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。     

新型抗uPAR人源化抗体的表达和活性检测

目的制备抗尿激酶型纤溶酶原激活物受体（uPAR）人源化抗体并初步检测它们与抗原的亲和能力。方法通过计算机辅助设计的结果,合成新型抗uPAR抗体的轻链和重链可变区基因序列,通过重叠PCR方法,拼接成完整的轻链和重链基因并克隆入pIRES双向表达载体。瞬时转染293T细胞,收取细胞上清,rProtein A亲和层析法纯化目的抗体,并进行SDS-PAGE和免疫印迹鉴定,采用Biacore3000技术检测抗体与抗原的结合能力。结果成功构建5种表达载体S1~S5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和55×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合。Biacore3000实验结果表明,S2、S4和S5抗体与抗原具有良好的亲和活性,且亲和活性分别为1.74×10-8,1.49×10-8和1.05×10-8mol/L。结论成功构建并表达了5种抗uPAR人源化抗体,其中S2、S4和S5具有良好的抗原结合能力。     

基因工程技术制备抗IL-2蛋白抗体Fab片段及其鉴定方法的建立

目的利用基因工程技术制备人源性抗IL-2蛋白的可溶性Fab抗体片段,并鉴定其抗原结合活性及特异性。方法用固相化的IL-2蛋白从人天然噬菌体抗体库中筛选出表达抗IL-2蛋白Fab抗体片段的阳性克隆,酶切及连接反应构建可溶性表达噬菌粒转化至大肠杆菌BL21(DE3)pLysS中,IPTG诱导表达,并用SDS-PAGE鉴定抗体表达情况、ELISA鉴定其抗原结合活性和特异性。结果成功筛选并表达了抗IL-2蛋白的Fab段抗体,在SDS-PAGE中形成47kD的条带,Western blot证明为抗人Fab段抗体;ELISA证实该抗体片段具有良好的抗原特异性和抗原结合活性,与牛血清白蛋白、IL-4无交叉反应。结论成功表达并鉴定了人源性抗IL-2蛋白的可溶性Fab片段,构建了一种制备基因工程抗体的有效途径,为基因工程抗体在肿瘤免疫治疗方面的临床应用研究奠定了基础。     

新型抗EGFR人源化抗体基因的构建和表达

目的：构建并表达进一步人源化且效果显著增强的抗EGFR抗体。方法：通过计算机辅助设计的结果,合成新型抗EGFR抗体的轻链和重链可变区序列,拼接成完整的轻链和重链基因并克隆入pIRES双表达载体。瞬时转染293T细胞,用rProteinA亲和层析柱从细胞培养上清中纯化抗体,并进行SDS-PAGE和免疫印迹鉴定。采用Biacore3000技术检测抗体结合抗原的能力;细胞侵袭实验初步检测抗体的功能。结果：成功构建3种不同突变体表达载体C2、C3和C5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和50×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合。Biacore3000实验结果表明,C3抗体与抗原具有良好亲和活性（亲和力为6.13×10-10mol/L）;细胞侵袭实验结果表明,C2和C3抗体对肿瘤细胞生长迁移均具有一定的抑制作用。结论：成功构建、表达了3种抗EGFR人源化抗体C2、C3和C5,其中C3具有良好的抗原结合能力和抑制肿瘤细胞生长迁移能力。     

