Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备

[目的]小麦赤霉病菌产生的毒素不仅在病害发展过程中具有加重赤霉病的作用,而且污染谷物导致严重的食用安全性问题.由于赤霉病的普遍发生,有必要建立快速、灵敏、有效的毒素检测方法,本试验旨在制备可用于检测被脱氧雪腐镰刀菌烯醇污染的粮谷类特异性单克隆抗体.[方法]本实验首先将脱氧雪腐镰刀菌烯醇(DON)的衍生物3-半琥珀酰-脱氧雪腐镰刀菌烯醇(3-HS-DON-OVA)与卵清蛋白(OVA)采用碳化二亚胺法进行偶联得到人工抗原,以此人工抗原免疫BALB/C小鼠,取该鼠脾细胞与SP2/O鼠骨髓瘤细胞融合,经筛选和克隆,得到了1株能稳定分泌DON抗体的单克隆细胞株(382),并制备单克隆抗体腹水.[结果]经检测382的抗体类型及亚类均为IgG1,其轻链为κ链.腹水通过间接酶联免疫吸附测定效价在1×10-7以上.该单克隆抗体与脱氧雪腐镰刀菌烯醇特异性结合反应的50%抑制质量浓度为29 μg/L,除与3-acetyldeoxynivalenol(3-Ac-DON)的交叉反应率为78.38%,与其他脱氧雪腐镰刀菌烯醇结构类似物无交叉反应.[结论]本实验所制备的单克隆抗体有较高的灵敏度和特异性,具有较好的应用价值.     

Characterization of an Anti-Thioredoxin Monoclonal Antibody

An anti-E. coli thioredoxin monoclonal antibody, IMM-3C6, which showed high specificity to thioredoxin as assessed by indirect ELISA, was generated using hybridoma technology. The affinity constant of IMM-3C6 to thioredoxin was 0.40×10⁹ m⁻¹ and its sensitivity to thioredoxin fusion protein in dot blotting was 50 ng. In sandwich ELISA, it detected thioredoxin fusion protein between 16 and 150 ng/ml. By using IMM-3C6 as the ligand, thioredoxin fusion protein was successfully purified by affinity chromatography. IMM-3C6 was confirmed to be a useful tool for immunoassay and purification of thioredoxin fusion proteins. These authors contributed equally to the work. Received 21 September 2005; Revisions requested 7 October 2005; Revisions received 10 November 2005; Accepted 11 November 2005     

OAS1蛋白单克隆抗体的制备及其表征

目的:用OAS1蛋白免疫小鼠,获得OAS1特异性单克隆抗体,为OAS1的含量测定提供基础。方法:通过全基因合成的方法获得目的基因序列,转化大肠杆菌BL21细胞诱导His-OAS1及OAS1蛋白表达,纯化后用作抗原免疫小鼠,取脾融合,筛选稳定分泌抗体的阳性细胞株,制备并纯化单抗,通过SDS-PAGE,ELISA,Western blot等方法进行检测。结果:体外高效表达OAS1蛋白,并成功制备特异性单克隆抗体,效价在5×10^-11mol/L以上,亲和常数为3.37×10⁸ L·mol^-1。结论:获得高亲和力OAS1单克隆抗体,为其含量的检测奠定了基础。     

Generation and Characterization of a Neutralizing Monoclonal Antibody Against Rabies Virus

Russian Journal of Bioorganic Chemistry - Rabies is a zoonotic disease, for which effective treatment methods after the onset of clinical symptoms have not been developed yet. Polyclonal sera, both...     

单核细胞增生李斯特菌单克隆抗体的制备与鉴定

以单核细胞增生李斯特菌细胞碎片免疫BALB/c小鼠,间接ELISA法成功筛选获得2株稳定分泌抗LM的单克隆杂交瘤细胞株4A7、4H11.抗体效价为1∶160 000以及1∶20 000,亚型为IgG1、IgG2a,Dot-ELISA结果表明4A7和4H11单克隆抗体具有很好的属特异性,Western blot分析表明4A7、4H11抗体分别与单核细胞增生李斯特菌62 kDa以及32 kDa外膜蛋白抗原表位结合,胶体金免疫电镜实验进一步确证以上抗体可有效识别单核细胞增生李斯特菌细胞表面抗原.     

