SPR技术在免疫学研究中的应用

表面等离子共振(Surface Plasmon Resonance,SPR)技术是研究生物分子相互作用的强有力工具之一,该技术使生物分子之间相互作用的实时检测成为可能,并且灵敏度高、无需标记.通过分析传感图谱及分子相互作用的响应值获取分子相互作用的模式和动力学常数等方面的信息,并且获得的信息是能够定性和定量.SPR技术现在已广泛应用于生物、化学、免疫学研究及新药开发等领域.本文主要就SPR技术在免疫学研究中抗体活性检测、抗原表位预测等方面的应用进行了综述.     

SPR技术分析生物分子相互作用的研究方法

生物分子的活性功能是通过分子之间的相互作用来实现的,了解这种相互作用的关系时生命科学的研究及揭示生命发生发展的基本机制具有着重要的意义.基于表面等离子共振(SPR)的分析分子相互作用(BIA)的技术是新型的生物传感技术,其无需标、能实时跟踪检测生物分子间结合、解离的整个过程,通过分析传感图谱获取分子相互作用的模式和动力学常数等方面的信息.SPR是研究生物分子相互作用的强有力工具,SPR技术已被广泛应用于生命科学领域的研究,并且显示出广阔的应用前景.概述了SPR技术原理、分析方法及其评述了其存在的问题.     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

AlphaLISA技术的发展及应用

AlphaLISA技术是一种基于微珠的化学发光的新型均相检测技术,与传统的ELISA技术相比具有更高的敏感性、精确性、均一性、背景低、广泛的动态检测范围,而且无需洗涤及样本需求量极少等特点.该技术可用于多种组织、细胞来源的生物分子的检测,如细胞因子、抗原抗体的检测、蛋白相互作用及蛋白质与核酸相互作用的检测以及药物分析等研究.随着AlphaLISA技术的发展,该技术已在医学及分子生物学领域得到了广泛的应用.     

化学交联技术在蛋白质相互作用研究中的应用

蛋白质相互作用是生命科学研究的一个重要领域.随着生物质谱的出现,利用化学交联技术研究蛋白质的相互作用已经成为切实可行的策略.文章介绍了化学交联反应的相关内容,及其在蛋白质相互作用研究中的应用,并简单探讨了甲醛作为交联剂的应用价值.     

串联亲和纯化（TAP）技术在蛋白质组学中的应用

蛋白质是各种生命活动的主要执行者,因此构建蛋白质相互作用的网络图对于准确理解蛋白质功能、揭开各种细胞活动的奥秘十分重要.串联亲和纯化（TAP）,是近年来发展出来的一种能够快速研究在生理条件下蛋白质相互作用,揭示蛋白质复合体相互作用网络的新技术,已成为研究蛋白质组学的一个重要工具.随着该技术的不断完善,TAP技术在认识蛋白质相互作用的过程中必将发挥越来越重要的作用.     

基于质谱技术的蛋白质互作研究进展

蛋白质作为生命活动的执行者,其功能往往体现在与其他蛋白质的相互作用中,研究蛋白-蛋白相互作用对于人们深入了解和预防传染病、靶向治疗多基因疾病、阐明蛋白质的分子作用机制及各种复杂的生命现象具有重要意义。目前,有多种技术被用来研究蛋白间的相互作用,研究难点在于实时捕获瞬时或弱蛋白质间的相互作用,质谱技术（mass spectrometry, MS）可在某种程度上解决该难点。由于质谱技术可研究简单的蛋白质复合物再到大规模的蛋白质组实验,基于质谱技术研究蛋白质间相互作用被越来越多地应用于科学研究中。综述了蛋白质间相互作用检测方法的研究进展,重点介绍了氢氘交换质谱法和化学交联质谱法研究蛋白质间相互作用的优缺点及其应用,最后对基于质谱技术研究蛋白质间相互作用进行了总结与展望,以期为深入开展相关研究提供借鉴。     

基于雷帕霉素与FRB结合的蛋白质间相互作用的相关技术

FKBP12-rapamycin复合物的结合位点(FKBP1 2-rapamycin binding,FRB)为雷帕霉素(rapamycin,RAP)与哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)结合的结构城,基于RAP介导FK506结合蛋白12(12 kD FK506-binging protein,FKBP12)与FRB蛋白质相互作用相关研究技术的发展与应用,使人们对小分子介导的蛋白质相互作用有了更多的认识.就研究RAP作用于FRB域及研究FKBP12-RAP-FRB三元复合物形成的相关技术作一综述,为确认新的mTOR抑制剂的作用机制及认识其他蛋白质间相互作用提供参考.     

