五厂家EB病毒抗体检测试剂盒血清学诊断鼻咽癌的比较

目的 通过比较EB病毒抗体检测试剂盒血清学诊断鼻咽癌的准确性和检测结果的一致性,为试剂盒在临床上的使用选择和性能改进提供依据.方法 使用五厂家的EB病毒衣壳抗原IgA和IgG抗体检测试剂盒（VCA IgA和VCA IgG试剂盒）、核抗原I IgA和IgG抗体检测试剂盒（EBNA1 IgA和EBNA1IgG试剂盒）、早期抗原IgA和IgG抗体检测试剂盒（EA IgA和EA IgG试剂盒）以及Zta IgA抗体检测试剂盒,分别检测33例鼻咽癌患者（NPC）、30例健康体检者（HD）和41例非鼻咽癌的其他肿瘤患者（NNPC）血清或血浆样本.结果 A厂家的VCA IgA试剂盒灵敏度高于其他厂家同品种试剂盒,但对于NNPC特异度最低（36.6％）;而D厂家VCA IgA试剂盒的特异度最高（97.6％）,且对HD的特异度均大于90％.B和D厂家的EBNA1 IgA试剂盒间阳性、阴性符合率分别为92.1％和100.0％.A和E厂家的EA IgA试剂盒的灵敏度均较低而特异度高,试剂盒间阳性符合率低（39.4％）,阴性符合率高（98.6％）;而VCA IgG试剂盒的灵敏度高但特异度低.A和C厂家的EBNA1IgG试剂盒的灵敏度高（100.0％,97.0％）但特异度低（3.3％,13.3％）.C厂家EA IgG试剂盒检测所有样本结果均为阴性.结论 五个不同厂家VCA LgA、EA IgA试剂盒诊断鼻咽癌的准确性和检测结果的一致性存在差异,特别是A厂家和其他国内厂家同品种试剂间差异明显,需根据临床目的进行选择.三家国产VCA IgA试剂盒的灵敏度需进一步提高.相反,EBNA IgA试剂盒诊断鼻咽癌的准确性和结果一致性较好.单独使用VCA IgG和EBNA1 IgG试剂盒血清学诊断鼻咽癌的特异度差,其判读界值可能需根据检测且的进行调整.     

安泰吉(北京)生物技术有限公司

<正>大鼠、小鼠抗体亚型快速检测卡媲美ELISA灵敏度,用于检测杂交瘤培养上清和纯化抗体的重链亚类和轻链亚型。小鼠亚型检测卡可鉴定IgG1、IgG2a、IgG2b、Kappa、Lambda和IgM。大鼠亚型检测卡可鉴定IgG1、IgG2a、lgG2b、IgG2c、Kappa、Lambda、IgA和IgM。     

沙眼衣原体CPAF免疫活性区域重组蛋白的表达及在临床诊断中的应用

目的分析沙眼衣原体(Ct)蛋白酶样活性因子(CPAF)免疫优势区的免疫活性,探讨其在Ct感染诊断中的应用价值。方法构建原核表达重组质粒pGEX-6p-2/CPAF′,异丙基硫代半乳糖苷(IPTG)诱导表达,用该纯化的表达产物包被酶标板,建立间接ELISA检测病人血清中的Ct特异性IgM和IgG抗体,同时与晶美公司Ct ELISA试剂盒检测结果进行对比分析。结果含pGEX-6p-2/ CPAF′的大肠杆菌BL21大量表达相对分子质量(M_r)约为43×10~3的目的蛋白,用该重组蛋白包被酶标板,建立间接ELISA检测Ct IgM、IgG阴性和阳性参考血清各50份,符合率均为100%;同时检测250份临床血清标本,与晶美公司ELISA试剂盒检测结果的符合率IgM为96.8%,IgG为98.4%。结论表达的Ct CPAF免疫优势区(AA_(200)～AA_(338))重组蛋白具有良好的免疫反应活性,可望用于Ct感染的临床诊断。     

