抗体芯片的应用进展

抗体芯片作为蛋白芯片的一种,具有微型化、集成化、高通量化的特点,可以用于检测某一特定的生理或病理过程相关蛋白的表达丰度,目前主要用于信号转导、蛋白质组学、肿瘤及其他疾病的相关研究。本文就抗体芯片的原理、基本技术环节、检测内容、应用领域及存在的问题等方面进行综述。     

肌红蛋白压电免疫芯片抗体固定方法学研究

目的:构建一种检测心肌肌红蛋白的压电免疫芯片,探讨压电免疫传感技术用于诊断急性心肌梗死等疾病的可行性。方法:设计四通道芯片微阵列,分别应用巯基自组装法、戊二醛交联法、蛋白A法固定心肌肌红蛋白单克隆抗体,从抗体固定量、响应特性等方面比较三种固定方法的优缺点,探讨抗体的最佳固定方法,并对抗体固定条件进行优化。观察该芯片检测心肌肌红蛋白的灵敏度、线性范围、精密度与重复性及非特异性反应等。检测临床标本并与酶联荧光法进行对比,评价该方法的临床应用价值。结果:Sulfo-LC-SPDP巯基自组装法芯优于其他两种方法,以此法构建的压电免疫芯片可用于半定量检测心肌肌红蛋白。但限于现有的实验条件,目前尚无法实现精确定量检测。结论:构建的压电免疫芯片具有灵敏度高、特异性好等特点,具有一定的临床应用价值。     

抗体芯片技术研究进展

抗体芯片是蛋白质组学的新兴技术,属于蛋白芯片的一个分支,具有高通量、高特异性、平行性分析的优点。目前抗体芯片技术在癌症标记物发现、药物发展、毒素检测、细胞因子研究、信号转导、表达产物分析、RNAs检测以及其它的基础和应用蛋白质组学研究等领域具有广泛的应用,并且在临床上有补充或取代传统免疫学方法的趋势。     

抗体芯片技术研究进展

抗体芯片是蛋白质组学的新兴技术,属于蛋白芯片的一个分支,具有高通量、高特异性、平行性分析的优点.目前抗体芯片技术在癌症标记物发现、药物发展、毒素检测、细胞因子研究、信号转导、表达产物分析、RNAs检测以及其它的基础和应用蛋白质组学研究等领域具有广泛的应用,并且在临床上有补充或取代传统免疫学方法的趋势.     

抗体芯片技术研究进展

抗体芯片是蛋白质组学的新兴技术,属于蛋白芯片的一个分支,具有高通量、高特异性、平行性分析的优点.目前抗体芯片技术在癌症标记物发现、药物发展、毒素检测、细胞因子研究、信号转导、表达产物分析、RNAs检测以及其它的基础和应用蛋白质组学研究等领域具有广泛的应用,并且在临床上有补充或取代传统免疫学方法的趋势.     

桃过敏原组分检测蛋白质芯片的制备及验证

目的制备用于检测血清中针对几种桃过敏原组分抗体的蛋白质芯片,为针对桃过敏原组分过敏患者的临床诊断提供快速可靠的方法。方法合成6种常见的桃过敏原组分的编码基因,分别将其插入p GAPZαA酵母表达载体,AvrⅡ单酶切线性化后,电转毕赤酵母SMD1168,表达和纯化相应的桃过敏原组分蛋白,并制备蛋白质芯片;用蛋白芯片检测临床血清样本中针对桃过敏原组分蛋白的抗体,与ImmunoCAP法相比,验证蛋白质芯片检测的灵敏度和特异性。结果用酵母表达系统成功表达并纯化了4种桃过敏原组分Pru p 1、Pru p 3、Pru p 4和Pru p 7,制备了能进行质量控制的检测血清桃过敏原组分抗体的蛋白质芯片。检测了疑似桃过敏患者血清41例,结果以ImmunoCAP法为金标准,蛋白芯片检测血清Pru p 1、Pru p 3和Pru p 4抗体的灵敏度分别为40%、100%和100%,特异性为78%、50%和100%;对上述3种组分抗体的综合检测灵敏度为86%,特异性为96%。针对缺乏ImmunoCAP检测结果的Pru p 7抗体,蛋白芯片检测出6份阳性的血清样本,均为桃过敏患者。结论桃过敏原组分检测芯片在灵敏度和特异性方面与ImmunoCAP相当,血清用量少,优化后有望成为临床桃过敏检测的一种新方法。     

