CN1013586B - 制备能产生用于回收人外周血中白细胞的单克隆抗体的杂交瘤细胞系的方法和利用所述单克隆抗体的回收方法 - Google Patents
制备能产生用于回收人外周血中白细胞的单克隆抗体的杂交瘤细胞系的方法和利用所述单克隆抗体的回收方法Info
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Abstract
一种鼠的单克隆抗体,它与暴露在红细胞膜上的血型糖蛋白A的决定簇位点或特异抗原决定簇选样性地结合而不与其它血型糖蛋白结合。将这种单克隆抗体包被在一个微球体或一个合适的单分散种类的基质上并在无红细胞溶解时回收白细胞亚型的分离程序中利用这种单克隆抗体。在样品的白血细胞群体不相反减少时回收被结合的微球体。所用的微球体或珠粒与细胞之比使本发明非常适用于商业。因此本发明使得不需红细胞溶解就能精确检测循环外周血中的白细胞亚型。
Description
本发明是关于一种鼠的单克隆抗体,这种抗体特别适用于检测无红细胞溶解的循环外周血中的白细胞亚型。其次,本发明的目的在于提供一套改进的检测方法,即利用被该单克隆抗体包裹的适宜单分散微球体来回收入血样品中的白细胞。
用对细胞或组织的抗原决定族具有特异性的单克隆抗体包被支持物或微球体,通常将这些支持物或微球体用于检测和诊断。这些都在先有技术中记载了。
为了将所选的细胞群体分类并使之与异源细胞群体分开,磁性粒子或微球体的使用也是众所周知的。Senyei等人的专利4,230,685,Graver的专利3,970,518,4,018,886,和4,115,535,DaVis的专利4,177,253,Molday的专利4,452,773,Gerlinski的专利4,454,234和Rernbaum的专利4,267,234都仅为这种技术的一些例子。Smilh等人的4,272,510和Forrest等人的4,141,687讲授过用磁性粒子达到检测目的的仪器。在挪威,奥斯陆的Sintef根据专利合作条约(PCT)提交的公开国际专利申请中也描述了磁性微球体对细胞分离的应用,其PCT申请号为83/00014,1983年4月22日提交,国际公布号为WO83/03920,1983年11月10日公布及PCT申请号为83/00016,1983年4月27日提交,国际公布号为WO84/02031,1984年5月24日公布。然而,申请人不
知道单克隆抗体可成功而精确地用于从人的外周血样中分离白血细胞亚型。
人的循环血是由细胞和液体成分所组成的。细胞成分包括红细胞(红血细胞)、血小板和白细胞(白血细胞)1毫升正常全血样品中含有5×106红细胞,3×105血小板和5×103白细胞。白血细胞群体又分为5个亚型或群体,称为中性白细胞,嗜曙红细胞,嗜碱细胞,单核白细胞和淋巴细胞。在白血细胞数为5×103的人血样品中,每毫升血含有3,075个中性白细胞,150个嗜曙红细胞,25个嗜碱细胞,250个单核白细胞和1500个淋巴细胞。淋巴细胞由8种不同的类型组成,表明有几种功能的种类存在。已知大多数控制人体内抗体产生并为抗体的产生作准备的细胞都是在称为淋巴细胞的白细胞群体中发现的。此外,经鉴定淋巴细胞亚型的数目很多。
容易看出对血样中白细胞群体的透彻分析是一个有用的诊断工具。获得这样的资料的常规方法包括自动化电子白血细胞分类仪,机械,电子和光散射细胞计数技术。这种昂贵仪器的使用在经济上受到一定的限制。此外,在无特异的单克隆抗体和特殊技术的情况下,不能用这样的方法来确定白细胞亚型。这也适用于荧光型技术的应用。某些先有技术也需要红血细胞的初溶。
还已知红血细胞(红细胞)缺少多数细胞器,而只有一个单层膜,即原生质膜。几乎所有细胞质成分都能通过渗透溶血作用释放出来,从而提供“血影细胞”,这种血影细胞是相当纯的原生质膜。通过已知的染色技术确定红血细胞膜含有几种富含碳水化合物的蛋白。此外,已确定这些蛋白是细胞表面蛋白。
红细胞含有一种跨膜蛋白,经鉴定是血型糖蛋白A,这个蛋白由16个寡糖和一条单肽组成。大约60%的糖蛋白主体是碳水化合物并且这些碳水化合物单位都位于细胞膜的外表面。由于血型糖蛋白A富含碳水化合物,因而使得它能够很好地着色。蛋白水解的化学修饰和电镜研究表明血型糖蛋白A具有(1)一个含有所有碳水化合物的氨基末端区,该区位于膜的外表面,(2)一个埋藏在烃中心的疏水中区和(3)一个位于细胞内并构成多肽链的羧基末端区。在分子氨基末端部分的碳水化合物单位富含带负电荷的唾液酸基团。
血型糖蛋白B,也叫糖蛋白δ,是与Ss同种抗原结合的糖蛋白。29位上蛋氨酸或苏氨酸的存在表明该同种抗原的区别。看来至今只有血型糖蛋白B的氨基末端部分已序列化了。血型糖蛋白C与称为糖蛋白β和γ的一些成分有关。
构成红细胞血型糖蛋白的糖蛋白链可作为同型和杂二聚体存在,同型和杂二聚体在聚丙烯酰胺电泳(PAGE)之后具有特征带,应该注意可以进一步根据糖基化和唾液酸化的类型及其程度的不同来描述血型糖蛋白。
