Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibody against ginsenoside Rb1 was produced by fusing splenocytes immunized with a ginsenoside Rb1- bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very small cross-reaction appeared with ginsenoside Rc and ginsenoside Rd. The full measuring range of the assay extends from 20 ngml-1 to 400 ngml-1 of ginsenoside Rb1. This revised version was published online in July 2006 with corrections to the Cover Date.     

埃博拉病毒NP蛋白的单克隆抗体制备及抗原肽的定位

埃博拉病毒(Ebola virus,EBOV)是一种能导致人类及脊椎动物出血热的致死性病毒,对公共卫生具有较严重的危害。EBOV的NP蛋白在病毒复制中具有重要作用,也是诊断该病重要的靶蛋白。文中原核表达重组扎伊尔型EBOV的NP蛋白,重组蛋白免疫bal/c小鼠,制备了一株小鼠抗EBOV-NP的单克隆抗体。利用Western blotting方法,该抗体能特异识别真核表达和原核表达的重组EBOV-NP,并能同莱斯顿型(RestonEbola virus,REBOV)、科特迪瓦型(Cote-d’Ivoire Ebola virus,CIEBOV)和本迪布焦型(Bundibugyo Ebola virus,BEBOV)埃博拉病毒产生交叉反应,而不与苏丹型(the Sudan Ebola virus,SEBOV)和马堡型(the Marburgvirus,MARV)埃博拉病毒产生反应。利用突变PCR和Western blotting方法,定位了该抗体识别的抗原决定簇序列,该序列(PPLESD)位于EBOV-NP蛋白的C端583-588aa。生物信息学研究表明,该序列在已经公布的ZEBOV、CIEBOV、BEBOV共16个型和REBOV的4个型中高度保守。研究结果为建立以上各型埃博拉病毒的检测方法提供了工具,也为研究埃博拉病毒复制及致病机理提供了基础。     

抗鸡白细胞介素4单克隆抗体的制备及鉴定

为了制备鸡白细胞介素4(chIL-4)单克隆抗体,将成熟的chIL-4基因亚克隆至原核表达载体pET-28a和pGEX-6P-1上,然后在大肠杆菌中分别诱导重组蛋白His-chIL-4和GST-chIL-4的表达,并纯化。将纯化后的His-chIL-4作为免疫原免疫BALB/c小鼠,经4次免疫后,取小鼠脾细胞与骨髓瘤细胞(SP2/0)融合。将纯化后的GST-chIL-4作为筛选抗原,利用间接ELISA筛选阳性克隆。阳性细胞株经3次亚克隆后,获得3株稳定分泌抗chIL-4蛋白的杂交瘤细胞株,分别命名为1G11-3B、2E5-3D和1G11-5H。经ELISA检测,3株单克隆抗体的亚型均为IgG1,亲和力解离常数(Kd)分别为1.79×10~(–9)、1.61×10~(–9)和2.36×10~(–9)。经Western blotting及间接免疫荧光试验鉴定,3株单克隆抗体均能特异性识别原核和真核表达的chIL-4蛋白。Western blotting试验证明1G11-3B、2E5-3D和1G11-5H识别的抗原表位区域分别为chIL-4蛋白N端的第1–40、80–112和40–80位氨基酸。该单克隆抗体的制备为chIL-4的检测和生物学功能研究奠定了基础。     

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.     

Preparation of monoclonal antibody against crocin and its characterization

Three crocin-carrier protein conjugates were synthesized and their hapten numbers were determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Three monoclonal antibodies against crocin were produced by hybridomas fused with the splenocytes immunized with crocin hemisuccinate-bovine serum albumin conjugate and HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. They were identified as IgG2a and IgG2b possessing light chain, respectively. Their wide reactivities against crocetin glycosides were discussed.     

