抗rhNDPK-A单克隆抗体的制备及鉴定

目的 :研制抗rhNDPK A (recombinanthumannucleosidediphosphatekinase A)单克隆抗体 (mAb) ,并鉴定其特性。 方法 :以纯化的rhNDPK A免疫BALB/c小鼠 ,采用杂交瘤技术制备抗rhNDPK AmAb ;用免疫双扩散鉴定Ig亚类 ;West ernblot鉴定mAb的特异性 ;间接ELISA检测mAb的腹水效价、亲和常数 ,并进行表位分析。结果 :获得 6株可分泌特异性mAb的抗rhNDPK A的杂交瘤细胞系 2D9、8C7、13E2、15D9、15E3和 2 0D9,Ig亚类均为IgG1;其效价为 1× 10 -4～5× 10 -6;亲和常数为 4 .5× 10 -9～ 2 .8× 10 -10 mol/L ;共有3个抗原表位。结论 :获得抗rhNDPK A的mAb ,为进一步用于临床诊断和实验研究创造了条件     

抗SARS冠状病毒N蛋白单克隆抗体的制备和初步应用

目的:研制抗SARSCoVN蛋白的单克隆抗体(mAb)。方法:以纯化的GSTN免疫BALB/c小鼠,采用杂交瘤技术制备抗SARSCoVN蛋白mAb;用免疫双扩散鉴定Ig亚类;Westernblot和免疫组化鉴定mAb的特异性;间接ELISA检测mAb的腹水效价、相对亲和常数。结果:获得1株可分泌特异性mAb的抗SARSCoVN蛋白的杂交瘤细胞系3E10H,Ig亚类为IgG2b;其腹水效价为8×10-5;其相对亲和力1.725×10-10mol/L,Westernblot和免疫组化阳性。结论:获得特异性抗SARSCoVN蛋白的mAb,为进一步用于临床诊断和实验研究创造了条件。     

抗死亡受体-5单克隆抗体的制备及特性鉴定

目的:研制抗DR5单克隆抗体(mAb),并鉴定其特性。方法:以纯化的DR5免疫BALB/c小鼠,采用杂交瘤技术制备抗DR5mAb。用Ig亚类ELISA试剂盒鉴定抗DR5mAb的亚类。用ELISA法测定sDR5对抗DR5mAb与DR5结合的阻断作用及流式细胞术检测与Jurkat细胞膜上DR5结合的阻断作用,以鉴定mAb的特异性。用间接ELISA法检测腹水mAb的效价、亲和常数并进行表位分析。结果:获得4株可分泌抗DR5mAb的的杂交瘤细胞系YM366EC、YM366ED、YM369F5和YM369E6。4株mAb的Ig亚类均为IgG1;腹水mAb效价为1×10-4~5×10-6;亲和常数为1×109水平,4株mAb可识别2种不同的抗原表位。结论:获得4株抗DR5的mAb,为进一步用于临床诊断和实验研究创造了条件。     

抗大豆Ⅱ号亚型钙调素单克隆抗体的制备与鉴定

目的:制备抗大豆Ⅱ号亚型钙调素(SCaM-2)单克隆抗体(mAb)并进行初步鉴定。方法:从含有pSCaM-2的大肠杆菌BL21中,提取并纯化SCaM-2。以其作为抗原免疫BALB/c小鼠后,取其脾细胞与小鼠骨髓瘤细胞经细胞融合筛选及克隆化,建立稳定分泌SCaM-2mAb的杂交瘤细胞株并对抗SCaM-2mAb进行鉴定。结果:经两次细胞融合,共获得10株可稳定分泌抗SCaM-2mAb的杂交瘤细胞株。对其中3株杂交瘤细胞制备的腹水mAb(6F11F3、7C5E9、7E10E2)用间接ELISA及免疫印迹实验表明,在高稀释度时,mA6F11F3可识别SCaM-1和SCaM-2,而mAb7C5E9和7E10E可分别识别SCaM-4和SCaM-5。Ig亚类鉴定结果表明,mA6F11F3和7C5E9均为IgG2a,mAb7E10E2为IgG1。相加实验的结果显示,mAb6F11F3和7C5E9可能识别SCaM-2相同的抗原表位;而mAb7E10E2可能识别不同的抗原表位mAb6F11F3对SCaM-2的亲和常数为(2.127±0.418)106M-1,mAb7C5E9对SCaM-4的亲和常数为(9.69±2.406×106M-1,mAb7E10E2对SCaM-5的亲和常数为(1.2030.429)×107M-1。结论:制备了可识别不同SCaM亚型的mAb6F11F3、7C5E9及7E10E2,为研究SCaM的特性及功能提供了重要的制剂。     