申请人已发展了一种仅对血型糖蛋白A上的一个特异抗原决定簇,即与一个唯一的结合位点结合的单克隆抗体。为了从人的全血样品中回收白细胞,在磁性分离程序中进行了用抗血型糖蛋白A单克隆抗体包被市售磁性微球体的尝试。在一例研究中,使用了从加利福尼亚,San Diego的分子生物系统中获得的蛋白A磁性微球体,其分类号为MMOO2。如美国专利4,230,685所述,这些粒子经过多分散具有0.15-2.0微米的平均大小并发现需要一个表面活性剂。适宜的培养期所需的珠粒与细胞之比和红细胞减少的百分比证明不能用于商业。此外,还遇到了对白细胞回收的不利干扰。
在一次试验研究中,利用了从宾夕法尼亚,费城的公爵科研所获得的磁性微球体,其分类号为9420A,在这里,这些粒子也经过了多分散,从0.2-0.9微米并且也需要表面活性剂。可以看到这些颗粒的凝集和聚集。白细胞的回收不充分是由于在此过程中随着红细胞的减少导致了白细胞的丧失。
申请人以前对利用这种市售多分散磁性微球体从人的全血样中分离并回收白细胞的尝试表明在商业上不能使用这种磁性粒子分离程序。利用这种颗粒基质使本技术商业化而满足上述普通磁性微球体大量生产的需求是行不通的。然而,现在申请人已能够进行该方法学的改进,这种改进产生了预想不到的,令人吃惊的成功结果。申请人也已确定了哪种磁性微球体能与其唯一的单克隆抗体一起使用。
本发明阐述了一种由申请人发展起来的鼠的单克隆抗体,这种单克隆抗体选择性地与暴露在人红细胞表膜上的血型糖蛋白A的一个决定簇位点相结合。这个单克隆抗体被称为“KC-16”。与抗血型糖蛋白A结合的特异性使得可通过从血液中分离红细胞而不需其本身溶解的程序,将KC-16单克隆抗体用于人外周血中白细胞的商业可行性检测。此外,
在不改变试验血样中白细胞的形态学及无白细胞群体的不利减少情况下得到了分离。
一种特别成功的检测程序包括在大小分别为1.0和4.5微米的单分散磁性微球体或珠粒上包被KC-16单克隆抗体。在每个例子中,都是将人血样放在一支试管中,在室温下培养一段时间,微球体与红血细胞之比在一例中为7∶1,在另一例中为14∶1,将其引入到试验容器中。通过在试验容器的底部应用磁力,把与微球体结合的红细胞从血样中分离出来,轻轻倒出剩余的样品上清液,用注册商标为EPICS
的佛罗里达,Hialeah Coulter公司的细胞分类仪,通过光散射技术进行分析。
随着白血细胞的回收红血细胞成功地减少。红血细胞减少的百分数为99.5%-99.99%,剩余的白细胞群体为97%-98%。5-30分钟的培养期就获得了这些结果。为了显出一个无孔聚合表面选样了所用的磁性微球体,这种磁性微球体无表面活性剂。其特征为球形,单分散的并且磁性材料的均匀度是合乎要求的。
为了从人的外周血样中分离红细胞,然后用电子血细胞计数和分类技术检测白细胞群体也可将KC-16单克隆抗体连接到具有合适特征的非磁性微球体上,这种电子白细胞计数和分类技术是在一台COULTER COUNTQR
型仪器上实现的,该仪器是由佛罗里达,Hialeah的Coulter电器公司,即本专利申请代理人所拥有的附属机构销售的。KC-16单克隆抗体的独特的专一性使得借助于体外诊断仪器的使用,采用商业上可行的检测技术成为可能。
本发明提供能选择性地与暴露在红细胞表膜上的血型糖蛋白A的一个特异抗原决定簇结合的鼠的单克隆抗体。对血型糖蛋白A有此特异性的所有单克隆抗体都可称为“KC-16”。
一种能够产生KC-16单克隆抗体的杂交细胞或杂交瘤的具体制备方法如下:取肺腺癌活体的完整细胞给Balb/c鼠注射,用这种鼠的脾细胞与鼠的浆细胞瘤细胞系,Sp210-Aa14融合;从而产生杂交瘤。每隔两周给鼠注射3次,在最后一次注射后四天将鼠处死。按照KOhler和Milstein的程序(自然256∶495-497,1975)进行细胞隔合。
在此融合过程中,在30%的聚乙烯乙二醇(PEG)和Dulbecco改良Eagles′培养液(DMEM)中将7×107个脾细胞与3.6×107个骨髓瘤细胞融合。细胞融合后,将细胞分布到大约1000个小容器中并在含有次黄嘌呤、氨基蝶呤和胸苷的选样性培养基(HAT)中进行培养。在倒置显微镜下定期观察这些容器中细胞的生长情况。然后收集有细胞生长的上清液,用Elisa技术对产生鼠免疫球蛋白的上清液进行初步筛选。再通过间接免疫过氧化酶染色,用肺腺瘤和正常肺的冰冻和石蜡包埋组织切片筛选来自那些对鼠免疫球蛋白显阳性细胞集落的上清液。
含有KC-16集落的上清液表明对红细胞的特异反应性而其它组织成分不着色。为了保证得到专一的克隆,BC-16,将这种集落在软琼脂糖中再克隆两次。由克隆所产生的KC-16抗体通过双重免疫扩散确定是一种鼠免疫球蛋白1gG1。利用人的细胞类型,即O-阴性,O-阳性,A-阳性,A-阴性,B-阳性,B-阴性,AB-阳性和AB-阴性血型进行了另外的筛选。
