重金属汞单克隆抗体的制备及鉴定

通过双功能螯合剂(异硫氰基.苯甲基-乙二胺四乙酸,ITCBE)使汞离子与载体蛋白(匙孔血蓝蛋白,KLH)偶联,获得了免疫抗原KLH-ITCBE-Hg(Ⅱ).以该抗原免疫BALB/C小鼠,取免疫小鼠的脾细胞与骨髓瘤细胞融合,杂交瘤细胞用EIJSA方法筛选及有限稀释法亚克隆化,获得了5株可稳定分泌抗汞单克隆抗体的杂交瘤细胞株(E/H9、E/B2、1/F11、1/H7和B/B11).杂交瘤细胞染色体数目在97-104之间,所分泌的抗体均为IgM、k型,其中杂交瘤细胞株E/H9、1/F11和B/B11细胞培养上清液效价分别达1:640、1:320、1:160,腹水效价分别达1:6400、1:3200、1:6400.B/B11的亲和常数为2.981×108L/mol,特异性试验表明:B/B11与其它金属离子的交叉反应率均小于2%,具有较高的特异性.高亲合力和高特异性的抗汞单克隆抗体在该研究中的成功制备,为建立农产品和环境中重金属汞的快速免疫检测方法奠定了基础.     

对抗氯霉素单克隆抗体4D10特性鉴定研究

目的对抗氯霉素单克隆抗体4D10进行纯化与特性鉴定,以获得可用于检测氯霉紊残留试剂盒的单克隆抗体。方法用两步硫酸铵法和G蛋白亲和层析法纯化单克隆抗体,用间接酶联免疫吸附试验、竞争ELISA等方法测定抗体效价、特异性、敏感性与亲和性以及抗体与其它氯霉素结构类似物的交叉反应率。结果纯化后抗体的纯度高达95％,对抗体进行相关性质的鉴定得出：4D10效价为1：2．56×105,其亲和力常数可达2×108M-1,该抗体与CAPBSA,CAPOVA有反应,与BSA、OVA无反应。与氯霉素琥珀酸钠、氯霉素反应,而与其它氯霉索结构类似物无交叉反应。结论抗氯霉索单克隆抗体4D10效价高、特异性强、亲和力高,可利用该抗体制备检测氯霉索残留的ELISA试剂盒。     

鳗弧菌油乳化二价口服疫苗免疫养殖大菱鲆的免疫应答及免疫效果的研究

以鳗弧菌M3和SMP1为细菌抗原制备了油乳化二价疫苗,用饵料包埋后连续7天口服免疫大菱鲆鱼,评价鱼的免疫应答和疫苗的保护效果.结果显示,乳化疫苗在鱼后肠刺激产生的溶菌酶和抗蛋白酶活性以及抗体水平均高于未乳化疫苗(P＜0.05);并且在乳化疫苗免疫的鱼血清检测到明显升高的M3抗体效价(P＜0.05).原位杂交结果显示,乳化疫苗免疫鱼的后肠IgM的产生水平高于未乳化疫苗免疫鱼.攻毒实验显示,乳化疫苗免疫的鱼对M3和SMP1的感染分别获得100%和50%的免疫保护率,而未乳化疫苗获得的免疫保护率分别为57.9%和0%.结果表明,鳗弧菌油乳化二价口服疫苗能引起大菱鲆后肠的非特异性和特异性免疫应答并有效地抵抗鳗弧菌的感染,适合用作水产口服鱼用疫苗.     

鳗弧菌金属蛋白酶单克隆抗体的制备及鉴定

鳗弧菌M3胞外产物经纯化得到分子量为36.5kDa的金属蛋白酶，浓缩后作为抗原免疫BALB/C小鼠，经细胞融合及筛选。得到三株稳定分泌抗金属蛋白酶的杂交瘤细胞株。分别命名为P2B1，P4A3和P4B1。亚型鉴定结果表明，三株细胞株产生的单抗均为IgG3型。蛋白质印迹（Wes tern Blot)分析显示，所得到的单抗只与分子量为36.5kDa的金属蛋白酶反应，具有较高特异性。三株单抗均能有效抑制蛋白酶活性。     