两株登革2型病毒E基因重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 用含登革2型病毒(Dengue type 2 virus,DEN2)B株和NGC株E基因部分序列pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 用两株含DEN2 E基因部分序列(1～476 bp)的pcDNA3.1重组质粒与含有佐剂的重组质粒共同免疫BALB/c小鼠,初次免疫后第14天、28天分别加强免疫1次,共免疫3次.收集初次免疫后第14、28、42、70和98天外周血标本,间接ELISA法测定小鼠血浆特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异性抗体水平.结果 不同DEN2毒株E基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类抗体的产生存在差异,B株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DEN2两毒株E基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.     

阪崎肠杆菌单克隆抗体的制备与特性鉴定

目的:制备抗阪崎肠杆菌的单克隆抗体并对其生物学特性进行鉴定。方法:采用灭活的阪崎肠杆菌菌体为抗原,免疫BALB/c小鼠,取血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞,mAb亚类检测试剂盒鉴定单克隆抗体的亚型,通过Western blot和间接ELISA法鉴定该单克隆抗体的特性、效价及mAb相对亲和力。结果:获得2株能稳定分泌抗阪崎肠杆菌mAb的杂交瘤细胞株,分别命名为1H7、2B12,抗体Ig亚类分别为IgG1和IgG2b;交叉反应显示单抗具有良好的特异性;ELISA分析表明制备的单抗效价在1×107～2×107,相对亲和常数达109L/mol,染色体鉴定分别为104和106条,符合杂交瘤细胞的特性。结论:抗阪崎肠杆菌单克隆抗体的成功制备为其快速检测方法的建立奠定了基础。     

AsiaⅠ口蹄疫病毒VP1蛋白单克隆抗体的制备与鉴定

目的:制备AsiaI口蹄疫病毒VP1蛋白单克隆抗体(mAb)并进行鉴定。方法:用纯化的AsiaI型口蹄疫病毒VP1表位重组蛋白为抗原免疫6～8周龄的雌性BALB/c小鼠,经过3次免疫后,取其脾细胞与Sp2/0骨髓瘤细胞融合。采用有限稀释法和间接ELISA克隆和筛选阳性杂交瘤细胞株,用SDS-PAGE电泳、间接ELISA以及微量细胞中和试验对所获得的mAb的特异性进行鉴定。结果:成功获得3株能稳定传代并分泌抗AsiaImAb的杂交瘤细胞株,分别命名为:1B8、5E1、5E2,其分泌的mAb为IgG1(1B8)和IgG2a(5E1、5E2)亚类,他们均能特异性的识别VP1重组蛋白和AsiaI型全病毒,其腹水效价在1:105～1:106。微量细胞中和试验表明该mAb能很好地识别灭活的FMDV,中和效价达1:1024以上。交叉试验表明该mAb具有高度特异性,型间无交叉反应,证明所获得的mAb均完全针对AsiaI FMDV抗原决定簇。结论:在口蹄疫病毒mAb的研究中,VP1重组蛋白有望代替活病毒来制备mAb。AsiaI口蹄疫病毒VP1mAb的成功制备,为进一步研究和开发新型口蹄疫的检测方法和抗原表位奠定了基础。     

