鼠抗粒细胞集落刺激因子单克隆抗体的研制

本文运用杂支瘤技术，成功地建立了６株能稳定分泌小鼠杭重组人粒细胞集落刺激因子（Ｇ－ＣＳＦ）单克隆抗体的杂交瘤细胞１Ｂ９Ｄ１０、１ＧＳＦ１０、２Ｃ８Ｃ１、２Ｅ４Ｆ６、３Ｃ６Ｅ１１、７Ｄ２Ｃ７）。试验结果表明，６株单抗均为１ｇＧ类，且特异性强，能分别识别不同的抗原结合位点，相对亲和力２Ｅ４Ｆ６单抗最高，１ＧＳＦ１０最低，为今后Ｇ－ＣＳＦ单抗的应用打下了基础。     

Definition of glomerular antigens by monoclonal antibodies produced against a human glomerular membrane fraction.

Experimental animal models of glomerulonephritis (GN) produced by direct antibody binding to non-basement membrane glomerular capillary wall antigens do not to date have human parallels. To examine the potential for this form of humoral glomerular injury in man, we sought to define discrete human non-GBM glomerular antigenic targets using hybridoma technology. Mice were immunised intraperitoneally with 20-100 micrograms of a human glomerular membrane fraction (HGMF). Six fusions have yielded 12 stable reagents defined by positive glomerular indirect immunofluorescence (IF) and microELISA using HGMF as the screening antigen. Subclass analysis of ascitic McAbs indicated several IgG1, one IgG2b, and three IgM reagents. Distinctive IF patterns of reactivity with epithelial, endothelial or mesangial structures have been observed, with or without peritubular capillary, tubular basement membrane and vessel wall reactivity. Seven normal non-renal human organs and the kidneys of rat, rabbit and sheep have shown patterns characteristic of each individual McAb, restricted to human or with species cross reactivity. To partially characterise McAb-reactive antigens, detergent-solubilised renal cortex and collagenase-solubilised GBM (CS-GBM) extracts have been probed by immunoblot. A unique McAb 7-5Q, reactive with glomerular and tubular epithelial structures, binds major bands of approximately 107 KD and 93 KD in detergent solubilised cortex and a single band of similar size by immunoprecipitation (110 KD). 5-3A (a human-restricted linear-reacting McAb) binds bands of 20-200 KD (major band 58 KD) in CS-GBM. In conclusion, distinct species-restricted and more broadly disposed glomerular epitopes are definable in man by McAbs and are potential targets for humoral injury. Purification of these antigens will allow assay for circulating putative nephritogenic auto-antibody and potentially, McAbs may be useful in screening urine for evidence of occult structural renal disease.     

抗HCMV不同多肽McAb的鉴定

目的为了将单克隆抗体 (Mc Ab)用于诊断技术 ,我们首先建立了 13株抗人巨细胞病毒 (HCMV)的 Mc Ab,选取 6株 Mc Ab作进一步鉴定。方法这 6株 Mc Ab的鉴定主要采用间接免疫荧光试验、免疫印迹试验等方法。结果间接免疫荧光试验表明 :Mc Ab8B8相应的多肽为早期抗原 ;而 Mc Ab7B4、7D7、7E7、7E11、8D6的相应病毒多肽为晚期抗原。免疫印迹试验的结果表明 :Mc Ab7B4、7D7、7E7、7E11、8B8、8D6相应的病毒多肽分子量分别为 46、15 0、38、5 1、72、6 5 kd。结论 Mc Ab8B8可不用于 HCMV的快速诊断。     