能够产生KC-16单克隆抗体的杂交细胞系样品贮存在美国典型培养物保藏所,指定号码为CRL8994。能够产生KC-16单克隆抗体的杂交瘤细胞系已于1986年8月20日寄存于中国典型培养物保藏中心(中国,武汉大学),保藏号为C-86003。
KC-16单克隆抗体也可用下列程序通过免疫鼠腹水液的亲和纯化来获得。在37℃水浴中使冰冻腹水液融化并除去凝块。将肝素加到融化的腹水液中使腹水液的终浓度达到2mg/ml并在25℃下混合30分钟。然后加入氯化镁溶液(1.5M)使浓度为每毫升腹水液含0.344毫升氯化镁溶液并在25℃下混合30分钟,生成液在15℃以下,以105G超速离心30分钟分出上清液,然后用pH为9.0并由1m甘氨酸和4M氯化钠组成的结合缓冲液以1∶1体积比进行稀释。用结合缓冲液平衡载体上的蛋白A-琼脂糖以此来制备亲和柱。把用缓冲液稀释的上清液倾注到柱上,比例为1ml上清液与1/2ml悬浮在3×CSA中的蛋白A-琼脂糖。用结合缓冲液把柱子洗到它的基线,用0.1M醋酸钾(pH6.0)从柱上将KC-16抗体作为第一个峰洗脱下来。将洗脱下来的KC-16抗体浓缩并对0.1%磷酸盐缓冲盐水(PBS)透析。
由于KC-16单克隆抗体选样性地与暴露在红细胞表膜上的一个特异血型糖蛋白A抗原决定族结合,而不与白细胞结合,所以可从人的全血样品中将红细胞和白细胞群体分开,在一种完成这样的血细胞分离的方法中,KC-16单克隆抗体可包在合适的磁性微球体上,然后将这种磁性微球体引入全血样品中;
借助于KC-16单克隆抗体,使微球体选样性地与红细胞结合,这些红细胞可通过将一个磁场加到微球体-血液混合物中与白细胞分开。在不使红细胞溶解,不改变适于进一步检测和分析的白细胞的形态学和不因结块或其它原因而使白血细胞相反减少的情况下完成最终的分离。
通常是单分散的或直径分别为4.5和1.0微米的均匀的无孔磁性微球体(有时称为“珠粒”)通过预先用兔子或山羊的抗鼠免疫珠蛋白(RAM或GAM)包被微球体(珠粒)而使KC-16单克隆抗体很好地结合在它上面。操作如下:将250mg珠粒分散在3ml蒸馏水中并进行2-3分钟声波处理。在4℃下使水中珠粒的混合物冷却几小时。然后将珠粒进行磁化分离并弃去水。再将珠粒重新悬浮在5mgRAM中并用500ml磷酸盐缓冲盐水(PBS)稀释;将此混合物在室温下培养并彻底混合4-5小时。此后,用4mlPBS-1%BSA混合物将珠粒洗涤6次。然后在4mlPBS-1%BSA中重新悬浮这些珠粒并彻底混合,在24小时内更换3次PBS-1%BSA。
将5×109的悬浮珠粒吸量到一支硅化试管中。从弃去的液体中将珠粒磁化分离出来。然后将珠粒重新悬浮在PBS中并将KC-16单克隆抗体加到此溶液中,使最终浓度达0.5mg/ml;然后将此溶液在室温下边混合边培养1小时。然后用PBS-1%BSA将珠粒洗涤6次并重新悬浮在1mlPBS-1%BSA中。
用KC-16包被的磁性微球体使全血中的红细胞(RBCs)与白细胞(WBCs)分离是通过将微球体与10-100μl的一系列全血样品混合制备微球体与红细胞的比例为7∶1-20∶1的混合物来实现的。将微球体与全血的混合物在各自的试管中于室温下边轻轻混合边培养2分钟。将这些试管放在一个磁场中,从而使在试管底部与磁性中心球结合的红细胞分离,白细胞则随着上清液一起被轻轻倒出,用注册商标为EPICS
的,佛罗里达,Hialeah的Coulter公司的细胞分类仪通过光散射技术分析上清液。这些分离的结果列在表Ⅰ中,表Ⅰ表明10∶1-20∶1的微球体与红细胞或细胞比例使得全血样品中的红细胞可减少99.99%。
表Ⅰ
微球体与红细胞的比例
比例 RBCs减少的百分比 WBCs剩余的百分比
7∶1 99.21 90
10∶1 99.99 73.6
14∶1 99.99 61.2
20∶1 99.99 78.4
在第二次分离过程中,将来自6个不同供体的7个全血样品与KC-16包被的微球体在每个混合物中以14∶1的相同的微球体与细胞的比例混合。1分钟后,倾出第一部分上清液,再过1分钟,倾出第二部分上清液;用EPICS
仪测定在两次上清液中红细胞减少的百分比和残存白细胞的百分比并显示在表Ⅱ中。表Ⅱ表明在两次连续的一分钟培养后使红细胞平均减少了99.97+0.02%。
表Ⅱ
用KC-16标记的4.5微米的磁性微球体进行两次连续的一分钟培养在微球体与红细胞的比为14∶1时红细胞的减少
第一部分上清液 第二部分上清液
RBCS减少的 残存WBCs的 RBCS减少的 白细胞的
百分比 百分比 百分比 百分比
99.79 78.6 99.95 71.4
99.95 96.6 99.98 90.9
99.94 100 99.99 100
99.80 100 99.94 100
99.85 100 99.95 100
99.94 100 99.99 94.8
99.97 100 99.98 94.8
99·89·08 96·58·0 99·97·02 93·10·0
申请人确定是实用的并使用的磁性微球体有两个来源。所用珠粒的一个例子是在上文所提到的Sintef的专利申请中描述的。