抗HCV HVR1模拟合成肽单克隆抗体的制备及特性鉴定

目的:制备抗-HCV高变区1(HVR1)合成肽单克隆抗体(mAb),并对其特性进行鉴定。方法:以固相合成的HCV HVR1合成肽与牛血清白蛋白(BSA)偶联后,免疫8周龄的BALB/c小鼠。采用杂交瘤技术,取免疫小鼠的脾细胞与骨髓瘤细胞融合,制备抗-HCV HVR1 mAb。经HAT、HT选择培养及有限稀释法进行克隆化后,用相关的试剂盒鉴定小鼠mAb的Ig亚类;经中和抑制后用ELISA法检测mAb的特异性;用间接ELISA法检测mAb腹水的效价及相对亲和常数。结果:获得1株可分泌抗-HCV HVR1合成肽特异性mAb的杂交瘤细胞1A9G9F11。该株mAb的Ig亚类为IgG3;腹水效价为3.125×10-5;相对亲和常数为1.0×106L/mol。该株mAb与HBsAg、HBeAg、HBcAg、HAAg、牛血清白蛋白、酪蛋白和胸腺5肽均无交叉反应,与HCV HVR1合成肽-牛血清白蛋白出现特异性反应。结论:成功地建立了1株可分泌抗-HCV HVR1 mAb的杂交瘤细胞1A9G9F11,为进一步的实验研究提供了重要的制剂。     

抗ICAM-1单克隆抗体的制备与特性鉴定

目的:制备有生物学活性的抗人细胞间黏附分子-1(ICAM-1)单克隆抗体(mAb)并进行特性鉴定。方法:以纯品ICAM-1为抗原免疫BALB/c小鼠4次。采用杂交瘤技术,经3次亚克隆筛选稳定分泌抗人ICAM-1 mAb的杂交瘤细胞株。采用动物体内诱生的方法大量制备mAb。以protein G对其进行纯化后,用间接ELISA法测定mAb的效价并鉴定其Ig亚类。用Western blot鉴定mAb的特异性。结果:筛选出4株可稳定分泌抗人ICAM-1 mAb的杂交瘤细胞株,分别命名为B9株、F1株、C11株和D6株。4株mAb的Ig亚类均为IgG1。4株mAb培养上清的效价均为1∶1 000;腹水的效价B9株与D6株为1∶2×105,F1株与C11株为1∶4×105。纯化后mAb的蛋白浓度为1.2 g/L,均可与ICAM-1特异性结合。结论:成功制备出效价高、特异性良好的4株抗ICAM-1 mAb,为进一步研究ICAM-1的生物学功能和临床应用奠定了基础。     

抗李斯特菌溶血素单克隆抗体的制备及初步鉴定

目的: 研制抗李斯特菌溶血素(LLO)的单克隆抗体(mAb).方法: 通过诱导重组大肠杆菌BL21(pGEX-6p-1-hly), 以SDS-PAGE分离菌体蛋白, 切割目的蛋白LLO-GST条带, 研磨粉碎后免疫BALB/c小鼠, 取免疫鼠脾细胞与Sp2/0细胞融合.利用亲和层析法纯化目的蛋白, 作为检测抗原进行杂交瘤细胞的筛选.采用间接ELISA法测定腹水效价, Dot-ELISA、 Western blot分析mAb的特异性.结果: 获得3株稳定分泌抗LLO mAb的杂交瘤细胞株3B6、 4D1、 5D10, 其Ig亚类均为IgG1.3B6、 4D1、 5D10腹水的ELISA效价分别为1:2×105、 1:2×105、 1∶1×105.Western blot试验中, 3株mAb均能与融合蛋白LLO-GST发生反应出现特异性条带, 而与纯化蛋白GST不反应.在Dot-ELISA试验中, 3株mAb均能与表达LLO的细菌发生特异性反应.结果表明, mAb 3B6、 4D1、 5D10是针对LLO的特异性mAb.结论: 成功地制备了抗LLO的mAb, 为进一步研究LLO的生物学特性、产单核细胞李斯特菌的致病机制, 以及建立产单核细胞李斯特菌(LM)快速检测技术奠定了基础.     