大规模酵母双杂交技术研究蛋白质相互作用的应用

简介了酵母双杂交技术原理, 总结了酵母双杂交技术大规模筛选蛋白质相互作用的基础、应用及存在的问题。因为大规模酵母双杂交技术结果有大量假阳性及假阴性问题, 因此, 有条件情况下有必要同时开展其他方法的大规模蛋白相互作用研究, 以构建规模更大可信度更高的蛋白质相互作用网络图。     

免疫沉淀技术的最新进展

免疫沉淀(Immunoprecipitation,IP)技术是以抗体和抗原之间的专一性作用为基础的用于研究蛋白质相互作用的经典方法.也是确定两种蛋白质在完整细胞内生理性相互作用的有效方法.免疫沉淀技术现已广泛应用于基因、蛋白质以及它们之间相互作用等领域的研究,并且其可与多种技术联合应用,也可合并一些实验方法进行多方面探索.本文将主要介绍染色质免疫沉淀技术、蛋白质免疫沉淀技术和放射免疫沉淀技术的原理和方法,并对它们相应的应用作简要的说明.     

Mechanisms of Action of Carbamazepine and Its Derivatives,Oxcarbazepine, BIA 2-093, and BIA 2-024

Carbamazepine (CBZ) has been extensively used in the treatment of epilepsy, as well as in the treatment of neuropathic pain and affective disorders. However, the mechanisms of action of this drug are not completely elucidated and are still a matter of debate. Since CBZ is not very effective in some epileptic patients and may cause several adverse effects, several antiepileptic drugs have been developed by structural variation of CBZ, such as oxcarbazepine (OXC), which is used in the treatment of epilepsy since 1990. (S)-(–)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-093) and 10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide (BIA 2-024), which were recently developed by BIAL, are new putative antiepileptic drugs, with some improved properties. In this review, we will focus on the mechanisms of action of CBZ and its derivatives, OXC, BIA 2-093 and BIA 2-024. The available data indicate that the anticonvulsant efficacy of these AEDs is mainly due to the inhibition of sodium channel activity.     

Serial Measurements of Body Composition in Obese Subjects During a Very-Low-Energy Diet (VLED) Comparing Bioelectrical Impedance with Hydrodensitometry

Bioelectrical impedance analysis (BIA) is a convenient, inexpensive, and noninvasive technique for measuring body composition. BIA has been strongly correlated with total body water (TBW) and also has been validated against hydrodensitometry (HD). The accuracy and clinical utility of BIA and HD during periods of substantial weight loss remain controversial. We measured body composition in moderately and severely obese patients serially using both methods during a very-low-energy diet (VLED). Mean initial weight in these patients was 116 (± 30) kg (range, 74–196 kg). Mean weight loss was 24 (± 13) kg with a decrease in fat mass (FM) by HD of 20 kg (p<0.001) and a decrease in fat-free mass (FFM) of 3.6 kg (p<0.05). Loss of FFM is best predicted by the rate (kg/wk) of weight loss (r² = 0.86, p<0.0001). FFM, as predicted from BIA equations, was highly correlated with FFM as estimated by HD during all testing sessions (r=0.92-0.98). Although highly correlated, BIA overestimated FFM relative to HD and this difference appeared to be more pronounced for taller patients with greater truncal obesity. Although the discrepancy was no greater during weight-loss treatment, the level of disagreement was considerable. Therefore, the two methods cannot be used interchangeably to monitor relative changes in body composition in patients with obesity during treatment with VLED. The discrepancy between BIA and HD may be caused by body mass distribution considerations and by perturbations in TBW which affect the hydration quotient for FFM (BIA) and/or which affect the density constants for FFM and FM (HD).     