Characterization of a Monoclonal Antibody and a cDNA for Polyubiquitin of Amoeba proteus

A monoclonal antibody was obtained that reacts with many different proteins (14-200 kDa) of Amoeba proteus. By indirect immunofluorescence microscopy we found the antigens to be dispersed throughout the cytoplasm but were more concentrated in the nucleus. The antibody cross-reacted with proteins of Tetrahymena, Xenopus embryo, and mouse macrophages. Using the antibody as a probe we cloned a cDNA of 1.2 kb coding for ubiquitin in five repeats. Amino acid sequences of ameba's polyubiquitin showed the most variations among the nineteen polyubiquitins of other organisms compared. The well-conserved ²⁰Ser and ⁵⁵Thr residues were replaced with Gly and Ser. respectively. The ²⁸Ala residue found in most organisms was replaced with Gln or Glu in the amoeba. Amoebae contained two ubiquitin-mRNAs that could be detected by Northern blot analysis using the cDNA as a probe. In an analysis for specificity, the antibody reacted with polyubiquitin and ubiquitin-fusion proteins larger than 14 kDa but not with monomeric ubiquitin. The antibody is a useful probe in the detection and characterization of proteins ubiquitinated in response to cellular stresses.     

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K_D of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.     

抗甲肝病毒基因工程单抗分离纯化与鉴定

为克服血源免疫球蛋白制品的不足,开发了抗甲肝病毒基因工程单克隆抗体anti-HAV IgG。用无血清培养基培养rCHO工程细胞株,上清液经过rProtein A SFF亲和层析→脱盐→离子交换层析→超滤换液纯化后,所得anti-HAV IgG纯度达99%以上,比活性约100IU/mg,anti-HAV IgG活性回收率40%。所纯化的anti-HAV IgG分子量150kD,等电点8.4～9.3。免疫印迹实验证实anti-HAV IgG为人源全抗体分子。亲和层析介质rProtein A SFF确实存在亲和配基脱落问题,但通过后续纯化步骤可有效除去。在亲和层析过程中加入高盐清洗步骤,可有效降低宿主DNA残留量水平。对样品中自由巯基含量进行了测定,认为非还原电泳图谱中低分子量条带是由于抗体分子内存在自由巯基引起。用该工艺制备的anti-HAV IgG各项纯度检测指标均达到我国对基因工程产品的质量要求。     

小鼠抗人胰岛素受体单克隆抗体的制备、纯化及鉴定

 M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。     

猪细小病毒单克隆抗体的制备和鉴定

目的制备猪细小病毒（PPV）杂交瘤细胞株,并对其分泌的PPV单克隆抗体进行鉴定。方法按常规方法制备并获得2株杂交瘤细胞。用染色体分析对杂交瘤细胞进行鉴定,用间接ELISA、免疫过氧化物酶单层试验（IPMA）和间接免疫荧光试验（IFA）对其分泌的单克隆抗体进行效价测定、亚型鉴定和特异性鉴定。结果得到2株分泌单克隆抗体的杂交瘤细胞株2H9、1F9,染色体数目介于90～110之间。细胞上清效价均达1∶1×104,腹水效价均达1∶1×107,其亚型分别为IgG1、IgM,均为kappa链。2H9、1F9单抗与猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）、猪圆环病毒I型（PCV-1）、猪圆环病毒Ⅱ型（PCV-2）、乙脑病毒（JEV）等均无交叉反应。IPMA和IFA检测结果显示2H9、1F9单抗均能与接种于PK-15细胞的PPV发生特异性反应。结论成功制备了2株抗PPV杂交瘤细胞株,证实其产生的单克隆抗体具有良好的特异性和敏感性。     

