抗盐酸克伦特罗人源ScFv抗体筛选与鉴定

目的 筛选盐酸克伦特罗(CLB)人源单链可变区(ScFv)抗体,为研究快速检测试剂(盒)提供基础依据.方法 采用噬菌体表面展示技术,以CLB作为抗原,从噬菌体单链可变区抗体库中经过3轮"吸附-洗脱-扩增"筛选过程,随机挑选抗CLB的60个克隆,利用酶联免疫吸附法(ELISA)、交叉反应及竞争抑制实验,对其进行免疫学检测和鉴定,获得与CLB结合活性较强的ScFv阳性克隆,从噬菌体抗体阳性克隆中提取质粒,经酶切鉴定SfiⅠ/NotⅠ后,亚克隆到pCANTAB5E载体;转化大肠埃希菌XLI-Blue,提取质粒进行DNA序列测定;并对CLB ScFv的编码基因序列分析.结果 经过筛选60个克隆中有15株克隆ELISA的吸光度(A490nm)值较高,将这些噬菌体上清与牛血清白蛋白(BSA)进行交叉反应,确定有7株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆.亚克隆到pCANTAB5E载体;转化大肠埃希菌XL1-Blue,提取质粒进行DNA序列测定;DNA为750 bp;经BLAST分析,此片段属于新抗体基因序列,GenBank注册号为:EU681272.结论 本实验用噬菌体抗体库技术能够初步获得抗CLB的ScFv抗体,为下一步其特异性亲和力的研究创造了条件.     

HIV-gp120人源单链可变区抗体筛选与鉴定

目的 通过制备抗人类免疫缺陷病毒(HIV)gp120蛋白人源单链可变区抗体(seFv),探索新型抗人获得性免疫缺陷病毒(HIV)治疗方法.方法 采用噬菌体表面展示技术,将HIV-gp120蛋白固相包被于Nunc板,应用半合成的人源单链可变区抗体文库技术,从噬菌体单链可变区抗体库中经过5轮吸附-洗脱-扩增筛选过程,随机挑选96个克隆,利用酶联免疫吸附法(ELISA)、交叉反应和竞争抑制实验,进行免疫学检测和鉴定,获得与HIV-gp120蛋白结合活性较强的scFv阳性克隆,并对HIV-gp120蛋白ScFv的编码基因进行序列测定分析.结果 经过筛选96个克隆中有39株克隆ELISA的吸光度(A450 nm)值较高,将这些噬菌体上清与牛血清白蛋白(BSA)进行交叉反应,确定其中有7株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆.提取质粒,进行DNA序列测定,DNA为699 bp.结论 用噬菌体抗体库技术成功地获得抗HIV-gp120蛋白的scFv,为抗gp120蛋白的ScFv防治艾滋病研究创造了条件.     

抗氯菊酯人源scFv抗体筛选与鉴定

目的 筛选氯菊酯人源单链可变区（scFv）抗体,为研究快速检测试剂盒奠定基础.方法 采用噬菌体表面展示技术,以氯菊酯作为抗原,固相包被于Nunc板,应用半合成的人源单链可变区抗体文库技术,从噬菌体单链可变区抗体库中经过3轮＂吸附-洗脱-扩增＂筛选过程,随机挑选抗氯菊酯的100个克隆,利用酶联免疫吸附法（ELISA）、交叉反应及竞争抑制实验,对其进行免疫学检测和鉴定,获得与氯菊酯结合活性较强的scFv阳性克隆,从噬菌体抗体阳性克隆中提取质粒,经酶切鉴定Sfi Ⅰ/Not Ⅰ后,亚克隆到pCANTAB5E载体;转化大肠杆菌XL1-Blue,提取质粒进行酶切鉴定分析.结果 经过筛选100个克隆中有18株克隆ELISA的吸光度（A值）-490 nm波长（A490nm）值较高,将这些噬菌体上清与牛血清白蛋白（BSA）进行交叉反应,确定有5株交叉反应较弱,结合3次ELISA重复实验的A值及竞争抑制实验结果,最后确定1株阳性克隆.亚克隆到pCANTAB5E载体;转化感受态细胞XL1-Blue.结论 提取质粒酶切片段与目的相符;为下一步其特异性亲和力的研究创造了条件.     