恙虫病东方体Sxh951株56kD蛋白的表达及其在ELISA中的应用

目的　构建含OrientiatsutsugamushiSxh95 1株 (Ot.Sxh95 1)相对分子质量 (Mr)为 5 6×10 3外膜蛋白基因 (sxh5 6 )的重组质粒pQE30 5 6 ,表达Mr5 6× 10 3蛋白并观察其在ELISA中的应用。方法　IPTG诱导sxh5 6重组质粒 ;观察SDS PAGE和免疫印迹结果 ;经Ni NTA亲和层析纯化后的重组蛋白分别与Ot.Gilliam、Ot.Karp、Ot.Kato感染鼠血清进行ELISA和免疫印迹检测。结果　SDS PAGE和免疫印迹检测显示有一Mr 约为 5 6× 10 3的特异蛋白带 ;ELISA和免疫印迹结果显示该重组蛋白只与Ot.Gilliam感染鼠血清呈阳性反应 ;重组蛋白用作ELISA包被抗原检测小鼠血清抗体IgG ,特异性为 10 0 % ,敏感性 96 .6 7% ;检测人血清抗体IgG的敏感性为 88.0 8% ,特异性 96 .36 %。结论　表达的重组Sxh5 6蛋白具有良好的免疫反应性 ;其作为ELISA包被抗原 ,具有良好的特异性和敏感性。     

基于HSV-1型特异性表位的血清抗体ELISA检测方法建立与初步应用评价

目的建立并评价HSV-1型特异性抗体鉴别诊断的ELISA方法。方法以HSV-1-gG112-127型特异性表位的串联重组表达蛋白作为包被抗原，建立检测血清HSV．1特异性IgG的间接ELISA方法。同时以免疫印迹检测作为“金标准”，评价检测方法的真实性和可靠性。结果采用棋盘法确定了包被抗原、抗体的最佳浓度，建立ELISA检测方法。血清特异性IgG检测的灵敏度为91％，特异度为97．6％，阳性预测值为97％，阴性预测值为93％，符合率为94．5％，试验的一致率为98％。结论用串联重组蛋白作为包被抗原对HSV-1感染的血清进行ELISA分型检测，其特异性好。     

巨噬细胞源性趋化因子对NTHiP6蛋白疫苗免疫效果的影响

观察巨噬细胞源性趋化因子(MDC)佐剂对NTHiP6蛋白疫苗免疫效果的影响。将原核表达质粒PGEX-6P2/P6转入E.coli XL1-Blue,IPTG诱导P6蛋白的表达并进行纯化。将BALB/c小鼠随机分为A-D四组,分别为PBS对照组、MDC对照组、P6蛋白组、P6蛋白联合MDC组。分别于0、14、28d经黏膜免疫,末次免疫后14d,每组12只小鼠取血和肺泡灌洗液,ELISA检测血清中IgG抗体和肺泡灌洗液中IgA抗体水平。每组取3只小鼠,制备脾淋巴细胞,ELISA检测IL-4、IL-17和IFN-γ水平。用10LD50NTHi攻击每组剩余15只小鼠,观察免疫保护作用。在大肠杆菌中成功表达P6蛋白。第三次免疫后,D组诱导的IgG抗体、IgA抗体、IL-4、IL-17和IFN-γ水平显著高于其他各组(P0.05)。经NTHi攻击后,D组生存率达80%,与A组、B组相比差异有统计学意义(P0.05),但C组、D组之间无显著性差异(P0.05)。MDC作为佐剂可以使NTHiP6疫苗获得较好的免疫效果。     

人DcR3融合蛋白的表达、纯化及多克隆抗体的制备

目的:制备DcR3融合蛋白及其多克隆抗体,并鉴定其特异性。方法:将亚克隆构建的pET28a( )/DcR3重组表达质粒转化入大肠杆菌(E.coli)BL21菌株,IPTG诱导表达,镍柱亲和层析法纯化目的蛋白,SDS-PAGE及Western blot分析蛋白产物,将纯化的目的蛋白免疫家兔制备多克隆抗体,并对其进行纯化及鉴定。结果:pET28a( )/DcR3重组表达质粒在E.coli中诱导表达相对分子质量(Mr)为33000的目的蛋白,表达量约占菌体蛋白总量的38%,纯化后的目的蛋白纯度达98%。Western blot显示纯化蛋白与抗DcR3单克隆抗体(mAb)具有良好的反应性。纯化后多克隆抗体效价达1.28×10-6。结论:DcR3蛋白在E.coli中得到高效表达,成功制备高纯度DcR3蛋白及高效价抗DcR3多克隆抗体,为研究DcR3在组织中的表达、分布,研制ELISA试剂盒以检测恶性肿瘤及自身免疫性疾病等患者血清DcR3表达水平提供实验基础。     

