CN1025219C - 制备可产生特异性抗hiv抗原的单克隆抗体的杂交瘤细胞系的方法 - Google Patents
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Abstract
提供了产生能与HIV核心抗原P55即gag基因编码之前体蛋白、核心蛋白P24及部分分解产物P39和P33结合的单克隆抗体的杂交细胞系。本发明的单克隆抗体并不与P18核心蛋白或任何HIV被膜抗原结合。本发明的细胞系是经特殊的免疫程序建立的，其中使用一组选择的免疫原经一系列多重输注免疫BALB/C小鼠，并用一常规使用的骨髓瘤细胞系与收获的小鼠脾细胞融合。
该单克隆抗体被鉴定为KC-57抗体。其特别适用于固相免疫检测法中，其中测定的是与病人血清或血浆样品中HIV抗原结合的单克隆抗体。产生KC-57单克抗体的细胞系已寄存于美国典型培养物保藏中心(Rockville，Maryland)且登记号为A.T.C.C.№.HB9585。
Description
本发明涉及以杂交瘤技术生产的单克隆抗体，特别是涉及产生一种独特之单克隆抗体的细胞系，所说的单克隆抗体识别一组自不同地区分离的人免疫缺陷病毒（HIV）gag编码蛋白的抗原决定基。
已经认识到，目前大多称为人免疫缺陷病毒（“HIV”）的Ⅲ型人T细胞白血病病毒（HTLVⅢ）是人类免疫缺陷综合症（即爱滋病，ADIS）的致病因子。鉴于爱滋病在美国和其它一些国家造成一定比例的流行这一慢性感染特征，而对其作了广泛的研究并努力开发了检测人外周血中爱滋病病毒抗原及相应抗体的可靠而稳定的免疫诊断方法。在实际检测中，也已反过来将单克隆抗体用于发展这类免疫检测法。
HIV属于还原病毒类。还原病毒携带一正链RNA并在其核心中有一特异的逆向转录酶，其被用于将病毒RNA转化为DNA。与该过程相反的典型细胞转录过程则是将DNA转化为RNA。
已知HIV结合于T4淋巴细胞上，因为T4淋巴细胞表面上的T4蛋白质可作为HIV的受体或结合位点（Dalgleish AG等，Nature 321：763-767（1985））。HIV还能结合并攻击其他细胞，如单核细胞、组织巨噬细胞和脑、脊髓及外周神经系统中的细胞。HIV的生活史要求病毒通过性活动或输血等途径进入宿主病人的体内，然后结合于单核细胞或淋巴细胞的受体上。病毒穿透细胞并脱去其被膜或蛋白衣壳，以使其病毒RNA核心暴露。逆转录酶使病毒RNA核心转化为DNA，进而整合到宿主细胞基因组中。在其中大量产生新的病毒颗粒，直至宿主细胞膜被破坏，以向人血循环中释放出新的病毒颗粒。
HIV系由形成封闭膜或被膜的蛋白质及包被病毒RNA抗原的核心组成。膜上和核心材料中有被表达的抗原;核心具有大部分组成病毒的蛋白质。虽然广泛使用并在理论上了解了生成单克隆抗体的常规技术，但也充分认识到了生产特异性抗体的尝试中所遇到的不少复杂情况和变化。涉及开发和生产特异性单克隆抗体的每项研究工作均会遇到各自不同的障碍而难于取得成功。任何一个特定课题能否取得成功，在很大程度上与所用抗原的性质以及用于细胞融合和分离适宜杂交瘤细胞的技术有关。对于生产分泌抗HIV特异性单克隆抗体的细胞系来说，这些障碍特别明显。HIV系由具有无数抗原决定基的被膜及表达无数抗原位点的核心蛋白所组成，由此便可很容易地了解到，骨髓瘤细胞与被免疫小鼠的脾细胞的寻常融合将产生多达用天文数字计数的杂交瘤细胞，且此后筛选特异性抗体的工作将更为繁复。
Ferns等人（J.Gen.Virol.，68：1543-1551（1987-Great Britain））研究了识别HIV分离物之病毒蛋白的单克隆抗体。制得了抗HIV之gag蛋白的单克隆抗体。用Western吸印法鉴定了一组单克隆抗体，证实由这些单克隆抗体识别的HIVgag蛋白是分子量为55，000道尔顿（p55）、24，000道尔顿（p24）、18，000道尔顿（p18）的蛋白质，其均为核心抗原。
在Serki等人（Proc.Natl.Acad.Sci.，80：
3618-3622（1983））的研究中，已测定了HIV整个基因组的核苷酸顺序。这一研究表明，HIV被膜基因表达不同的糖基化蛋白，且核心蛋白不是糖基化的蛋白。Ferns等人（上述文献）的研究则表明，HIV被膜基因的糖基化蛋白被鉴定为分子量为160，000道尔顿的gp160、分子量为120，000道尔顿的gp120和分子量为41，000道尔顿的gp41。
这期间利用单克隆抗体检测人体是否患有爱滋病或是否与能被鉴定的病毒有过免疫学上的接触，但这些商业上可行的诊断试验尚未完全获得成功。因为在疾病表现出症状之前，需要进行这期间的保温处理，而使得对该病的诊断变得极为复杂。鉴于该病的高度感染性质以及就目前的科学水平尚不能治疗该疾病这一事实。也增加了研究活病毒及其对人类免疫系统之有害影响以发展成功的诊断试验的困难。
为此，本发明提供了一种产生能与病毒核心中选择的主要蛋白结合的新的单克隆抗体的杂交瘤或杂交细胞系。该单克隆抗体可稳定而可靠地识别某些HIV核心抗原的共同抗原决定基，故可将所说的抗体用于免疫检测法中，以高度准确地追踪人生理性血清样品中的HIV病毒抗原。该单克隆抗体不与HIV被膜抗原结合。