新型人源化抗CD20单克隆抗体的表达和活性检测

目的：制备新型人源化抗CD20单克隆抗体并检测其与CD20抗原的亲和力和抗肿瘤活性。方法通过计算机模建技术,在蛋白质结构数据库中找到与利妥昔单抗（ rituximab）空间结构最相近的人IgG1为骨架,将利妥昔单抗的互补决定区（ complementarity determining region ,CDR）进行移植和改造,通过重叠PCR方法,扩增出目的基因片段。分别将轻链（L）、重链（H）基因片段克隆到载体pcDNA3．3、pOptiVEC质粒上。瞬时转染至293F细胞,收取细胞上清,用rProteinA亲和层析法纯化目的抗体,并用十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）检验抗体的纯度和表达量,采用Fortebio技术检测抗体与抗原的结合能力,最后通过裸鼠移植瘤生长抑制实验检测抗体的抗肿瘤活性。结果构建了3种抗CD20人源化抗体。抗体在还原SDS-PAGE中表现为两条相对分子质量约为25×103和55×103的条带,与预计条带大小相符;Fortebio实验结果表明,3组抗体与抗原具有良好的亲和活性：利妥昔单抗、L4H7抗体、L5H5抗体和L5H7抗体的Kd值分别为6．48×10－9、1．91×10－9、7．35×10－10和1．91×10－9 mol／L。裸鼠移植瘤生长抑制实验表明,L5H7抗体的抗肿瘤活性优于利妥昔单抗。结论成功构建并表达了3种抗CD20人源化抗体,其中L5H7抗体具有良好的抗原结合能力和抗肿瘤活性。     

基因工程抗角蛋白人抗体的制备与鉴定

目的　制备亲和力高、性能良好的人源性抗角蛋白抗体。方法　以人表皮角蛋白为抗原从噬菌体抗体库中克隆特异性抗角蛋白抗体 ,采用链替换 (chain shuffling)技术对所获抗体进行亲和力成熟 ,并对其生物学性能进行一系列鉴定。结果　经过筛选半合成噬菌体抗体库 ,成功获得了能与角蛋白特异性结合的人源性抗角蛋白抗体 ;选择其中活性最好的一株抗体 ,通过轻链替换和重链替换使抗体亲和力得到了有效提高 ,达到8× 10 1 0 mol的理想水平。结论　用基因工程的方法可以制备高质量的人源性抗角蛋白抗体 ,为其功能和临床应用的进一步研究打下了基础     

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。     

盐酸克仑特罗单克隆抗体的制备及特性鉴定

目的:制备抗盐酸克仑特罗(clenbuterol ,CL)的单克隆抗体并进行免疫学特性鉴定.方法:用重氮化法将盐酸克仑特罗偶联于载体牛血清白蛋白(BSA),形成的结合抗原BSA-CL作为免疫原免疫BALB/c小鼠,同时将CL与卵清白蛋白(OVA)偶联制备检测原(OVA-CL).应用杂交瘤技术建立分泌抗盐酸克仑特罗单克隆抗体的杂交瘤细胞株,体内诱生腹水并进行纯化.用竞争性ELISA法测定抗体的亲和常数及交叉反应性.结果及结论:获得了18株稳定分泌抗盐酸克仑特罗单克隆抗体的杂交瘤细胞株,挑选其中E9-C11、C11-E3、E7-G8三株高亲和力的细胞株进行免疫学特性鉴定.三株抗体的亚类均为IgG1.细胞上清的间接ELISA效价分别为1:1 280、1:1 000、1:800,三株腹水效价都约为1×10-6.亲和常数分别为2.4×10-8mol/L、2.83×10 -8 mol/L 、3.28×10-8 mol/L .E9-C11单抗对CL的IC50为6.1035μg/L;对沙丁胺醇的IC50大于781.25 μg/L,交叉反应性小于0.78%;对肾上腺素、去甲肾上腺素、异丙肾上腺素、莱克多巴胺及部分维生素和抗生素均无交叉反应性.蛋白免疫印迹分析证明三株单抗特异性均很高,可用于与其相关的免疫检测或应用研究.     

分泌抗人IgA单克隆抗体杂交瘤细胞株的建立与鉴定

利用常规杂交瘤技术制备了一株抗人IgA杂交瘤PA_(229) ELISA结果表明,它分泌的抗体与人IgA有特异性反应,而与其它免疫球蛋白重、轻链无交叉反应,为小鼠IgG_1亚类。免疫转印结果只显示针对IgAα链的一条电泳带。用改良ELISA法测得PA_(229)的亲和常数为1.33×10~8L/mol。     

