哇巴因多克隆抗体F（ab）2片段的制备及其分子量测定

目的：制备哇巴因多克隆抗体F（ab)2片段,并对其进行分子量的测定。方法：免疫家兔制备哇巴因多克隆抗体,在适宜的条件下,用胃蛋白酶酶解哇巴因多克隆抗体,制备F（ab)2片段,酶解产物经HPSEC分析,纯化,ELISA法检测其免疫学活性,并在适宜的色谱条件下,分别测定三种标准蛋白的洗脱体积,绘制标准曲线,测定其分子量。结果100mg哇巴因多克隆抗体在2mg胃蛋白酶消化18h,反应体系pH3.0的条件下消化,制备出了活性的F（ab)2片段,其相对分子质量为107kD。结论：用胃蛋白酶消化喹巴因多克隆抗体,可制备出活性的F（ab)2片段,同时其相对分子质量的测定为其进一步研究提供了依据。     

高纯度抗五步蛇毒F(ab′)_2血清的制备

目的摸索高纯度抗五步蛇毒血清的制备方法,以降低抗血清治疗毒蛇咬伤时因纯度不高发生的过敏和其他不良反应。方法从免疫后的马血浆中先提取IgG,然后酶解,通过疏水层析得到F(ab′)2片段,以SDS-PAGE电泳确定纯度,以小鼠体外中和试验确定F(ab′)2活性片段的中和能力。结果制备的抗五步蛇毒血清F(ab′)2冻干品纯度质量分数为91.3%,中和五步蛇毒干粉质量比为15∶1。结论采用新方法制备的F(ab′)2活性片段纯度质量分数能从70%提高到90%以上。     

酶法制备牡蛎抗氧化肽研究

目的优化牡蛎酶解制备抗氧化肽的工艺。方法通过正交试验法确定胃蛋白酶、胰蛋白酶及这两种蛋白酶的酶解液抗氧化(Fenton体系)的最佳工艺条件;采用Sephadex G-25葡聚糖凝胶柱层析法对酶解液中抗氧化肽进行分离纯化。结果胃蛋白酶最佳酶解条件:温度35℃、加酶量2.5%、时间6h、pH 2.0,在此条件下清除率达到50%时的酶解液浓度(EC50)为1.929mg.mL-1;胰蛋白酶最佳酶解条件:温度45℃、加酶量2%、时间8h、pH 7.5,EC50为1.165mg.mL-1;胰蛋白酶、胃蛋白酶分步酶解法的酶解液EC50为0.869mg.mL-1。双酶酶解液经纯化后,得相对分子质量为751的抗氧化活性肽组分(BPO-Ⅱ),其EC50为0.529mg.mL-1。结论双酶酶解法优于单酶酶解法,从双酶酶解液中分离纯化得抗氧化肽BPO-Ⅱ。     

载多柔比星人血清白蛋白毫微粒和两种免疫毫微粒的体内外靶向性研究

目的 构建载多柔比星的普通毫微粒、全抗体免疫毫微粒和抗体F(ab’)2片段免疫毫微粒，比较它们对肿瘤的体外和体内靶向性，寻找对肿瘤组织具有特异性靶向作用的制剂。方法 采用异型双功能交联剂琥珀酰亚胺基-3-(2-吡啶二硫)丙酸酯(SPDF)，将兔抗大鼠乳腺癌细胞系Walker-256细胞的多克隆抗体(Wab)和其F(ab’)2片段即WF(ab’)2与载多柔比星(Doxorubicin，DRB)的人血清白蛋白毫微粒(DRB-HSA-NP)交联，制备全抗体免疫毫微粒Wab-DRB-HSA-NP和F(ab’)2片段免疫毫微粒WF(ab’)2-DRB-HSA-NP，比较它们的体外肿瘤细胞靶向性和体内组织器官靶向性。结果 与DRB-HSA-NP相比，两种免疫毫微粒均有较好的体外肿瘤特异靶向性，瘤体内给药后在瘤体内的滞留量也显著增高，特别是WF(ab’)2-DRB-HSA-NP，由于所偶联的抗体切除了FC段，降低了抗体与巨噬细胞的结合。结论 免疫毫微粒，特别是WF(ab’)2-DRB-HSA-NP具有较好的肿瘤肥向性，是一种很有价值的靶向制剂。     

我国马免疫球蛋白制品现状及展望

马免疫球蛋白(equine immunoglobulin)是用目标抗原免疫马匹后,产生抗该种抗原的抗体免疫球蛋白,经胃酶消化、纯化提取制得,分别用于各相应疾病的预防(被动免疫)和治疗. 基本原理是采用无致病性的致病物质多次免疫马匹,待免疫抗体达到较高滴度后,采血提取抗体免疫球蛋白IgG, 利用胃蛋白酶切掉无中和活性的Fc段,仅保留具有中和活性的抗体V形F(ab')2片段,利用其特异性中和致病原的功能进行疾病预防与治疗.     