Comparison of body composition methods in obese African-American women

Objective : To compare the accuracy of percentage body fat (%BF) estimates between bioelectrical impedance analysis (BIA) and DXA in obese African‐American women. Research Methods and Procedures : Fifty‐five obese African‐American women (mean age, 45 years; mean BMI, 38; mean %BF, 48%) were studied. BF was assessed by both BIA (RJL Systems BIA 101Q; RJL Systems, Clinton Township, MI) and DXA (Hologic QDR‐2000 Bone Densitometer; Hologic Inc., Bedford, MA). Generalized and ethnicity‐ and obese‐specific equations were used to calculate %BF from the BIA. Bland‐Altman analyses were used to compare the agreement between the BIA and the DXA, with the DXA serving as the criterion measure. Results : Two of the generalized equations provided consistent estimates across the weight range in comparison with the DXA estimates, whereas most of the other equations increasingly underestimated %BF as BF increased. One of the generalized and one of the ethnicity‐specific equations had mean differences that were not significantly different from the DXA value. Discussion : The findings show that the Lukaski equation provided the most precise and accurate estimates of %BF in comparison with the QDR 2000 and provide preliminary support for the use of this equation for obese African‐American women.     

Comparison of bioelectrical impedance and BMI in predicting obesity-related medical conditions

Objective : To determine the relative validity of specific bioelectrical impedance analysis (BIA) prediction equations and BMI as predictors of physiologically relevant general adiposity. Research Methods and Procedures : Subjects were >12, 000 men and women from the Third National Health and Nutrition Examination Survey population. We examined the correlations between BMI and percentage body fat based on 51 different predictive equations, blood pressure, and blood levels of glucose, high‐density lipoprotein cholesterol, and triglycerides, which are known to reflect adiposity, while controlling for other determinants of these physiological measures. Results : BMI consistently had one of the highest correlations across biological markers, and no BIA‐based measure was superior. Percent body fat estimated from BIA was minimally predictive of the physiological markers independent of BMI. Discussion : These results suggest that BIA is not superior to BMI as a predictor of overall adiposity in a general population.     

Bias and limits of agreement between hydrodensitometry, bioelectrical impedance and skinfold calipers measures of percentage body fat

Previous research has often used correlations as a statistical method to show agreement; however, this is not a valid use of the statistic. The purpose of this study was to investigate the bias and limits of agreement for three methods of estimating percentage body fat for 117 male and 114 female university athletes: hydrodensitometry (HYD), bioelectrical impedance (BIA) and skinfold calipers (SKF). The mean (SD) percentage body fat for males as assessed by HYD, BIA and SKF methods, respectively, were 13.2 (3.3)%, 14.1 (3.3)% and 13.0 (3.2)%. Female body fat measurements were 22.5 (3.9)%, 23.7 (4.3)% and 23.8 (4.2)%, respectively. Pearson product moment correlations for male and female body fat percentages between the three methods were high, ranging from 0.81 to 0.86 (P < 0.05). However, compared to the criterion measure of body fat percentage (HYD), the magnitude of agreement BIA and SKF revealed a different pattern. The mean absolute difference between HYD and BIA measurements of body fat for males was −0.8 (2.0)% fat, and between HYD and SKF was it was 0.2 (1.7)% fat. The mean absolute difference for females between HYD and BIA was −1.2 (2.5)%; for HYD and SKF it was −1.4 (2.2)%. Compared to the HYD measures for males and females, the BIA and SKF measures were as much as a 3.8% underestimation and a 6.2% overestimation of body fat. This study provides evidence that the strength of a correlation does not indicate agreement between two methods. In future, reliability and validity studies should examine the absolute differences between two variables and calculate limits of agreement around which a practitioner can appreciate the precision of the methodologies. Accepted: 26 August 1997     

Bioelectrical impedance vs. four-compartment model to assess body fat change in overweight adults