人宫颈癌基因蛋白单克隆抗体的制备、鉴定及初步应用

目的:制备人宫颈癌基因蛋白单克隆抗体并初步用于肝癌血清检测。方法:重组表达HCCR-1蛋白胞外区(第167th-360th氨基酸)进行动物免疫,用含有HCCR蛋白抗原表位的融合蛋白筛选阳性克隆。通过ELISA、Western Blot、免疫组化鉴定抗体性质,并用于肝癌血清样本检测。结果:获得3株抗人HCCR蛋白单克隆抗体,其中B3抗体特异性、灵敏度较好。ELISA结果表明B3只识别GST-HCCRep蛋白和His-HCCR蛋白,与其他蛋白没有交叉反应;B3可检测到10 mg/L的GST-HCCRep蛋白;B3抗体亲和力常数Kaff=1.86×107(L/mol);Western Blot结果表明该抗体可识别天然HCCR蛋白;免疫组化检测表明肝癌组织中有HCCR蛋白表达而正常组织中未见表达;肝癌患者血清HCCR阳性率达到75%。结论:成功制备了HCCR单克隆抗体,为建立新的肝癌早期诊断方法奠定了基础。     

抗登革病毒2型E蛋白单克隆抗体的制备和生物学特性的鉴定

为了制备可用于胶体金快速检测试纸条的抗登革病毒2型（DEN2）E蛋白单克隆抗体（mAb）,通过基因克隆获得E基因与质粒pET32a（＋）的重组质粒,将重组质粒转化入大肠杆菌BL21,IPGT诱导表达重组蛋白。以DEN2重组E蛋白免疫BALB／c小鼠,采用杂交瘤技术制备抗DEN2E蛋白的mAb,以间接ELISA法和Western blot进行mAb特异性鉴定;同时采用间接ELISA法鉴定mAb的Ig亚类。结果表明获得1株可分泌特异性mAb的杂交瘤细胞（7C7）,其抗体亚类为IgG1。Western blot显示该株mAb能特异识别重组pET32a-DEN2E蛋白。因此,成功制备出抗DEN2E的1株mAb,为建立快速特异橙测登革病毒感染的实验方法提供了有力的工具。     

Characterization and Application of a New Monoclonal Antibody with High Specificity for Helicobacter hepaticus

Background and Aims:  Infection with Helicobacter hepaticus is suggested to play a role in the pathogenesis of chronic liver disease in humans. However, reactive antigens among Helicobacter species make the development of an H. hepaticus ELISA test with high specificity difficult. A new monoclonal antibody from a hybridoma clone (HRII-51) showed high specificity to H. hepaticus without cross-reaction to other gastrointestinal bacteria.
Methods:  The molecular weight of HRII-51 immunoreactive antigen was examined by Western blot of H. hepaticus probed with the monoclonal antibody HRII-51. A HRII-51-immunoreactive antigen capture ELISA was prepared in which the specific antigen was anchored by HRII-51-immobilized ELISA plate. Accuracy of HRII-51 antigen capture ELISA was examined using sera obtained from mice inoculated with Helicobacter species. Specificity of HRII-51 antigen capture ELISA was compared to that of H. hepaticus antigen-based ELISA using human sera with absorption by H. pylori cell lysate.
Results:  HRII-51 immunoreactive antigen had a molecular weight of 15 kDa. Sensitivity and specificity of HRII-51 antigen capture ELISA were 87.0% and 97.6% in mice inoculated with Helicobacter species. In human sera, modification of the results by absorption with H. pylori lysate was smaller in HRII-51 antigen capture ELISA comparing with H. hepaticus -antigen-based ELISA.
Conclusion:  Use of the HRII-51 antigen capture ELISA would be a useful approach for the serodiagnosis of H. hepaticus infection in both experimental animals and humans.     