抗体F(ab′)_2片段靶向的载多柔比星免疫毫微粒的构建及其抗肝癌作用

目的　构建抗体F(ab′) 2 片段靶向的载药免疫毫微粒 ;评价F(ab′) 2 片段免疫毫微粒对肿瘤的特异结合性及杀伤作用。方法　采用异型双功能交联剂琥珀酰亚胺基 3 (2 吡啶二硫 )丙酸酯 (SPDP)将抗人肝癌单抗HAb18或对照抗体 4E3的F(ab′) 2 片段与多柔比星 (ADR)人血清白蛋白毫微粒 (ADR HAS NP)共价交联构建抗体F (ab′) 2 片段靶向的载药免疫毫微粒 (HAb18F(ab′) 2 ADR HSA NP ,4E3F (ab′) 2 ADR HSA NP) ;采用玻片凝集试验 ,免疫荧光染色实验确定F(ab′) 2 片段是否结合在毫微粒表面 ;采用花环形成实验 ,花环形成阻断实验 ,扫描电镜分别从光镜、电镜水平观察F(ab′) 2 片段免疫毫微粒是否同人肝癌细胞株SMMC 772 1特异性结合 ;采用MTT比色分析法研究F(ab′) 2 片段免疫毫微粒的体外细胞毒作用 ;采用皮下荷人肝癌裸鼠模型 ,经瘤体注射观察免疫毫微粒的抗人肝癌作用。结果　F (ab′) 2 片段免疫毫微粒圆球状 ,粒径1 2 μm左右 ,免疫荧光染色阳性而ADR HSA NP为阴性 ,提示F (ab′) 2 片段已偶联到ADR HSA NP表面 ;HAb18F(ab′) 2 ADR HSA NP能有效结合于人肝癌细胞株SMMC 772 1,该结合能被HAb18抗体的F(ab′) 2片段阻断 ,未见该免疫毫微粒与对照细胞 (SW 1116)有效结合 ,这些结果提示 ,HAb18F(ab′)     

中国毛虾ACE抑制产物的酶法制备及其降低原发性高血压大鼠血压的效果(英文)

目的利用中国毛虾制备具有ACE抑制活性及抗高血压活性的酶解产物。方法采用体外试验法检测中国毛虾5种常用蛋白酶酶解产物的ACE抑制活性,筛选出一种蛋白酶作为酶法制备中国毛虾ACE抑制产物用酶,并对其作用条件进行正交优化,利用原发性高血压大鼠评估酶解产物的抗高血压效应。结果胃蛋白酶优化后的酶解条件是:pH 2.4,温度41℃,酶解时间3 h,酶/底物比(E/S)3%,底物浓度8%。以该条件制备的酶解产物对ACE的抑制活性为IC500.65 mg/ml,以1.0 g/kg体重的剂量喂食原发性高血压大鼠4 h后收缩压降低3 886 Pa(29mmHg)。结论中国毛虾的胃蛋白酶酶解产物具有较强的ACE抑制活性及抗高血压活性。     

用胃蛋白酶消化全血清蛋白制备F(ab′)_2的简易方法

[Poulscn F et al:Acta Path MicrobiolScand(C)88(5):241,1980(英文)] 在某些免疫实验系统中用F(ab')_2片段比用完整的IgG抗体更有利。这主要是因为应用F(ab')_2能避免IgG分子的Fc段的非特异性结合。F(ab')_2通常是用纯化的IgG经胃蛋白酶消化再用凝胶过滤法来制备。1976年Madsen等曾使用全球蛋白组份消化的方法制备F(ab')_2,但是,这些方法不适用于从大量血清中制备F(ab')_2。     

用类风湿因子包板检测HTLV-Ⅲ抗体的特异性酶免疫测定法

Sachers等报道了用酶标抗原(ELA)和人类风湿因子(RF)包被微量滴定板检测抗体的酶免疫测定法.本文作者对这种RF-ELA试验加以修改,用酶标免疫复合物(ELIC)代替ELA,检测HTLV-Ⅲ抗体,称为RF-ELIC试验.方法如下:从人血清中分离出丙种球蛋白,用胃蛋白酶消化后获取F(ab')_2片段.并     

酶解条件对透明质酸提取及其分子量分布的影响

使用胃蛋白酶和胰蛋白酶从鸡冠中提取透明质酸,考察不同酶解条件对透明质酸质量与分子量分布的影响.结果显示,胃蛋白酶用量30 g/kg,胰蛋白酶用量3 g/kg,各酶解2h,胰蛋白酶酶解pH 8.5,酶解温度45℃时,透明质酸得率为0.5％～0.6％,葡萄糖醛酸含量为41.2％～44.3％,透明质酸平均相对分子质量大于1.0× 106,分子量分布指数接近1.     