另外，本发明描述了以杂交瘤技术制得的细胞系，该细胞系可产生与HIV核心抗原p55（其为gag基因编码的前体蛋白）、核心蛋白p24及部分分解产物p39和p33结合的单克隆抗体。本发明的单克隆抗体不结合p18核心蛋白或任何HIV被膜抗原。单克隆抗体识别本文鉴定为“KC-57”抗原的p55、p24、p39和p33抗原上的共同抗原决定基。
本发明的细胞系是用独特的免疫程序建立的，其中使用一组选择的免疫原，经一系列多点输注免疫BALB/C小鼠并以常用的骨髓瘤细胞系与收获的小鼠脾细胞进行融合。
寄存声明
产生本发明之KC-57单克隆抗体的细胞系已于1987年11月6日提交本专利申请的同时寄存于美国典型培养物保藏中心（Rockville，Maryland 20852）。该细胞系的寄存登记号为A.T.C.C.N.H.B9585。该细胞系于1988年6月30日保藏于中国典型培养物保藏中心（CCTCC），保藏号为CCTCC No.C88011。
病毒分离物
由淋巴腺病毒（LAV）感染的细胞系中制备用于免疫小鼠的抗原。以分子排阻层析法由培养上清中分离全病毒。并用Triton X-100溶解LAV，然后过小扁豆植物凝血素亲合柱吸附之，以由培养上清中制备病毒提取物。经Western印迹分析显示由甲基-α，D-吡喃甘露糖苷洗脱的抗原主要是病毒被膜蛋白（gp160/120，另外存在某些核心抗原（p55、p24）。
杂交瘤细胞系的生产
交分离的LAV感染的细胞系与完全弗氏佐剂乳化后用于腹腔内免疫雄性BALB/C小鼠。然后再给小鼠腹腔内注射三次纯化的病毒（在不完全弗氏佐剂的乳悬液中），每次间隔一周。经这些注射后使小鼠每隔一周接受一次gp160/120病毒提取物免疫，共三次。第三次注射gp160/120后三天，切除一只小鼠的脾脏并制备脾细胞悬液。然后在使脾细胞在聚乙二醇1500存在下与骨髓瘤细胞融合。将融合的细胞铺敷于96小井组织培养板上，并用HAT（次黄嘌呤-氨基蝶呤-胸苷）培养基选择抗体生成杂交瘤细胞（Nature 256：495-497，1975）。
细胞群落的筛选
用俘获ELISA法鉴定产生抗HIV核心抗原之抗体的细胞群落。将识别HIV核心抗原的人抗体吸附于96小井聚苯乙烯检测板上。用牛血清白蛋白封闭非特异性结合位点后，将去污剂破坏的LAV与检测板一起保温。然后洗涤平板并在检测平板之小井内加入各杂交瘤细胞群落的条件培养基。保温后，洗涤平板，加入过氧化物酶连接的羊抗小鼠IgG+IgA+IgM并保温。再次洗涤各小井，并加入底物四甲基联苯胺（TMB）。用H2SO4终止反应并测定450nm处吸光率。经鉴定。含KC-57的阳性细胞群落之吸光率比阴性细胞群落的吸光率至少高3倍。克隆到软琼脂胶中以扩充阳性细胞群落，然后注入姥鲛烷（pristane）刺激的BALB/C小鼠体内，以
使之产生含抗体的腹水液。
KC-57的特性
经Ouchterlony免疫双扩散法测知KC-57的免疫球蛋白亚类为IgCl。完成Western吸印实验以测定被KC-57识别的抗原的分子量。用LAV感染之细胞的溶胞产物作为这一分析的抗原来源。抗原阴性对照是未感染T细胞的溶胞产物。证明对照制剂与KC-57无反应活性。使用的内部分子量标志物有血纤维蛋白原（340，000）、纤维结合素（440，000）、肌球蛋白（200，000）、β-半乳糖苷酶（116，000）、磷酸化酶B（92，500）、牛白蛋白（66，000）、卵白蛋白（43，000）、碳酸酐酶（30，000）、胰蛋白酶抑制剂（20，100）和α-乳清蛋白（14，000）。将硝化纤维素滤膜条与KC-57一起保温后，用125I标记的羊抗小鼠抗体检测被单克隆抗体识别的HIV抗原。KC-57可识别HIV相关核心抗原p55和P24，以及可能是p55之裂解产物的p39和p31。没有检出与p18及各HIV被膜抗原的反应活性。
用EPICS流动细胞计数装置（Coulter公司，Healeah，Florida）分析KC-57识别的HIV相关抗原在细胞表面的表达。LAV感染的细胞与KC-57一起保温，然后与羊抗小鼠FITC结合物保温。结果证明KC-57与活的HIV感染的细胞表面上的抗原决定基无反应活性。
使用丙酮固定的LAV感染的细胞完成脆质HIV核心相关抗原的表达。另外，得到并以相同方式制备未感染的、HTLV-Ⅰ感染的和HTLV-Ⅱ感染的淋巴细胞。与KC-57保温后，用羊抗小鼠FITC结合物检测特异反应活性。结果证明KC-57与未感染的、HTLV-Ⅰ感染的或HTLV-Ⅱ感染的细胞无反应活性，而只与存在于固定的HIV感染的淋巴细胞胞质中的核心相关抗原产生特异性反应。
建立一使用KC-57作俘获抗体的抗原俘获酶免疫检测法（EIA）。在96小井检测平板上将纯化的KC-57制成固相并用BSA封闭。以去污剂溶解的LAV感染之淋巴细胞的培养物上清作为HIV抗原来源。保温并洗涤后，加入生物素标记的人抗HIV核心抗体，再次保温并洗涤。加入抗生蛋白链菌素（streptavidin）过氧化物酶并继续保温。末次洗涤后加入TMB并保温。加硫酸终止反应。读出450nm处的吸光率。使用这一分析方法，生长于组织培养物中的9个个别的HIV分离物均获得阳性结果。用以检测这一反应活性的抗原是去污剂溶解的培养物上清。与未感染之细胞的上清未见有反应活性。下列表3中给出了分析数据。