抗Ig融合蛋白Fc段单克隆抗体的制备、鉴定与应用研究

目的：制备并鉴定抗Ig融合蛋白Fc段的单克隆抗体(mAb)，建立用于检测Ig融合蛋白的夹心ELISA法和纯化Fc融合蛋白的亲和层析法。方法：以hBCMA—Ig融合蛋白为抗原免疫BALB／c小鼠，通过细胞融合制备抗Fc段mAb，用ELISA等方法鉴定mAb的Ig亚类、表位以及种属特异性，建立用于检测Ig融合蛋白的夹心ELISA法;Western blot检测mAb对变性Ig融合蛋白的反应性。将mAb与Sepharose4B交联，制备亲和层析柱，对LAIR1-Ig融合蛋白进行纯化。结果：获得7株稳定分泌抗Fc段mAb的杂交瘤(FMUFcl-FMUFc7)。利用FMUFc4作为包被mAb，FMUFc5作为酶标记mAb，成功地建立了检测Ig融合蛋白的ELISA法，敏感度达到2μg／L；在7株mAb中，FMUFc6可用于Ig融合蛋白的western blot检测。用FMUFc 6mAb制备的亲和层析柱，可有效地纯化LAIRl—Ig融合蛋白。结论：成功地制备了抗Ig融合蛋白Fc段的mAb，建立了可用于Ig融合蛋白检测和纯化的方法，为Ig融合蛋白的应用提供了有力手段。     

Signal transduction via a protein associated with a glycosylphosphatidylinositol-anchored protein, decay-accelerating factor (DAF/CD55)

Decay-accelerating factor (DAF/CD55) is a glycosylphosphatidylinositol- anchored protein which is known to have signal transducing capacity and to be associated with several proteins. To determine the signal transducer in the DAF-forming complex, we purified DAF-associated proteins from Raji B cells using an anti-DAF mAb (1C6)-bound affinity column and established five mAb against them. Among these, mAb 2E12- G7(IgM/kappa) reacted with a variety of intact cells, including peripheral blood mononuclear cells (PBMC), as well as cells from T and B cell lines, as shown by cytofluorimetric analyses. The Mr of 2E12-G7 antigen was estimated to be 43 kDa by surface biotinylation and immunoblotting analysis. This antigen was demonstrated in 1C6 immunoprecipitates, but not in anti-CD59 (another GPI-anchored complement regulatory factor)-immunoprecipitates. Sequential treatment with 1C6 F(ab')2 and then with anti-mouse Ig F(ab')2 stimulated PBMC to induce tyrosine phosphorylation on proteins of 45, 72, 78 and approximately 100 kDa. Also, mAb cross-linked to 2E12-G7 stimulated PBMC to induce tyrosine phosphorylation on proteins of 72, 78 and approximately 100 kDa. Furthermore, when 2E12-G7 and 1C6 immunoprecipitates were incubated with [gamma-32P]ATP, the main constituents detected in both were phosphorylated proteins of 26, 32 and 62 kDa. Thus, DAF-associated 2E12-G7 antigen transduces a signal, similar to the DAF molecule.      

不同免疫方案制备抗HAb18G/CD147胞外区多克隆抗血清的比较

目的: 制备针对肝癌相关抗原HAb18G/CD147胞外区不同表位的抗血清, 比较不同免疫方案的免疫效果。方法: 以原核表达的GST -HAb18GEF融合蛋白、重组真核表达质粒pcDNA3 /HAb18G及HCC细胞为免疫原, 分别采用蛋白常规免疫、DNA肌肉免疫及pcDNA3 /HAb18G -HCCbooster(DNA -cellbooster)免疫接种BALB/c小鼠。采用间接ELISA和细胞ELISA, 同时测定免疫血清中抗变性和天然HAb18GEFIgG抗体的滴度和Ig亚类。用Westernblot检测各免疫方案制备的抗血清, 与变性HAb18GEF抗原结合的特异性。用免疫荧光法验证DNA- cellbooster免疫接种法制备的抗血清, 与细胞膜上天然HAb18G抗原的结合特异性。结果:以GST- HAb18GEF常规免疫后, 可诱导高滴度的IgG1抗体产生, 但针对的多为HAb18GEF的变性或线性表位; 以pcD -NA3 /HAb18G肌肉免疫后, 可诱导针对其天然表位的IgG2a抗体产生, 但滴度较低; 以DNA -cellbooster免疫后, 可诱导中等滴度、针对其天然表位的IgG2a和IgG1抗体产生。结论: 不同免疫方案可诱导针对HAb18GEF不同表位、不同滴度的多克隆抗血清, 为淘筛针对其不同表位的多样性抗体奠定了基础。     

