盐析法与低温乙醇法制备抗人T细胞猪免疫球蛋白工艺的对比

目的两种常用抗人T细胞猪免疫球蛋白(anti-human T lymphocyte porcine immunoglobulin)制备工艺的对比。方法以人T淋巴细胞免疫的健康猪血浆为原料,采用硫酸铵-Sephadex A-50和Cohn低温乙醇法,经醛化人健康红细胞、醛化人胎盘渣及混合人血浆吸收后,制备抗人T细胞猪免疫球蛋白,参考《中国药典》三部(2010版)相关附录,进行中间品及成品检定。结果用硫酸铵盐析法与低温乙醇法连续分别制备了3批产品,每批投浆量约5 000 ml;3批中间品鉴别试验及3批成品检定结果均符合《中国药典》三部(2010版)相关要求,两种工艺方法均适用于制备抗人T细胞猪免疫球蛋白,但在质量和收获率上,低温乙醇法更具优势。结论低温乙醇法制备抗人T细胞猪免疫球蛋白更适用于生产。     

一种抗人T淋巴细胞免疫球蛋白淋巴细胞毒效价检测方法的建立

目的 建立基于人白血病T细胞株Jurkat的抗人T淋巴细胞免疫球蛋白(anti-human T lymphocyte immunoglobulin,ALG)淋巴细胞毒效价定量检测方法,并进行验证。方法 采用人Jurkat细胞作为靶细胞,抗人T细胞兔免疫球蛋白(商品名：Grafalon)作为待检样品,建立其淋巴细胞毒效价定量检测方法。优化反应体系,考察细胞培养密度、细胞代次对待检样品效价的影响,并对方法的精密度进行验证。结果 优化的反应体系组成为50μL待检样品+50μL补体(1∶5稀释)+50μL细胞(5×10⁶个/mL);细胞传代密度为2×10⁵个/mL传代培养72 h或3×10⁵个/mL传代培养48 h用于效价测定最佳;当细胞生长状态较好且一致时,从P10到P25代次的细胞效价测定结果较一致。采用该方法板内重复测定待检样品效价,变异系数(CV)为6.32%,重复性良好;不同代次Jurkat细胞测定待检样品效价6次,CV为4.15%,中间精密度良好。结论 建立了一种便捷、精确的测定ALG淋巴细胞毒效价的定量检测方法,为...     

抗人淋巴细胞免疫球蛋白制品中的热原处理工艺

目的 研究热原不合格的抗人淋巴细胞免疫球蛋白（ALG）半成品再制的条件。方法 将热原不合格的ALG半成品通过碱、酸以及DEAE　Sephadex　A-50处理,并检测其热原和质量的变化。结果 经处理后,细菌内毒素的含量明显降低,用家兔法和鲎试剂法检测热原均合格,制品的免疫效价仍不低于1:4000,并且其他各项主要指标也均达到2000年版《中国生物制品规程》的要求。结论 这种处理方法能有效地除去ALG制品中的热原。     

人破伤风免疫球蛋白国家标准品的研制

目的　研制人破伤风免疫球蛋白国家标准品。方法　按WHO有关要求制备并检测标准品 ,以人破伤风免疫球蛋白国际标准品为标准进行协作标定。结果　冻干人破伤风免疫球蛋白国家标准品经外观、水分、无菌、分装误差、效力检测 ,均符合要求 ,在 4℃保持 6 0个月效力稳定。协作标定平均值为 10 5 5IU 支 ,CV值为4 7%。结论　该批人破伤风免疫球蛋白标准品可以作为国家标准品 ,效价定为 10IU 支 ,批号为 0 0 1     

病毒灭活后抗淋巴细胞免疫球蛋白的稳定性

目的 观察经过病毒灭活后抗淋巴细胞免疫球蛋白(ALG)的稳定性。方法 取各项指标均符合国家暂行规程要求的ALGc放2-8℃保存,在不同时间进行质量测试。结果 24个月时,制品的花环抑制效价仍不低于1:4000,淋巴细胞毒效价不低于1:1 000。其纯度和IgG各组分含量与试验之前相比,差异无显著意义,且其他各项主要指标也均达到国家制检规程的标准。结论 经过病毒灭活后制品的稳定性良好。     

