CN87107275A - 对中性白细胞特异的单克隆抗体 - Google Patents
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Abstract
一种能够产生对人中性白细胞具有专一特异性之单克隆抗体的杂交细胞系。该单克隆抗体与其它人外周血细胞无反应性，实际上与骨髓中的粒细胞前体或其它细胞也无反应性，进而。与人急性白血病细胞无反应性。以使用经高度纯化的人粒细胞作免疫接种剂诱导的小鼠脾细胞作为杂交瘤融合时的参与者之一。该单克隆抗体的进一步的特征在于其可用于计数和分离正常人外周血和急性白血病患者外周血的中性白细胞。
Description
本发明涉及一种可产生结合到中性白细胞群体上的单克隆抗体的杂交瘤细胞系。因此，本发明可通过该单克隆抗体的特异结合能力直接免疫测定人外周血中的中性白细胞，进而可使外周血样中的中性白细胞选择性地减少。
人循环系统中的外周血主要由红血细胞，即红细胞，和白血细胞，即白细胞所组成。白细胞的功能及其临床相关性已经引起科学界的极大的兴趣。白血细胞由中性白细胞，单核细胞，嗜曙红细胞，嗜碱细胞和淋巴细胞所组成，白血细胞有许多亚型。
已知中性白细胞，单核细胞，嗜曙红细胞和嗜碱细胞都是吞噬细胞，因为它们在人免疫系统中的主要功能是吞噬或消化细菌和其它微生物。这些细胞是在人的骨髓中产生的。然而，这些吞噬细胞作为既相关又独立的系统又各有其不相同的功能。虽然这些吞噬细胞起源于骨髓，但它们都进入外周血中并循环。
吞噬细胞的一般功能是吞噬那些作为外来物被识别的物质并有助于产生对抗外来物的免疫反应。中性白细胞作为吞噬已包被有抗体的细菌的吞噬细胞是很有效的，但对未包被抗体的细菌不那么有效。中性白细胞的主要功能是在病原微生物侵袭后，通过将其限制于局部并杀伤之来阻止病原微生物的侵入。为了成功地攻击侵入的微生物，中性白细胞将离开外周血，迁移到受侵染的组织区域，然后识别，杀死并消化微生物。
中性白细胞是骨髓中最常见的细胞和外周血中最常见的白细胞。1微升正常全血样品中平均含有5×103个白细胞，其中有3，075个是中性白细胞，150个是嗜曙红细胞，25个是嗜碱细胞，250个是单核细胞，1500个是淋巴细胞。外周血中中性白细胞偏离正常浓度常伴有临床病变。中性白细胞的浓度增加有时称为“中性白细胞增加症”其可以是某种疾病或身体状况的指征，而外周血中中性白细胞的浓度减小（有时称为“中性白细胞减少症”）可出现不同的临床关联。例如，中性白细胞绝对计数少于每微升500个细胞时机体便处于一种生命受到威协的状况下，因为具有这样异常中性白细胞计数的患者，对危及生命的细菌或真菌感染是高度敏感的。中性白细胞减少症可能起因于胞毒药物，如用于治疗癌症或白血病的药物。还有许多与中性白细胞功能异常相关的先天和后天疾病。外周血中中性白细胞的测定是总的白细胞计数的重要部分。
本发明提供一种杂交细胞系，其可产生对人外周血中性白细胞所表达的表面抗原有特异性的单克隆抗体。经鉴定该表面抗原为1D3抗原，其特征是在处于还原条件下时分子量约为48，000道尔顿，而在非还原条件下则约为48，000～60，000道尔顿。
本发明提供一种可与中性白细胞表面上的抗原位点特异结合的单克隆抗体，该抗原位点被鉴定为1D3抗原，所说的抗-1D3单克隆抗体的进一步的特征是不与人的急性白血症细胞、其它的人外周血细胞结合，并且不与骨髓中的粒细胞前体形成明显的结合。
本发明的抗-1D3单克隆抗体是通过杂交瘤细胞系产生的，参加融合的细胞之一是用纯化的人粒细胞免疫接种得到的。
可从抗-1D3单克隆抗体在免疫测定应用中看出这种特异性抗体的优点。这种单克隆抗体可执行各种不同的功能，其功能取决于如何使用它，即如何进行标记，如用荧光素或生物素标记，或如何结合到小珠或微球上，用于鉴定和计数的中性白细胞珠粒或微球可以是磁性的或非磁性的。
本发明的目的在于提供一种用杂交癌技术生产的细胞系，该细胞系具有寄存在美国典型培养物保藏中心（马利兰州，Rockville）的样品（登记号为A、T、C、C、HB9445）的主要特征，即可产生抗中性白细胞上1D3表面抗原的抗体。
本发明的单克隆抗体被命名为1D3。它是使用由Kohler和Milstein介绍的标准技术〔Nature，256，495-497（1975）〕通过经用纯化的人粒细胞免疫的鼠脾细胞与鼠骨髓瘤细胞融合而产生的。
用于免疫接种程序的人粒细胞是以特定方法制备的。经静脉穿刺得到人血，通过Ficoll-Hypaque密度梯度沉淀使单核细胞与粒细胞和红血细胞分离（1.077g/cc）。然后用Hanks平衡盐溶液稀释红细胞/粒细胞片状沉淀物并加入1/10体积的4%高分子量葡聚糖使红血细胞和粒细胞分离。将粒细胞悬浮于磷酸盐缓冲盐水（PBS）中。
将106个这一高度纯化的人粒细胞注射到经麻醉的BalbC小鼠的脾脏中。脾内免疫接种后十天，用10×106个同样纯化的粒细胞经尾静脉注射对小鼠作加强免疫。三天后，回收鼠脾并用常规技术收集脾细胞。
按下述方法融合形成杂交瘤。在无血清培养基中以8个脾细胞对1个NS-1细胞的比例用NS-1浆细胞瘤细胞系洗涤脾细胞。将细胞离心形成片状沉淀物并悬浮于0.5ml30%聚乙二醇中，25℃，8分钟，然后，倾去聚乙二醇，用HAT培养液稀释细胞并将其分配到微滴盘中。
通以间接免疫荧光法和流动血细胞计数方法检测单克隆抗体1D3的反应性，筛选与纯化的人粒细胞的反应性。在鉴定对粒细胞具有绝对选择性之抗体的努力中选择了1D3单克隆抗体并进而根据其结合有很高密度的抗原而定性之。1D3单克隆抗体与90%以上得自于25个受试供血者的粒细胞反应，1D3不与纯化的单核细胞、T细胞（E+细胞），B细胞/NK细胞（E-细胞）反应;它只与3%正常骨髓单核细胞反应。1D3不与嗜曙红细胞或嗜碱细胞反应，因而它确实是对中性白细胞特异的。
