用同系抗独特型单克隆抗体亲和层析法提纯小鼠单克隆抗体

1C_6McAb_2是抗7A_4McAb_1独特型的同系小鼠单克隆抗体。应用硫酸铵盐析,1C_6McAb_2—Sepharose 4B柱亲和层析法提纯了小鼠腹水7A_4McAb_1。获得的样品经二巯基乙醇还原,在SDS—PAGE上为两条带,分别是IgG的重链和轻链,免疫双扩散检查只与兔抗鼠IgG_1血清有沉淀反应,免疫电泳检查与兔抗鼠全血清反应只形成一条沉淀弧,纯化的McAb的PHA效价达125000/mg。本法提纯的McAb具有产量高,纯度好,特异性强等优点。     

应用鸡卵细胞大量制备多克隆抗体

用三种不同来源的抗原(基因工程HBVe、HCV核心区抗原和GM—CSF)免疫鸡,观察血清及卵黄中特异性抗体的应答,结果经免疫的鸡对这3种抗原均有免疫应答,所产卵的卵黄中可测出大量的有活性的抗体。提示鸡源性抗体在生物制品开发应用方面有一定的前景。     

Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA

The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.     

抗白念珠菌人源性单链抗体库的构建及筛选

目的构建多样性良好的抗白念珠菌人源性单链抗体库,筛选抗白念珠菌特异性的噬菌体抗体。方法从20份人外周血淋巴细胞(包括正常成年人5份、新生儿5份和白念珠菌感染恢复期患者10份)中提取总RNA,反转录为cDNA,PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,以重叠延伸PCR法将VH和VL拼接成scFv基因,克隆入噬菌粒载体pCANTAB-5E中,电转化大肠杆菌XL1-Blue,构建人源抗白念珠菌天然噬菌体抗体库,并从中筛选阳性克隆抗体。结果构建的人源性抗白念珠菌天然噬菌体抗体库库容为2.8×109,多样性良好。共筛选出18个阳性克隆。结论已成功构建了1个多样性良好的抗白念珠菌人源性噬菌体抗体库,为筛选有效治疗白念珠菌和耐药性白念珠菌感染的药物提供了条件。     

麻疹活疫苗免疫后血凝抑制抗体与中和抗体的比较

本文对无麻疹干扰的人工封闭地区麻疹活疫苗免疫后的 HI 及 Nt 抗体进行了比较。结果显示,免疫成功后,HI 抗体阴转持续≤5年和6～10年者,分别有82%和43.8%的人均可测出不同水平的 Nt 抗体,证明 HI 抗体降至<1∶2仍具有免疫力。初免与再免者,在 HI 抗体阴转持续时间与 Nt 抗体之间关系没有差别,说明只要初免成功,其 Nt 抗体比较牢固。免疫成功者再显性或隐性感染后3年,仍有较高的 HI 和 Nt 抗体,这与人工再免后1～2年内,大部分人 HI 抗体还原到再免前水平有明显的不同。     

肿瘤坏死因子α单克隆抗体和多克隆抗体的研制和鉴定

以rhTNF－α作为免疫原，通过杂交瘤技术建立了15株稳定分泌rhTNF－α单克隆抗体的细胞株，对其所分泌单抗的生物学特性进行了鉴定，并探索了其纯化工艺。制备了高纯度兔抗rbTNF－α多克隆抗体，其中和细胞毒能力达到或超过SIGMA公司同类产品水平。该抗体对LPS所致的免发热有明显的抑制作用。利用单抗和多抗建立了ELISA双抗体夹心法，敏感性高，稳定性好，特异性强，适宜于临床检测大量样品。     

抗狂犬病病毒抗体的制备及酶联免疫法的建立

目的制备具有中和活性的抗狂犬病病毒(Rabiesvirus,RV)抗体,并建立检测RV抗原的ELISA法。方法以RV全病毒免疫2只新西兰家兔,制备抗RV多克隆抗体。以RV全病毒免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞SP2/0融合,建立稳定分泌抗RV单克隆抗体的杂交瘤细胞株。以小鼠中和试验(MNT)检测抗体的中和活性。以ELISA双抗体夹心法和ELISA竞争法检测RV抗原。结果2只家兔的多克隆抗体ELISA效价分别为1∶6.0×103和1∶1.2×104,纯化的兔抗RVIgG中和活性分别为46.3和29.2IU/ml。获得了9株稳定分泌抗RV的单克隆抗体杂交瘤细胞株,属于IgG1或IgG2b亚型,腹水的抗体ELISA效价为1∶1.0×105~1∶1.0×107。单克隆抗体3E5、4C2和4F8具有中和活性,Westernblot分析提示,单克隆抗体4C2是针对RV糖蛋白线性表位的抗体。以单克隆抗体4C2作为捕捉抗体,兔多克隆抗体作为检测抗体,建立了ELISA双抗体夹心法,检测RV抗原。同时建立了另一种ELISA竞争法,加入固定工作浓度的单克隆抗体4C2,与RV病毒液孵育,以ELISA间接法检测RV病毒抗原。结论所获得的狂犬病毒多克隆和单克隆抗体具有中和活性,可在ELISA中用于检测RV抗原。     