人血清中猪抗人淋巴免疫球蛋白双抗体夹心ELISA检测法的建立及验证

目的建立人血清中猪抗人淋巴免疫球蛋白(anti-human T lymphocyte porcine immunoglobulin,ALG)双抗体夹心ELISA检测法,并进行验证。方法以兔抗猪Ig G包被酶标板,HRP酶标记的鼠抗猪Ig G为二抗,建立人血清中ALG含量的双抗体夹心ELISA检测法,并对方法中包被浓度及二抗浓度进行优化,同时验证该方法的重复性、敏感性、准确性、特异性及稳定性。采用优化后的方法对14位严重型再生障碍性贫血(severe aplastic anemia,SAA)患者进行ALG的血药浓度监测(therapeutic drug monitoring,TDM)。结果最佳包被浓度为1∶2 000,最佳二抗稀释度为1∶8 000。参考品浓度在4.9~5 000μg/ml范围内标准曲线的四参数Logistic曲线拟合较好,R2值为0.991 1,在4.9~156μg/ml范围内的线性拟合也较好,R2值为0.991 5,批内重复性CV值均10%;其检测底限为0.39μg/ml;浓度为500、50及5μg/ml参考品的回收率为91.2%~107.0%;该方法可特异性检测出注射ALG患者血清中ALG含量;包被好的ELISA板及二抗、底物于4及37℃条件下分别保存1~3周及1~3 d后检测结果均较稳定。经DAS 3.1.4软件分析,14名经ALG治疗的SAA患者血清中ALG的曲线下面积(AOC)为(7.3±5.1)mg/(L·d)(95%CI),半衰期(t1/2)为(20.65±38.67)d(95%CI)。结论该方法具有良好的重复性、准确度及稳定性,可用于ALG的TDM。     

静注巨细胞病毒人免疫球蛋白原料血浆的筛选及制备

目的筛选获得高效价血浆并制备静注巨细胞病毒人免疫球蛋白(intravenous immunogloblin against cytomegalovirus,CMV-IVIG)。方法分别使用酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)法及微量细胞中和法对113份人血浆巨细胞病毒(cytomegalovirus,CMV)IgG抗体进行结合效价及中和效价的检测,将两种方法获得的高效价血浆分别制备CMV-IVIG,采用常规微量细胞中和法检测抗CMV中和效价,并与IVIG中和效价进行比较,确定符合要求的免疫球蛋白成品,确立血浆筛选方法。结果对113份血浆进行效价检测,将结合效价≥20 IU/mL以及中和效价≥1∶32的血浆分别收集并制备5%CMV-IVIG,成品批号分别20190401和20190402;20190401批CMV-IVIG成品中和效价为498.28 IU/mL,20190402批CMV-IVIG成品中和效价为2 357.56 IU/mL,而5%IVIG中和效价为259.53 IU/mL。结论 ELISA法筛选获得的血浆制备的CMV...     

高效价巨细胞病毒人免疫球蛋白原料血浆的筛查

目的分析普通健康供浆员中人巨细胞病毒(human cytomegalovirus,HCMV)免疫球蛋白(IgG)抗体效价分布情况,筛查富含高效价HCMV IgG抗体的原料血浆。方法采用HCMV IgG定量检测试剂,检测本公司下属广西地区五个单采血浆站采集的血浆样本的HCMV IgG抗体效价,检测抗体效价15 PEI-U/ml的血浆样本混合后的HCMV IgG抗体效价,并对血浆中富含HCMV抗体的供浆员血浆进行采集,分析效价分布变化情况。结果健康供浆员28 607份血浆样本的HCMV IgG抗体阳性率为85.2%,其中效价分布在1~10、10~15、15 PEI-U/ml的各占64.5%、8.7%和11.9%;1 318名血浆中富含HCMV抗体的供浆员8 426份血浆样本HCMV IgG抗体阳性率为98.1%,抗体效价15 PEI-U/ml占43.8%,约为普通健康人血浆样本的3.7倍,3个月内平均抗体效价可维持在15 PEI-U/ml以上的有586人,占44.5%;不同浆站抗体效价15 PEI-U/ml的血浆样本混和后的平均效价为33.72 PEI-U/ml。结论健康供浆员中高效价HCMV IgG抗体的比例较高,部分供浆员HCMV IgG抗体高效价水平可稳定维持3个月,从健康供浆员中采集含高效价HCMV IgG抗体的原料血浆,用于制备巨细胞病毒人免疫球蛋白具有可行性。     