胸腺肽α1单克隆抗体的制备及其鉴定

目的:研制胸腺肽α1(Tα1)单克隆抗体，为Tα1作用机制的阐明奠定基础。方法:用戊二醛将Tα1和牛血清白蛋白进行交联，以其交联物为抗原，按常规淋巴细胞杂交瘤技术制备单克隆抗体，并采用间接酶联免疫吸附(ELISA)法和Western免疫印迹来鉴定单克隆抗体的特异性。结果:建立了分泌抗Tα1抗体的杂交瘤细胞株，能特异性地与Tα1结合，但与单纯牛血清白蛋白、酵母抽提物和转染Tα1酵母未诱导表达产物无反应。其抗体效价为1：10240，Ig亚类为IgG2b。结论：本研究建立了针对Tα1高效价、高特异性单克隆抗体细胞株。     

抗华支睾吸虫CAg噬菌体抗体表达与活性鉴定

目的对从抗华支睾吸虫噬菌体抗体库中筛选到的阳性克隆进行诱导表达和鉴定。方法对阳性克隆株1305、C10和E38用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达，经SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹试验(Western blot)观察有无Fab抗体条带。然后，用卫氏并殖吸虫(Pw)、肝片吸虫(Fh)、姜片吸虫(Fb)、华支睾吸虫(G)、日本血吸虫(Sj)脱脂全虫粗抗原和华支睾吸虫代谢抗原(C5-Mhg)进行SDS-PAGE，并分别和B05、C10和E38噬菌体抗体进行Western blot，鉴定抗体活性。结果Western blot结果表明，3株阳性克隆的培养上清和细菌冻融上清在约50kD和27kD处均有特异抗体条带出现。B05噬菌体抗体与C5-MAg和C5-Ag在约25kD处出现特异性反应条带，而与其它4种吸虫抗原无反应条带的出现。C10除与C5-MAg和华支睾吸虫全虫抗原(C5-Ag)在约38kD处出现反应条带外，还与Sj-Ag在该处有弱阳性反应；而与其它3种吸虫抗原无反应条带出现。E38与C5-MAg和C5-Ag在约16kD处出现特异性反应条带，而与其它4种吸虫抗原无反应条带出现。结论3株阳性克隆均可表达抗华支睾吸虫循环抗原(CAg)噬菌体抗体，其中B05和E38具有特异结合C5-CAg活性。     

抗桔青霉素单克隆抗体的研制与鉴定

目的制备抗桔青霉素的单克隆抗体。方法采用活性酯法、甲醛加成法和羰基二咪唑法制备了4种桔青霉素与载体蛋白的偶联物(A、B、C和D),免疫小鼠制备抗桔青霉素单克隆抗体。结果免疫原性鉴定证明偶联物C可刺激BALB/c小鼠产生抗桔青霉素的抗体,细胞融合后筛选到一株抗桔青霉素的单克隆抗体,单抗与赭曲霉毒素A、黄曲霉毒素B1和展青霉素等毒素交叉反应低于0.01%,在此基础上建立了竞争ELISA检测方法,线性范围为20~10,00ng/ml,检测下限为10ng/ml。在小麦样品中的加标回收率为95%~112%,变异系数为9.1%~18.6%。结论本研究成功筛选到抗桔青霉素单克隆抗体。     

265例HIV-1抗体阳性者抗体蛋白印迹带型分析

目的通过对本实验室以蛋白印迹（WB）法确认的HIV-1抗体阳性者抗体WB条带的分析，了解不同人群抗体WB带型分布的差异。方法WB法确认HIV-1抗体阳性。结果抗pol基因编码蛋白的抗体WB带型分布以及P31抗体的阳性率在不同传播途径的HIV-1抗体阳性者问差异有统计学意义（P〈0．01）；年龄（age，A）≤40岁的HIV-1抗体阳性者P66和P24抗体阳性率分别明显高于相应A〉40岁以上者，（P〈0．05）；抗pol基因编码蛋白的抗体WB带型分布以及P66抗体阳性率在不同地域的HIV-1抗体阳性者闻差异有统计学意义（P〈0．01）。结论HIV-1抗体WB带型分布在不同性别、不同年龄人群间差异无统计学意义。但在不同传播方式、不同地域的人群间差异有统计学意义；HIV-1 pol、gag基因编码的某些蛋白的抗体产生在不同传播方式、不同年龄及不同地域的人群间差异有统计学意义。     

Engineering filamentous phage carriers to improve focusing of antibody responses against peptides