亲和层析纯化的特异性免疫球蛋白及其片段对VX中毒的保护作用

用Ｓｅｐｈａｒｏｓｅ４Ｂ－６－氨基己酰－氨基ＶＸ亲和凝胶层析分离纯化兔抗ＶＸ抗血清中对ＶＸ特异的免疫球蛋白（Ｉｇｓ），再用胃蛋白酶裂解为Ｆ（ａｂ）２片段．用酶联免疫吸附试验法测定抗体活性，在吸光度１．０（４９２ｎｍ）时，抗血清，Ｉｇｓ和Ｆ（ａｂ）２的蛋白质浓度分别是１．７５，０．２５和０．１７ｍｇ·Ｌ－１．在试管内保护电鳐乙酰胆碱酯酶活性５０％所需的浓度，Ｉｇｓ和Ｆ（ａｂ）２只需抗血清的２．６％和３．８％．ｉｖ抗血清，Ｉｇｓ和风Ｆ（ａｂ）２对抗小鼠１×ＬＤ９５ＶＸ中毒死亡的最小全保护剂量分别为每鼠７００，３２和３７μｇ，在此剂量下对小鼠脑胆碱酯酶活性的保护申分别为６４％，６７％和７６％．     

Cross clade prophylactic and therapeutic efficacy of polyvalent equine immunoglobulin F(ab′)2 against highly pathogenic avian influenza H5N1 in mice

BackgroundHighly pathogenic avian influenza H5N1 virus (HPAI H5N1) has the potential to cause a new pandemic, which may lead to disasters in the world. However, we cannot predict the HPAI H5N1 strain that might cause the pandemic. Therefore, broad-spectrum prophylactic or therapeutic preparations for containment of a possible future pandemic are urgently needed. Polyvalent equine immunoglobulin F(ab′)2 may be a promising candidate.MethodsWe prepared four pepsin digested immunoglobulin F(ab′)2 from the horses immunized with purified VNH5N1-Puerto Rico/8/34 (PR8)/CDC-RG (VNRG, Clade 1), A/Indonesia/05/2005(H5N1)-PR8-IBCDC-RG2 (INRG, Clade 2.1), and A/Anhui/01/2005(H5N1)-PR8-IBCDC-RG5 (AHRG, Clade 2.3.4) and PBS (negative control), respectively. The protective effect of the monovalent or polyvalent F(ab′)2 against A/Ostrich/SZ/097/04 (clade 0) infection was determined by cytopathic effect (CPE) in cultured Madin–Darby canine kidney (MDCK) cells. The prophylactic and therapeutic efficacy of the polyvalent F(ab′)2 was further evaluated by observing survival, weight loss and viral load when the polyvalent F(ab′)2 was introduced into mice one day prior to-, three days post-lethal challenge with A/Ostrich/SZ/097/04.ResultsThe half neutralization doses (ND50) of purified monovalent equine F(ab′)2 prepared by the VNRG, INRG or AHRG-immunized horses and polyvalent one against A/Ostrich/SZ/097/04 were 320, 1280, 1280 and 2560 in cultured MDCK cells, respectively. 10 μg polyvalent F(ab′)2 could completely protect mice infected with 100 half lethal doses (LD50) of A/Ostrich/SZ/097/04 in preventive settings. In therapeutic settings, even when injected three days post lethal infection, mice were still completely protected, although 200 μg of polyvalent F(ab′)2 was required.ConclusionsOur work has provided experimental supports for testing the broad-spectrum protective efficacy of polyvalent equine immunoglobulin F(ab′)2 for the future large trials.     

Preparation and preliminary bioevaluation studies of 68Ga‐NOTA‐rituximab fragments as radioimmunoscintigraphic agents for non‐Hodgkin lymphoma

