伪狂犬病病毒单克隆抗体制备及特征分析

目的:制备伪狂犬病病毒(PRV)单克隆抗体,为PVR防控奠定基础。方法:用PRV疫苗毒株Bartha-k61免疫小鼠,取免疫小鼠脾脏B淋巴细胞与SP2/0细胞融合,用酶联免疫吸附实验筛选阳性杂交瘤细胞并制备腹水,用病毒中和实验检测单克隆抗体(腹水)对PRV的中和作用。结果:通过细胞融合,共得到11株针对PRV的杂交瘤细胞。中和实验结果显示,无论是对于PRV弱毒株Bartha-k61还是强毒株AV25,2株杂交瘤细胞产生的腹水都表现为明显的中和效应;但2株单抗的中和能力不同,其中1株杂交瘤细胞腹水的中和效价为1∶16~1∶32,另1株的效价为1∶4。交叉反应显示,11株单克隆抗体中,2株与单纯性疱疹病毒存在明显的免疫反应,1株与猴B病毒的gD蛋白存在明显交叉反应。结论:制备了PRV的单克隆抗体,并获得了对PRV具有中和作用的单克隆抗体,以及能与其他疱疹病毒交叉反应的单克隆抗体。     

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。     

番茄花叶病毒单克隆抗体的制备及检测应用

用番茄花叶病毒(ToMV)免疫的BAL B/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗ToMV单克隆抗体(Mab)的杂交瘤细胞,其中2株能同时检测ToMV和烟草花叶病毒(TMV),各单克隆抗体腹水ELLSA效价在1∶32 000～1∶1 024 000之间。经TASELISA测定,4株单克隆抗体检测病汁液的稀释度均能达到1∶2 000倍以上。4株单克隆抗体与其他病毒无交叉反应。Westernblot分析表明,其中两株与ToMV176kD的外壳蛋白亚基有特异反应,而另两株无反应,推测它们是针对构象决定簇的抗体。     

大豆脂肪氧化酶同工酶全缺失种质的创新

大豆脂肪氧化酶(Lipoxygenase,Lox)是豆腥味因子。利用脂肪氧化酶双缺失的优质大豆新品系96P17(Lox-2.3)作母本,93704(Lox-1．3)作父本进行有性杂交,采用等电聚焦聚丙烯酰胺凝胶电泳(IEF—PAGE)技术,对杂种后代脂肪氧化酶3种同T酶进行缺失检测及多年辅助选择,创造大豆脂肪氧化酶3种同工酶全缺失(Lox-1．2．3)的大豆优质新种质,为大豆品质育种及食品加工提供优异的种质材料。     

中国大豆资源脂肪氧化酶缺失类型研究

作利用改进的IEF-PAGE电泳方法,对中国26个省(自治区)的1726份栽培大豆资源(包括南方多作区资源16ll份、黄淮海夏作区资源62份,北方春作区资源53份)进行了脂肪氧化酶(Lox)缺失类型鉴定,并研究了Lox缺失与种植区域、播种期类型及籽粒颜色的关系。结果表明：在中国大豆资源中发现了4种Lox缺失类型,共99份Lox缺失突变体,占鉴定资源总数的5．73％,其中80份缺失Lox-3,12份缺失Lox-2,6份缺失Lox-2．3及1份缺失Lox-1,这些缺失体主要分布在南方多作大豆区(97份)和黄淮海夏作大豆区(2份);缺失频率较高的有四川(Lox缺失体占本省鉴定资源数的9．77％)、贵州(7．09％)、浙江(6．86％)等省。筛选出的99份Lox缺失材料中,夏播大豆65份,春播大豆33份,秋播大豆1份;黄种皮大豆64份、绿种皮大豆15份、黑种皮大豆10份、褐种皮大豆9份、双色种皮大豆1份。     