嵌合抗原受体T细胞及T细胞抗原耦合器修饰T细胞技术的研究进展

目前,恶性肿瘤已成为严重威胁人类健康的公共问题之一。除手术、放疗、化疗、靶向疗法等方法外,随着分子生物学的发展,免疫疗法得到迅速发展,成为肿瘤治疗的一种新兴方法。临床免疫疗法采用最多的免疫细胞分别为DC、NK、CIK、CTL及嵌合抗原受体T细胞(chimeric antigen receptor T cell,CAR-T)等。其中CAR-T技术全球研究最早,但由于其存在脱靶、神经毒性、转染载体缺陷等问题,在临床应用方面具有一定局限性。T细胞抗原耦合器修饰T细胞(T cell antigen coupler modified T cell,TAC-T)技术是在CAR-T技术基础上发展的一种利用天然T细胞受体(T cell receptor,TCR)修饰T细胞并重新靶向肿瘤细胞抗原的新技术。本文就CAR-T技术的研究现状及TAC-T技术的研究进展作一综述,以期为TAC-T技术作用机制及其临床应用安全性的深入研究提供参考。     

记忆性T细胞亚类的研究进展

T细胞免疫对于调节抗体的生成及清除受感染的细胞均发挥相当重要的作用。记忆性T细胞的特点是在初次免疫反应完成后仍能长期存活,并形成不同的亚类,每一亚类细胞又分为中枢记忆性T细胞和效应记忆性T细胞。这些记忆性T细胞在宿主体内长期存在,当宿主受到再次感染时可提供有效的保护作用。本文结合最新研究进展,对主要的记忆性T细胞(如CD8+和CD4+记忆性细胞)类型及其功能作一综述。     

Inhibition of cytolytic T lymphocyte activity by oxysterols

The objective of this study was to investigate the effects of oxysterols (OS), namely 5α-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10⁻⁵ M 5α-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h⁵¹Cr release. At a concentration of 5×10⁻⁶ M, 5α-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.     

Humanization of mouse anti-human IL-2 receptor antibody B-B10

Mouse monoclonal anti–human IL–2 receptor antibody(BB10) inhibits EL–2–dependent human T–cellproliferation. It has been used in clinical trials in the transplantationfield and promising results are being accumulated. Mouse B–B10antibody was humanized by grafting all CDRs and some frameworkamino add residues onto human antibodies, KAS for V_H and PAYfor V_x. Nine humanized B–BlOs with differently graftedframework residues were constructed and assessed for their biologicalactivities. One of these humanized B–B10, M5, showed nearlythe same activity as the mouse B–B10. The 49th residueof V_x was demonstrated to play a crucial role in the antigen–antibodyinteraction by 3–D structure analysis with a computermodeling system.     

抗人细胞间粘附分子-1嵌合抗体的制备及其生物学活性

目的构建抗人细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)嵌合抗体的真核表达质粒,在CHO-dhfr-细胞中进行表达,并检测其生物学活性。方法采用RT-PCR法从特异性分泌抗人ICAM-1单克隆抗体的杂交瘤细胞株3F2中扩增抗体VH和VL基因,从人淋巴细胞RNA中扩增人κ、IgG1的轻、重链恒定区序列,再通过重叠延伸PCR连接鼠源性可变区基因片段与人源性恒定区基因片段,获得人-鼠嵌合抗体基因;将轻、重链基因连接至pIRES双表达载体,构建嵌合抗体真核表达质粒;将质粒在脂质体LipofectAMINE介导下转染CHO-dhfr-细胞进行表达,表达的嵌合抗体经Protein A-Sepharose 4B亲和层析柱纯化后,紫外分光光度法测定抗体浓度,ELISA法检测嵌合抗体的特异性抗原结合活性及人源性,并进行还原性SDS-PAGE分析、Western blot分析及抑制细胞间黏附活性分析。结果嵌合抗体真核表达质粒pIRES-anti-ICAM-1经双酶切鉴定构建正确;嵌合抗体在真核细胞CHO-dhfr-中高效表达,培养上清中表达量可达0.5 mg/L;纯化的嵌合抗体经10%还原性SDS-PAGE分析,可见相对分子质量分别约为25 000的IgG轻链和50 000的IgG重链,相对分子质量约25 000的蛋白可被羊抗人IgGκ链所识别,而相对分子质量约50 000的蛋白可被羊抗人IgGγ链所识别;纯化的嵌合抗体可与羊抗人κ链或羊抗人IgG多抗呈强阳性反应,可与人ICAM-1抗原特异结合;该嵌和抗体具有良好的抑制内皮细胞与单核细胞黏附的活性。结论成功制备了抗人ICAM-1嵌合抗体,并在真核细胞中实现高表达,该抗体减少了鼠源性成分,降低了其免疫原性,为ICAM-1相关的炎性疾病的抗体治疗奠定了基础。     