双抗体间接夹心ELISA法检测乳腺癌患者外周血MUC1蛋白水平的评价

目的优化双抗体间接夹心ELISA试剂盒,并探讨其在乳腺癌患者中检测MUC1黏蛋白水平的应用价值。方法用基因重组MUC1-GST和MUC1-MBP融和蛋白免疫家兔和大鼠,获得抗MUC1血清,并对其纯化,获得纯化的家兔抗人及大鼠抗人MUC1多克隆抗体;经不同的筛选确立了以家兔抗人MUC1抗体作为包被抗体、大鼠抗人MUC1抗体作为检测抗体的双抗体间接夹心试剂盒,敏感度可达到0.2 ng/ml。结果应用建立的试剂盒对40例乳腺癌,18例乳腺良性疾病和120健康对照者血清中MUC1蛋白水平的进行检测,检测结果绘制ROC曲线,分析得出以2.75 ng/ml为乳腺癌患者与乳腺良性疾病患者的临界值,以1.86 ng/ml为乳腺疾病与正常人为临界值,检测结果表明本研究对乳腺癌诊断的阳性率高达97.5%,乳腺良性疾病的阳性率为66.7%,正常人特异性为96.7%。对于乳腺癌同一病例样本用酶联免疫法CA15-3诊断试剂盒进行对比检测,其检出率为3.33%,特异度为100%。绘制ROC曲线对比显示,本研究所建立的双抗体夹心ELISA方法对乳腺癌诊断的准确度明显高于CA15-3试剂盒。结论本研究成功建立了特异性强,灵敏度良好的双抗体间接夹心ELISA试剂盒,有望开发为临床辅助诊断的常规试剂盒,尤其有望应用于乳腺癌的大规模筛查及早期诊断。     

肠道病毒71型重组VP1蛋白的表达和EV71型手足口病血清学诊断方法的建立

目的 获得纯化的具有免疫活性的肠道病毒71型VP1蛋白,建立EV71感染早期、快速和准确的ELISA血清学诊断方法.方法 通过PCR方法扩增出VP1基因,定向克隆到原核表达载体pET-21b(+),阳性质粒转化入E.coli B121(DE3)感受态细胞经IPTG诱导,SDS-PAGE电泳和蛋白免疫印迹分析目的 蛋白的表达水平.纯化的VPI蛋白用作包被抗原,建立手足口病(HFMD)患者抗-EV71-IgM和IgG的血清学诊断方法.结果 成功表达和纯化了VP1重组蛋白,所表达的蛋白能被EV71型手足口病患者血清所识别.调查发现,与正常人和EV71阴性手足口病患儿比较,EV71阳性手足口病血清中抗-EV71-IgM和IgG中A值显著升高.差异具有统计学意义(P<0.05).与RT-PCR结果比较,发现该方法IgM的诊断敏感性和特异性分别为73%和77%;该IgG诊断方法的敏感性和特异性分别为82%和83%.完成该试验仪需4 h.结论 利用pET原核表达系统成功克隆、表达和纯化了肠道病毒71型重组外壳蛋白VPI,且具有良好的抗原性.该抗原可用于研制EV71血清学诊断试剂盒.     