三株流行性出血热病毒免疫原性的比较研究

三株病毒用不同方法制备的疫苗，免疫家兔后都能诱导产生免疫荧光抗体（IF）。其中Z10和L99株用细胞冻融以β-丙内酯灭活制备的疫苗还可以测到HI抗体。以β-丙内酯灭活的疫苗所产生的中和抗体，以Z10株最高（1：80～1：160），L99株较低（1：10），而LR1株未测出。但三株病毒感染的细胞培养上清液，用两种灭活剂均未产生HI或NT抗体。     

流行性出血热病毒不同结构蛋白单克隆抗体的免疫学特性研究

本文比较了12株出血热病毒不同结构蛋白的单克隆抗体的免疫学特性,结果大多数抗外膜糖蛋白G_1和G_2单抗具有较高的中和及血凝抑制(HI)活性,一株有低HI滴度而无中和滴度,表明与HA有关的位点不一定有中和活性。抗核蛋白(NP)单抗,除一株(3G_1)外均无中和活性,但具有低的HI滴度,表明核蛋白上亦存在HA位点。所有NP单抗均显示对A_(35)(抗NP)致敏血球的凝集抑制作用(RPHI),而G_1和G_2单抗则均无抑制作用。 3G_1株显示较高的中和及HI活性,但又与A_(35)(抗NP)单抗有明显的交叉RPHI反应,因此可能是针对二种结构蛋白的特殊单抗。     

Improvement of Biophysical Properties and Affinity of a Human Anti-L1CAM Therapeutic Antibody through Antibody Engineering Based on Computational Methods

The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)—which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue—exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.     

纳米抗体用于肿瘤诊断与治疗的研究进展

骆驼体内天然存在缺失轻链的重链抗体,其可变区是最小的功能性抗原结合片段,因其分子大小在纳米级而被称为纳米抗体,具有稳定性强、可溶性好、免疫原性低、容易表达等特点。在介绍这种新型纳米抗体的生物学特性及获得途径的基础上,着重综述了在近几年来纳米抗体在肿瘤诊断与治疗的研究方面取得的重大进展及存在的问题,并对其未来的应用提出了展望。     

H5N1人用禽流感疫苗免疫原性检测方法的建立

目的确定人用禽流感疫苗免疫原性检测方法。方法用禽流感病毒R1203株制备疫苗,以不同剂量免疫家兔,检测血清中的血凝抑制抗体和中和抗体,并进行交叉血凝抑制试验、交叉单向免疫扩散试验和交叉中和试验。结果R1203株疫苗接种家兔1针后14 d,中和抗体和血抑抗体滴度均较低;第2针后14 d均明显升高。血抑抗体量-效反应不明显,而中和抗体量-效反应明显。交叉血凝抑制试验显示,异型之间普遍有交叉反应,滴度大部分不低于1∶40。单向免疫扩散试验显示异型之间无交叉反应。禽流感疫苗抗血清不能中和人流感病毒,但能中和同型病毒(R1194),滴度可达1∶240。结论中和抗体能正确反映疫苗的免疫原性;检测中和抗体的方法可用于禽流感疫苗的效力试验。     

牛血清白蛋白单克隆抗体的筛选与双抗体夹心ELISA检测方法的建立

目的筛选高效抗牛血清白蛋白(BSA)单克隆抗体细胞株,并建立BSA抗原检测的双抗体夹心ELISA法。方法分别采用辛酸-硫酸铵法和葡萄球菌A蛋白亲和层析法纯化单抗,并进行抗体类型、腹水效价、特异性和相对亲和力测定;应用纯化的单抗建立BSA双抗体夹心ELISA检测法,并进行初步应用。结果筛选出4株可稳定分泌抗BSA单抗的杂交瘤细胞株,抗体类型为IgG,抗体滴度均可达10-6,特异性良好,相对亲和力较高。建立的双抗体夹心ELISA法线性范围为1.25～20ng/ml,R2>0.98,灵敏度为1.25ng/ml。结论已筛选出高效抗BSA的单抗,并建立了BSA双抗体夹心ELISA检测方法。     

Epigenetic Modification of PD-1/PD-L1-Mediated Cancer Immunotherapy against Melanoma

Malignant melanoma is one of the representative skin cancers with unfavorable clinical behavior. Immunotherapy is currently used for the treatment, and it dramatically improves clinical outcomes in patients with advanced malignant melanoma. On the other hand, not all these patients can obtain therapeutic efficacy. To overcome this limitation of current immunotherapy, epigenetic modification is a highlighted issue for clinicians. Epigenetic modification is involved in various physiological and pathological conditions in the skin. Recent studies identified that skin cancer, especially malignant melanoma, has advantages in tumor development, indicating that epigenetic manipulation for regulation of gene expression in the tumor can be expected to result in additional therapeutic efficacy during immunotherapy. In this review, we focus on the detailed molecular mechanism of epigenetic modification in immunotherapy, especially anti-PD-1/PD-L1 antibody treatment for malignant melanoma.     

