CN1144045C - 用免疫测定技术鉴别和定量聚合物的方法 - Google Patents
用免疫测定技术鉴别和定量聚合物的方法 Download PDFInfo
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- CN1144045C CN1144045C CNB971022720A CN97102272A CN1144045C CN 1144045 C CN1144045 C CN 1144045C CN B971022720 A CNB971022720 A CN B971022720A CN 97102272 A CN97102272 A CN 97102272A CN 1144045 C CN1144045 C CN 1144045C
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Abstract
公开了一种使用免疫测定技术定量鉴别水溶液系统中的聚合物的方法,其中至少有一部分聚合物含有可检测末端。这对水处理系统特别有用。还公开了表达可特异性识别该可检测末端的MAbs的新的杂交瘤细胞系。
Description
发明背景
本发明涉及一种使用免疫测定技术鉴别和定量水溶液系统中的聚合物的方法。具体地讲,单克隆抗体(Mab’s)的制备是针对于:用于开始聚合过程的引发剂(或其片段);用于在聚合过程中制备预测定聚合物的链转移剂;或是一个能在随后连接到链转移剂上的功能基。本发明还涉及表达这些MAbs的新的杂交瘤(不死的细胞系)。本发明对于测定水处理应用中的聚合物浓度特别有用。
水溶性聚合物被用于许多水溶液系统,例如:作为矿物分散剂;作为锅炉用水、冷凝塔、反渗透应用、糖精制、纸生产、地热过程、和油井的水处理添加剂;作为去污添加剂,起助洗剂、抗薄膜剂、分散剂、分离剂、包鞘抑制剂的作用。在这些类型的应用中,多聚物与矿物质或其它物质形成配合物使它们从水中消除,以防止在水处理应用中产生腐蚀和矿物质沉淀(水垢)。一段时间后,随着聚合物上配合位点的饱和,聚合物开始失活或分解,从而必须加入更多的活性聚合物。特别重要的是,在水处理应用中,活性聚合物不足可导致溶解的矿物质对水处理的破坏,引起严重的腐蚀或水垢沉淀;而维持高于所需的活性聚合物的浓度则昂贵而浪费。因此,希望能够方便地测定在不同时间下系统内的聚合物浓度,以确定是否需要另外加入聚合物。
关于测定水溶液系统中的聚合物的问题之一在于,传统的检测方法(例如比色或荧光分析)缺乏灵敏度,因为聚合物通常以极低的水平存在,从百万分之(“ppm”)500至5以下。关于测定水溶液中的聚合物的另一个问题在于,检测方法常常缺乏选则性,可能对水溶液系统中的非聚合物成分给出错误的结果。
克服这些问题的尝试包括,针对聚合物产物的链节制造MAbs、然后用这些MAbs通过免疫测定技术检测聚合物产物的存在或浓度的方法。在例如EP 540314 A1(Wetegrove等)、EP 535347 A2(Wetegrove等)、和EP 59249 A1(Weatherby等)中公开和讨论了这种方法。在所有这些方法中,MAbs结合于聚合物的选定位点上,但是,由于聚合物链内有大量的重复单元,因而MAbs和具体的聚合物链之间不可能一一对应;此外,由于聚合物具有变动的链长度,因而结合于各聚合物上的MAbs的数量会有变化。因此,关于MAbs和聚合物浓度的比例的测定是批次依赖性的(即该比例将依据所制备的聚合物的具体批次而变化),并且这种测量的精确度的变化可能相对较大。