6种虫媒病毒蛋白芯片检测方法的建立

目的 建立针对6种虫媒病毒的蛋白芯片检测方法,用以检测流行性乙型脑炎病毒、蜱传脑炎病毒、登革病毒(1～4型)、西尼罗病毒、西部马脑炎病毒和东部马脑炎病毒的特异性抗体.方法 将病毒特异性抗原作为捕获抗原点样制备蛋白芯片,利用双抗夹心ELISA原理检测血清中的病毒特异性抗体.首先利用免疫兔血清进行特异性诊断抗原的筛选,并对抗体芯片检测条件进行优化,然后采用56份临床疑似的阳性血清标本及阴性对照标本对该方法进行验证,并与常规ELISA方法进行比对.结果 共筛选出11个特异性较好的重组诊断抗原.抗原点样浓度在0.125 ～0.900mg/ml时可获得良好的检测效果,血清检测范围为1:100～1:1000.对26份临床疑似的蜱传脑炎病毒血清标本,22份登革病毒血清标本及8份流行性乙型脑炎病毒临床血清标本的检测结果为:共检测出蜱传脑炎病毒IgG阳性血清标本20份,阳性检出率为76.9％,IgM阳性血清标本17份,阳性检出率65.3％,与ELISA检测符合率分别为96.1％和84.6％.乙型脑炎病毒IgG阳性血清4份,阳性检出率50.0％,IgM阳性血清5份,阳性检测率62.0％,与ELISA检测符合率分别为87.5％和100％.登革病毒IgG阳性血清标本13份,阳性检出率63.6％,IgM阳性血清标本14份,阳性检测率68.1％,与ELISA检测符合率分别为86.3％和90.1％,结果经一致性Kappa检验后,与ELISA检测结果一致性良好.阴性对照血清结果显示检测特异性为100％.结论 本研究建立的虫媒病毒抗体芯片检测方法具有较高的特异性和可靠性,可用于6种虫媒病毒抗体的临床检测.     

Pain and Effusion and Quadriceps Activation and Strength

Context:

Quadriceps dysfunction is a common consequence of knee joint injury and disease, yet its causes remain elusive.

Objective:

To determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion affect the magnitude of quadriceps dysfunction.

Design:

Crossover study.

Setting:

University research laboratory.

Patients or Other Participants:

Fourteen (8 men, 6 women; age = 23.6 ± 4.8 years, height = 170.3 ± 9.16 cm, mass = 72.9 ± 11.84 kg) healthy volunteers.

Intervention(s):

All participants were tested under 4 randomized conditions: normal knee, effused knee, painful knee, and effused and painful knee.

Main Outcome Measure(s):

Quadriceps strength (Nm/kg) and activation (central activation ratio) were assessed after each condition was induced.

Results:

Quadriceps strength and activation were highest under the normal knee condition and differed from the 3 experimental knee conditions (P < .05). No differences were noted among the 3 experimental knee conditions for either variable (P > .05).

Conclusions:

Both pain and effusion led to quadriceps dysfunction, but the interaction of the 2 stimuli did not increase the magnitude of the strength or activation deficits. Therefore, pain and effusion can be considered equally potent in eliciting quadriceps inhibition. Given that pain and effusion accompany numerous knee conditions, the prevalence of quadriceps dysfunction is likely high.Key Words: arthrogenic muscle inhibition, central activation failure, voluntary activation, muscles

Key Points

• Knee pain and effusion resulted in arthrogenic muscle inhibition and weakness of the quadriceps.
• The simultaneous presence of pain and effusion did not increase the magnitude of quadriceps dysfunction.
• To reduce arthrogenic muscle inhibition and improve muscle strength, clinicians should employ interventions that target removing both pain and effusion.