重组人α干扰素单克隆抗体的研制及其应用

目的　研制抗干扰素的单克隆抗体 (McAb)及其应用。方法　应用杂交瘤技术 ,获得 4 0株抗重组人α干扰素的单克隆抗体细胞株 ;用ELISA方法检测腹水滴度 ,用辛酸法纯化McAb。结果　 4 0株细胞中 2E9、4G1能稳定分泌抗重组人α1b干扰素的单克隆抗体 ,2C9为抗重组新型干扰素 ,2A7、4G10分泌抗重组人α干扰素 (α1b、α2b、α2a、)和重组集成干扰素、重组新型干扰素的单克隆抗体细胞系 ;体外连续传代 6个月 ,分泌抗体能力不变 ,特异性专一。 5株单克隆抗体均为IgG。采用辛酸方法提纯抗体纯度达到 95 %以上。粗制的重组人α干扰素经 4G10单克隆抗体亲和层析柱提纯后 ,获得的干扰素纯度 95 %以上 ,平均收率 93% ,残余鼠IgG含量 <10 0ng 剂量 (5 0 μg)。 2C9单克隆抗体亲和层析柱适用于纯化新型干扰素。结论　 4G10单克隆抗体亲和层析柱可以应用于大规模重组人α干扰素生产。     

人巨细胞病毒单克隆抗体的研制检定和应用

采用人巨细胞病毒（ＨＣＭＶ）ＡＤ１６９株作为免疫原，制备出１３株鼠－鼠杂交瘤细胞系。对其中的６株进行了检定．免疫印迹试验结果表明：单克隆抗体（ＭｃＡｂ）７Ｂ４、７Ｄ７、７Ｅ１１、８Ｅ８和８Ｄ６相对应的ＨＣＭＶ多肽分子量分别为４６、１５０、３８、５１７２和６５ｋＤ．ＨＣＭＶ感染人胚肺二倍体细胞（２ＢＳ）后不同时间制成抗原片，与ＭｃＡｂ作间接免疫荧光试验。结果表明：ＭｃＡｂ８Ｂ８相应的病毒多肽为即刻早期抗原，其它５株ＭｃＡｂ相应的病毒多肽均为晚期抗原，６株ＭｃＡｂ等量混合后，标上辣根过氧化物酶，用于ＩｇＭ抗体捕获法ＥＬＩＳＡ（ＭａｃＥＬＩＳＡ）中，并与间接ＥＬＩＳＡ（ＩＥＬＩＳＡ）同时检测ＨＣＭＶ－ＩｇＭ．在未经选择的１００份脐带血中，两法均为阳性的３份，两法均为阴性的９４份；ＭａｃＥＬＩＳＡ阳性而ＩＥＬＩＳＡ阴性的２份血清的特异性试验证明，ＨＣＭＶ－ＩｇＭ确为阳性．ＩＥＬＩＳＡ阳性而ＭａｃＥＬＩＳＡ阴性的１份血清的特异性试验证明，它是由ＲＦ引起的假阳性。     

鼠抗丙型肝炎病毒（HCV）NS3区单克隆抗体的研制及特性分析

用淋巴细胞杂交瘤技术，制备出５株抗ＨＣＶ－ＮＳ３区单克隆抗体（ＭｃＡｂ）分别命名为Ｃ７－１，３，６，８，９。单抗特性测定结果显示５株单抗均为ＨＣＶ－ＮＳ３区特异性，与ＨＣＶ其它区域的抗原及宿主菌成份均无交叉反应，此５株单抗识别ＮＳ３区上两个不完全相关的抗原位点，其中Ｃ７－１，３，６株识别同一抗原决定簇，Ｓ７－９的识别位点与此３株有一定的相关，Ｃ７－８则识别与此４株不完全相关的位点。ＨＣＶ－ＮＳ３抗     

Antigenic phenotype of chronic granulocytic leukaemia progenitor cells in chronic phase and in blastic transformation characterized by means of monoclonal antibodies