单中心两种方法检测280例群体反应性抗体的对比分析

背景：液态芯片技术(Luminex)检测方法是近年来兴起的检测方法,应用液相芯片技术检测抗群体反应抗体,具有灵敏度高,特异性强,检测干扰少,高通量等优点。目的：比较ELISA和Luminex两种方法对肾脏病患者血清标本中群体反应性抗体的检测差异及灵敏度。方法：选取280例肾脏病患者血清标本,同时应用ELISA和Luminex两种方法检测群体反应性抗体阳性率,并运用配对四格表资料的χ²检验进行统计学分析。结果与结论：ELISA法检测群体反应性抗体阳性率为18.9%,Luminex法检测群体反应性抗体阳性率为33.6%。ELISA法检出抗HLA-Ⅰ类抗体和抗HLA-Ⅱ类抗体阳性率分别为12.8%和12.5%;Luminex法检出抗HLA-Ⅰ类抗体和抗HLA-Ⅱ类抗体阳性率分别为25.0%和20.7%。Luminex法的阳性检出率明显高于ELlSA法且Luminex法能准确检测到低浓度抗体。配对四格表资料的χ²检验P < 0.01,显示两种方法检测肾脏病患者群体反应性抗体结果差异有显著性意义。结果显示与ELISA法相比,Luminex技术具有更高的灵敏度和准确性,更适合应用于临床检测。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

利用蛋白质芯片技术筛查多发性硬化患者的自身抗体

目的用高通量蛋白质芯片技术筛选多发性硬化(Multiple Sclerosis,MS)患者血清中可能存在的自身抗体,为筛选新的疾病辅助性诊断的指标奠定基础。方法 15例MS患者作为疾病组,选取同样数量MS阴性并无其他自身免疫疾病的性别和年龄相匹配的健康人15位作为健康对照组。两组组内随机分为3个小组,血清充分混匀后,采用高通量蛋白质芯片法检测MS患者血清中可能存在的自身抗体。结果通过高通量蛋白质芯片技术检测及与对照组分析比对,共筛出MS患者血清27种自身抗体。其中阳性率较高为SLC16A4、NOL3和DTX1,提示其可能是MS患者血清中特异性自身抗体的靶蛋白。阳性蛋白数量上,MS组高于HC组。功能聚类结果显示,自身抗体主要针对神经相关的蛋白质。结论采用高通量蛋白质芯片技术,可以较为有效筛选出MS血清中的自身抗体的靶抗原,为进一步验证MS相关自身抗体的功能及作用机制奠定了基础。     

用于制备抗体芯片的抗人甲胎蛋白单克隆抗体的制备及特性分析

目的：制备抗人甲胎蛋白（AFP）单克隆抗体（mAb），并用于制备抗体芯片。方法：利用杂交瘤技术建立能稳定分泌抗人AFPmAb的杂交瘤细胞株；对抗体的亲和力、特异性等特性进行分析；利用抗体相加实验，双抗体夹心ELISA、胶体金免疫层析实验以及抗体芯片技术对抗体表位及其配对情况进行分析研究。结果：共获得16株稳定分泌抗人AFPmAb的杂交瘤细胞株，从中筛选出多组性能优良的配对抗体，结合抗体芯片技术，最终筛选出适合制备抗体芯片的配对抗体。结论：当抗体包被材料或包被方法发生改变时，会引起mAb构象发生变化，从而影响抗体的配对效果。因此，筛选适合制备抗体芯片的配对mAb时，需要对mAb的特性进行综合分析，选取特异性好，亲和力高的mAb进行配对筛选。     

单克隆抗体2A10在蛋白芯片技术检测黄病毒属病毒中的应用

目的 本研究拟探讨1株黄病毒属特异的单克隆抗体2A10作为检测抗体在同时检测5种黄病毒属病毒的蛋白芯片中的应用价值.方法 将病毒的特异性单抗作为捕获抗体进行点样,制备用于检测5种黄病毒属病毒的抗体芯片,然后分别加入拟检测的病毒灭活抗原,捕获病毒颗粒后,加入荧光染料CY3标记的2A10检测抗体进行孵育,通过扫描并分析芯片上不同抗体的荧光强度,确定2A10检测抗体的效价和可检测的病毒滴度,并对2A10的检测效果进行评价.结果 蛋白芯片检测结果表明,2A10可作为检测抗体用于对5种黄病毒属病毒的检测,CY3标记的2A10效价为1∶160,以脾传脑炎(TBE)病毒为例,可检测的病毒滴度为1250 PFU.结论 单克隆抗体2A10作为一株黄病毒属特异的单抗,可以用于蛋白芯片技术检测黄病毒属病毒.     