对1D3单克隆抗体的进一步的反应性试验证明，它不与扁桃体细胞或植物血凝素激活的外周血T细胞反应。检测了抗体与几种人细胞系的反应性。发现HL60细胞系具有较弱的反应性，即大约26%。U937，KG1，K562和Daudi细胞系不与1D3反应。已证明1D3抗体与6份人急性淋巴母细胞白血病细胞样品和5份人急性成髓细胞白血病细胞样品均无反应性。
由于1D3单克隆抗体对人中性白细胞具有极好的特异性，所以它是独一无二的。该抗体与其它的人外周血细胞无反应性且实际上与骨髓中的粒细胞前体或其它细胞也无反应性。由于该抗体对中性白细胞具有高度特异性并且以如此高的抗原密度存在，所以用任何方法（如免疫荧光法或免疫组织化学法）检测粒细胞都是极其便利的。这种单克隆抗体对于鉴定和计数人中性白细胞将是有用的。1D3与人白血病细胞无反应活性具有潜在的临床关联性。实际上，所有以前报道过的可与中性白细胞反应的单克隆抗体都也可与某些急性成髓细胞白血病细胞反应。因此，1D3抗体在急性白血病情况下具有正确计数中性白细胞的能力。
能够产生1D3单克隆抗体的杂交细胞样品寄存在美国典型培养物保藏中心（A、T、C、C），登记号为HB9445。
以生化方法定性由1D3单克隆抗体所鉴定的抗原的尝试曾遇到了异常的困难。曾采用了如下三种不同的方法：（1）对已溶解和经提取的中性白细胞做Western吸印实验;（2）对得自中性白细胞的1D3抗原进行亲和层析纯化;（3）在提取前对中性白细胞进行碘化处理，然后对得自中性白细胞的抗原作亲和层析纯化。分离生产杂交瘤中所用免疫接种细胞的所有同样的标准实验室方法均可用于从全血中分离中性白细胞。但当时未能用这些方法测出1D3抗原的分子量。因此，1D3抗原可能是一种碳水化合物或脂类抗原。
用细胞学方法定性由1D3单克隆抗体所识别的细胞表面受体或抗原的尝试是通过将其从外周血细胞样品中选择性地除去而完成的。为此目的使用了以下两种方法：
Ⅰ.使用EPICS
装置（佛罗里达Coulter Corporation of Hialeah公司销售）并利用流动细胞计数和细胞分选技术。在与1D3单克隆抗体反应和GAM-FITC荧光标记后将显示荧光阳性的那些白细胞通过细胞分选技术除去。用Wright氏染色剂对筛分的细胞染色并进行形态学分类，每份受试样品最少计数到500个白细胞。
用这种方法共计数了20份正常成人外周血样品。在这些样品中，至少有92%的成熟分裂的中性白细胞显示有1D3抗体阳性标记。
Ⅱ.在该方法中，用与1D3抗体结合的磁性微球进行细胞分离。通过溶胞或其它技术处理正常成人外周血样品以除去红血细胞，同时对剩余的白细胞不产生有害影响。在用Wright氏染色剂染色后对白细胞进行细胞形态学分析。将结合了1D3的微球引入到样品中，从样品中移除白细胞中与之结合的中性白细胞。然后以形态学方法计数剩余阴性细胞中存在的中性白细胞。
用这一方法测试了9份外周血样品。在这些剩余的细胞样品中观察到中性白细胞平均不足0.2%。而且最少计数到500个白细胞。
由这些试验所获得的数据证实了与1D3单克隆抗体特异结合的白血细胞群体是中性白细胞。
已在还原和非还原条件下鉴定了由1D3单克隆抗体识别的细胞表面受体或抗原的细胞特性。在还原条件下，1D3抗原的分子量为48，000道尔顿。在非还原条件下，1D3抗原具有一条分子量范围为48，000～60，000道尔顿的宽带。从循环粒细胞（通过密度梯度离心得自加肝素的人外周血）中分离出了1D3抗原并鉴定其具有单一的形式。
在不连续垂直平板凝胶上进行SDS-聚丙烯酰胺电泳（5-15%的梯度）检测了4×107个细胞的等价物。使用了U.K.Laemole〔Nature，227，680（1980）〕所述方法的改良法。电泳后，将丙烯酰胺平板中的蛋白质电吸印到一张硝化纤维素滤纸上。用氨基黑B对硝化纤维素滤纸上的分子量标记物作蛋白质染色。硝化纤维素滤纸的剩余部分用含有无脂乳粉的缓冲剂阻断，以便用与放射活性标记物碘125（I125）结合的抗-1D3抗体进行免疫检测。抗-1D3-125I与硝化纤维素作用，与1D3抗原结合。在洗掉未结合的放射碘化的抗体之后，使用Kodak XAR胶片通过放射自显影技术显现免疫印迹。Kodak XAR胶片显影后，可见在1D3抗原位置上的带已移动。用这种方法，即可确定与1D3单克隆抗体如此特异结合的1D3的性质。
Claims (13)
1、一种用杂交瘤技术产生的细胞系，该细胞系产生一种可与中性白细胞的1D3抗原特异结合的单克隆抗体。
2、根据权利要求1的细胞系，其中所说的抗体产生细胞得自于用纯化的人粒细胞免疫的小鼠。
3、根据权利要求1的细胞系，其中所说的抗体生成细胞是用纯化的人粒细胞免疫的小鼠脾细胞。
4、根据权利要求1的细胞系，其中所说的单克隆抗体是根据其不与其它人外周血细胞反应并且与骨髓中的粒细胞前体没有显著反应性而定性的。
5、权利要求4的细胞系，其中所说的单克隆抗体与人急性淋巴母细胞白血病细胞无反应性。
6、权利要求4的细胞系，其中所说的单克隆抗体与人急性成髓细胞白血病细胞无反应性。
7、权利要求1的细胞系，其中所说的单克隆抗体与人急性白血病细胞无反应性。
8、一种用杂交瘤技术产生的细胞系，该细胞系具有寄存于美国典型培养物保藏中心的可产生抗中性白细胞上1D3抗原之抗体的样品（ATCC HB9445号）的主要特征。
9、一种可与中性白细胞表面上的抗原特异结合的称为1D3的单克隆抗体，所说的1D3单克隆抗体的进一步的特征是不与下列细胞结合：
A.人急性白血病细胞
B.其它人外周血细胞
并且不与骨髓中的粒细胞前体有效地结合。
10、权利要求9的通过杂交瘤技术获得的单克隆抗体，其中小鼠脾细胞是用纯化的人粒细胞免疫的。