鲆鲽类主要病原菌抗血清的制备及应用

用甲醛灭活8种鲆鲽类主要病原菌,鳗弧菌(Vibrio anguillarum)、创伤弧菌(Vibrio vulnificus)、哈维弧菌(Vibrio harveyi)、溶藻弧菌(Vibrio alginolyticus)、嗜水气单胞菌(Aeromonas hydrophila)、豚鼠气单胞菌(Aeromonas caviae)、迟缓爱德华菌(Edwardsiella tarda)、鮰爱德华菌(Edwardsiella ictaluri),制备成灭活疫苗后肌肉注射大菱鲆获得各病原菌抗血清。结果显示,抗鳗弧菌血清、抗哈维弧菌血清、抗豚鼠气单胞菌血清、抗鮰爱德华菌效价为1:6 400,抗创伤弧菌血清、抗溶藻弧菌血清、抗嗜水气单胞菌血清效价为1:12 800,抗迟缓爱德华菌血清效价为1:102 400。应用ELISA分析各抗血清与8种病原菌及迟缓爱德华菌蛋白的免疫交叉反应,结果显示,各病原菌与其本身的抗血清反应最为强烈,而与其他抗血清间有程度不等的交叉反应;菌蛋白与其相应的菌抗血清反应最为强烈,与其他病原菌抗血清反应较弱。本研究所制备的病原菌抗血清效价较高,且具有特异性,可应用于鲆鲽类病原菌的检测。     

拟除虫菊酯类农药单克隆抗体的制备及鉴定

以间苯氧基苯甲酸(PBA)为半抗原,与牛血清白蛋白(BSA)偶联后作为抗原,免疫Balb/c小鼠,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合,使用PBA与鸡卵清白蛋白(OVA)偶联物作为包被物,间接ELISA法筛选到4株特异性针对PBA的杂交瘤。其中两株细胞2F7和7B1制备腹水并用ProteinG柱纯化抗体,抗体效价均达到1:625000。单抗7B1用于间接竞争ELISA法,PBA检测下限为0.025μg·mL-1,IC50为0.57μg·mL-1,对溴氰菊酯、甲体氯氰菊酯、甲氰菊酯、氰戊菊酯均有特异性识别,IC50分别为0.79μg·mL-1、0.74μg·mL-1、0.63μg·mL-1、0.8μg·mL-1,对联苯菊酯识别弱,IC50为99.7μg·mL-1。该单抗可用于多种菊酯类农药的检测。     

HIV-1核心蛋白的表达与纯化

将编码人I型免疫缺陷病毒(HIV1)核心蛋白p24gag的基因序列克隆到原核表达载体pET28(b)中,高效表达了N,C端融合His·Tagp24蛋白,所表达的重组p24蛋白占菌体总蛋白的46%。在变性条件下,使用NiNTA亲和层析法纯化了p24蛋白,纯度为94%。菌体中及纯化、复性后的目的蛋白均能与抗HIV1p24单克隆抗体发生特异性反应。用纯化的p24蛋白免疫小鼠,4周时小鼠血清抗p24抗体效价达1∶400。实验结果表明:大肠杆菌表达的HIV1p24蛋白纯化后可用作HIV1检测试剂的原料。     

口蹄疫病毒二价DNA疫苗的构建及实验免疫研究

通过PCR方法从本室已构建的克隆载体pGEMT-P1-2A获得O型口蹄疫病毒主要保护性抗原VP1基因,以基因突变获得A型VP1基因。将这两种血清型的VP1基因串联后连接到真核表达载体PVAX1 PCMV启动子下游,构建成口蹄疫二价核酸疫苗pVAX1-OA。经Western blot和IFA检测,目的蛋白在HeLa细胞中获得正确表达。动物实验表明,免疫小鼠T淋巴细胞明显增殖,特异性CTL杀伤活性较对照组显著提高;血清抗体能分别与O型和A型抗原反应,抗体效价均高于空白对照组,但较灭活苗低。     