Simultaneous analysis of eight human Th1/Th2 cytokines using microarrays

The adaptive immune system induces T cells to change from a naive phenotype to a Th1/Th2 phenotype each of which produce characteristic types of cytokines. Knowledge of whether a specific immune response is Th1 or Th2 is a useful indicator for diseases with basis in immune function disorder. An assay that can rapidly analyze multiple cytokines indicative of these two cell types from small sample quantities can be an extremely useful research and diagnostic tool. Silanized glass slides were printed with multiple arrays of capture antibodies to detect eight different cytokines involved in the Th1/Th2 response along with control proteins for assessing assay performance. Arrays were developed by sequential addition of known antigen amounts, detector antibodies and a fluorescent detection system followed by imaging and quantification. These arrays were used to determine the specificity, sensitivity and reproducibility of the assay and the performance compared with conventional ELISA. This multiplexed assay is able to measure human Th1/Th2 cytokines in sample volumes lower than 20 microl. The assay sensitivity for the eight cytokines range from 0.3 microg/l for IL-4 to 6.4 microg/l for IL-5 which are either comparable to or higher than those reported for conventional ELISA or bead-based multiplex ELISA methods. This assay can be automated to measure expression levels of multiple Th1/Th2 cytokines simultaneously from tens to hundreds of biological samples. This assay platform is more sensitive and has a larger dynamic range as compared to a conventional ELISA in addition to significantly reducing the time and cost of assay. This platform provides a versatile system to rapidly quantify a wide variety of proteins in a multiplex format.     

Sensitive biosensor array using surface plasmon resonance on metallic nanoslits

Chip-based biosensor arrays for label-free and high-throughput detection were fabricated and tested. The sensor array was composed of a 150-nm-thick, 50-nm-gap, and 600-nm-period gold nanoslits. Each array size was 100 mumx100 mum. A transverse-magnetic polarized wave in these metallic nanostructures generated resonant surface plasmons at a wavelength of about 800 nm in a water environment. Using the resonant wavelength shift in the nanoslit array, we achieved detection sensitivity up to 668 nm per refractive index unit, about 1.7 times larger than that reported on an array of nanoholes. An antigen-antibody interaction experiment in an aqueous environment verified the sensitivity in a surface binding event.     

Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.

DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.     

Lipopolysaccharide microarrays for the detection of antibodies

Lipopolysaccharide (LPS) is the major component of Gram-negative bacterial outer membrane. LPS are immunogenic and show species/strain specificity. The demonstration of anti-LPS antibodies in clinical samples is of diagnostic value in certain Gram-negative bacterial infections. In the present study we explored the possibility of immobilizing LPS isolated from different bacteria in a microarray format for the detection of anti-LPS antibodies. LPS was successfully immobilized on nitrocellulose-coated glass slides, preserving the accessibility of epitopes for antibody binding. Specificity of the LPS arrays was established using four different monoclonal antibodies specific for Escherichia coli O111, E. coli O157, Francisella tularensis and Salmonella typhimurium O-antigens and a panel of LPS preparations. The detection limit of antibodies was found to be 10 ng/ml, which is about a 100-fold greater sensitivity compared to conventional immunofluorescence assays. Furthermore, using LPS arrays, tularemia positive canine serum samples could be differentiated from negative samples based on the presence of significantly higher levels of anti-F. tularensis LPS antibodies in positive samples. LPS arrays will facilitate simultaneous screening of samples against multiple antigens and are expected to find applications in diagnostics and seroepidemiology.     