所用珠粒的另一个例子是由宾夕法尼亚。费城的公爵科研所出售的并经鉴定为1.0微米的胶乳-丙烯酰胺。
我们认为KC-16单克隆抗体的独特专一性使它适用于其它非磁性的支持物或珠粒。在这种应用中,将KC-16单克隆抗体包被在聚合性的,即一种大小均匀的合适的合成塑料制成的支持物或珠粒上。在红细胞与KC-16包被的支持物结合后,使血样的上清液通过一个金属网,珠粒被网眼挡住,而上清液则通过网眼,从而达到分离的目的。然后可进行白血细胞的检测。
已知血型糖蛋白A是红细胞膜的主要唾液酸化糖蛋白成分。我们认为糖蛋白A分子的抗原决定簇或抗原位点是一个特异的碳水化合物组分,我们的
KC-16单克隆抗体选样性地与之结合,获得了如上所述的令人满意的结果。
本领域专业人员可能想到的显而易见的修饰都在本发明的范围之内。
Claims (9)
1、一种生产小鼠单克隆抗体的方法,其特征在于所述的单克隆抗体只与暴露于人红细胞膜表面上的血型糖蛋白A的特异性抗原决定簇(称为KC-16抗原决定簇)结合,而不与血型糖蛋白B结合,所述单克隆抗体用于从人血样中回收白血细胞而不使红细胞溶解,所述单克隆抗体进一步的特征在于它缺乏与白细胞或血小板的结合特异性,红细胞具有O,A,B和AB型,Rh阳性和Rh阴性两种,单克隆抗体不改变样品中白血细胞的形态学,该方法包括:
(1)给Ba1b/c小鼠接种得自人实体瘤的肺腺瘤细胞并从接种小鼠中收集产生的脾细胞,
(2)使小鼠浆细胞瘤细胞系SP2/0-Ag14与所收集的脾细胞融合生成杂交瘤细胞,
(3)分离通过在选择性培养基上培养而产生的杂交瘤细胞以除去所收集的其它细胞,
(4)筛选所分离的杂交瘤细胞以鉴定并收集可产生与所述KC-16抗原决定簇特异性结合的单克隆抗体的杂交瘤细胞系,该细胞系具有1986年8月20日储存于中国典型培养物保藏中心,保藏号为G-86003的样品的特征,
(5)在适当的培养基中培养所收集的杂交瘤细胞以产生对KC-16抗原决定簇特异的单克隆抗体。
2、一种从人外周血样品的红细胞群体中分离白细胞群体而不使样品的红细胞群体溶解的方法,其特征在于:
(1)以预定的微球体与细胞的比例,将一定量的包被有由具有C.C.T.C.C.保藏号为C-86003的杂交瘤细胞系产生的单克隆抗体的基本上单分散的微球体引入到所述血样所含的样品中,
(2)使被包裹的微球体与所述的血样充分接触一段时间,从而使基本上所有红细胞与单克隆抗体包被层结合而不使白细胞群体减少到不利的程度,和
(3)从剩余的血样中分离与抗体结合的微球体。
3、权利要求2的方法,其中微球体的特征为磁性的并且从剩余的血样中分离与抗体结合的微球体是通过在容器的外面接近所述的微球体处加一个磁场而使微球体形成一个凝集物来实现的。
4、权利要求2的方法,其中微球体的特征为非磁性的聚合支持物并且从剩余的血样中分离与抗体结合的微球体是借助于一个机械的网或筛子,其孔径大小应使当倾析样品时将所述的微球体截住。
5、权利要求2的方法,其特征在于所述微球体的直径基本上都是4.5微米。
6、权利要求2的方法,其特征在于引入到血样中的微球体与红细胞之比在大约7∶1到20∶1的范围之内。
7、权利要求6的方法,其特征在于所述比例约为14∶1。
8、权利要求6的方法,其特征在于所述比例约为7∶1。
9、权利要求2的方法,其特征在于所述微球体的直径基本上都是1.0微米。
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AU (1) | AU590615B2 (zh) |
BR (1) | BR8606994A (zh) |
CA (1) | CA1294233C (zh) |
DE (1) | DE3685023D1 (zh) |
DK (1) | DK374987D0 (zh) |
ES (1) | ES2001973A6 (zh) |
IE (1) | IE59433B1 (zh) |
IL (1) | IL80082A (zh) |
NO (1) | NO169243C (zh) |
WO (1) | WO1987003096A1 (zh) |
ZA (1) | ZA867150B (zh) |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
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US5076950A (en) * | 1985-12-20 | 1991-12-31 | Syntex (U.S.A.) Inc. | Magnetic composition for particle separation |
NO162946C (no) * | 1987-08-21 | 1990-03-14 | Otto Soerensen | Anordning for magnetisk separasjon av celler. |