抗H5N1亚型禽流感病毒血凝素单克隆抗体的制备及鉴定

目的建立稳定分泌抗H5N1亚型禽流感病毒血凝素单克隆抗体的杂交瘤细胞系,为进一步研究禽流感诊断技术奠定基础。方法以纯化的H5亚型禽流感病毒按常规方法免疫BALBc小鼠,最后一次免疫后第3天取其脾细胞与SP20细胞在聚乙二醇作用下融合,用选择性培养、有限稀释法克隆和血凝抑制试验进行筛选,对获得阳性克隆株用ELISA方法进行亚型鉴定,并用37株H5、H7、H9亚型AIV测定其特异性、覆盖性。结果最后获得了3株分泌特异性抗体的杂交瘤细胞,命名为1E5、4A4、4B1,经长期体外培养和冻存后复苏能稳定地分泌抗体。经鉴定,其亚型均为IgG1、kappa链。腹水HI效价1∶210～1∶216,细胞培养上清HI效价1∶26～1∶28。3株杂交瘤所分泌的单克隆抗体均能与本中心保存的全部20株H5亚型禽流感病毒分离株发生反应,而与15株H9亚型禽流感病毒分离株、2株H7亚型禽流感病毒分离株以及H1H4、H6H15亚型禽流感病毒标准毒株均不反应,与鸡新城疫病毒、鹅新城疫病毒、鹅腺病毒和鸡产蛋下降综合征病毒等均无交叉反应。结论所获3株单克隆抗体可用于禽流感病毒特异性诊断试剂的研制。     

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。     

P2X7受体短肽单克隆抗体的制备及鉴定

旨在制备与鉴定鼠抗P2X7受体的蛋白单克隆抗体.以人P2X7受体的胞外段制备短肽作为抗原,皮下注射免疫Balb/c小鼠.分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定P2X7其生物学效应.结果显示,获得1株稳定分泌抗人P2X7受体的单克隆抗体的杂交瘤细胞株,所分泌的单抗类型重链为IgG1,轻链为κ;该株杂交瘤细胞腹水效价为1∶6.4×104;传30代及液氮中保存6个月,抗体效价稳定;Western blotting检测证明该单抗与人细胞表面的P2X7受体蛋白特异地结合.所制备的抗人P2X7受体的单克隆抗体具有高度的特异性及稳定性,为针对P2X7受体为靶点的抗体药物的开发应用、疾病的辅助诊断奠定了基础.     

幽门螺杆菌尿素酶B单克隆抗体的制备与鉴定

目的:制备稳定分泌抗幽门螺杆菌尿素酶B单克隆抗体(mAb)的杂交瘤细胞系,并对其分泌的mAb进行鉴定。方法:用初步纯化的重组幽门螺杆菌尿素酶B免疫BALB/c小鼠,利用杂交瘤技术制备抗尿素酶B的mAb,用间接ELISA检测mAb的特异性和亲和力,检测mAb腹水效价,鉴定Ig亚类并测定其抗原决定簇。结果:获得8株能稳定分泌抗尿素酶B的mAb杂交瘤细胞系,这8株单抗与能产生尿素酶的小肠结肠耶尔森氏菌、肺炎克雷伯氏菌和普通变形杆菌均无交叉反应,相对亲和力为1.13×10-8～4.66×10-10,腹水mAb效价可达2×104～3.2×105。其中2株单抗属IgG1亚类,3株单抗属IgG2a亚类。8株单抗分属于3种不同的抗原决定簇。结论:获得了IgG1和IgG2a类型的针对3种不同抗原决定簇的特异性幽门螺杆菌尿素酶B的mAb,为进一步用于幽门螺杆菌的临床诊断和实验研究创造了条件。     

大豆脂肪氧化酶的研究动态

本文介绍了大豆种子脂肪氧化酶（Lox）同工酶的生物化学特性、缺失体的分子结构、氨基酸同源性等的分子生物学特性以及Lox与生长发育、调节分子合成、衰老和逆境反应等生理学功能的相关性的研究动态。     

Monoclonal antibodies to rat brain astroglial cells—Their characterization and application for isolation of astroglial subpopulations

Three monoclonal antibodies (MAbs) specifically recognizing rat astrocyte cell surface proteins have been characterized and their antigen binding specificities determined. One of these MAbs has been employed to isolate a distinct subpopulation of astroglial cells using immunoaffinity chromatography.

MAbs to rat astroglial cell surface proteins, generated by fusion of mouse Sp2/O-Ag 14 myeloma cells and spleen cells from Balb/C mice immunized with purified astroglial cells, were screened for their cell binding specificities using ELISA and indirect immunofluorescence assay. The antigen binding specificity of three of these clones, which displayed specific binding to astrocytes, was determined by radioiodination of whole astrocytes and precipitation of the iodinated surface proteins by the MAbs. Immunoaffinity chromatography, using IgG from one of the clones coupled to CNBr activated Sepharose 6MB, demonstrated the potential usefulness of such MAbs in isolating a specific subpopulation of astroglial cells.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA

In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen.     