The filamentous bacteriophage are highly immunogenic particles that can be used as carrier proteins for peptides and presumably other haptens and antigens. Our previous work demonstrated that the antibody response was better focused against a synthetic peptide if it was conjugated to phage as compared to the classical carrier, ovalbumin. We speculated that this was due, in part, to the relatively low surface complexity of the phage. Here, we further investigate the phage as an immunogenic carrier, and the effect reducing its surface complexity has on the antibody response against peptides that are either displayed as recombinant fusions to the phage coat or are chemically conjugated to it. Immunodominant regions of the minor coat protein, pIII, were removed from the phage surface by excising its N1 and N2 domains (Δ3 phage variant), whereas immunodominant epitopes of the major coat protein, pVIII, were altered by reducing the charge of its surface-exposed N-terminal residues (Δ8 phage variant). Immunization of mice revealed that the Δ3 variant was less immunogenic than wild-type (WT) phage, whereas the Δ8 variant was more immunogenic. The immunogenicity of two different peptides was tested in the context of the WT and Δ3 phage in two different forms: (i) as recombinant peptides fused to pVIII, and (ii) as synthetic peptides conjugated to the phage surface. One peptide (MD10) in its recombinant form produced a stronger anti-peptide antibody response fused to the WT carrier compared to the Δ3 phage carrier, and did not elicit a detectable anti-peptide response in its synthetic form conjugated to either phage carrier. This trend was reversed for a different peptide (4E10_L), which did not produce a detectable anti-peptide antibody response as a recombinant fusion; yet, as a chemical conjugate to Δ3 phage, but not WT phage, it elicited a highly focused anti-peptide antibody response that exceeded the anti-carrier response by ∼65-fold. The results suggest that focusing of the antibody response against synthetic peptides can be improved by decreasing the antigenic complexity of the phage surface.     

环丙沙星单克隆抗体制备、纯化及鉴定

目的制备抗环丙沙星单克隆抗体.方法用碳二亚胺(EDC)法合成的环丙沙星-牛血清白蛋白完全抗原(CPFX-BSA)免疫BALB/c小鼠,通过杂交瘤技术筛选分泌抗环丙沙星单克隆抗体的杂交瘤细胞株.结果筛选出3株分泌抗环丙沙星单克隆抗体的杂交瘤细胞株1D10、3C6和3G7,其抗体类型分别为IgM、IgG1和IgG1.其中3C6腹水抗体纯度、抗体浓度、亲和常数分别为80.04%、3.96 g/L和1.01×108 L/mol.该单抗与恩诺沙星、诺氟沙星的交叉反应率分别为125.89%、19.50%,与青霉素、卡那霉素和庆大霉素等无交叉反应.结论该单抗可用于动物性食品中CPFX残留ELISA检测方法的建立.     

Immunogenicity of synthetic HIV-1 V3 loop peptides by MPL adjuvanted pH-sensitive liposomes

A successful HIV-1 vaccine should be capable of generating humoral and cellular immune responses at the same time. The only response shown to be effective in this regard is virus-neutralization antibodies and virus-specific cytotoxic T-lymphocytes (CTL) directed against the viral antigens. In the present study, it is shown that V3 peptides encapsulated pH-sensitive liposomes elicit the virus neutralization antibodies and virus specific CTL response at the same time in Balb/c mice. None of the immunization protocols elicited an antibody response and CTL response when R15K and T26K was used as immunogen without liposomes. In contrast, antibodies and CTL response were detectable in the mice which were immunized with peptide encapsulated pH-sensitive liposomes. Antibody production was confirmed by virus neutralizing assay. CD4+ T-cells are involved in target cell lysis to some degree but CTL activity is mainly due to the CD8 + T-cells. The consistency of the antibody and CTL response was related to the V3 loop peptides size. The T26K (26mer) peptide induced a stronger antibody and CTL response than R15K (15mer) in vivo. Based on the results of this study, T26K was used as a potentially effective HIV-1 vaccine component and T26K encapsulated pH-sensitive liposomes composed of phosphatidylethanolamine-beta-oleoyl-gamma-palmitoyl (POPE)/cholesterol hemisuccinate (CHOH)/monophosphoryl lipid A (MPL) (7:3:0.1, mole ratio) may be used as a potentially immunomodulating adjuvant system for the development of HIV and other viral vaccines.