PCT US94/09264(Garner等)公开了一种标记产品以用于随后的鉴别的方法。该方法包括:制备一种低分子量半抗原,将该半抗原共价结合于一个载体化合物上,然后将这种半抗原标记的化合物与产物缔合,这样,半抗原作为标记物可以在随后用免疫测定技术测定。载体化合物可以是聚合物,这样,可以将半抗原连接到已形成的聚合物上,或者将半抗原在聚合之前连接到单体上。如想用于该方法,半抗原标记的化合物(“标记物化合物”)应对产物是无影响的,对产物而言是惰性的,并且事先没有缔合在产物上。通常通过将标记物化合物与产物混合使其与产物缔合,但它可以在包装或标记产物时存在。
尽管Garner等公开了“以与未标记化合物固定的比例使用这种标记的化合物可以示踪标记的化合物”,但他们的方法的性质使其不可能快速而方便地测定聚合物产物的浓度。Garner等指出,可以将半抗原缔合于已形成的聚合物上,或在聚合之前缔合于单体上;然而,在所有情况下,由于各个聚合物的链长度是不固定的,缔合于个具体的聚合物链上的半抗原数量会随之变动,所以,不可能绝对地测定出半抗原:聚合物的比例。因此,半抗原:聚合物的比例是批次依赖性的,使聚合物的浓度难于测定:对于各批的标记聚合物均需作出标准浓度曲线;加入系统中的聚合物不可能与先前加入的聚合物来自同一批次,可能会影响测定的校准和精确度;系统中总的聚合物浓度可能由来自于不同批次而不是仅仅一个批次的聚合物组成,也可能影响测定的校准和精确度。
发明陈述
一种鉴别和定量水溶液系统中的聚合物的方法,其中至少有一部分聚合物含有选自于链转移剂、引发剂或其片段、或连接于链转移剂上的基团的可检测末端,该方法由以下步骤组成:a)制备可检测末端的单克隆或多克隆抗体;b)获得要测定的水溶液系统的样品,将该样品与单克隆或多克隆抗体一起孵育;c)检测和测量单克隆或多克隆抗体的结合程度以鉴别聚合物并测定水溶液系统中的聚合的浓度。
附图简述图1是图解聚合物的浓度(μg/ml)对反应性(%抑制)的曲线,说明不同聚合物在TNT-Tag免疫测定中的反应性。曲线的图标如下:-◆-=TNT-CYS-PAA;-■-=CYS-PAA;-▲-=PAA-PAA;-X-=Acusol445N。
发明详述
本说明书中使用的下列术语具有以下定义,除非文中另有明确的指示。“致免疫抗原”或“致免疫分子”指一种物质,当将其引入到免疫系统具有功能的动物体内时,可激发免疫反应而导致免疫状态。“抗原决定蔟”或“表位”指致免疫分子上可被特异抗体鉴别的部分。“半抗原”是一种不能在体内致免疫的低分子量分子,但它可以作为致免疫分子上的抗原决定簇的一部分;也就是说,如果事先将这些半抗原偶联于一个致免疫抗原,就可以形成抗配合物的不同部分的抗体,包括抗某些可特异性结合于半抗原的部分的抗体。“标记”指聚合物链上已形成单克隆或多克隆抗体的可检测末端,其中的可检测末端选自链转移剂、引发剂(或其片段)、或连接于链转移剂上的基团;“标记的”指聚合物,用于表示所指的聚合物含有标记。除非特别说明,指定的范围均包括端值。
在使用链转移剂的自由基聚合过程中,聚合反应由链转移剂中止。由此产生的自由基成为新聚合物链的开端。当仅有有限量的聚合分支时〔例如通常在低分子量(小于50000)聚合物中出现的情况-代表性的是anti-scalants〕,链转移剂与聚合物链的比例大约是1∶1;因此,检测标记的聚合物浓度的方法将导致对系统内聚合物的总浓度的直接测定。通过使用灵敏和选择性的免疫测定技术对标记进行测定,本发明的方法提供了一种简便、有效的检测聚合物的方法。
在水溶液系统内使用的所有聚合物均可被标记,或者是某一特定部分的聚合物可以被标记。优选仅标记系统内的一小部分聚合物。当仅有一部分聚合物被标记时,未标记的聚合物无需与标记的聚合物相同,但是优选未标记的聚合物和标记的聚合物在系统内的性质相似,从而使标记的聚合物的排除率能够精确地反映整个系统的排除率。