The molecular nature of Fucus serratus sperm surface antigens recognised by monoclonal antibodies FS1 to FS12

Sperm of the brown alga Fucus serratus are highly differentiated, biflagellate, naked cells. Immunolocalisation studies, employing monoclonal antibodies (MAbs — designated FS1 to FS12) raised against antigens of these sperm cells, have revealed that some sperm surface components are distributed over the entire cell, whereas others are restricted to, or occur preferentially on, the surface of the anterior flagellum or cell body. This report describes the use of these MAbs in Western-blot procedures and antigen-modification binding assays to determine the nature of these sperm surface components. Monoclonal antibodies which bind to antigens found on the cell body and both flagella (FS3, FS4, FS6, FS8, FS10) recognise carbohydrate epitopes of a high-molecular-weight glycoprotein (M_r=205 kDa). These MAbs were initially chosen at random from a much larger number of antibodies which bound to sperm in a similar fashion, indicating that this glycoprotein is an immunodominant antigen. Though these MAbs compete under conditions of limited antigen availability, differences in the effects of periodate on antibody binding and differences in other binding data indicate that the MAbs recognise epitopes of this glycoprotein which are neighbouring or overlapping, rather than common. The MAb FS9, which has a similar binding pattern to the above antibodies, also seems to bind to carbohydrate epitopes, but the antigen recognised by this antibody could not be identified in Western-blotting procedures. The MAbs FS7 and FS12, which bind to the mastigonemes on the anterior flagellum and to the cell body and posterior flagellum, recognise a set of glycoproteins in the molecular-weight range 40–250 kDa. The evidence indicates that the antibodies are binding to N-linked carbohydrate side chains of these glycoproteins. Three MAbs that bind to the anterior flagellum (FS2, FS5 and FS11) recognise protein antigens in the molecular-weight range 90–250 kDa; it is not known whether these antigens are glycosylated. The MAb FS1, which binds primarily to the sperm cell body, could not be used in enzyme-linked immunosorbent assays or Western-blotting procedures and the antigen recognised by this antibody is so far uncharacterised.Abbreviations ELISA enzyme linked immunosorbent assay - HRP-RAMIG horseradish-peroxidase-labelled rabbit anti mouse immunoglobulin - Ig immunoglobulin - kDa kilodalton - MAb monoclonal antibody - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis We are grateful to AFRC for financial support under the cell signalling initiative.     

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.     

Comparison of indirect immunofluorescence (IF) test with enzyme-linked immunosorbent assay (ELISA) in screening of hybridomas to very virulent Marek's disease virus.

For identifying virus-specific antigens of Marek's disease virus (MDV), monoclonal antibodies (MAbs) against strain Md5 of serotype 1, which is known to be a very virulent MDV (vvMDV), were isolated. Fifty-eight hybridoma clones that secreted MAbs against vvMDV were obtained. Of these MAbs, 36 gave positive reactions in an immunofluorescence (IF) test, and 22 gave positive reactions on enzyme-linked immunosorbent assay (ELISA). None of these MAbs gave positive reactions in both the IF test and ELISA. Of the MAbs that gave positive reactions in the IF test, 33 clones reacted with MDV1-specific epitopes, the other three reacting with MDV1-HVT intertypic epitopes. None of the clones reacted with MDV1-MDV2 intertypic epitopes. Three virus-specific polypeptides were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting. These polypeptides were recognized by 12 MAbs giving positive reactions by IF, but by none of those giving positive reactions by ELISA. In addition, size heterogeneity of the MDV1-specific phosphorylated polypeptides in the MDV1 strains was shown using the MAbs against Md5.     

蚕豆萎蔫病毒单克隆抗体制备及检测应用

:用蚕豆萎蔫病毒(BBWV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得6株能稳定传代并分泌抗BBWV单克隆抗体(Mab)的杂交瘤细胞株,单抗腹水ELISA滴度为1:320000～1:640000,各单抗抗体类型均为IgG1。6株单抗与BBWV不同分离物均有反应,而与其它植物病毒无交叉反应。经Westernblot印迹分析表明,此6株单克隆抗体均是针对BBWV447kD的外壳蛋白大亚基的特异性抗体。这是国内外首次报道获得BBWV单克隆抗体     

Studies of murine malaria antigens using monoclonal antibodies. Production, selection, and characterization of antibodies

A panel of ten monoclonal antibodies made against Plasmodium chabaudi and Plasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs to P. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted with P. chabaudi antigens. Of the MAbs to P. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.