Objective: The Tanita TBF‐305 body fat analyzer is marketed for home and clinical use and is based on the principles of leg‐to‐leg bioelectrical impedance analysis (BIA). Few studies have investigated the ability of leg‐to‐leg BIA to detect change in percentage fat mass (%FM) over time. Our objective was to determine the ability of leg‐to‐leg BIA vs. the four‐compartment (4C) model to detect small changes in %FM in overweight adults. Research Methods and Procedures: Thirty‐eight overweight adults (BMI, 25.0 to 29.9 kg/m²; age, 18 to 44 years; 31 women) participated in a 6‐month, randomized, double‐blind, placebo‐controlled study of a nutritional supplement. Body composition was measured at 0 and 6 months using the Tanita TBF‐305 body fat analyzer [using equations derived by the manufacturer (%FM_T‐Man) and by Jebb et al. (%FM_T‐Jebb)] and the 4C model (%FM_4C). Results: Subjects in the experimental group lost 0.9%FM_4C (p = 0.03), a loss that did not reach significance using leg‐to‐leg BIA (0.6%FM_T‐Man, p = 0.151; 0.6%FM_T‐Jebb, p = 0.144). We observed large standard deviations (SDs) in the mean difference in %FM between the 4C model and the Tanita_Manufacturer (2.5%) and Tanita_Jebb (2.2%). Ten subjects fell outside ±1 SD of the mean differences at 0 and 6 months; those individuals were younger and shorter than those within ±1 SD. Discussion: Leg‐to‐leg BIA performed reasonably well in predicting decreases in %FM in this group of overweight adults but resulted in wide SDs vs. %FM_4C in individuals. Cross‐sectional determinations of %FM of overweight individuals using leg‐to‐leg BIA should be interpreted with caution.     

Crystal structure of norcoclaurine‐6‐O‐methyltransferase,a key rate‐limiting step in the synthesis of benzylisoquinoline alkaloids

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate‐limiting enzymes and negative feedback inhibition by end‐products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)‐norcoclaurine‐6‐O‐methyltransferase (6OMT), a key rate‐limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end‐products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S‐adenosyl‐l ‐homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time‐dependent sensitivity toward sanguinarine.     

Assessing Body Composition among 3‐ to 8‐Year‐Old Children: Anthropometry,BIA, and DXA

Objective: To examine the inter‐relationships of body composition variables derived from simple anthropometry [BMI and skinfolds (SFs)], bioelectrical impedance analysis (BIA), and dual energy x‐ray (DXA) in young children. Research Methods and Procedures: Seventy‐five children (41 girls, 34 boys) 3 to 8 years of age were assessed for body composition by the following methods: BMI, SF thickness, BIA, and DXA. DXA served as the criterion measure. Predicted percentage body fat (%BF), fat‐free mass (FFM; kilograms), and fat mass (FM; kilograms) were derived from SF equations [Slaughter (SL)1 and SL2, Deurenberg (D) and Dezenberg] and BIA. Indices of truncal fatness were also determined from anthropometry. Results: Repeated measures ANOVA showed significant differences among the methods for %BF, FFM, and FM. All methods, except the D equation (p = 0.08), significantly underestimated measured %BF (p < 0.05). In general, correlations between the BMI and estimated %BF were moderate (r = 0.61 to 0.75). Estimated %BF from the SL2 also showed a high correlation with DXA %BF (r = 0.82). In contrast, estimated %BF derived from SFs showed a low correlation with estimated %BF derived from BIA (r = 0.38); likewise, the correlation between DXA %BF and BIA %BF was low (r = 0.30). Correlations among indicators of truncal fatness ranged from 0.43 to 0.98. Discussion: The results suggest that BIA has limited utility in estimating body composition, whereas BMI and SFs seem to be more useful in estimating body composition during the adiposity rebound. However, all methods significantly underestimated body fatness as determined by DXA, and, overall, the various methods and prediction equations are not interchangeable.     

生物分子相互作用分析技术应用实例（一）——配体垂钓和抗体测定

BIA技术(biomolecular interaction analysis)——即生物分子相互作用分析技术的应用范围相当广泛.先介绍利用BIAcore分离得到了ECK-酪氨酸激酶受体的蛋白配体.另一应用实例是利用BIAcore直接从杂交瘤细胞上清液中测定单克隆抗体的活性、亲和力和动态常数.