大菱鲆疾病早期快速检测方法——胶体金免疫层析试纸的研制与建立

使用成分单一的牛血清白蛋白(BSA)为模拟病原,以胶体金标记兔抗血清(即大菱鲆免疫球蛋白多抗)作为检测示踪物,并分别将BSA和葡萄球菌A蛋白印记到硝酸纤维素膜上制成检测线和对照线,通过一系列工艺创制与组装配套,首次成功制备了一套完整的大菱鲆抗体快速检测试纸。采用大菱鲆抗BSA血清作为阳性样本,以健康大菱鲆血清作为阴性样本,用以检验试纸的性能,并与酶联免疫吸附实验(ELISA)法检测结果相比较。结果表明:本试纸检测抗体的特异性与敏感性均很高,与ELISA方法相当,而且使用方便,不需专业技能和额外的试剂与辅助仪器设备,5 min内即可用裸眼获得观察结果,很适合于基层生产操作及户外调研使用。以该实验为基础建立起来的抗体检测试纸,亦可推广应用于其他病害抗体的检测,可为鱼类疾病早期发生提供简易、快捷和操作性强的诊断方法。     

分泌抗Bt Cry1Ac蛋白单克隆抗体杂交瘤细胞株的建立

从苏云金芽孢杆菌HD-73中提取Bt Cry1Ac蛋白,电镜观察Bt Cry1Ac蛋白为标准的菱形。用SP2／0-Ag14骨髓瘤细胞与经该Bt Cry1Ac蛋白免疫的BALB／c小鼠的脾细胞融合,经3次克隆化,筛出两株稳定分泌Bt Cry1Ac单克隆的杂交瘤细胞株1C3、2F3。两株细胞均具有抗Bt Cry1Ac特异性,与Bt Cry1Ab和Bt Cry2A无明显的交叉反应,经亚型鉴定均为IgG1。腹水滴度均为1：1024000。     

五香型冷熏大菱鲆工艺研究

大菱鲆是食疗滋补品,也是理想的保健和美容食品。本文研究了大菱鲆的冷熏加工工艺,结果表明:烟熏时间为7 h、盐度为10%、浸渍时间为18 h、五香粉浓度为1.0%时,冷熏大菱鲆色泽好、熏香浓郁、组织紧密、苯并(a)芘和肉毒梭菌的含量符合相关标准。烟熏是一种传统的集加热、熏制和干燥共同进行的复杂加工和贮藏方法,可以提高产品的风味和附加值,是值得推广的熏制技术。     

Monoclonal antibodies: formulations of marketed products and recent advances in novel delivery system

Monoclonal antibodies (mAbs) are extensively employed for disease diagnosis and treatment because of their high homogeneity and antigen specificity. In recent years, important outcomes have been achieved with mAbs due to their admirable therapeutic efficacy and relatively rare side effects. In clinical practice, several mAb products have been approved by regulatory entities, but their formulations have been highly specific given the complex structure and proteinaceous nature of mAbs. Thus, more attention has been given on formulations. An increasing number of novel delivery systems have been exploited to optimize the application of mAbs. In this article, the formulations, dosages, origins and administration routes of available mAbs approved by the Food and Drug Administration (FDA) are summarized and categorized. Key issues involved in formulation, processing and storage are addressed as well as other challenges in achieving effective mAb delivery. Finally, recent advances in delivering mAbs in their most bioavailable forms are also briefly reviewed.     

Preparation of a single-chain variable fragment and a recombinant antigen-binding fragment against the anti-malarial drugs, artemisinin and artesunate, and their application in an ELISA

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.     

General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies

Ligand binding assays (LBAs) are widely used for therapeutic monoclonal antibody (mAb) quantification in biological samples. Major limitations are long method development times, reagent procurement, and matrix effects. LC-MS/MS methods using signature peptides are emerging as an alternative approach, which typically use a stable isotope labeled signature peptide as the internal standard (IS). However, a new IS has to be generated for every candidate, and the IS may not correct for variations at all processing steps. We have developed a general LC-MS/MS method approach employing a uniformly heavy-isotope labeled common whole mAb IS and a common immunocapture for sample processing. The method was streamlined with automation for consistency and throughput. Method qualification of four IgG(2) and four IgG(1) mAbs showed sensitivity of 0.1 μg/mL and linearity of 0.1-15 μg/mL. Quality control (QC) data of these eight mAbs were accurate and precise. The QC performance of the whole molecule labeled IS was better than those of synthetic labeled IS peptides tested. The pharmacokinetic results of two mAbs (an IgG(2) and IgG(1) candidate) dosed in rats were comparable to those of LBA. The general LC-MS/MS method approach overcomes the limitations of current methods to reduce time and resources required for preclinical studies.     