用亲和层析法测定P81抗体所抗细胞表面抗原的分子量

P81是抗人全部T细胞及部分B细胞的单克隆抗体,其特异性类似于Leu-1或OKT_1,但又与它们不完全相同。测定P81抗体所抗细胞表面抗原的分子量将有助于对抗体特异性进行鉴定,也有助于研究相应抗原的性质。本文报道用亲和层析法测定P81抗体所抗抗原的分子量,并与常规的免疫沉淀法所得结果进行比较。材料和方法 Protein A-Sepharose CL-4B,CNBr-Sepharose 4B及测分子量用的标准蛋白均购自     

重组人抗HBsAg Fab抗体的纯化方法比较研究

目的研究重组人抗HBsAg Fab抗体的纯化条件。方法采用羊抗人Fab抗体亲和层析柱、14F7单克隆抗体亲和层析柱、离子交换-分子筛层析柱,分别纯化由酵母工程菌(Gs115／Fab)发酵的重组人抗HBsAg Fab抗体,并对3种纯化方法所得Fab抗体的纯度、收率、与HBsAg的结合活性进行比较。结果3种纯化方法中,14W单克隆抗体柱纯化的Fab抗体的纯度达98％左右,Fab抗体柱纯化的Fab抗体的纯度为95％,但这两种亲和柱的目的蛋白收率都不高,分别为35％、55％。而离子交换柱纯化的Fab抗体的纯度为93．8％,经分子筛柱进一步纯化后,可达98％以上,Fab抗体蛋白收率可达80％以上。经ELISA分析,3种方法纯化的Fab抗体均具有较高的HBsAg抗原结合力和特异性。结论通过对3种纯化方法的比较得出,离子交换一分子筛层析法是重组人抗HBsAg Fab抗体的最佳纯化方法,这为抗HBsAg Fab抗体的产业化生产和临床研究打下良好基础。     

Mathematical analysis of equilibrium on 2-site immunoradiometric assay using anti-human TSH monoclonal antibody

A mathematical analysis of equilibrium was adapted on a practical 2-site immunoradiometric assay (IRMA) using 10 murine monoclonal antibodies (Mabs) to human TSH. The affinity constant of 10 Mabs was from 2.5 x 10(8) to 3.3 x 10(10) M-1. The dose-response curves using appropriate combinations of Mab-coated bead and 125I-labeled Mab were compared with the theoretical curves calculated from mathematical analysis of equilibrium. Theoretical curves were directly affected by the affinity constants in which antibodies with higher affinity constants showed higher binding activity of tracer. However, the curves which antibodies have more than 1 x 10(9) M-1 and 1 x 10(10) M-1 for capture antibody and tracer antibody did not show remarkable change, but were similar with the curve obtained from unlimited affinity constant. From these curve, the maximum and minimum detectable dose were approx. 2 x 10(-10) M and 4 x 10(-14) M, respectively. Seven out of 10 experimental curves for TSH showed good consistency with the corresponding theoretical curves which indicated that a mathematical analysis of equilibrium was useful to presume experimental results using monoclonal antibody.     

The labeling of high affinity sites of antibodies with 99mTc

Labeling of F(ab')2 with 99mTc was investigated. The best labeling procedure for F(ab')2 was then applied to IgG and Fab. Stannous ion was used as the reducing agent for 99mTcO-4 and free DTPA was used as a competing reagent to prevent colloid formation and loosely bound 99mTc. The competition reactions revealed two 99mTc binding sites for F(ab')2 and IgG. One is a high capacity, low affinity site. This accounts for 76 and 84% of total IgG and F(ab')2 binding sites. The labeling of these sites can be prevented if the antibody is labeled in the presence of free DTPA. The second site is a low capacity, high affinity site. The labeling of these sites cannot be prevented by free DTPA. Fab, unlike IgG and F(ab')2, does not have an appreciable percentage of high affinity sites. The determination of sulfhydryl groups using Ellman's reagent indicates the production of 5.5, 4.2 and 0.9 sulfhydryl groups when IgG, F(ab')2 and Fab were exposed to 56 micrograms/mL SnCl2 X 2H2O. These sulfhydryl groups may be the source of the high affinity binding. Biodistribution in mice for 99mTc labeled F(ab')2 and F(ab')2-DTPA, both prepared in the presence of excess free DTPA, was similar to that of F(ab')2-DTPA-111In.     