We studied the surface antigens of "early" and "late" granulocyte-macrophage progenitor cells (CFU-GM) from bone marrow and peripheral blood of 18 patients with chronic granulocytic leukaemia in chronic phase (CGL-CP) using 14 selected murine monoclonal antibodies (McAbs) in complement dependent cytotoxicity assay followed by culture in methyl cellulose. The same panel of McAbs was used to determine the antigens on leukaemic blast cells from peripheral blood of 15 patients with CGL in blastic transformation (BT) by complement mediated lysis and in vitro culture technique for clonogenic blasts (CFU-L) and immunofluorescence assay for total blast population. McAb for HLA-DR antigens (L243) and McAbs MY9, S3-13, S17-25 and 53/6 reacted with CFV-GM and CFU-L. In contrast, McAbs PM81 and AML-2-23 recognizing antigens on more mature myeloid cells did not react with these progenitor cells. McAb S4-7 reacted with the majority of CFU-L and a small proportion of "early" and "late" CFU-GM. This McAb may be useful for the prediction of blastic transformation in CGL patients. Generally, the reactivity of most McAbs was more heterogeneous with CFU-L than with CFU-GM in individual patients. The majority of McAbs included in our study reacted with a higher percentage of CFU-L and CFU-GM than predominant blast cell population in individual patients perhaps because the detected antigens are expressed more strongly on dividing progenitors than on relatively nonproliferative progeny. Thus we interpret the results of these studies showing that the antigenic phenotypes of the blast colony progenitor cells in CGL-BT are very similar to but not identical with those of CFU-GM from CGL-CP patients.     

应用免疫金标记技术分析福氏2a痢疾杆菌膜抗原

本文报告应用抗福氏２ａ痢疾杆菌膜成份ＭｃＡｂ及胶体金探针对细菌膜抗原进行了定位。结果显示４种抗脂多糖ＭｃＡｂ（３Ａ６、２Ｅ６、４Ｆ１．２Ａ３）可不均一的识别位于细菌表面的抗原，而另外一种抗ＬＰＳＭＣＡｂ（１Ｇ８）和一抗膜蛋白ＭｃＡｂ（１Ａ１）识别的抗原未暴露于细菌表面。应用细菌膜碎片包埋前染色成功地定位了抗胰蛋白ＭｃＡｂ１Ａ１位于细菌内膜的抗原。与ＭｃＡｂ的生物学活性比较表明。阳性标记细菌表面抗原的ＭｃＡｂ的抗原与其阻断志贺氏菌接触性溶血试验和对小鼠的被动保护力密切相关。提示免疫电镜标记技术确定抗原表位在细菌表面的可及性和拷贝数对构建和筛选痢疾工程菌苗具有重要意义。     

通过HIT_(8a)和HIT_(8b)两个单克隆抗体鉴定发现CD_8抗原有不同的表位

用间接免疫荧光法、免疫组化技术鉴定了HIT_(8a)和HIT_(8b)两个单克隆抗体(单抗)对各种正常组织、细胞系和各种白血病患者细胞的反应性,证明其反应模式与CD8单抗相似。HIT_(8a)、HIT_(8b)识别的胸腺细胞上抗原分子量分别为 59和60KD(未还原),也与CD8近似。然而,竞争抑制试验显示,9个国际命名的 CD8单抗中只有5个可以完全抑制 HIT_(8a)、HIT_(8b)-FITC与靶细胞结合,4个无作用。证实HIT_(8a)和 HIT_(8b)是 CD8样抗体,识别同一抗原位点。但也揭示 CD8抗原有不同表位。本文对此进行了讨论。     

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.     

14株抗乙型肝炎表面抗原单克隆抗体与变异表面抗原的反应模式及应用

目的制备抗乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)的单克隆抗体,检测单克隆抗体与15种变异HBsAg的反应模式。用筛选出的单抗建立快速检测变异HBsAg的ELISA实验方法,并做初步评价。方法用中国乙型肝炎病毒感染者血清中分离的HBsAg免疫BALB/c小鼠,通过杂交瘤细胞融合技术制备抗-HBs单克隆抗体。检测不同单克隆抗体与野生及变异HBsAg的反应性。筛选出两种可以较好识别变异HBsAg的单克隆抗体McAb2和McAb3,建立两种抗体ELISA检测HBsAg的方法。结果制备了14株抗-HBs单抗。经过初筛,有4种可以较好识别包括G145R在内的大多数变异HBsAg。优化了McAb2和McAb3检测HBsAg的条件,检测HBsAg的灵敏度较好,检测变异HBsAg的能力优于2种现行国产HBsAg检测试剂盒。结论用本实验制备的单抗可以很好地识别包括G145R在内的大多数变异HBsAg。     