Low doses of antigen coupled to anti-CR2 mAbs induce rapid and enduring IgG immune responses in mice and in cynomolgus monkeys

The complement system and B cell complement receptor 2 (CR2), specific for C component C3dg, play important roles in both the innate and adaptive immune response. We used hapten and protein conjugates of anti-CR2 mAbs as models for C3dg-opsonized antigens and immune complexes to examine the handling of and immune response to these reagents in mice and in non-human primates (NHP). Mice immunized and boosted i.v. with only 100 ng of Alexa 488 rat anti-mouse CR1/2 mAb 7G6 had strong IgG immune responses to the Alexa 488 hapten and to rat IgG, compared to very weak immune responses in mice treated with a comparable isotype control; larger doses of Alexa 488 mAb 7G6 did not increase the immune response. A vaccine constructed by cross-linking anthrax protective antigen to mAb 7G6 proved to be effective at low doses in generating sufficiently high titer serum IgG antibodies to neutralize anthrax lethal toxin in vitro and to protect mice from i.v. challenge with anthrax lethal toxin. When biotinylated HB135, a mouse mAb specific for human CR2, was injected i.v. into NHP, the probe manifested the same initial marginal zone B cell binding and subsequent localization to follicular dendritic cells as we have previously reported for comparable experiments in mice. Moreover, i.v. immunization of NHP with 1 microg/kg of Alexa 488 mAb HB135 promoted an IgG immune response to the Alexa 488 hapten and to mouse IgG. Taken together, these results demonstrate the efficacy of using anti-CR2 mAbs as antigen carriers for i.v. immunization with small amounts of antigens without adjuvant.     

IgE antibody response to mite antigen in the mouse. Suppression of an established IgE antibody response by chemically modified antigen

The antibody response to mite antigen in several mouse strains was studied. BALB/c, CBA, C3H/He, and C57B1 strains showed good responses against mite antigen. The AKR strain, on the other hand, showed a relatively poor response. When BALB/c mice were immunized with DNP-mite conjugate in aluminum hydroxide gel (alum), anti-DNP IgE antibody and IgG1 antibody were induced. When these mice were boosted with mite antigen alone in alum, both anti-mite IgE antibody and IgG1 antibody were induced, although these antibodies were not observed after the first immunization. Both IgE antibody responses were high and persistent. Mite antigen was denatured by alkylation and reduction in the presence of 8 M urea. Native antigen remaining in the chemically denatured antigen was removed by an immunoadsorbent method. The modified mite antigen could stimulate the carrier-specific helper cells, at least when it was injected with alum. It was also found that repeated injections of the modified mite antigen resulted in the suppression of an established IgE antibody response against mite antigen. These findings suggest possible clinical application for hyposensitization therapy.     

抗人LSECtin单克隆抗体的制备与初步鉴定

目的:制备抗肝脏、淋巴结窦内皮细胞C型凝集素(LSECtin)单克隆抗体(mAb),并进行特性鉴定。方法:采用原核表达的LSECtin免疫BALB/c小鼠,以间接ELISA法筛选分泌特异性mAb的杂交瘤细胞,采用蛋白印迹、间接免疫荧光、流式细胞术和免疫组化染色法鉴定mAb的特异性。结果:共获得8株可稳定分泌mAb的杂交瘤细胞株。mAb的Ig亚类均为IgG,效价达1∶106~1∶107。这些mAb均可识别转染3T3细胞膜上的人LSECtin,6株mAb可特异识别肝脏窦内皮细胞。结论:成功地制备8株抗LSECtin的mAb,经免疫印迹、流式细胞术和免疫组化染色检测,这些mAb的特异性良好,为研究LSECtin的功能提供了有力的试剂。     

Characterization of monoclonal immunoglobulin a and g against shiga toxin binding subunits produced by intranasal immunization