Platinum nanowire nanoelectrode array for the fabrication of biosensors

Platinum nanowire arrays can be grown by electrodeposition in polycarbonate membrane, with the average diameter of the nanowires about 250 nm and the height about 2 microm. The nanowire array prepared by the proposed method can be considered as nanoelectrode array (NEA) with nanoelectrode density of 5 x 10(8)cm(-2). While the NEA can improve the signal-to-noise ratio and decrease the detection limit, the high surface area of the platinum NEA circumvents the problem of conventional platinum electrodes associated with the limited electroactive site. The platinum NEA can direct response to hydrogen peroxide at low potential of 0 V with wide linear range (1 x 10(-7)-6 x 10(-2)M) and sensitivity 50 times larger than that of the conventional platinum electrode. With the absorption of glucose oxidase onto the ordered NEA surface, the spatially patterned glucose oxidase improves greatly the resulting biosensor. The biosensor can achieve interference free determination of glucose with wide linear range (10(-6)-3 x 10(-2)M). The sensitivity of the glucose biosensor is one-fifth of the sensitivity toward hydrogen peroxide, indicating high efficiency of signal transduction. The biosensor was used to determine glucose in real blood samples, and the glucose contents determined by the present biosensor were in agreement with the results of existing method.     

A review of enhancers for chemiluminescence enzyme immunoassay

There is increasing interest for chemiluminescence (CL) detection with the characteristics of simplicity, low cost and high sensitivity, especially wide application of enhancersin CL detection to increased signals, prolonged luminescence time and enhanced intensity. In this review, the applications of primary enhancers and secondary enhancers were mainly described in the horseradish peroxidase–luminol system of CL enzyme immunoassay for light delay in the course of the reaction with improved sensitivity. The present review on enhancers covers the papers since 1983. Future research needs to develop novel enhancers with less interference and better performance. With wide utilization of enhancers in the CL system, the CL immunodetection technology showed a good potential and wide development prospects in food and medicine fields.     

Challenges and opportunities for pathogen detection using DNA microarrays

DNA microarrays offer the potential for simultaneous detection of many pathogens that are of interest to homeland security, public health, medicine, and veterinary diagnostics. These tools are best suited for detecting the presence or absence of genetic sequences characteristic of specific pathogens, but microarrays are poorly suited for determining pathogen viability, and current methods provide only limited potential for pathogen enumeration. Two basic strategies have been described for pathogen detection: using enzymatic amplification to generate targets for interrogation with a microarray, or using direct interrogation of DNA or RNA without pre-amplification. Multiplex PCR has the advantage of a high degree of sensitivity and specificity, but associated microarrays are necessarily limited in scope. PCR-independent, whole-genome amplification eliminates biases inherent in PCR amplification and can accommodate more extensive microarrays, but assay sensitivity is compromised and these methods are probably of limited use when testing tissue samples. Direct hybridization of DNA or RNA provides the least bias in gene detection, but also the lowest level of analytic sensitivity. Ultimately, cost and limited sample throughput make it unlikely that planar microarrays will play a significant role in future pathogen detection schemes. Alternative microarray formats such as bead arrays, however, may circumvent the cost and throughput limitations and permit us to apply what we have learned from planar microarrays to develop robust pathogen detection systems. Assay validation and sample preparation will continue to be significant challenges for these detection systems.     

A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

Detection of unknown single nucleotide polymorphism (SNP) relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos), the other includes oligos specific for known SNP within the same genomic region (variant-oligos). Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.     

Optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests

Recent studies have shown the public health importance of identifying individuals with acute human immunodeficiency virus infection (AHI); however, the cost of nucleic acid amplification testing (NAAT) makes individual testing of at-risk individuals prohibitively expensive in many settings. Pooled NAAT (or group testing) can improve efficiency and test performance of testing for AHI, but optimizing the pooling algorithm can be difficult. We developed simple, flexible biostatistical models of specimen pooling with NAAT for the identification of AHI cases; these models incorporate group testing theory, operating characteristics of biological assays, and a model of viral dynamics during AHI. Pooling algorithm sensitivity, efficiency (test kits used per individual specimen evaluated), and positive predictive value (PPV) were modeled and compared for three simple pooling algorithms: two-stage minipools (D2), three-stage hierarchical pools (D3), and square arrays with master pools (A2m). We confirmed the results by stochastic simulation and produced reference tables and a Web calculator to facilitate pooling by investigators without specific biostatistical expertise. All three pooling strategies demonstrated improved efficiency and PPV for AHI case detection compared to individual NAAT. D3 and A2m algorithms generally provided better efficiency and PPV than D2; additionally, A2m generally exhibited better PPV than D3. Used selectively and carefully, the simple models developed here can guide the selection of a pooling algorithm for the detection of AHI cases in a wide variety of settings.