Rituximab is used for the treatment of non‐Hodgkin lymphoma (NHL). This study focuses on development of ⁶⁸Ga‐labeled rituximab fragments, (⁶⁸Ga‐NOTA‐F (ab′)‐rituximab and ⁶⁸Ga‐NOTA‐F (ab′)₂‐rituximab, as PET‐imaging agents for NHL. Rituximab was digested with immobilized pepsin and papain to yield F (ab′)₂ and Fab fragments respectively that were characterized by size exclusion HPLC (SE‐HPLC) and SDS‐PAGE. They were conjugated with p‐SCN‐Bn‐NOTA, labeled with ⁶⁸Ga and characterized by SE‐HPLC. Intact rituximab was labeled with gallium‐68 for comparison. Specificity of ⁶⁸Ga‐labeled immunoconjugates was ascertained by immunoreactivity and cell binding studies in Raji cells, while biodistribution studies were performed in normal Swiss mice. Gradient SDS‐PAGE under nonreducing condition showed molecular weights of F (ab′)₂‐rituximab and F (ab′)‐rituximab as approximately 100 and 40 Kd, respectively. Radiochemical purity (RCP) of ⁶⁸Ga‐NOTA‐F (ab′)₂‐rituximab and ⁶⁸Ga‐NOTA‐F (ab′)‐rituximab were 98.2 ± 0.5% and 98.8 ± 0.2% respectively with retention times of 17.1 ± 0.1 min and 19.3 ± 0.1 min in SE‐HPLC. ⁶⁸Ga‐labeled rituximab fragments were stable in saline and serum up to 2‐hour post preparation and exhibited specificity to CD20 antigen. Immunoreactivity of ⁶⁸Ga‐labeled immunoconjugates was greater than 80%. Clearance of the fragmented radioimmunoconjugates was predominantly through renal route. Preliminary results from this study demonstrate the potential of ⁶⁸Ga‐ NOTA‐F (ab′)₂‐rituximab and ⁶⁸Ga‐NOTA‐F (ab′)‐rituximab as PET imaging agents for NHL.     

脑型一氧化氮合酶钙调蛋白结合区的克隆表达与活性鉴定

应用PCR法扩增了脑型一氧化氮合酶（nNOS)的钙调蛋白CaM结合区基因,将扩增出的0．6kBDNA片段经NdeI/EcoRI酶切,重组于表达载体pET/28a( )质粒中,用电穿孔法导入E.Coli,筛选获得基因工程菌。在IPTG诱导下,目的蛋白在工程菌的表达量为菌体蛋白的15％－20％,应用His、Tagsepharose金属螯合亲和层析和SDS－PAGE纯化,获得电泳纯的目的蛋白、相对分子质量为22000,以它为抗原,制备了免疫克隆抗血清,经纯化获得高度专一性的nNOS CaM结合区蛋白的抗体,实验证实所得抗体与nNOS抗原特异结合,与诱导型一氧化氮合酶（iNOS)无交叉反应。     

生长抑素一级结构的电喷雾质谱分析

目的生长抑素的分子量及其一级结构的测定。方法应用电喷雾质谱法(ESI-MS)测定了环肽药物生长抑素的分子量,用2-巯基乙醇将生长抑素的双硫键还原,再用源内碰撞诱导解离技术进行测定,根据其典型碎片离子确证其氨基酸序列。结果测得生长抑素准分子离子峰［M+H］⁺ m/z 1 637.8, ［M+Na］⁺ m/z 1 659.5及［M+2Na-H］⁺ m/z 1 681.5,［M+2H］²⁺ m/z 819.5及［M+H+Na］²⁺ m/z 830.3,表明生长抑素的分子量为1 636.7,与理论值一致。将双硫键还原后,用CID技术进行测定,得到了一系列y和b系列的特征碎片离子,证实生长抑素还原产物的氨基酸序列为A-G-C-K-N-F-F-W-K-T-F-T-S-C。结论用ESI-MS法测得的生长抑素的分子量及一级结构与理论值相符。     

Combination of two antibody fragments F(ab')(2)/Fab: an alternative for scorpion envenoming treatment

Immunotherapy is the only effective treatment for scorpion stings. However the efficiency of this treatment varies depending on the forms of the antibodies and route of administration used. The antibodies are mostly injected as F(ab )(2) fragments. In this study, we investigated damage to the heart and lung tissue and the inflammatory response caused by Androctonus australis hector venom, its toxic fraction after molecular filtration or the isolated main alpha toxin (Aah II) in the presence or absence of different antibody molecules. A mixture of antibody fragments, F(ab )(2) and Fab, significantly reduced local leukocytosis, hemorrhage and inflammatory oedema induced by the A. australis hector venom and its toxins.     

Canine immunoglobulin F(ab′)2 fragments protect cats against feline parvovirus virus infection

Feline parvovirus virus (FPV) causes severe diarrhea and leukopenia in felines, and threatening the health of wild and domestic felines. Currently, specific drugs to treat FPV have not yet been developed. In this study, IgG was extracted from inactivated FPV-immunized dog sera. Canine F(ab′)₂ fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. The results showed that canine immunoglobulin F(ab′)₂ fragments showed efficient neutralizing activity in vitro against FPV and had therapeutic and prophylactic effects in FPV infected cats. The anti-FPV-specific F(ab′)₂ fragment can significantly alleviate the clinical symptoms of FPV infected cats and reduce the viral loads of the intestinal tract. These results indicated that the F(ab′)₂ fragment prepared from inactivated FPV-immunized felines may be used as a prophylactic and therapeutic agent for diseases caused by FPV.     

Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield

Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.