KC-57单克隆抗体特别适用于固相免疫检测，其中所说的单克隆抗体包被于固相表面（如微量滴定板的小井）上并在一控制的程序中与人生理体液样品反应。检测法可以是酶联免疫吸附检测法（ELISA），其中所检测的是与样品中HIV抗原结合的KC-57单克隆抗体。
表3
KC-57HIV抗原俘获 ELISA
HIV分离物 分离病毒的地区 结果
LAV 法国 +
BAGAL 纽约城 +
1265 D13 亚特兰大 +
Z34 托伊尔 +
SDR 旧金山 +
2153 D16 纽约城 +
121886-1 Bethesda +
1272 D21 芝加哥 +
表3所示的结果表明，用KC-57单克隆抗体作俘获相、人抗HIV抗体作检测相的HIV抗原检测法，鉴定上列所有病毒株结果均呈阳性反应。
表4
KC-57单克隆抗体 的特异性
感染因子 检测数 阳性数
EBV 2 0
HTLV Ⅰ 2 0
HTLV Ⅱ 2 0
HSV Ⅰ 2 0
HSV Ⅱ 2 0
CMV 2 0
Chlamydia 2 0
上表数据表明了当进行上述的HIV抗原检测时KC-57抗体与被测的其他感染因子均未见有阳性反应活性。
上表证明KC-57单克隆抗体在所说的HIV抗原检测法中用作俘获抗体时的抗其他感染因子的反应活性。
Claims (2)
1、一种制备杂交瘤细胞系KC-57(CCTCCNo.88011)的方法，其特征在于所说的杂交瘤细胞系产生一种可识别具有共同抗原决定基的p55，p24和其它核心抗原，且基本上不识别p18核心抗原或HIV被膜抗原的单克隆抗体，所述方法包括以下步骤：
(a)用一系列不同的免疫原，包括分离的LAV感染的细胞，纯化的HIV病毒和gp120腹腔内免疫雄性BALB/C小鼠并从经过免疫的小鼠中收集脾细胞；
(b)将SP2/0-Ag14小鼠骨髓瘤细胞与所说的脾细胞融合；
(c)通过在选择性培养基中培养分离所产生的杂交瘤细胞以排除所收集的其它细胞；和
(d)筛选所分离的杂交瘤细胞以鉴定和收集能产生与所说的具有共同抗原决定基的p55，p24和其它核心抗原特异性结合，而基本上不与p18核心抗原或HIV被膜抗原结合之单元隆抗体的杂交瘤细胞。
2、一种生产可与核心蛋白p24，p33，和p55结合，而不与核心蛋白p18或被膜蛋白结合的小鼠单克隆抗体的方法，该方法包括：在合适的培养基中培养杂交瘤细胞系KC-57（CCTCCNo.88011）。
Applications Claiming Priority (2)
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| US07/118,145 US4888290A (en) | 1987-11-06 | 1987-11-06 | Monoclonal antibody specific to HIV antigens |
| US118,145 | 1987-11-06 |
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| CN1033071A CN1033071A (zh) | 1989-05-24 |
| CN1025219C true CN1025219C (zh) | 1994-06-29 |
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| US (1) | US4888290A (zh) |
| EP (1) | EP0415920B1 (zh) |
| JP (1) | JP2710972B2 (zh) |
| KR (1) | KR960016304B1 (zh) |
| CN (1) | CN1025219C (zh) |
| AU (1) | AU612686B2 (zh) |
| CA (1) | CA1339350C (zh) |
| DE (1) | DE3854502T2 (zh) |
| ES (1) | ES2016427A6 (zh) |
| IE (1) | IE61241B1 (zh) |
| IL (1) | IL87535A (zh) |
| WO (1) | WO1989004376A1 (zh) |
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| US5104790A (en) * | 1987-06-29 | 1992-04-14 | Genetic Systems Corporation | Monoclonal antibodies to specific antigenic regions of the human immunodeficiency virus and methods for use |
| US4886742A (en) * | 1987-06-15 | 1989-12-12 | Coulter Corporation | Enzyme immunoassay for detecting HIV antigens in human sera |
