非竞争性ELISA法测定人源抗HBaAg Fab功能性亲和常数

目的 测定完全人源化基因工程抗体HBsAg Fab的亲和常数。方法 采用非竞争性ELISA同相法，经确定最佳抗原包板浓度、最佳抗原包板时间及最佳抗原与抗体结合反应时间后，得到了HBsAg与抗体片段抗HBsAg Fab及完整抗体抗HBsAg IgG的抗原抗体结合反应曲线，计算出抗HBsAg Fab及抗HBsAg IgG的亲和常数。结果 人源基因工程抗体抗HBsAg Fab的功能性亲和常数在10^7～10^8M^-1水平，比完整抗HBsAg IgG仅仅小约1个数量级(10^8～10^9M^-1)。结论 该基因工程抗体与抗原结合能力较强，为今后开发府用Fab进行牛物导向治疗提供了理论基础。     

丙型肝炎病毒核心蛋白人源抗独特型单链抗体在大肠杆菌中的表达

目的 在大肠杆菌中表达丙型肝炎病毒核心蛋白人源抗独特型单链抗体。方法 采用噬菌体表面展示技术,将丙型肝炎病毒(HCV)核心蛋白单克隆抗体固相包被于Nunc板。从人源噬菌体单链可变区抗体库中经过3轮“吸附-洗脱-扩增”的筛选,获得结合活性较强的人源HCV核心蛋白抗独特型(抗-Id)scFv阳性克隆。从阳性克隆中提取质粒,经SfiI／Not I酶切鉴定后,亚克隆到pCANTAB5E载体,转化大肠杆菌XL1-Blue,应用异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达可溶性的HCV核心蛋白抗独特型单链可变区抗体。酶联免疫吸附法(ELISA)证实表达的HCV核心蛋白抗-Id scFv具有与HCV核心蛋白单克隆抗体反应的特异性。对转化的大肠杆菌XL1-Blue上清中表达的HCV核心蛋白抗-Id scFv进行硫酸铵沉淀,并进行SDS-PAGE电泳。结果 筛选得到的HCV核心蛋白抗-Id scFv片段基因由774bp组成。SDS-PAGE电泳表明,大肠杆菌XL1-Blue中表达的可溶性HCV核心蛋白抗-Id scFv分子量约28kD。结论 HCV核心蛋白抗-Id scFv与HCV核心蛋白抗-Id scFv单克隆抗体具有较强的结合活性和特异性。HCV核心蛋白抗-Id scFv的筛选和表达成功,为今后HCV核心蛋白抗-Id scFv的研究和应用奠定了基础。     

大肠癌噬菌体抗体库的构建及抗CEA抗体的筛选

目的构建及初步鉴定大肠癌噬菌体抗体Fab呈现库,筛选人CEA单克隆抗体,并进行序列分析。方法分离大肠癌患者外周血淋巴细胞,提取淋巴细胞总RNA,逆转录成cDNA。用PCR扩增人全套抗体基因片段,克隆于pComb3载体,再经电穿孔转化大肠杆菌XL1-Blue菌株,形成噬菌体抗体库。以固相化CEA抗原淘筛抗体库,ELISA鉴定噬菌体抗体。其中一个阳性克隆进行测序。结果逆转录PCR分别扩增出约680bp大小的κ、λ和Fd基因。PCR产物和载体经纯化、双酶切后进行连接转化,成功地构建了人源性Fab抗体基因库,库容量达2.1×107,Fab基因重组率为50%。以单抗捕获的CEA抗原淘洗4轮,出现特异性富集,阳性克隆经直接ELISA和交叉反应ELISA实验证实具有良好的抗CEA抗原特异性。DNA测序表明该抗体重链属IgG亚类并含有一条IgL亚类的轻链。结论成功构建了大肠癌患者自然致敏抗体Fab段噬菌体呈现库,从中获得可与CEA抗原结合的噬菌体抗体,由此为大肠癌早期诊断及基因治疗提供了一种新的思路和方法。     