Quadriceps weakness is a common consequence of traumatic knee joint injury^1,2 and chronic degenerative knee joint conditions.^3,4 Arthrogenic muscle inhibition (AMI), a neurologic decline in muscle activation, results in quadriceps weakness and hinders rehabilitation by preventing gains in strength.⁵ The inability to reverse AMI and restore muscle function can lead to decreased physical abilities,⁶ biomechanical deficits,⁷ and possibly reinjury.⁵ Furthermore, researchers^8,9 have suggested that quadriceps weakness resulting from AMI may place patients at risk for developing osteoarthritis in the knee. In light of the substantial influence of quadriceps AMI on these clinically relevant outcomes, we need to improve our understanding of the factors that contribute to this neurologic decline in muscle activity so efforts to target and reverse it can be implemented and gains in strength can be achieved more easily.Joint injury and disease are accompanied by numerous sequelae (ie, pain, swelling, tissue damage, inflammation), so ascertaining which one ultimately leads to neurologic muscle dysfunction is difficult. Whereas a joint effusion can result in AMI,^10–12 the effects of pain are less understood despite many clinicians attributing AMI to pain. Using techniques that introduce knee pain without accompanying injury may provide insights into the role of pain in eliciting AMI.The degree of knee joint damage may play a role in the quantity of AMI that manifests. Hurley et al^13,14 demonstrated that quadriceps AMI, measured using an interpolated-twitch technique, was greater in patients with extensive traumatic knee injury (eg, fractured tibial plateau, ruptured medial collateral ligament, and medial meniscectomy) than patients with isolated joint trauma (ie, isolated anterior cruciate ligament [ACL] rupture). Similarly, patients with more knee joint symptoms (ie, greater number of symptoms and increased severity of symptoms) may present with greater magnitudes of quadriceps inhibition. Recently, investigators¹⁵ have suggested that patients with more pain display less quadriceps strength, supporting this tenet. Given that effusion and pain often present simultaneously with joint injuries and diseases, such as ACL injury and osteoarthritis, examining both the isolated and cumulative effects of these sequelae appears warranted to determine if they influence the magnitude of muscle inhibition.Experimental joint-effusion and pain models are safe and effective experimental methods that allow for the isolated examination of their effects on muscle function. The effusion model, whereby sterile saline is injected directly into the knee joint capsule,⁷ produces a clinically relevant magnitude of the joint effusion that may be present with traumatic injury. Effusion is thought to activate group II afferents responding to stretch or pressure,^16–18 which in turn may facilitate group Ib interneurons and result in quadriceps AMI.⁵ The pain model involves injecting hypertonic saline into the infrapatellar fat pad to produce anteromedial knee pain similar to that described in patients with patellofemoral pain syndrome.¹⁹ Pain is considered to initiate AMI through activation of group III and IV afferents that act as nocioceptors to signal damage or potential damage to joint structures.^16–18 The firing of these afferents then may lead to facilitation of group Ib interneurons, the flexion reflex, or the gamma loop, ultimately resulting in quadriceps inhibition.²⁰ Thus, these models allow us to create symptoms that are associated with knee injury and have the added benefit of providing a way to examine their effects in isolation.Therefore, the purpose of our study was to determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion would affect the magnitude of quadriceps dysfunction. We hypothesized that pain alone would result in quadriceps inhibition and that the magnitude of inhibition would be greater when effusion and pain were present simultaneously.     

即早基因c-fos与脑血管病及学习记忆

即早基因c-fos是广泛存在于原核细胞和真核细胞的高度保守基因.在正常情况下,c-fos基因参与细胞生长、分化、信息传递、学习和记忆等生理过程,而在病理情况下c-fos基因表达及调控变化与多种疾病的发生和发展有关.C-fos在中枢神经系统的某些部位可有基础水平的表达,但表达很低,当受到如脑缺血、脑出血、痫性发作、应激等刺激后,其在数十分钟内做出反应,在对外界刺激-转录耦联的信忠传递过程中起着核内第三信使的重要作用.     

Opportunities and challenges in the prevention and control of cancer and other chronic diseases: children's diet and nutrition and weight and physical activity

OBJECTIVE: The purpose of this article is to review the role of behavioral research in disease prevention and control, with a particular emphasis on lifestyle- and behavior-related cancer and chronic disease risk factors--specifically, relationships among diet and nutrition and weight and physical activity with adult cancer, and tracking developmental origins of these health-promoting and health-compromising behaviors from childhood into adulthood. METHOD: After reviewing the background of the field of cancer prevention and control and establishing plausibility for the role of child health behavior in adult cancer risk, studies selected from the pediatric published literature are reviewed. Articles were retrieved, selected, and summarized to illustrate that results from separate but related fields of study are combinable to yield insights into the prevention and control of cancer and other chronic diseases in adulthood through the conduct of nonintervention and intervention research with children in clinical, public health, and other contexts. RESULTS: As illustrated by the evidence presented in this review, there are numerous reasons (biological, psychological, and social), opportunities (school and community, health care, and family settings), and approaches (nonintervention and intervention) to understand and impact behavior change in children's diet and nutrition and weight and physical activity. CONCLUSIONS: Further development and evaluation of behavioral science intervention protocols conducted with children are necessary to understand the efficacy of these approaches and their public health impact on proximal and distal cancer, cancer-related, and chronic disease outcomes before diffusion. It is clear that more attention should be paid to early life and early developmental phases in cancer prevention.