| ZA903492B (en) * | 1989-05-15 | 1991-02-27 | Akzo Nv | T-lymphotropic retrovirus monoclonal antibodies |
| US5210181A (en) * | 1989-05-15 | 1993-05-11 | Akzo N.V. | T-lymphotropic retrovirus peptide |
| DE69112059T2 (de) * | 1990-06-01 | 1996-02-29 | Lepetit Spa | Monoklonale antikörper gegen gag-protein-vorläufer-p55 von hiv-virus. |
| US5164293A (en) * | 1990-09-25 | 1992-11-17 | Coulter Corporation | Monoclonal antibody for detecting HTLV-I, HTLV-II and STLV-I viruses |
| EP0519866A1 (en) * | 1991-06-18 | 1992-12-23 | Ciba-Geigy Ag | Novel anti-HIV antibodies |
| US5451525A (en) * | 1992-02-14 | 1995-09-19 | Coulter Corporation | Method and materials for determining particle count in a flow cytometer |
| US6319665B1 (en) | 1994-06-07 | 2001-11-20 | Inverness Medical Technology, Inc. | Home test kit and method with telephone verification of results |
| AU5082698A (en) * | 1996-10-23 | 1998-05-15 | Oravax, Inc | Heat inactivation of viruses in antibody preparations |
| US5891994A (en) * | 1997-07-11 | 1999-04-06 | Thymon L.L.C. | Methods and compositions for impairing multiplication of HIV-1 |
| US5902722A (en) * | 1997-12-05 | 1999-05-11 | The Perkin-Elmer Corporation | Method of detecting organisms in a sample |
| US5915307A (en) * | 1998-01-29 | 1999-06-29 | Suncast Corporation | Sports shelf |
| NO311807B1 (no) * | 1999-03-04 | 2002-01-28 | Bionor Immuno As | HIV-peptider, antigener, vaksinepreparater, immunoassay- testsett og en metode for påvisning av antistoffer fremkalt av HIV |
| US6399067B1 (en) * | 2000-04-28 | 2002-06-04 | Thymon L.L.C. | Methods and compositions for impairing multiplication of HIV-1 |
| US6818392B2 (en) * | 2000-12-06 | 2004-11-16 | Abbott Laboratories | Monoclonal antibodies to human immunodeficiency virus and uses thereof |
| EP1851247A2 (en) | 2005-02-15 | 2007-11-07 | Thymon L.L.C. | Methods and compositions for impairing multiplication of hiv-1 |
| US20080108054A1 (en) * | 2006-08-28 | 2008-05-08 | Jayasri Basu | Detecting early HIV infection in genital tract cells and secretions |
| WO2011119920A2 (en) | 2010-03-25 | 2011-09-29 | Oregon Health & Science University | Cmv glycoproteins and recombinant vectors |