非竞争性ELISA法测定人源抗HBsAg Fab功能性亲和常数

目的　测定完全人源化基因工程抗体HBsAgFab的亲和常数。方法　采用非竞争性ELISA固相法 ,经确定最佳抗原包板浓度、最佳抗原包板时间及最佳抗原与抗体结合反应时间后 ,得到了HBsAg与抗体片段抗HBsAgFab及完整抗体抗HBsAgIgG的抗原抗体结合反应曲线 ,计算出抗HBsAgFab及抗HBsAgIgG的亲和常数。结果　人源基因工程抗体抗HBsAgFab的功能性亲和常数在 10 7～10 8M 1水平 ,比完整抗HBsAgIgG仅仅小约 1个数量级 (10 8～ 10 9M 1)。结论　该基因工程抗体与抗原结合能力较强 ,为今后开发应用Fab进行生物导向治疗提供了理论基础。     

抗丙型肝炎病毒核心蛋白单链抗体的制备及免疫组织化学研究

目的 制备丙型肝炎病毒核心蛋白单链抗体并进行免疫组织化学研究。方法以大肠杆菌表达的重组丙型肝炎病毒(HCV)核心蛋白为固相抗原,利用抗原抗体亲和性结合的原理,从半合成的人源可变区噬菌体抗体库中经过3轮“吸附-洗脱-扩增”的筛选过程及酶联免疫吸附试验(ELISA)和DNA序列分析,获得HCV核心蛋白的人源单链抗体;用该抗体对7721转染全长HCV cDNA后筛选的阳性克隆细胞中的HCV核心蛋白抗原进行免疫组化鉴定。结果 ELISA结果表明,制备的HCV核心蛋白人源单链抗体能与HCV核心蛋白抗原特异性结合;免疫组化结果表明,该抗体能够特异性识别HCV核心蛋白抗原,与正常肝细胞无交叉反应。结论此法制备单链抗体方法简便,周期短,可为HCV核心蛋白病原的检测提供新的检测手段。     

基因工程人抗角蛋白抗体对角质形成细胞增殖的影响

目的：研究一株基因工程人源性抗角蛋白Fab抗体在无血清培养的角质形成细胞中的免疫学活性，观察其对角质形成细胞增殖的影响。方法：利用从噬菌体抗体库中筛选到的特异表达抗角蛋白Fab片段的质粒转化大肠杆菌，异丙基-D-硫代半乳糖苷(Pm)诱导表达出Fab抗体，用免疫组化观察对角质形成细胞的反应性，免疫印迹法检测所识别的角蛋白组分及用四甲基偶氮唑蓝比色法(MTT法)测定对细胞增殖的影响。结果：基因工程人源性抗角蛋白Fab抗体仅识别角质形成细胞中相对分子质量为46000的角蛋白区带，呈现为一条很强的染色带；免疫组化染色呈明显胞浆阳性；该抗体明显抑制培养的角质形成细胞增殖，并且存在剂量依赖关系。结论：特异性抗相对分子质量为46000的基因工程人源性抗角蛋白Fab抗体可能是角蛋白抗体家族中影响角质形成细胞增殖的有效成分。     

人PD-L1胞外区基因的克隆、原核表达及抗体制备

目的 克隆人PD-L1胞外区基因并进行原核表达及相应蛋白的抗体制备,为进一步研究PD-1/PD-L1通路及针对该通路进行疾病治疗和药物筛选奠定基础.方法 用PCR方法扩增编码人PD-L1胞外区的基因序列,克隆到原核表达载体pET28a(+)中并进行表达,用His抗体为一抗进行Western blotting鉴定并验证结合活性.用纯化的hPD-L1胞外区蛋白作为抗原免疫日本大耳白兔,制备多克隆抗体.通过酶联免疫吸附实验(ELISA)和流式法检测抗体滴度及其特异性.结果 原核表达质粒pET28a(+)-hPD-L1成功构建,并可在大肠埃希菌BL21(DE3)中诱导高效表达,得到的hPD-L1胞外区蛋白经SDS-PAGE和Western blotting鉴定正确.用纯化蛋白免疫日本大耳白兔,制备的多克隆抗体具有较强的免疫结合活性,抗体滴度可达105.结论 鉴定和纯化了原核表达的hPD-L1蛋白,制备的相应多克隆抗体能够检测真核细胞表达的hPD-L1蛋白,为进一步研究PD-1/PD-L1生物学活性奠定了实验基础.     