US5238812A (en) * | 1987-03-13 | 1993-08-24 | Coulter Corporation | Method and apparatus for rapid mixing of small volumes for enhancing biological reactions |
US5641622A (en) | 1990-09-13 | 1997-06-24 | Baxter International Inc. | Continuous centrifugation process for the separation of biological components from heterogeneous cell populations |
AU660003B2 (en) * | 1990-11-23 | 1995-06-08 | Coulter International Corporation | Method and apparatus for screening microscopic cells utilizing light scatter techniques |
WO1992014812A1 (en) * | 1991-02-22 | 1992-09-03 | Coulter Corporation | Method and apparatus for screening microscopic cells utilizing polarized light scatter techniques |
US5227369A (en) * | 1991-07-11 | 1993-07-13 | The Regents Of The University Of California | Compositions and methods for inhibiting leukocyte adhesion to cns myelin |
US5449619A (en) * | 1992-04-16 | 1995-09-12 | Sybron Chemical Holdings, Inc. | Drain opener formulation |
US5763204A (en) * | 1992-09-14 | 1998-06-09 | Coulter Corporation | Preparation of preserved, non-infectious control cell for use in the identification of a disease through blood testing |
US6140040A (en) * | 1995-10-06 | 2000-10-31 | Advanced Minerals Corporation | Method of mechanically separating microparticles suspended in fluids using particulate media |
US5594116A (en) * | 1995-11-08 | 1997-01-14 | Promega Corporation | Tryptase polyclonal antibody and purification method for use in human tryptase immunoassay |
JP2000500337A (ja) * | 1995-11-13 | 2000-01-18 | コールター コーポレイション | 細胞の集団又は亜集団のバルク濃縮のための方法および装置 |
BR9706998A (pt) * | 1996-01-16 | 1999-07-20 | Sybron Chemical Holding Inc | Fomulação líquida limpadora e higienizadora |
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 |
US6153113A (en) * | 1999-02-22 | 2000-11-28 | Cobe Laboratories, Inc. | Method for using ligands in particle separation |
AU2001259449A1 (en) * | 2000-05-03 | 2001-11-12 | Eligix, Inc. | Whole blood separator apparatus and method of use |