抗人IL-11单克隆抗体的研制及生物学特性研究

以rhIL 11作为免疫原常规免疫BALB/c小鼠 ,采用细胞融合、ELISA检测和杂交瘤细胞克隆筛选等方法获得了 2株鼠抗人IL 11的单克隆抗体 (5A4、 1G12 ) ,分别属于IgG1亚类和IgG2a亚类。MTT法和细胞增殖试验分析表明 ,5A4、 1G12均不同程度地抑制IL 11对其依赖株细胞B9 11的增殖效应。表明成功地研制成了 2株鼠抗人IL 11的中和功能性单克隆抗体     

Use of monoclonal antibodies reactive with secretory epithelial cells for the immunocytochemical identification of plasma cells

Six monoclonal antibodies (McAbs) were identified as plasma cell-reactive when screened on sections of human tonsil. They were all produced following immunisation of mice with cells of a human plasmacytoid line. Three of the antibodies also stained the cytoplasm (but not the surface) of blood B cells and were unreactive with other leucocytes; one McAb showed broad lymphocyte reactivity and two were completely unreactive with blood leucocytes; on testing with a panel of cell lines specificity for the plasmacytoid line was demonstrated by three of the McAbs. In spite of the marked restriction shown by the reactivity of these antibodies in tests on cells of haemopoietic origin, tests on other human tissues - including thyroid and pancreas - showed that a related antigen was present in the cytoplasm of secretory epithelial cells. The overall patterns of reactivity of the individual McAbs on various tissues and blood lymphocytes were different. Comparisons were made with the established McAb OKT10, which binds to plasma cells, early stem cells and activated lymphocytes; its binding to plasma cells was confirmed and it was shown that it did not stain secretory epithelia. The potent reactions obtained with the new McAbs suggest that antibodies to antigens associated with epithelial cell secretory apparatus provide potentially useful reagents for studying plasma cells.     

O1群小川型霍乱弧菌单克隆抗体的制备与鉴定

目的制备抗O1群小川型霍乱弧菌(VibriocholeraeO1serotypeOgawa)特异性单克隆抗体(McAb),为霍乱的早期快速诊断提供有力的抗体工具。方法以灭活的O1群小川型霍乱弧菌免疫Balbc小鼠,通过杂交瘤技术制备针对O1群小川型霍乱弧菌的McAb,以间接ELISA法对所需的杂交瘤细胞株进行筛选,分析其亚类,检测其效价及相对亲和力,以间接ELISA和Westernblot鉴定McAb特异性,并进行McAb结合表位分析。结果融合了602株能分泌抗O1群小川型霍乱弧菌McAb的杂交瘤细胞株,最后得到5株能稳定分泌特异性的针对该McAb的细胞株,其抗体亚类分别为3株IgG1,1株IgG2b,1株IgG3;腹水效价均达1×10-6;亲和常数在1×108～1×109之间。间接ELISA法及Westernblot证实所获的McAb可与O1群小川型霍乱弧菌发生特异性反应。ELISA相加实验结果显示除有2株McAb识别相同的抗原表位外,其余均识别不同的抗原表位。结论获得霍乱弧菌O1群小川型特异性McAb,为O1群小川型霍乱早期快速诊断和发病机理的研究提供基础。     

Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen

Three monoclonal antibodies (MAbs), designated 7C5, 7C9, and 5G8, against a cytoplasmic antigen of Cryptococcus neoformans were produced. MAbs 7C5 and 7C9 recognize culture filtrate antigen (exoantigen) of both encapsulated and nonencapsulated isolates of this pathogen, which suggests that they do not recognize capsular polysaccharide material. This is supported by immunofluorescence data which show reactivity of all 3 MAbs to cytoplasm and cell membranes only. MAb 7C9 also recognized C. neoformans var. gattii antigens but no other fungal pathogens tested in an enzyme-linked immunosorbent assay, while 7C5 and 5G8 recognized antigens of the cross-reactive pathogen Trichosporon beigelii but did not recognize either C. neoformans var. gattii isolates or any other fungal antigens. By Western blot (immunoblot), 7C9 detected antigen at 110 to 120, 65 to 70, 45 to 50, and 36 to 38 kDa; in addition to the latter band, the other two MAbs recognized a band at approximately 30 kDa. All three MAbs were of the immunoglobulin G1 subclass. The two MAbs which are capable of reacting with noncapsular culture supernatant antigen have possible uses in serodiagnosis, particularly in AIDS patients infected with C. neoformans, since in this group the present latex agglutination test has some limitations.     

抗膜抗原的单抗对痢疾杆菌入侵HeLa细胞的影响

目的 观察抗痢疾杆菌膜蛋白和LPS的单抗对痢疾菌入侵HeLa细胞的影响。方法 采用不同膜抗原的单抗处理痢疾杆菌后进行HeLa细胞入侵及阻断实验，观察不同膜抗原单抗对痢疾杆菌入侵的作用。结果 观察到抗菌膜蛋白IpaB的单抗和LPS的单抗均不能阻断细菌的入侵且能促进细菌的入侵，在HeLa细胞胞浆内出现成簇的S．flexneri-2a菌，入侵菌数远远超过未经单抗处理的S．.flexneri-2a菌感染的HeLa细胞组。结论 提示痢疾杆菌的抗菌膜蛋白单抗和LPS单抗所识别的表位，均具有调节S．flexneri-2a菌进入HeLa细胞的能力。     

Detection of Legionella pneumophila antigens in patients' sera using monoclonal antibodies

Three monoclonal antibodies (McAb) were produced against soluble antigens of Legionella pneumophila serogroup 1 which was cultured on BCYE agar. The McAbs were all of the IgM isotype. The McAbs were used in the McAb-based ELISA for detection of circulating L. pneumophila antigens in 186 sera collected from patients with symptoms and signs suggestive of atypical pneumonia. The normal reference optical (OD) density value of each of the McAbs was determined using 44 sera collected from healthy blood donors. The antigen positivity rates for the McAbs 1C7.2B, 2B2.10F and 2B2.11E were 11.3%, 7.7% and 22.2% respectively. Antigen positivity of the McAb 2B2.10F was significantly higher in the younger age group (p < 0.05). There is no significant association between the antigen positivity with age and sex for all the McAbs. There was no cross-reaction demonstrated between the McAbs with other bacterial antigens.     

单克隆抗体在沙门氏菌血清学鉴定中的应用研究

应用肠杆菌科共同抗原单抗AA9、沙门氏菌属特异单抗试剂(CB_8+de7)、沙门氏菌O、H抗原单抗对1271株菌株进行鉴定,试验结果表明,AA9和CB_8+de7可用于沙门氏菌科、属的初步鉴定;沙门氏菌O、H抗原单抗只能选择性地与具有相应抗原的菌株的发生反应,而不与其它菌株产生交叉反应,其敏感性和特异性优于常规血清,完全可以取代后者用于沙门氏菌血清群、型的鉴定。可见,沙门氏菌单抗的应用为沙门氏菌的鉴定提供了新方法,文中进一步讨论了上述单抗的应用前景。     

抗淋球菌IgA蛋白酶单克隆抗体特性的初步研究

本文对以重组淋球菌ＩｇＡ蛋白酶为抗原制备的７株特异性ＭｃＡｂ的特性进行了初步研究。结果有５株（１Ｃ８、１Ｅ５、１Ｆ１１、１Ｇ３和２Ｅ６）能中和淋球菌ＩｇＡ蛋白酶活性。经相加试验初步证明，其中１Ｆ１１、２Ｇ３与其它３株ＭｃＡｂ的作用位点不同。因此，淋球菌ＩｇＡ蛋白酶至少存在３个中和表位。