Immunoglobulin A (IgA) is considered to play a major role in protection of the mucosal surface. However, its immunological and biological properties have not been extensively studied because the production of IgA class monoclonal antibodies (mAbs) is difficult. We compared the properties of IgA and IgG mAbs against Shiga toxin B subunits (Stx1B). These mAbs were secreted from hybridomas that had been produced from mice after intranasal immunization with recombinant Stx1B and cholera toxin. The dose response curves for the binding of the IgA (clone G2G7) and IgG (clone D11C6) mAbs to immobilized Stx1B were similar, as revealed on ELISA. The majority of the IgA mAb formed dimers while the IgG mAb was monomeric, as judged by immunoblot analysis. The IgG mAb completely inhibited the binding of Stx1B to Burkitt's lymphoma cell line Ramos, while the inhibition by the IgA mAb was only partial. The IgG mAb was able to neutralize the cytotoxicity of Stx1 holotoxin towards Vero cells, whereas the IgA mAb was not. The binding affinity of each binding site was compared by means of surface plasmon resonance analysis involving a capture method, with which the binding of soluble Stx1B to immobilized mAb was detected. The association rate was similar but the dissociation rate was twofold faster in the case of the IgA mAb, resulting in twofold higher affinity of the IgG mAb. These results suggest that one can obtain high affinity IgA mAb but toxin neutralization is another challenge as to therapeutic antibodies of the IgA class.     

抗弓形虫P30单克隆抗体的制备及其抗虫作用观察

制备抗弓形虫天然P30、重组P30的单克隆抗体(mAb),并研究它们对虫体的影响,为弓形虫病诊断及保护性抗原鉴定奠定基础。采用弓形虫天然P30、重组P30及全虫抗原(对照)分别免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,筛选稳定分泌高滴度mAb的杂交瘤细胞株。用ELISA法测定mAb亚类和效价;通过SDS-PAGE和West-ern blot进行特异性鉴定;Giemsa染色观察杂交瘤细胞的染色体数目;利用扫描电镜及间接荧光抗体试验(IFAT)观察mAb对弓形虫的杀伤作用及其靶抗原定位。结果表明,筛选出2株(2B3、1H6)特异性较好、能稳定分泌mAb的杂交瘤细胞株。Western blot结果显示,2B3、1H6产生的mAb与弓形虫全抗原的阳性反应带均在30 000 Mr处;mAb亚类均属IgM,其效价(ELISA)分别为:2B3 1∶102 400、1H6 1∶51 200;2株细胞的染色体数均在100条以上。电镜观察与mAb作用后的弓形虫虫体聚集、肿胀,膜变厚,表面出现缺口和空洞,甚至虫体变形、破碎。抗弓形虫天然P30的mAb能识别重组体pET-30a-P30的表达蛋白。成功制备了抗弓形虫天然P30、重组P30的mAb,可进一步用于弓形虫病诊断及保护性抗原鉴定研究。     

抗人红细胞膜抗原非凝集型单克隆抗体的研制及特性鉴定

目的:制备抗人红细胞膜抗原的非凝集型单克隆抗体(mAb)并鉴定其特性。方法:应用杂交瘤技术,以人O型红细胞膜抗原免疫BALB/c小鼠。取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,用聚凝胺试管法筛选识别红细胞表面共同抗原的抗体,玻片凝集实验剔除凝集型抗体(完全抗体),再将分泌非凝集型mAb(不完全抗体)的杂交瘤细胞株用有限稀释法克隆化3次。对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果:获得1株可稳定分泌mAb的杂交瘤细胞2E8。mAb2E8为IgG1类,可特异性地识别红细胞膜上的H抗原,没有种属交叉血凝反应。杂交瘤细胞的培养上清与人的A、B、AB和O型红细胞均能产生强凝集,凝集效价为1∶1024,腹水mAb的凝集效价达到1∶64×106。mAb的亲和力用凝集试验检测,出现血凝的时间为7s,3min以内凝块>1mm。结论:成功地制备了针对红细胞膜H抗原的非凝集性mAb,此mAb的凝集效价、相对亲和力及特异性均较良好,为构建双特异性抗体奠定了基础。     

抗艰难梭菌A毒素单克隆抗体的制备及特性分析

目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具