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.     

玉米赤霉烯酮抗独特型单抗的制备及特异性分析

目的制备并鉴定玉米赤霉烯酮(Zearalenone,ZEN)抗独特型单抗Ab2β-1D5。方法将ZEN单抗1G4与载体蛋白KLH偶联作为免疫原,免疫BALB/c小鼠,取免疫小鼠脾细胞,与小鼠骨髓瘤细胞SP2/0融合后,以ZEN单抗Fab片段作为包被抗原,间接ELISA法筛选阳性杂交瘤细胞。经小鼠腹腔注射杂交瘤细胞,制备腹水型单抗,并经Protein G亲和层析柱进行纯化。间接ELISA法检测ZEN抗独特型单抗的抗体效价及特异性;间接竞争ELISA法检测ZEN抗独特型单抗类型、灵敏度及其与ZEN毒素间的相关性。结果共获得6株稳定分泌ZEN抗独特型单抗的杂交瘤细胞株,腹水型抗体效价为1∶1.2×105～1∶2.0×105;6株单抗均为β型抗独特型抗体(Ab2β),其中Ab2β-1D5抗体灵敏度最高,对ZEN的IC50值达10.09 ng/ml,其与ZEN毒素呈线性相关(r=0.990);ZEN抗独特型单抗与莱克多巴胺、重金属铅、铬及虾过敏原单抗均无交叉反应,特异性良好。结论已成功制备玉米赤霉烯酮抗独特型单抗,该抗体与ZEN间存在"内影像"关系,可以替代ZEN毒素标准品,用于建立ZEN的无毒免疫学检测技术。     

A functional antibody mutant with an insertion in the framework region 3 loop of the VH domain: implications for antibody engineering

We have studied the effects of a four residue insertion intothe FR3 loop of the heavy chain variable region from the anti-NPantibody Bl-8. The insertion mutant is obtained as secretedantibody without major defects in biosynthesis, indicating thatantibody variable domains can accommodate length variation notonly in complementarity determining regions (CDRs), but alsoin framework region (FR) loops. The Bl-8 antigen binding siteis not affected by the change in a neighbouring loop. FR3 insertionsrepresent a new method of antibody engineering with a potentialto obtain strong antigen binding by designing additional antigencontacting residues.     

从半合成噬菌体抗体库筛选抗狂犬病毒人单链抗体

目的　应用纯化的狂犬病毒抗原从半合成噬菌体抗体库中筛选针对狂犬病毒的人单链抗体(ScFv)。方法　用固相化的狂犬病毒抗原对半合成抗体库进行 3轮“吸附 洗脱 扩增”的筛选 ,从第 3轮洗脱下来的克隆中获得一株有可溶性表达且特异性结合狂犬病毒抗原的ScFv ,并进行基因序列测定。结果　所获氨基酸序列经blast数据库搜索 ,与一种抗狂犬病毒免疫球蛋白的氨基酸序列同源性最高 ( 82 % )。经检索kabat数据库 ,发现其轻、重链可变区分别属于VkⅠ型、VHⅢ型。结论　从噬菌体抗体库可以方便快捷地分离到针对狂犬病毒的单链抗体 ,对狂犬病毒的预防具有重要意义     

重组抗体高效表达的研究进展(Ⅰ)

重组抗体在哺乳动物细胞中的高效表达有赖于高效表达载体的构建、抗体基因序列的优化、抗体稳定细胞株的筛选、表达宿主细胞的改造及细胞培养工艺的优化等。本文对上述问题的研究进展进行综述。     

三价脊髓灰质炎活疫苗在不同地区人群的基础免疫应答

应用同一批三价脊髓灰质炎活疫苗(TOPV),于夏季分别在我国吉林、湖北和广西壮族自治区观察人群的基础免疫应答.3次免疫后发现,Ⅰ、Ⅲ型,吉林儿童的血清中和抗体阳性率高于广西儿童,且有显著性差异,抗体几何平均滴度(GMT)北方高于南方,吉林与湖北、广西之比,除了吉林与广西Ⅲ型之比外,其他皆有显著性差异。