SIVp27单克隆抗体的制备和鉴定

[目的]建立SIVp27杂交瘤细胞株,并对其分泌的SIVp27单克隆抗体进行初步鉴定。[方法]使用基因重组的SIVp27蛋白免疫BALB/c小鼠,采用杂交瘤技术使用半固体培养基法建立杂交瘤细胞株,制备单克隆抗体。通过染色体核型对杂交瘤细胞株进行鉴定;采用Westernblot、免疫荧光法、酶联免疫吸附法确定单克隆抗体的交叉反应性、相对亲和力、抗原识别表位、免疫球蛋白的类型和亚类,对单克隆抗体进行鉴定。[结果]获得四株可稳定分泌SIVp27单克隆抗体的杂交瘤细胞,1C3、2B6为IgG1类,2E12为IgG2b类,3G3为IgG2a类。四株单抗均能识别SIV的p27蛋白,与逆转录病毒SRV、STLV无交叉反应,2B6、2E12与HIVp24有交叉反应。免疫荧光法检测腹水效价为1:10240~40960。1C3、2B6、2E12、3G3染色体平均数分别为103、97、96、101。2E12与3G3识别不同的抗原表位。[结论]成功地制备出四株SIVp27单克隆抗体,均具有良好的特异性和亲和力,为进一步建立免疫分析方法,进行SIV/SAIDS及其艾滋病相关研究,奠定了基础。     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

Preparation and characterization of a human aurora-A kinase monoclonal antibody

We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an Elisa (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.     

Human Immunodeficiency Virus Type 1 (HIV-1) Infection and Transcytosis Activity of a HIV-1 Susceptible Clone from HeLa Cell

In order to clarify the transmission process of human immunodeficiency virus type 1 (HIV-1) through the epithelial cell barrier, HeLa cells susceptible and non-susceptible to HIV-1 were cloned and designated as P6 HeLa and N7 HeLa cells, respectively. P6 HeLa cells could be infected with the LAI strain of HIV-1 and mediated HIV-1 transcytosis. In contrast, N7 HeLa cells exhibited neither HIV-1 infection nor transcytosis. CD4 and galactosylceramide as the receptors for HIV-1 were not detected on P6 HeLa cells, although an anti-CD4 monoclonal antibody (mAb) blocked HIV-1 infection. Since HIV-1-infected P6 HeLa cells exhibited no fusion and survived, we speculated that the P6 HeLa cells expressed molecules other than CD4 which facilitated HIV-1 infection. Two mAbs (A-14 ITK and C57 a9-9) which inhibited the HIV-1 infection of P6 HeLa cells were generated. Each mAb recognized distinct molecule(s) as shown by Western blotting. Transcytosis by the P6 HeLa cells was inhibited by C57 a9-9 but not by A-14 ITK or anti-CD4 mAb. Both infection and transcytosis may be responsible for HIV-1 transmission through epithelial cells in a complex manner. Although infection and transcytosis occurred via different mechanisms, the molecule(s) recognized by C57 a9-9 mAb may be associated with both processes.     

Formation of monoclonal antibody against a major ginseng component, ginsenoside Rg1 and its characterization. Monoclonal antibody for a ginseng saponin

The ratio of hapten and bovine serum albumin in an antigenconjugate was determined by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry. Ahybridoma secreting monoclonal antibody against ginsenosideRg₁ was produced by fusing sprenocytes immunized with aginsenoside Rg₁-bovine serum albumin conjugate withHAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very smallcross-reaction appeared with ginsenoside Re. The full measuringrange of the assay extends from 0.3 g ml^-1 to 10 g ml^-1 ofginsenoside Rg₁.     

抗人血栓调节蛋白单克隆抗体的制备与鉴定

为了制备特异性抗人血栓调节蛋白(human thrombomodulin,hTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含hTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株。将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA (cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水。用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定。我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgGl,McAb腹水效价为1×10~(-6),腹水抗体含量为20 mg/ml。NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的hTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础。     

Production and characterization of a monoclonal antibody for Pefloxacin and mechanism study of antibody recognition

In this report, an artificial antigen (PFLX–BSA: Pefloxacin connected bovine serum albumin) was successfully prepared. The monoclonal antibody against pefloxacin was produced and characterized using a direct competitive ELISA. The linear range of detection was 0.115–6.564 µg/L. The limit of detection defined as IC₁₅ was 0.170 ± 0.05 µg/L and the IC₅₀ was 0.902 ± 0.03 µg/L. The antibody variable region genes were amplified, assembled, and sequenced. A three–dimensional structural model of the variable region was constructed to study the mechanism of antibody recognition using molecular docking analysis. Three predicted essential amino acids, Thr53, Arg97 of heavy chain and Thr52 of light chain, were mutated to verify the theoretical model. Three mutants lost binding activity signi?cantly against pefloxacin as predicted. These may provide useful insights for studying antigen–antibody interaction mechanisms to improve antibody affinity maturation in vitro.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