抗CD20嵌合抗体的表达与活性检测

表达了基因重组抗CD20嵌合抗体并对其生物学活性进行了初步鉴定。设计合成轻、重链可变区序列;提取血液RNA,通过RT-PCR得到人κ、IgG1的轻、重链恒定区序列。运用重叠延伸PCR,连接可变区与恒定区,将轻、重链基因连接至pIRES双表达载体。将质粒以阳离子脂质体转染CHO细胞,ELISA挑选阳性克隆,共获得7株表达较高的克隆,表达量约为2mg/L。扩大培养阳性克隆anti-CD20-1B3,收获上清,以蛋白A进行亲和层析纯化表达蛋白。SDS-PAGE检测表明纯化纯度达到95％,蛋白相对分子量与理论值吻合。以CD20₊细胞Raji、Daudi、Ramous检测,表明该抗体能与CD20抗原特异性结合,体外杀伤试验说明抗体能够杀伤CD20₊淋巴瘤细胞。     

群特异性蓝舌病病毒单克隆抗体的制备和鉴定

目的：制备群特异性抗蓝舌病病毒（BTV）单克隆抗体,并对其特性进行鉴定,为建立检测BTV抗原及抗体的ELISA方法奠定基础。方法：用纯化的BTV颗粒为免疫抗原免疫BALB/c鼠,以大肠杆菌表达的VP7蛋白作为筛选抗原,用间接ELISA法筛选杂交瘤细胞株;选取抗体效价最高的一株制备BTV单克隆抗体,以该抗体为捕获抗体与8种不同血清型BTV进行ELISA反应,结果与细胞病变反应进行比对;以该抗体为竞争抗体,与12种不同血清型绵羊BTV抗血清进行竞争ELISA反应,并将结果与参比c-ELISA试剂盒结果进行比对。结果：筛选出5株稳定分泌BTV单克隆抗体的杂交瘤细胞株,并选其中一株（3E2）制备了高纯度的单克隆抗体;该单抗用于检测不同血清型BTV,与细胞病变反应结果完全相符;用于检测不同血清型绵羊BTV抗血清,其结果与参比c-ELISA试剂盒符合率为100%,与鹿流行性出血热病毒抗原和抗体均无交叉反应。结论：制备的BTV单克隆抗体具有良好的群特异性,可用于检测不同血清型BTV抗原及BTV抗体。     

抗结核分枝杆菌RpfB结构域单克隆抗体的制备及鉴定

目的:制备抗结核分枝杆菌Rpf B结构域单克隆抗体。方法:将p PRO-EXHT-Rpf B domain原核表达载体接种于大肠杆菌DH5中,用IPTG诱导表达Rpf B结构域蛋白,以纯化的Rpf B结构域蛋白作为免疫原,皮下包埋免疫小鼠3次,每次间隔2周;分离小鼠的脾细胞,与Sp2/0细胞融合,克隆化制备抗Rpf B结构域单抗,ELISA检测其效价,鉴定其特异性和相对亲和力,观察制备的抗Rpf B结构域单抗对Rpf家族其他蛋白的识别能力及其对结核分枝杆菌和藤黄微球菌的生长抑制作用。结果:制备了3株抗Rpf B结构域单抗,特异性高,亲和力较强,均能特异性识别Rpf B结构域。经小鼠腹腔注射制备腹水并纯化,获得了较高纯度的单抗,所制备的抗Rpf B结构域多肽的单克隆抗体可以识别多种Rpf样蛋白及其结构域蛋白。在抗体滴度为1∶1000时可有效抑制Rpf B结构域对结核分枝杆菌H37Ra和藤黄微球菌的生长促进作用,提示抗Rpf B结构域单抗可能会抑制进入机体内生长停滞或潜伏感染的结核分枝杆菌的再次激活,可能具有预防隐性感染复发的作用。结论:抗Rpf B结构域单抗的制备为进一步研究Rpf B结构域的生物学和免疫特性提供了实验工具。