WO2007092028A2 (en) * | 2005-04-08 | 2007-08-16 | Medical Discovery Partners Llc | Method for enriching rare cell subpopulations from blood |
CN101558309B (zh) * | 2006-09-28 | 2014-02-26 | 麦克法兰布奈特医疗研究与公共健康研究所有限公司 | 一种诊断方法和用于此的试剂盒 |
US20080171399A1 (en) * | 2006-10-09 | 2008-07-17 | Independent Forensics, Inc. | Forensic Test for Human Blood |
FR2963108B1 (fr) * | 2010-07-21 | 2017-06-23 | Diagast | Procede magnetique d'immunodiagnostic et kit pour la mise en evidence de complexe anticorps/antigene de groupe/phenotype sanguin |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
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US3700555A (en) * | 1970-10-12 | 1972-10-24 | Technicon Instr | Method and apparatus for lymphocyte separation from blood |
US4230685A (en) * | 1979-02-28 | 1980-10-28 | Northwestern University | Method of magnetic separation of cells and the like, and microspheres for use therein |
US4598051A (en) * | 1980-03-12 | 1986-07-01 | The Regents Of The University Of California | Liposome conjugates and diagnostic methods therewith |
US4375407A (en) * | 1981-06-22 | 1983-03-01 | The Franklin Institute | High gradient magnetic separation device |
US4429008B1 (en) * | 1981-12-10 | 1995-05-16 | Univ California | Thiol reactive liposomes |
NO155316C (no) * | 1982-04-23 | 1987-03-11 | Sintef | Fremgangsmaate for fremstilling av magnetiske polymerpartikler. |
GB8318575D0 (en) * | 1983-07-08 | 1983-08-10 | Cobbold S P | Antibody preparations |
US4594327A (en) * | 1983-11-02 | 1986-06-10 | Syntex (U.S.A.) Inc. | Assay method for whole blood samples |
US4695553A (en) * | 1985-11-01 | 1987-09-22 | Becton Dickinson And Co., Inc. | Method for increasing agglutination of groups of cells to produce improved cell layer interface in centrifuged blood sample using antibodies |
US6450694B1 (en) * | 2000-06-20 | 2002-09-17 | Corona Optical Systems, Inc. | Dynamically configurable backplane |
-
1985
- 1985-11-19 US US06/799,489 patent/US4752563A/en not_active Expired - Lifetime
-
1986
- 1986-08-25 AT AT86905551T patent/ATE75317T1/de not_active IP Right Cessation
- 1986-08-25 BR BR8606994A patent/BR8606994A/pt not_active Application Discontinuation