Validity of the first-order fluid model

The general validity of the first-order fluid model is considered. Conditions are established for which a first-order fluid represents an acceptable approximation to the integral viscoelastic fluid from which it was derived as a Taylor series approximation. The results are applied to two flow problems: generation of flow by the application of a pressure gradient and the growth or dissolution of bubbles in viscoelastic liquids. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 547–552, 1999     

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.     

Effect of conjugated linoleic acid type, treatment period, and dosage on differentiation of 3T3 cells

This study was conducted to determine effect of CLA and linoleic acid (LA) on cell differentiation, cellular glycerol-3-phosphate dehydrogenase (GPDH) activity, and FA accumulation in differentiating 3T3-L1 cells (3 isomers×3 treatment periods×4 doses). The cells were cultured in 24-well plates for proliferation until confluence. Then they were treated with media containing 0, 10, 35, or 70 mg/L (0, 35, 125, or 250 mmol/L, respectively) of LA,cis9,trans11-ortrans10,cis12-CLA during early (day 0–2), intermediate and late (day 3–8), or overall (day 0–8) differentiation periods. Dexamethasone, methyl-isobutylxanthine, and insulin were supplemented to the media only for the early period to induce the differentiation. On day 8 of postconfluence the cells were harvested for Oil Red O staining, analysis of GPDH activity, and determination of the FA concentration. Cellular LA or CLA was found to accumulate in a dose-response manner, mainly during the intermediate/late period. Treatment withtrans10,cis12-CLA lowered (P<0.05) GPDH activity and the concentration of FA including palmitic acid (16∶0) and palmitoleic acid (16∶1), especially during the intermediate/late and overall periods, or whenever a high dose of 70 mg/L was applied. This also resulted in a higher (P<0.05) ratio of saturated FA to monounsaturated FA. Treatment with LA orcis9,trans11-CLA lowered cellular FA only when they applied during the early period at a dose of 70 mg/L. The results demonstrated that the inhibitory effects of CLA on differentiation, GPDH activity, and FA accumulation of 3T3-L1 cells are dependent on the isomer type, treatment period, and dose.     

Humanization of a mouse anti-human IgE antibody: a potential therapeutic for IgE-mediated allergies

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and,therefore, has potential as a therapeutic agent in patientswith IgE-mediated allergies such as hay fever, food and drugallergies and extrinsic asthma. The clinical usefulness of mouseantibodies is limited, however, due to their immunogenidty inhumans. Mouse C21 antibody was humanized by complementaritydetermining region (CDR) grafting with the aim of developingan effective and safe therapeutic for the treatment of IgE-mediatedallergies. The CDR-grafted, or reshaped human, C21 variableregions were carefully designed using a specially constructedmolecular model of the mouse C21 variable regions. A key stepin the design of reshaped human variable regions is the selectionof the human framework regions (FRs) to serve as the backbonesof the reshaped human variable regions. Two approaches to theselection of human FRs were tested: (i) selection from humanconsensus sequences and (ii) selection from individual humanantibodies. The reshaped human and mouse C21 antibodies weretested and compared using a biosensor to measure the kineticsof binding to human IgE. Surprisingly, a few of the reshapedhuman C21 antibodies exhibited patterns of binding and affinitiesthat were essentially identical to those of mouse C21 antibody.     

Characterization of porcine omental lipids

Porcine omental lipid extracts were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1, and 18∶2 fatty acids. Small quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry, was found to consist primarily of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by HPTLC and HPLC, were found to consist primarily of di-, tri-and tetraosylceramides. The complex glycolipid fraction, obtained from Folch upper phase solvent partition and characterized by HPTLC and immunoblotting, was found to consist primarily of ganglio-, globo-, and neolacto- neutral glycolipids and ganglio-, globo-, neolacto- and fucosylated gangliosides.