活化相关分泌蛋白1单克隆抗体的制备及其在保守结构域鉴定中的应用

目的:制备重组活化相关分泌蛋白1(ASP-1)的单克隆抗体,并用其鉴定保守结构域。方法:用原核表达并纯化的重组ASP-1不加佐剂免疫BALB/c小鼠,采用杂交瘤技术及有限稀释传代法筛选稳定分泌特异性抗体的杂交瘤细胞株,制备单抗腹水后用间接ELISA进行抗体特异性鉴定和效价检测,利用肽结合ELISA和Western印迹鉴定单抗识别的保守结构域。结果:获得5株能稳定分泌抗ASP-1单克隆抗体的杂交瘤细胞株,且5株单抗的识别区域均为21~28氨基酸残基的保守性结构域。结论:制备了抗ASP-1的单克隆抗体,为深入研究ASP-1佐剂的活性功能区及作用机制提供了有效工具。     

呼吸道合胞病毒融合抑制活性单克隆抗体的鉴定及应用

用杂交瘤技术制备了抗呼吸道合胞病毒 (RSV Long株 )的单克隆抗体F3细胞株。经IFA及ELISA证明 ,F3株McAb对RSV抗原特异 ,具中和活性及融合抑制活性 ,其腹水中和效价和融合抑制效价分别为 1∶12 8和 1∶6 4。将F3株McAb与商售混合单抗试剂盒进行临床诊断比较 ,二者阳性符合率 96 .9% ,阴性符合率 10 0 %。     

幽门螺杆菌尿素酶B单位DNA疫苗的构建及鉴定

报告了利用淋巴细胞杂交瘤技术制备抗呼吸道合胞病毒蛋白的鼠单克隆抗体。获得 7株分泌抗呼吸道合胞病毒单克隆抗体的杂交瘤细胞株 :IF12 ,4H10 ,1D8,2B2 ,1G8,4G6 ,2D2。其中IF12 ,4H10 ,1D8,2B2 ,4G6分泌的抗体类别为IgG1,1G8为IgG2a ,2D2为IgG2b。经免疫印迹分析证实单抗 1F12 ,2B2识别病毒 4 4ku的蛋白 ,单抗1G8识别 37ku蛋白 ,2D2识别 2 1ku的病毒蛋白 ,单抗4G6识别病毒 90ku蛋白。对单抗的生物学活性进行了分析 ,单抗 4G6对呼吸道合胞病毒有明显的中和作用 ,小鼠腹水的中和滴度为 1∶32 ,其余各单抗中和滴度均小于1∶4。…     

EpCAM蛋白的表达、纯化及其单克隆抗体的制备

目的:原核表达EpCAM蛋白并制备抗EpCAM特异性单克隆抗体,初步鉴定相应单克隆抗体的特性。方法:PCR扩增EpCAM基因胞外区,将目的基因亚克隆至载体pET-28a(+),转化至大肠埃希菌株BL21,IPTG诱导表达,组氨酸亲和层析法纯化表达产物。纯化蛋白免疫BALB/c小鼠,将成功免疫的小鼠脾细胞与骨髓瘤SP2/0细胞融合,经ELISA筛选得到分泌特异性抗EpCAM的单克隆抗体的细胞株,免疫BALB/c小鼠进一步制备相应的单克隆抗体,并通过Western blot(蛋白质印记)和FACS(流式细胞分析)鉴定单抗的特异性及生物学活性。结果:成功构建重组表达载体pET28a-EpCAM并在大肠杆菌中获得表达,经His-tag亲和层析法获得纯化的EpCAM重组蛋白。EpCAM重组蛋白免疫的BALB/c小鼠的脾细胞与SP2/0细胞融合、筛选,获得两株稳定分泌EpCAM抗体的杂交瘤细胞株,分别命名为4B2、2F2并免疫BALB/c小鼠获得相应的单克隆抗体。Western blot结果显示4B2腹水纯化所得单抗能够识别FaDu细胞系(人咽鳞癌细胞)中的EpCAM蛋白,但2F2未能识别FaDu细胞中的变性的EpCAM蛋白。FACS结果显示两者均能和FaDu细胞中天然的EpCAM蛋白结合。讨论:成功制备了抗EpCAM的单克隆抗体,并能够识别人咽鳞癌细胞系FaDu中表达的EpCAM,为进一步研究EpCAM抗体在肿瘤治疗中的作用提供基础。     