- 1986-08-25 AU AU62836/86A patent/AU590615B2/en not_active Ceased
- 1986-08-25 EP EP86905551A patent/EP0245291B1/en not_active Expired - Lifetime
- 1986-08-25 DE DE8686905551T patent/DE3685023D1/de not_active Expired - Lifetime
- 1986-08-25 JP JP61504547A patent/JPS63501332A/ja active Pending
- 1986-08-25 WO PCT/US1986/001734 patent/WO1987003096A1/en active IP Right Grant
- 1986-09-11 CA CA000517986A patent/CA1294233C/en not_active Expired - Lifetime
- 1986-09-18 ES ES8601990A patent/ES2001973A6/es not_active Expired
- 1986-09-19 ZA ZA867150A patent/ZA867150B/xx unknown
- 1986-09-19 IE IE250486A patent/IE59433B1/en not_active IP Right Cessation
- 1986-09-19 IL IL80082A patent/IL80082A/xx not_active IP Right Cessation
- 1986-10-14 CN CN86107203A patent/CN1013586B/zh not_active Expired
-
1987
- 1987-07-14 NO NO872934A patent/NO169243C/no unknown
- 1987-07-17 DK DK374987A patent/DK374987D0/da not_active Application Discontinuation
- 1987-07-18 KR KR870700622A patent/KR880700938A/ko not_active Application Discontinuation
-
1997
- 1997-02-10 JP JP9026706A patent/JPH09327287A/ja active Pending
Also Published As
Publication number | Publication date |
---|---|
ES2001973A6 (es) | 1988-07-01 |
DK374987A (da) | 1987-07-17 |
US4752563A (en) | 1988-06-21 |
DK374987D0 (da) | 1987-07-17 |
NO169243C (no) | 1992-05-27 |
IL80082A0 (en) | 1986-12-31 |
KR880700938A (ko) | 1988-04-13 |
AU6283686A (en) | 1987-06-02 |
EP0245291A4 (en) | 1988-06-23 |
IE862504L (en) | 1987-05-19 |
EP0245291B1 (en) | 1992-04-22 |
CA1294233C (en) | 1992-01-14 |
ATE75317T1 (de) | 1992-05-15 |
JPH09327287A (ja) | 1997-12-22 |
BR8606994A (pt) | 1987-12-01 |
EP0245291A1 (en) | 1987-11-19 |
DE3685023D1 (de) | 1992-05-27 |
CN86107203A (zh) | 1987-09-30 |
WO1987003096A1 (en) | 1987-05-21 |
NO872934D0 (no) | 1987-07-14 |
NO872934L (no) | 1987-07-14 |
AU590615B2 (en) | 1989-11-09 |
IE59433B1 (en) | 1994-02-23 |
NO169243B (no) | 1992-02-17 |
IL80082A (en) | 1990-12-23 |
ZA867150B (en) | 1988-05-25 |
JPS63501332A (ja) | 1988-05-26 |
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