| DK2691530T3 (en) | 2011-06-10 | 2018-05-22 | Univ Oregon Health & Science | CMV GLYCOPROTEIN AND RECOMBINANT VECTORS |
| AU2012216792A1 (en) | 2011-09-12 | 2013-03-28 | International Aids Vaccine Initiative | Immunoselection of recombinant vesicular stomatitis virus expressing HIV-1 proteins by broadly neutralizing antibodies |
| US9402894B2 (en) | 2011-10-27 | 2016-08-02 | International Aids Vaccine Initiative | Viral particles derived from an enveloped virus |
| ES2631608T3 (es) | 2012-06-27 | 2017-09-01 | International Aids Vaccine Initiative | Variante de la glicoproteína Env del VIH-1 |
| EP2848937A1 (en) | 2013-09-05 | 2015-03-18 | International Aids Vaccine Initiative | Methods of identifying novel HIV-1 immunogens |
| EP2873423B1 (en) | 2013-10-07 | 2017-05-31 | International Aids Vaccine Initiative | Soluble hiv-1 envelope glycoprotein trimers |
| EP3069730A3 (en) | 2015-03-20 | 2017-03-15 | International Aids Vaccine Initiative | Soluble hiv-1 envelope glycoprotein trimers |
| EP3072901A1 (en) | 2015-03-23 | 2016-09-28 | International Aids Vaccine Initiative | Soluble hiv-1 envelope glycoprotein trimers |
| CN110669133A (zh) * | 2019-10-18 | 2020-01-10 | 普健生物（武汉）科技有限公司 | 利用尾静脉注射进行基因免疫开发小鼠单克隆抗体的方法 |
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Also Published As
| Publication number | Publication date |
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| US4888290A (en) | 1989-12-19 |
| ZA884651B (en) | 1990-03-28 |
| KR890701771A (ko) | 1989-12-21 |
| AU612686B2 (en) | 1991-07-18 |
| KR960016304B1 (ko) | 1996-12-09 |
| IE881799L (en) | 1989-05-06 |
| IL87535A (en) | 1993-01-14 |
| CA1339350C (en) | 1997-08-26 |
| IE61241B1 (en) | 1994-10-19 |
| AU2133788A (en) | 1989-06-01 |
| WO1989004376A1 (en) | 1989-05-18 |
| EP0415920A4 (en) | 1993-03-17 |
| JP2710972B2 (ja) | 1998-02-10 |
| DE3854502T2 (de) | 1996-04-18 |
| EP0415920A1 (en) | 1991-03-13 |
| EP0415920B1 (en) | 1995-09-20 |
| JPH03500483A (ja) | 1991-02-07 |
| DE3854502D1 (de) | 1995-10-26 |
| ES2016427A6 (es) | 1990-11-01 |
| IL87535A0 (en) | 1989-01-31 |
| CN1033071A (zh) | 1989-05-24 |
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