茶尺蠖核型多角体病毒多角体蛋白单克隆抗体的制备

为了建立一种基于免疫反应检测茶尺蠖核型多角体病毒的方法,以纯化后的茶尺蠖核型多角体病毒作为抗原,免疫BALB/c小鼠,将小鼠脾脏细胞与小鼠骨髓瘤细胞Sp2/0融合,经间接ELISA筛选及克隆得到了一株稳定分泌单克隆抗体的杂交瘤细胞株,命名为7D3。同时克隆并在大肠杆菌中表达了EoNPV多角体蛋白基因,获得重组多角体蛋白。经Western blotting鉴定,该抗体可与EoNPV的多角体蛋白特异性结合。利用制备EoNPV多角体蛋白的单克隆抗体,建立了间接ELISA测定EoNPV的方法。     

登革病毒血清型特异单克隆抗体的制备和鉴定

登革病毒 (Dengue virus,DENV) 是全球传播最为广泛的虫媒病毒,由于缺乏快速鉴别感染病毒血清型的诊断技术,导致异型交叉感染引起重症登革出血热病例居高不下。为实现免疫学方法快速鉴别诊断不同血清型DENV感染,本研究采用哺乳动物细胞293T表达并纯化了4种DENV血清型NS1蛋白,免疫小鼠后通过杂交瘤技术制备了针对NS1蛋白的单克隆抗体。利用酶联免疫吸附方法 (Enzyme-linked immunosorbent assay,ELISA)、间接免疫荧光法 (Indirect immunofluorescence assay,IFA)、免疫斑点杂交试验 (Dot blotting) 以及蛋白质免疫印迹试验 (Western blotting) 确认所制备的单克隆抗体能够有效识别天然病毒NS1以及重组NS1蛋白。获得的单克隆抗体包含2株可识别1–4型DENV NS1蛋白的通用型抗体及3株分别针对DENV-1、DENV-2和DENV-4的血清型特异抗体。以所制备的DENV NS1抗体为基础,采用双抗体夹心ELISA可快速鉴别不同血清型DENV。DENV血清型特异单克隆抗体的制备和甄别DENV血清型ELISA方法的建立为快速鉴别感染DENV血清型的临床诊断奠定了基础。     

Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.     

抗rhEPO单克隆抗体的制备及初步应用

用重组人促红细胞生成素（ｒｈＥＰＯ）免疫Ｂａｌｂ／ｃ小鼠,取其脾细胞在ＰＥＣ４０００作用下与ＳＰ２／０小鼠骨髓瘤细胞融合,获得一株能分泌抗ｒｈＥＰＯ单抗隆抗体的杂交瘤细胞株２Ｆ１２,染色体数目大于１００条,间接ＥＬＩＳＡ法测定腹水和细胞培养上清效价,分别为１．６×１０＾－７和４×１０＾－４。测定抗体亚类时,则同时显示ＩｇＡ和ＩｇＧ１,其轻链为κ链;相对亲和力为５×１０％＾－１２ｍｏｌ／Ｌ。单抗２Ｆ     

抗鹅免疫球蛋白轻链单克隆抗体的鉴定及其应用

免疫球蛋白是机体固有免疫系统的组成部分,是机体防御的第一道防线。本研究对抗鹅免疫球蛋白轻链单克隆抗体进行了特征分析并将其应用到不同免疫试验中用以检测鹅免疫球蛋白。用此单克隆抗体制备的免疫亲和层析柱用以分离血清中的鹅免疫球蛋白;偶联辣根过氧化物酶 (Horseradish peroxidase,HRP) 后的单克隆抗体用作第二抗体来检测鹅特异性抗体。此外,该单克隆抗体可以识别和定位外周血淋巴细胞中的SIg+淋巴细胞。研究表明,该单克隆抗体可在多种条件下检测或分离鹅免疫球蛋白并作为研究鹅体液免疫的有力工具。     

Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity

Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC-MS. Fragmentation rates are monitored by SE-HPLC and SDS-PAGE over the pH range 4-6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 40°C, pH 4, ～60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was ～13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.