活性。结果:成功克隆SHP-2功能域,构建SHP-2-pEASY-E1原核表达载体,完成可溶性蛋白的表达;酶促动力学分析结果为:米氏常数Km=0.97mmol/L,Vmax为13.57mmol/L/s。结论:本研究成功构建SHP-2的原核表达载体,重组表达的SHP-2蛋白具有较高的磷酸酶活性。     

基于IgG检测为模型的玻璃介质定量蛋白质芯片的研制及评价

为研制高通量定量检测的蛋白质芯片,以人血清IgG为研究模型,选择戊二醛基修饰的玻片为载体,蛋白质样品溶解于20%甘油的PBS,由机械手将浓度为0.5g/L人IgG单克隆抗体点样在玻片上,人血清白蛋白为阴性对照,以1%的BSA为封闭液对蛋白质芯片进行封闭,并经相应处理,构建用于定量检测人血清IgG蛋白质芯片.先以不同浓度IgG标准品为待测样品,建立蛋白质芯片方法和ELISA方法的标准曲线,两条标准曲线的R2值分别为0.996和0.994(P<0.01).两种方法的检测人IgG结果有很好的相关性(R2=0.9937,P<0.01),其敏感性均在15.625μg/L.用两种方法分别检测10例人血清IgG,结果显示两种方法的检测的IgG浓度有很好的一致性,以玻片为载体的蛋白质芯片可用于微量生物样品的定量检测.     

A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R² = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R² = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.     

Detection of Autoantibodies to Annexin A11 in Different Types of Human Cancer

Introduction  Annexin A11 was previously identified as an autoantigen in 4.1–10.1% of patients with various systemic autoimmune diseases. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to investigate the occurrence and features of anti-annexin A11 autoantibodies in sera from patients with different types of cancer. Methods  The recombinant protein of GST fused to the N-terminal domain (1–175 residues) of human annexin A11 was expressed and used as antigen in ELISA. A total of 246 serum specimens were analyzed, which includes sera from healthy women (77), patients with ovarian cancer (72), breast cancer (18), colon cancer (19), pancreatic cancer (20), prostate cancer (20), and diabetes (20). Results  The overall titer of anti-annexin A11 autoantibodies in ovarian cancer patients (or primary tumors only) was found much higher than that in healthy controls (P < 0.05). At the cut-off value designating positive reaction, anti-annexin A11 autoantibodies were detected in 12.5% (5/40) of primary ovarian cancer patients with a significant difference from 2.6% (2/77) of the healthy controls (P < 0.05), but only in 6.25% (2/32) of recurrent tumors. ROC curve demonstrated the potential diagnostic value of anti-annexin A11 autoantibodies in primary ovarian cancer patients with an AUC of 0.62 (0.52–0.73). Anti-annexin A11 autoantibodies were also detected in 5.26% (1/19) of colon cancer and 10% (2/20) of diabetes patients but without significant difference from the healthy controls. Conclusion  A convenient assay to detect anti-annexin A11 autoantibodies in patients was developed, and the experimental data are promising but need to be expanded to address their biological/clinical relevance.     

Tyrosine 112 of Latent Membrane Protein 2A Is Essential for Protein Tyrosine Kinase Loading and Regulation of Epstein-Barr Virus Latency

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.     

基于流式细胞术快速检测人鼻黏膜组织淋巴细胞亚群的实验研究

目的：基于流式细胞术检测正常人的鼻黏膜组织中的淋巴细胞亚群(CD3+T、CD4+T、CD8+T、NK、CD19+B)的比例,初步探讨 鼻局部黏膜免疫功能的意义,为鼻局部黏膜免疫性疾病的研究提供更多的参考。方法：用鼻黏膜刮匙获取21 例正常人的鼻黏膜 组织,并抽取其外周血2 mL。采用流式细胞术分别检测其鼻黏膜和外周血中的淋巴细胞亚群(包括CD3+T、CD4+T、CD8+T、NK、 CD19+B)分别所占的比例。结果：鼻黏膜和外周血的淋巴细胞亚群存在很大差异,与外周血相比,CD3+T 比例增加（t=15.34,P<0. 0001),CD4+T 比例降低(t=5.952,P<0.0001)、CD8+T 比例增加(t=12.44,P<0.0001)、NK 比例降低(t=4.865,P<0.0001)、CD19+B 比例降 低(t=15.56,P<0.0001),CD4+T/CD8+T 降低。结论：流式细胞术可以用来检测鼻黏膜的淋巴细胞亚群,鼻黏膜的淋巴细胞亚群和外 周血的淋巴细胞亚群存在很大差异,这种差异体现鼻黏膜组织独特的局部黏膜免疫功能,本方法为变应性鼻炎的研究提供了新 的研究途径。