Imaging of HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice using (111)In-trastuzumab (Herceptin) Fab fragments

Trastuzumab (Herceptin) Fab were prepared by digestion of intact IgG with immobilized papain, derivatized with diethylenetriaminepentaacetic acid (DTPA) and radiolabeled with (111)In. The dissociation constant (Kd) for binding of Fab to HER2/neu-positive SK-BR-3 human breast cancer cells was two- to threefold higher than for intact IgG (14-36 vs. 8-14 nM). The binding affinity was not significantly decreased after DTPA derivatization (Kd=47 nM). (111)In-trastuzumab Fab localized specifically in HER2/neu-positive BT-474 human breast cancer xenografts in athymic mice with tumor uptake of 7.8+/-0.7% injected dose (ID)/g and tumor/blood ratio of 25.2+/-1.6 at 72 h postinjection compared with 2.7+/-0.7% ID/g and 7.0+/-0.9 for (111)In-HuM195 anti-CD33 Fab (significantly different, P<.001). Small (3-5 mm in diameter) BT-474 tumors were imaged with (111)In-trastuzumab Fab as early as 24 h postinjection.     

Complementary roles of antibody affinity and specificity for in vivo diagnostic cardiovascular targeting: How specific is antimyosin for irreversible myocardial damage?

BACKGROUND: Identification of irreversible myocyte injury with antimyosin antibody imaging depends on both antibody specificity and affinity. To characterize the role of antibody affinity, we performed studies in dogs with acute coronary occlusion followed by reperfusion using 3 monoclonal antimyosin antibodies with different affinities. METHODS AND RESULTS: Dogs with experimental reperfused acute myocardial infarction were injected with 2 high-affinity radiolabeled monoclonal antimyosin Fab fragments (R11D10 and 2G42D7), 1 low-affinity antimyosin Fab (3H31E6), and a nonspecific Fab. The left lateral gamma images at 5 H were used to assess the infarct (I) to blood (B) region of interest (ROI) count density ratios by computer planimetry. All infarcts were confirmed by in vivo imaging with 201Tl for perfusion defects as well as by postmortem histochemical staining. The mean I/B ROI (+/-standard deviation [SD]) for R11D10 (1.701+/-0.376) was not significantly different from that of 2G42D7 (1.501+/-0.267, P = NS), but both were significantly greater than that of 3H31E6 Fab (0.85+/-0.12, P = .0001 and .0012, respectively). The I/B ROI of 3H31E6 Fab was similar to that of nonspecific Fab (0.75 to 0.77 range). Radiolabeled R11D10 and 2G42D7 were unequivocally positive by gamma imaging in all infarcts by 5 H. No infarcts were visualized with 3H31E6 or nonspecific Fab. CONCLUSIONS: The low-affinity antibody, despite its specificity for cardiac myosin, cannot be used to image the infarct zone. Therefore immunoscintigraphic diagnosis of irreversible myocardial injury with radiolabeled antimyosin Fab is doubly specific because in vivo visualization required both specificity and high enough affinity of the antibody.     

人血浆脑利钠肽免疫放射分析

用免疫放射分析法（ＩＲＭＡ）测定人血浆脑利钠肽（ｈＢＮＰ），方法简便快速、灵敏度高，最低检测量为０１４ｐｍｏｌ／Ｌ。标准曲线及样品稀释线性良好。与心房利钠肽无交叉反应，能抗游离型和结合型胆红素及乳糜浊度的影响。回收率分别为９５０％和１００７％。样品效应误差关系曲线斜率为００３７，标准曲线各点的标准浓度平均误差（ＣＶ）与样品批内ＣＶ和批间ＣＶ均＜１０％。表明采用ＩＲＭＡ能有效地测定血浆ＢＮＰ浓度，可为临床诊断心功能不全、原发性高血压和糖尿病早期心室功能障碍提供依据。     