11、由杂交细胞系产生的权利要求9的单克隆抗体，其中所说的细胞系具有ATCC HB9445号样品的主要特征。
12、根据权利要求1的细胞系，其中所述1D3抗原的分子量在还原条件下限定大约为48，000道尔顿，在非还原条件下限定为48，000～60，000道尔顿。
13、包括于还原条件下限定分子量约为48，000道尔顿、于非还原条件下限定分子量为48，000～60，000道尔顿之所说的抗原的权利要求9的单克隆抗体。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US93886486A | 1986-12-08 | 1986-12-08 | |
| US938,864 | 1986-12-08 | ||
| US070,202 | 1987-07-06 | ||
| US07/070,202 US4931395A (en) | 1986-12-08 | 1987-07-06 | Monoclonal antibody specific to neutrophils |
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| Publication Number | Publication Date |
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| CN87107275A true CN87107275A (zh) | 1988-08-17 |
| CN1024285C CN1024285C (zh) | 1994-04-20 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN87107275A Expired - Fee Related CN1024285C (zh) | 1986-12-08 | 1987-12-07 | 对中性白细胞特异的单克隆抗体 |
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| US (1) | US4931395A (zh) |
| EP (1) | EP0338003B1 (zh) |
| JP (2) | JP2554153B2 (zh) |
| KR (1) | KR960013131B1 (zh) |
| CN (1) | CN1024285C (zh) |
| AT (1) | ATE88751T1 (zh) |
| AU (1) | AU611407B2 (zh) |
| CA (1) | CA1283066C (zh) |
| DE (1) | DE3785673T2 (zh) |
| DK (1) | DK440988D0 (zh) |
| IL (1) | IL84595A0 (zh) |
| MX (1) | MX9203237A (zh) |
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| AU618212B2 (en) * | 1987-06-30 | 1991-12-12 | Coulter International Corporation | Monoclonal antibody specific to a common determinant site of neutrophils and eosinophils |
| IE67038B1 (en) * | 1988-12-22 | 1996-02-21 | Patrick Joseph Joyce | Immunodiagnostic assays for use in the detection and determination of mastitis |
| US5348859A (en) * | 1990-11-23 | 1994-09-20 | Coulter Corporation | Method and apparatus for obtaining an absolute white blood cell subset count and white blood cell multipart differential |
| US5814468A (en) * | 1996-03-27 | 1998-09-29 | Coulter International Corp. | Methods of enumerating receptor molecules for specific binding partners on formed bodies and in solution |
| US6696243B2 (en) | 2001-01-23 | 2004-02-24 | Coulter International Corp. | Method for the analysis of soluble analytes |
| US6811777B2 (en) | 2002-04-13 | 2004-11-02 | Allan Mishra | Compositions and minimally invasive methods for treating incomplete connective tissue repair |
| US7608258B2 (en) * | 2002-04-13 | 2009-10-27 | Allan Mishra | Method for treatment of tendinosis using platelet rich plasma |
