单克隆抗体对感染流行性感冒病毒的小鼠的疗效观察

治疗流行性感冒(简称流感)对年老体弱者有着重要意义。由于流感病毒的一个新亚型出现后,往往要经历十几年至20多年的流行,因此选出具有亚型内共同抗原决定簇的McAb,用于治疗流感是可行的,文献也曾有过报道。前文已报道关于甲3型流感病毒McAb的建立,本文用抗甲3型流感病毒上海/32/84血凝素McAb,与1977年以来分离的甲3型流感     

猪戊型肝炎病毒重组蛋白ORF2-V1单克隆抗体的制备及抗原表位差异分析

用纯化的猪戊型肝炎病毒DQ株重组蛋白ORF2-V1免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0 骨髓瘤细胞进行融合,经3次克隆和间接ELISA 筛选,获得了αC11,αC12,γH1,γF8,BC4和CH8 6株稳定分泌单克隆抗体的杂交瘤细胞株。通过间接ELISA 测定,单抗效价为: 细胞培养上清1∶1.60×103～1∶3.20×103,腹水为1∶1.28×106～1∶2.56×106; 经ELISA法测定,6株单克隆抗体均与重组蛋白ORF2-V1反应, 而不与ORF2其它部分片段的蛋白反应; 将试验中制备的MAbs分别用HEV swDQ株感染的A549细胞涂片进行IFA检测,同时分别用猪的阳性、阴性血清和SP2/0细胞上清做对照,制备的6株MAbs对感染细胞均呈现阳性反应,说明其可以与天然的病毒发生反应,并且与阳性多克隆血清比较,以单克隆抗体为一抗,IFA反应表现出良好的特异性。单抗的亚类鉴定结果表明,所有MAbs均为IgG1型,而且所有单克隆抗体的轻链均为κ链。单克隆抗体抗原识别位点分析结果表明,6株单抗分别针对4个不同抗原位点,McAb-γH1,BC4,CH8识别的位点各不相同,其中McAb-γH1,BC4识别的位点有部分重叠,McAb αC11,αC12,γF8识别同一位点,位于γH1,BC4识别位点的重叠区内。     

不同基因型戊型肝炎病毒存在多种类型抗原表位

以戊型肝炎病毒(HEV)ORF2重组蛋白p166Us为免疫原制备单克隆抗体(McAbs),采用间接ELISA和免疫印迹法,检测McAbs与不同基因型和亚型HEV重组蛋白p166Bur(Ⅰa型)、p166Pak(Ⅰb型)、p166Mor(Ⅰc型)、p166Mex(Ⅱ型)、p166Us(Ⅲ型)、p166Nz(猪HEV,Ⅲ型)和p166Chn(Ⅳ型)的反应性,采用抗原或抗体竞争ELISA分析p166蛋白与天然HEV颗粒之间抗原表位的关系。结果获得4D3、2E3、11E11、12H5、3A3和1F16株稳定分泌McAbs的杂交瘤细胞株。4D3分泌的McAb与7种p166均发生反应,其与免疫原p166Us的结合可被Ⅰ、Ⅱ、Ⅲ或Ⅳ型天然HEV颗粒或病人血清竞争抑制。2E3、11E11和12H5分泌的McAbs只与p166Us、p166Nz和p166Chn发生反应,它们与p166Us的结合仅能被Ⅲ和IV型病毒或血清所抑制。3A3分泌的McAb只与p166Us及p166Nz结合,1F1分泌的McAb只与p166Us结合,两者均能被Ⅲ型美国株竞争抑制,而Ⅰ、Ⅱ、Ⅳ型不能抑制它们与p166Us的结合。由此可见,不同基因型和亚型HEV ORF2编码蛋白p166上存在多种类型抗原表位,其中包括Ⅰ、Ⅱ、Ⅲ、Ⅳ基因型共同的,Ⅲ、Ⅳ基因型共有的和第Ⅲ基因型特异的等,这些表位与天然HEV颗粒上的抗原表位具有相同的免疫学特征。     

甲型H1N1流感病毒血凝素蛋白单抗杂交瘤细胞株的制备与初步鉴定

目的:建立具有高特异、高效价的甲型H1N1流感病毒血凝素蛋白(HA)单抗的杂交瘤细胞株。方法:以纯化的昆虫杆状病毒表达的甲型H1N1流感病毒HA蛋白为免疫原免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,通过有限稀释法筛选阳性克隆,经ELISA和Western blot分析单抗的特性和特异性。结果:获得6株甲型H1N1流感HA抗原特异单克隆抗体杂交瘤细胞株,抗原肽库ELISA检测结果表明其中3株(1E12,3F12,1C11)单抗只与甲型H1N1流感HA抗原肽库反应,不与H5N1病毒HA抗原肽库反应;Western blot分析表明,单抗1B3只特异识别甲型H1N1流感HA抗原,而与其他季节性甲流病毒(H1,H3)及人禽流感H5N1病毒不反应。结论:所获杂交瘤细胞株特异性强,效价高,分泌抗体性能稳定,为分析甲型H1N1流感病毒抗原性位点、建立诊断试剂奠定了基础。     

Characterization of monoclonal antibodies to cytochrome c: analysis of the antigenic structure of holo- and apo-cytochromes, and CNBr-peptide fragments of horse cytochrome c