抗人甲状腺球蛋白单克隆抗体的制备

目的:制备分泌特异性抗人甲状腺球蛋白(thyroglobulin,Tg)单克隆抗体的杂交瘤细胞株,为建立高灵敏度的Tg检测方法做准备。方法:以天然人源甲状腺球蛋白为抗原经皮下免疫BALB/c小鼠,通过细胞融合制备分泌抗人甲状腺球蛋白单克隆抗体,并对其进行特异性鉴定,建立检测Tg的双抗体ELISA(enzyme-linked immunosorbent assay)夹心法。结果:获得7株可稳定分泌抗人甲状腺球蛋白单克隆抗体的杂交瘤细胞株,经ELISA鉴定,筛选抗体可与Tg抗原有良好的特异性反应。建立的双抗体夹心ELISA方法敏感性可达1 ng/mL。结论:成功制备了抗人Tg单克隆抗体并建立了检测人Tg双抗体夹心ELISA方法,为进一步研发Tg快速诊断试剂盒提供了原料。     

抗发热伴血小板减少综合征病毒结构蛋白单克隆抗体的制备和功能分析

为制备抗发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)结构蛋白的单克隆抗体,本研究用灭活纯化的SFTSV病毒颗粒免疫BALB/c小鼠,利用杂交瘤技术获得分别分泌抗糖蛋白单抗和核蛋白单抗的杂交瘤细胞株。用免疫荧光法和免疫沉淀方法对制备的单克隆抗体的抗原特异性进行鉴定,并初步进行单抗效价、中和活性及亲和力等功能分析。结果显示,通过细胞融合和克隆化,共筛选出13株稳定分泌抗糖蛋白(Glycoprotein,GP)单抗和7株稳定分泌抗核蛋白(Nucleoprotein,NP)单抗的杂交瘤细胞株。免疫荧光和免疫沉淀鉴定显示获得的单抗有良好的抗原特异性。抗GP单抗中6株针对Gn,7株针对Gc,大部分的间接免疫荧光(Indirect immunofluorescence assay,IFA)滴度在1 280~20 480之间,其中4株抗Gn单抗具有中和活性。获得的7株抗NP单抗均与NP特异性结合,IFA滴度范围在5 120~20 480,均无中和活性。此外,经非竞争ELISA检测的两株抗GP单抗(1C8和1G8)均有较高亲和力。本研究为SFTS诊断方法的发展及SFTSV致病机制研究奠定了基础。     

人β-actin蛋白原核表达及其单克隆抗体制备

为获得分泌抗人β-actin蛋白单克隆抗体（McAb）的杂交瘤细胞,通过在大肠杆菌中原核表达人β-actin蛋白,以纯化的人β-actin蛋白作为抗原免疫BALB/c小鼠。经过细胞的融合及筛选获得1株能稳定分泌抗人β-actin蛋白McAb的杂交瘤细胞,命名为2B4。采用间接ELISA和Western blot方法对McAb的特异性、稳定性和适用范围进行鉴定。结果显示：蛋白的相对分子质量为43 kDa,可溶于8 mol/L尿素;杂交瘤细胞上清的抗体效价为1×10^5,腹水的抗体效价为1×10^7;间接ELISA结果表明,杂交瘤细胞在体外传20代或液氮冻存3个月后,分泌的抗体效价不变;37℃保存24 h后,抗体的效价开始下降。Western blot结果显示,单克隆抗体识别人、鼠、兔和鱼的β-actin蛋白,与其发生特异性反应。2B4分泌的单克隆抗体可以广泛的应用于细胞生物学和免疫学试验,具有良好的应用价值。     