抗甲状腺刺激性免疫球蛋白单链抗体在大肠杆菌中的表达

 目的 制备可溶性抗甲状腺刺激性免疫球蛋白(TSI)单链抗体。方法 构建的抗TSI单链抗体可溶性表达载体,转化E.coli XL 1-blue进行表达,表达产物经SDS-PAGE、Western blotting和ELISA检测,同时对表达条件作了初步探索。结果 抗TSI单链抗体可溶性表达于细菌培养上清和周质间隙,大小约30 KDa,具有抗TSI活性。温度、诱导物浓度及蔗糖对抗TSI单链抗体的表达产量有一定影响。结论 抗TSI单链抗体可溶性表达成功,为制备基因工程抗体奠定了基础。     

从人源大容量噬菌体抗体库中筛选抗生存素抗体

目的从大容量天然噬菌体抗体库中筛选抗生存素(survivin)的人源单链抗体(single chainfragment variable,scFv)并进行鉴定。方法以survivin为抗原,固化在抗原管上,通过吸附—洗脱—扩增过程,从大容量天然噬菌体抗体库中筛选特异性噬菌体抗体,并转染HB2151菌,经异丙基-β-D-硫代半乳糖苷诱导制备成可溶性抗体,采用酶联免疫吸附测定法对其抗原结合活性进行检测,指纹图谱分析法分析抗体基因的种类。结果经过4轮筛选,共获得21个与survivin结合的阳性克隆,其中10个特异性结合的克隆,指纹分析有8种不同的抗survivin的scFv基因,在这8种中有5种获得可溶性表达。结论利用噬菌体抗体库技术获得了特异性的人源抗生存素抗体,为其在今后肿瘤治疗中的应用奠定基础。     

抗平颏海蛇短链神经毒素全人源单克隆抗体的筛选、制备及生物活性研究

目的 重组表达3种平颏海蛇短链神经毒素,利用噬菌体抗体库筛选制备全人源抗毒素单克隆抗体并评估其生物活性.方法 以重组表达纯化的神经毒素蛋白作为抗原,从自主构建的噬菌体抗体库中筛选获得噬菌体抗体,经序列测定确定其抗体类型后构建完整抗体.利用真核表达系统表达纯化抗体后鉴定其抗原结合活性、生化特性及药代动力学特性.在体内验证抗体药物的抗毒素作用.结果 经过4轮筛选,获得2株能同时结合3种神经毒素的噬菌体单链抗体,并将其制备成完整的抗体,进一步证实所获得的抗体可特异性结合3种抗原.2株全人源抗体均可拮抗神经毒素对昆明小鼠的致死作用.结论 利用重组神经毒素和噬菌体抗体库技术成功获得了特异性抗平颏海蛇短链神经毒素的全人源治疗性抗体.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

The role of interventional radiology in trauma

Introduction Interventional Radiology has evolved into a specialty having enormous input into the care of the traumatized patient.In all hospitals,regardless of size,the Interventional Radiologist must consider their relationships with the trauma service in order to     

人体中的镭-226、镭-228、钋-210、铅-210

本文报道了广东阳江高本底地区6名、对照地区8名人尸体的骨²²⁶Ra、²²⁶Ra的浓度以及部分居民内脏器官中。²¹⁰Po、²¹⁰Pb的浓度。结果轰明阳江高本底地区和对照地区居民骨镭-226、镭-228的浓度分别为29.9pCi/kg, 26.9pCi/kgl 8.7pCi/kg, 8.2pCi/kg.由此估算出阳江高本底地区屠民骨中²²⁶Ra、²²⁸Ra的负薄璧及对骨衬、骨髓所产生的剂量当量分别为对照地区民民的3.4倍, 3.3倍。两地区居民内脏器官中^{210Po、210Pb的测定分析铡数较少但仍看出, 高本底地区均明显高于对照地区.}     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).