| WO2005065269A2 (en) * | 2003-12-29 | 2005-07-21 | Am Biosolutions | Compositions and method for decreasing the appearance of skin wrinkles |
| US20070122906A1 (en) * | 2003-12-29 | 2007-05-31 | Allan Mishra | Method of culturing cells |
| US7678780B2 (en) * | 2003-12-29 | 2010-03-16 | Allan Mishra | Method of treating cancer using platelet releasate |
| US7462268B2 (en) * | 2004-08-20 | 2008-12-09 | Allan Mishra | Particle/cell separation device and compositions |
| WO2010042658A1 (en) | 2008-10-07 | 2010-04-15 | Bioparadox, Llc | Use of platelet rich plasma composition in the treatment of cardiac conduction abnormalities |
| US20140356893A1 (en) | 2013-06-04 | 2014-12-04 | Allan Mishra | Compositions and methods for using platelet-rich plasma for drug discovery, cell nuclear reprogramming, proliferation or differentiation |
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| AU618212B2 (en) * | 1987-06-30 | 1991-12-12 | Coulter International Corporation | Monoclonal antibody specific to a common determinant site of neutrophils and eosinophils |
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1987
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- 1987-11-17 AU AU10460/88A patent/AU611407B2/en not_active Ceased
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- 1987-11-17 KR KR1019880700945A patent/KR960013131B1/ko not_active IP Right Cessation
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Also Published As
| Publication number | Publication date |
|---|---|
| NO883495L (no) | 1988-08-05 |
| WO1988004320A1 (en) | 1988-06-16 |
| EP0338003A1 (en) | 1989-10-25 |
| ATE88751T1 (de) | 1993-05-15 |
| DK440988A (da) | 1988-08-05 |
| DE3785673T2 (de) | 1993-08-19 |
| AU1046088A (en) | 1988-06-30 |
| JPH02501029A (ja) | 1990-04-12 |
| JP2554153B2 (ja) | 1996-11-13 |
| EP0338003A4 (en) | 1990-01-26 |
| NO174855C (no) | 1994-07-20 |
| JPH08275775A (ja) | 1996-10-22 |
| KR960013131B1 (ko) | 1996-09-30 |
| CA1283066C (en) | 1991-04-16 |
| JP2667140B2 (ja) | 1997-10-27 |
| KR890700161A (ko) | 1989-03-10 |
| DK440988D0 (da) | 1988-08-05 |
| EP0338003B1 (en) | 1993-04-28 |
| AU611407B2 (en) | 1991-06-13 |
| NO174855B (no) | 1994-04-11 |
| US4931395A (en) | 1990-06-05 |
| MX9203237A (es) | 1992-07-01 |
| IL84595A0 (en) | 1988-04-29 |
| CN1024285C (zh) | 1994-04-20 |
| NO883495D0 (no) | 1988-08-05 |
| DE3785673D1 (de) | 1993-06-03 |
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