抗人B7-H1单克隆抗体的制备和鉴定

目的:采用杂交瘤技术制备抗人B7-H1单克隆抗体,并对其进行鉴定。方法:经抗原免疫的小鼠脾细胞与小鼠骨髓瘤细胞以常规方法融合;用间接ELISA法筛选分泌抗体的杂交瘤细胞株;阳性克隆用有限稀释法获得稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株;扩增杂交瘤细胞注射进小鼠腹腔后制备腹水;纯化腹水中的单克隆抗体并对其亚型进行鉴定;用间接ELISA法测抗体效价;将肺癌组织制成石蜡切片,用抗人B7-H1抗体进行免疫组化染色。结果:获得1株稳定分泌抗人B7-H1单克隆抗体的杂交瘤细胞株,所分泌的单抗类型为IgG1;抗体效价为1×108,纯化后的抗体含量为6.76g/L;免疫组化实验中,单抗可与肺癌组织表面的B7-H1蛋白特异地结合。结论:制备了人B7-H1单克隆抗体,为B7-H1检测试剂盒的研制奠定了基础。     

抗心型脂肪酸结合蛋白单克隆抗体制备、鉴定和在侧向免疫层析方法中的应用

目的:制备抗心型脂肪酸结合蛋白(H-FABP)单克隆抗体(mAb),并建立侧向免疫层析方法检测血浆中H-FABP。方法:用H-FABP蛋白免疫纯系Balb/c小鼠,采用杂交瘤技术建立能稳定分泌抗人H-FABP的单克隆杂交瘤细胞株。常规制备腹水,纯化后得到特异性抗H-FABP单克隆抗体,进行效价、特异性、亲和力的鉴定分析,并在ELISA平台进行抗体配对,用所筛选到的抗体对初步建立了检测H-FABP的侧向免疫层析方法。结果:成功获得12株稳定分泌抗体的阳性细胞株,并筛选出能相互配对,并应用于侧向免疫层析平台的抗体3D1和5F4,检测临床样品与对照试剂比较总符合率为100%。结论:筛选能稳定分泌抗体的细胞株,配对抗体应用于侧向免疫层析检测方法中,能快速、特异、灵敏的检测出临床样品中H-FABP,为临床应用快速检测H-FABP指标提供了方法和关键材料。     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

抗牙龈卟啉单胞菌血凝素2单克隆抗体的制备

目的制备抗牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)血凝素2(hemagglutinin-2,HA-2)的单克隆抗体(monoclonal antibody,mAb)。方法用重组HA-2(recombinant HA-2,rHA-2)免疫BALB/C小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,间接ELISA方法筛选杂交瘤细胞.用ELISA方法测效价。结果获得1株能够高效识别rHA-2的mAb,命名为4F11。此单克隆抗体的免疫球蛋白亚类为IgG1,效价达1?106。结论成功制备了重组牙龈卟啉单胞菌血凝素2的单克隆抗体mAb.将进一步用于牙龈卟啉单胞菌的诊断,并为牙周疾病的治疗研究奠定基础。     

Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.     

Passive Protection Assay of Monoclonal Antibodies Against Dengue Virus in Suckling Mice

Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are highly infectious diseases caused by dengue virus (DV). Specific monoclonal antibodies (mAbs) against DV are vital for diagnosis, pathological studies, and passive immune therapy. In this study, purified DV serotype 2 (DV2) was used as antigen and BALB/c mice were immunized to induce specific antibodies. We established five hybridoma cell lines, called 78#, 1E7, 7F7, 8F12, and 8H1, respectively, and evaluated them by enzyme-linked immunosorbent assay, indirect immunofluorescence assay, Western blot, plaque reduction neutralization test, and suckling mice protection assay. Lines 78#, 1E7, 7F7, and 8F12 showed a neutralizing effect, and lines 78#, 1E7, 8F12, and 8H1 recognized envelope glycoprotein of DV2. Among them, lines 78# and 8F12 had stronger neutralizing ability in vitro and could protect some suckling mice from virus challenge. Our results demonstrate that immunization with purified virion is efficient for the production of specific neutralizing mAbs against DV2, and these mAbs could be useful tools for studying or treating DV infection.     