131 I标记人源抗HBs Fab瘤内注射治疗荷人肝癌裸鼠的实验研究

目的　探讨13 1I标记人源抗乙肝表面抗原单克隆抗体Fab片段 (抗HBsFab)瘤内给药治疗荷人肝癌裸鼠移植瘤的合理性。方法　荷瘤裸鼠分为 5组 ,分别经瘤内注射13 1I 抗HBsFab、13 1I 无关Fab、13 1I、PBS及腹腔注射13 1I 抗HBsFab。 5d后每组各取 2只作组织分布测定 ,其余观察 3周 ,计算各组肿瘤生长抑制率。结果　瘤内注射13 1I 抗HBsFab组放射性计数瘤 /肝比值是腹腔注射组的 9倍 ,3周后前者肿瘤生长抑制率高于后者 ,分别为 62 .3%和 46.7%。结论　采用瘤内注射13 1I标记人源抗HBsFab导向治疗肝癌 ,具有低毒高效的治疗作用 ,临床实用价值大     

Iodine-125-NRLU-10 kinetic studies and bismuth-212-NRLU-10 toxicity in LS174T multicell spheroids

Alpha emitter-labeled antibodies (Abs) are of considerable interest in cancer therapy. Alpha particles are densely ionizing and therefore have a high radiobiologic effectiveness, and the cell killing produced is influenced very little by dose rate or hypoxic conditions. LS174T human colon adenocarcinoma spheroids were used in this study to evaluate the efficacy of alpha emitter-labeled Abs in a three-dimensional model. NRLU-10, an IgG2b Ab to a pancarcinoma antigen, and its Fab fragment were used. Initial kinetic studies using 125I-NRLU-10 revealed that a large number of binding sites/cell and high Ab affinity led to slow Ab penetration. This effect could be overcome by increasing the Ab concentration ten-fold for Fab but not for intact Ab. Bismuth-212-NRLU-10 therapy was very effective in killing single cells (over 3 log reduction in surviving fraction) but was ineffective in spheroids (less than 1 log reduction). This was likely due to inadequate penetration into the spheroids before the 212Bi decayed. The use of higher Ab concentrations, tumors with fewer antigenic sites/cell for the Ab being used, lower affinity Abs, alpha emitters with longer half-lives, and pretargeting with bifunctional Ab are all potential ways of increasing the efficacy of alpha emitter-labeled Abs for cancer therapy.     

三种方法纯化生物工程抗体片段的比较

比较3种不同纯化体系纯化生物工程抗－HBsFab的效率和效果，寻求高效，经济的生物工程产品批量纯化方法，采用增菌发酵抗－HBsFab阳性克隆，超声破菌法制备Fab上清，分别缓冲平衡抗Fab -Sepharose亲合胶，链球菌蛋白G（prot.G)-Sepharose胶和Ni-NTA离子螯合胶，对Fab上清进行过柱纯化，紫外光检测目的蛋白得率，SDS－PAGE鉴定产物的纯度并用HBsAg包被ELISA检测其生物活性，结果显示，等容积不同类别的胶体对目的蛋白的纯化效率依次为：Ni-NTA离子螯合胶＞prot.G-Sepharose胶＞抗－FabSepharose亲合胶；在各胶体饱和结合能力之内，3种方法中获高纯度产品，在SDS－PAGE中呈现单一区带：HBsAg包被ELISA检测结果为3种方法纯化制备的Fab在抗原结合能力上未显示明显差别，提示，3种纯化方法各有优势和应用限制，Ni-NTA法在经济性，实用性和纯化效率上明显优于其他两种方法。     

乙型肝炎病毒表面抗原定性检测阳性结果复检分析

目的　观察乙型肝炎病毒表面抗原 (HBsAg)检测结果的准确性 ,增强结果的可比性。 方法　对 30 6份HBsAg初检阳性结果进行复检。结果　 30 6份HBsAg初检阳性结果中 ,复检与初检一致者 2 82份 ,符合率为 92 16 % ;初检假阳性率为 7 84%。HBsAg结果复检符合率与初检OD值有关 ;HBsAg初检OD值越高 ,复检符合率亦越高。 结论　对HBsAg阳性结果进行复检 ,可以有效控制检测中影响结果的因素 ,对保证结果的准确性有重要意义。