牙鲆与大菱鲆病原迟钝爱德华氏菌生物学特性及系统发育学分析

对从7起牙鲆（Bastard halibut,Paralichthys olivaeeus L．）、3起大菱鲆（Turbot,Scophdudmus maximus L．）病害的病（死）鱼中分离到的相应病原菌较系统地进行了形态特征、理化特性等表观分类学指征的鉴定及代表菌株DNA中G＋Cmol％的测定。同时,择代表菌株进行了16SrRNA基因的分子鉴定,测定了16SrRNA基因序列,分析了相关细菌相应序列的同源性,构建了系统发生树。结果表明,分离鉴定的148株菌均为爱德华氏菌属（Edwardsiella Ewing and McWhorter 1965）的迟钝爱德华氏菌（E．tarda）,其中128株为迟钝爱德华氏菌野生型（E．tarda wild type）菌株,20株暂定为迟钝爱德华氏菌吲哚阴性变异株。代表菌株（HC010907—1及HC010830-1）的16SrRNA基因序列,与GenBank数据库中的迟钝爱德华氏菌的同源性均在99％。     

大菱鲆引种工程的综合效应及其发展前景

回顾了大菱鲆（Scophthalmus maximus L）引种、研究与开发的历程，探讨了引种工程的社会背景、学术意义、经济与社会效益及其所产生的综合效应，同时展望了这一引种工程潜在的影响力和发展前景。     

Immunopurification and mass spectrometric quantification of the active form of a chimeric therapeutic antibody in human serum

In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Following digestion with trypsin, specific peptides from light and heavy chains were monitored in the selected reaction monitoring (SRM) mode. Assay variability below 20% was provided through optimization of the digestion step and rigorous monitoring of the whole analytical process using an appropriate internal standard. The 20 ng/mL lower limit of quantification was similar to that of ELISA methods. These results show that this mass spectrometric approach is a potential alternative for pharmacokinetic evaluation of mAbs during clinical development.     

牙鲆发育过程中血清免疫球蛋白(IgM)含量的变化

从正常牙鲆(Paralichthys olivaceus)的血清中提取纯化牙鲆免疫球蛋白IgM,将提取的IgM免疫Wistar大鼠获得鼠多抗。用ELISA(酶联免疫吸附)法测定在牙鲆生长过程中,血清中IgM的含量与体重、全长的关系。实验结果表明,随牙鲆的体重、全长的变化,牙鲆IgM在血清中的含量增加。     

Protein arrays based on biotin-streptavidin system for the simultaneous detection of TORCH infections

In this paper, we developed a protein array based on biotin-streptavidin system (PABS) used in the identification of IgM antibodies against TORCH antigens, including toxoplasma gondii (TOX), rubella virus (RuV), cytomegalovirus (CMV) and herpes simplex virus types II (HSV-2) antigens. The detection signal intensities and sensitivities between the PABS and the direct labeling array system (DLAS) were compared. The linear ranges of detectable IgM antibodies in PABS were 0.485-1000 microg/mL, which was more sensitive than DLAS. Quantitatively, the lowest detectable amount for IgM antibodies on each spot of the PABS was 0.25 pg. Furthermore, sixty serum samples from patients were tested with the PABS in TORCH detection. All the results were correspondingly confirmed with ELISA assay. No significant differences in identifying TORCH specific IgM antibodies were found between the PABS and ELISA assay. There was a good concordance between PABS and ELISA in the classification of sera. The results suggested that the PABS was more sensitive, sample-saving and suitable for multi-pathogens parallel clinical detection.