人生长分化因子15单克隆抗体制备、鉴定及药用价值初探

目的:制备稳定分泌抗人生长分化因子15(GDF15)单克隆抗体(m Ab)的杂交瘤细胞系,并对其分泌的m Ab进行鉴定。方法:根据人GDF15氨基酸序列特征,设计合成了8条能够免疫产生GDF15特异性抗体的抗原多肽,与VLP载体偶联后,免疫雌性BALB/c小鼠,利用杂交瘤技术制备鼠源抗人GDF15的m Ab,用间接ELISA检测m Ab腹水效价。结果:获得针对7个抗原多肽的12株稳定分泌抗人GDF15的杂交瘤细胞系,腹水m Ab效价可达1×104~1×109。结论:获得了针对不同抗原多肽的抗人GDF15的特异性m Ab,为进一步研发以GDF15为靶点的单克隆抗体抗肿瘤药物奠定了基础。     

Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis^®), a humanized IgG₁ monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2–5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.     

Antibody-dependent cell-mediated cytotoxicity against cells infected with respiratory syncytial virus: characterization of in vitro and in vivo properties

An in vitro 51Cr release assay for human antibody-dependent cell-mediated cytotoxicity (ADCC) against HeLa cells infected with respiratory syncytial virus (RSV) has been characterized by using leukophoresed and adherent cell-depleted adult lymphocytes. Lymphoytes from RSV seronegative children were also competent as effector cells. Sera from children with :1) primary and recurrent natural RSV infections, or 2) live attenuated RSV vaccine infection were examined to characterize the behavior of ADCC antibody in vivo. After natural RSV infection ADCC antibody rose and fell more rapidly than neutralizing antibody. In two children undergoing primary RSV infection with attenuated vaccine, neutralizing antibody was formed in the absence of detectable ADCC antibody. The nonparallel behavior of ADCC and neutralizing antibodies suggests the heterogeneity of either the antigen involved or the mechanism of antibody production in the two antibody systems.     

A recombinant Fab neutralizes dengue virus in vitro.

A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11. The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced. The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs. The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e. nanomolar. Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro.     

A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV

In response to SARS-CoV infection, neutralizing antibodies are generated against the Spike (S) protein. Determination of the active regions that allow viral escape from neutralization would enable the use of these antibodies for future passive immunotherapy. We immunized mice with UV-inactivated SARS-CoV to generate three anti-S monoclonal antibodies, and established several neutralization escape mutants with S protein. We identified several amino acid substitutions, including Y442F and V601G in the S1 domain and D757N and A834V in the S2 region. In the presence of each neutralizing antibody, double mutants with substitutions in both domains exhibited a greater growth advantage than those with only one substitution. Importantly, combining two monoclonal antibodies that target different epitopes effected almost complete suppression of wild type virus replication. Thus, for effective passive immunotherapy, it is important to use neutralizing antibodies that recognize both the S1 and S2 regions.     

Effect of breast feeding on the development of anti-idiotype antibody response to F glycoprotein of respiratory syncytial virus in infant mice after post-partum maternal immunization

Groups of lactating BALB/c mice were immunized in the immediate postpartum period with high doses of mAb (Ab-1) to the F-glycoprotein (F-gp) of respiratory syncytial virus (RSV). This antibody possessed neutralizing activity against the whole virus. The immune response to F-gp was studied in the breast feeding infant mice of such mothers at regular intervals before and after weaning. All infant mice exhibited anti-F-gp activity in serum, which was detected until 6 wk of age. Splenic cells of such breast feeding infant mice collected after weaning exhibited in vitro synthesis of antibody against Ab-1, the antibody previously used for maternal immunization. Subsequent immunization with homologous purified RSV F-gp resulted in a booster response for IgG, IgM, and plaque-neutralizing antibody to the immunizing RSV protein and to the whole virus in the infants primed via breast feeding. The enhanced antibody response was specific for the antigenic epitopes of the virus protein recognized by the monoclonal antibody used for maternal immunization. Similar booster effect was seen in control infants of nonimmunized mothers who were immunized with a polyclonal Ab-2 prepared by repeated immunization with Ab-1 in DBA/2 mice. These data demonstrate induction of RSV-specific anti-idiotype antibody in the neonates via the process of breast feeding, secondary to maternal immunization after the delivery of the neonate. Use of idiotypic vaccines in the mother and the transfer of RSV-specific idiotypes may offer an alternate approach to the development of effective immunoprophylaxis for RSV infection in early infancy.