Five mouse hybridoma cell lines secreting SA, SB, SC, SD, and SE monoclonal antibodies (McAb) to cytochrome c have been produced. From the cross-reactivities of these McAb with various vertebrate cytochromes c, the antigenic sites for SA and SB McAb were proposed to be at Thr(89)-Glu(92)-Ala(96) and Asn(103), respectively. The binding site for other McAb have not been determined. Cross-reactivity studies based on enzyme-linked immunosorbent assays and dot immunobinding assays indicated that SA, SB, and SC McAb did not bind to apo-cytochrome c nor to any of the three CNBr-peptide fragments. This observation suggests that (i) the antigenic specificity of these McAb is dependent on the conformatiuon of the antigenic site which is inherent to the native holoprotein molecule and (ii) the ordered conformation in the C-terminal regions of holo-cytochrome c is destroyed during CNBr-peptide fragmentation. On the other hand, the lack of binding of SD and SE McAb to apo-cytochrome c indicates that these McAb are also specific for conformational sites. The binding of SD and SE McAb to the heme-containing A-peptide fragment (residues 1-65) suggests that the conformation around the heme, as possible antigenic sites, are stable because of the thioether linkages by the Cys residues.     

汉滩病毒S基因的分段表达及其线性和构象型抗原表位分析

构建汉滩病毒76—118N蛋白及其分别从N-端和C-端缺失的共6个突变体,在大肠杆菌BL-21中进行表达,并对其中一些蛋白进行了纯化。通过Western blot、酶联免疫吸附试验(ELISA)进行汉滩病毒N蛋白的抗原表位分析,N蛋白及6个缺失突变体都与组特异性抗体L13F3呈阳性反应,而缺失突变体与型特异性抗体AH30呈阴性反应。构建汉滩病毒76—118N蛋白及其6个缺失突变体的真核表达载体,并在COS-7细胞中进行表达。通过间接免疫荧光试验(IFA)进行汉滩病毒N蛋白的抗原表位分析,病人血清与真核表达的N蛋白及6个缺失突变体呈阳性反应。而仅有N蛋白及缺失N端1～30位氨基酸序列的NPN30与型特异性抗体AH30呈阳性反应。证实组特异性抗体L13F3结合的抗原表位位于N端1～30位氨基酸;而C端抗原表位对于型特异性抗体AH30与N蛋白的识别和结合具有重要意义,缺失N端100位氨基酸序列可能破坏羧基端构象型表位,也可以影响N蛋白与AH30的结合。     

抗肿瘤单抗3H11对应抗原cDNA片段的克隆

单克隆抗体３Ｈ１１能与多种肿瘤细胞特异结合,克隆其对应抗原无疑具重要意义．用胃癌细胞ＭＧＣ８０３构建ｃＤＮＡ表达文库,通过抗体３Ｈ１１对其进行原核表达筛选,获得一株能与３Ｈ１１特异反应的阳性克隆．其ｃＤＮＡ插入片段为５５４ｂｐ．ＧｅｎＢａｎｋ不含其同源序列．将此ｃＤＮＡ片段与谷胱甘肽转移酶表达质粒ｐＧＥＸ－４Ｔ重组,Ｗｅｓｔｅｒｎｂｌｏｔ和竞争抑制实验表明,表达产物依然保持同３Ｈ１１反应的特异性．可见它是３Ｈ１１对应抗原的ｃＤＮＡ．     

Characterization of monoclonal antibodies to the Zta and DNase proteins of epstein-barr virus

Two monoclonal antibodies (mAb) were derived and designated 4F10 and 311H. 4F10 was against the Epstein-Barr virus (EBV) Zta protein and 311 H specifically recognized EBV DNase enzyme. Using mAb 4F10 as a probe, the Zta protein could be detected as a 36-kD molecule in L5 cells and as a 38-kD molecule in B95-8 cells, reflecting the fact reported by other laboratories, using rabbit polyclonal antisera, that the Zta protein was variously modified in different host cells. 311H mAb was generated using antigens purified from one-step His-Bind column chromatography. The antigenic epitope recognized by this mAb was mapped within the residues 1–152 of EBV DNase by reacting the mAb with three distinct truncated mutants. Also, using 311H as a reagent to trace the kinetic expression of EBV DNase proteins in EBV-infected Akata cells, the Western blotting results indicated that DNase antigen could be detected at 12 h postactivation. The feasibility of applying these two mAb in the investigation of EBV biology is discussed.     

Monoclonal antibodies to Hammondia hammondi allowing immunological differentiation from Toxoplasma gondii

Five murine monoclonal antibodies (MAbs) were developed against purified sporozoites of Hammondia hammondi. Despite a large antigenic similarity between the 2 closely related coccidia, H. hammondi and Toxoplasma gondii, these MAbs only reacted with H. hammondi. Three MAbs, ID3, 3F2, and 4C9-7, recognized antigens of 38 kDa localized in rhoptries (1D3), in rhoptries and in oocyst and cyst walls (3F2), and in rhoptries and the apical region (4C9-7). Another MAb, 4C9-10, reacted with a 27-kDa antigen in dense granules of sporozoites and tachyzoites, and MAB 11B3 labeled an antigen of >94 kDa located in the pellicular membrane of the 3 stages of the parasite. These MAbs could be used for a rapid discrimination of the 2 coccidia in epidemiological studies or for diagnostic purposes in tissues.