本发明的标记的聚合物可以是可与链转移剂聚合的任何类型,一般是乙烯基聚合。在本申请的使用本发明的标记聚合物来测定水处理聚合物的实施方案中,优选使用多元羧酸的聚合物或共聚物,尤其是丙烯酸酯聚合物和其衍生物。特别有用的丙烯酸酯聚合物包括从下列单体制得的聚合物和共聚物,所述单体包括,每个分子含有3-5个碳原子的烯键不饱和的一元羧酸、每个分子含有4-8个碳原子的烯键不饱和的二元羧酸、以及它们的碱金属和铵盐、和顺式二元羧酸的酸酐。一元羧酸的例子包括但不仅限于:丙烯酸、甲基丙烯酸、乙烯基乙酸、巴豆酸、和丙烯氧基丙酸。其中的丙烯酸和甲基丙烯酸为优选。适当的二元羧酸的例子包括但不仅限于:马来酸、衣康酸、甲基富马酸、富马酸、柠康酸、四氢化邻苯二甲酸、四氢化邻苯二甲酸酐、和马来酸酐。在这些二元酸单体中,马来酸酐为优选。
起始聚合物还可以由最高为百分之70重量比(“wt%”)的不含酸的单烯键不饱和单体构成,只要聚合物仍然是水溶性的。这些单体包括但不仅限于:丙烯酸或甲基丙烯酸的烷基酯,例如丙烯酸甲酯、丙烯酸乙酯、丙烯酸丁酯、甲基丙烯酸甲酯、甲基丙烯酸乙酯、甲基丙烯酸丁酯、和甲基丙烯酸异丁酯;丙烯酸或甲基丙烯酸的羟烷基酯,例如丙烯酸羟乙基酯、丙烯酸羟丙基酯、甲基丙烯酸羟乙基酯、和甲基丙烯酸羟丙基酯;其它类型的丙烯酸或甲基丙烯酸酯,例如(甲)丙烯酸2-硫代乙基酯、(甲)丙烯酸3-硫代丙基酯、(甲)丙烯酸2-硫酸根合乙基酯、丙烯酸二甲氨基乙酯和甲基丙烯酸二氧磷基乙基酯;丙烯酰胺和烷基取代的丙烯酰胺,例如甲基丙烯酰胺、叔丁基丙烯酰胺、N-叔丁基丙烯酰胺、N-甲基丙烯酰胺、N,N-二甲基丙烯酰胺、和3-N,N,-二甲基氨基丙基丙烯酰胺;丙烯腈,例如甲基丙烯腈;烯丙醇;烯丙基磺酸;烯丙基膦酸;乙烯基膦酸;2-乙烯基吡啶;4-乙烯基吡啶;N-乙烯基吡咯烷酮;N-乙烯基甲酰胺;N-丙烯吗啉;丙烯醛;乙二醇二丙烯酸酯;三羟甲基丙烷三丙烯酸酯;邻苯二甲酸二烯丙基酯;乙酸乙烯酯;苯乙烯;乙烯基磺酸、对苯乙烯磺酸、和2-丙烯酰胺基-2-甲基丙磺酸;甲基丙烯基磺酸酯和1-丙烯氧基-2-羟丙基磺酸酯(COPS);丙烯酰胺基乙二酸;以及上述化合物的盐。
起始聚合物最为优选多元丙烯酸,或丙烯酸和马来酸和马来酸酐的共聚物。此外,起始聚合物或共聚物的数均分子量(“Mn”)优选在500-100000,更为优选1000-50000,最为优选2000-25000的范围内。
用于控制聚合物成分的分子量的链转移剂均可以用作本发明的标记物。这些链转移剂包括但不仅限于:硫醇、次膦酸酯(phosphinate)、膦酸酯、亚磺酸(例如苯基亚磺酸和对甲苯基亚磺酸)、和氨基硫醇。适当的次膦酸酯包括U.S.5294371(Clubley等)中所公开的分子式I(col.1)的化合物和U.S.5376731(Kerr等)中所公开的分子式III(col.4)的化合物,适当的氨基硫醇包括U.S.5298585(McCallum等)中所公开的类型。再此引入这三篇专利用于参考他们描述这些类型的链转移剂和制备它们的方法的范围。优选使用半胱氨酸(“CYS”)、氨基乙基硫醇(“AET”)、或苯基次膦酸(“PPA”)作为链转移剂。
用于引发自由基加成聚合反应的然后引发剂和引发剂的片段均可以用作本发明的标记物。这些引发剂或引发剂的片段包括但不仅限于:过氧酯,例如过氧苯甲酸叔丁酯和过氧苯甲酸叔戊酯;二烷基过氧化物,例如过氧化二枯基;二芳基过氧化物,例如过氧化苯甲酰、氢过氧化物,例如氢过氧化枯烯;偶氮化合物,例如2-苯基偶氮-4-甲氧基-2,4-二甲基戊腈、2,2’-偶氮二-2-甲基-N-苯基丙酰脒二盐酸盐、2,2’-偶氮二-2(N-(4-氯苯基)-2-甲基丙酰脒)二盐酸盐、2,2’-偶氮二-(2-(5-甲基-2-咪唑啉-2-基)丙烷)二盐酸盐、和2,2’-偶氮二(2-(2-咪唑啉-2-基)丙烷)二盐酸盐。
当标记物是连接到链转移剂上的基团时,可选则的链转移剂是氨基硫醇,尤其是CYS或AET。可连接到这些链转移剂上的基团是那些可以与所需基团发生特异性的反应、而不与聚合物的其它链节反应的基团。当链转移剂含有胺的侧基时,优选在它们上连接可与胺反应的基团作为标记物。优选的可与胺反应的基团包括但不仅限于:1-(二甲胺氨基)-5-萘磺酸和其卤化物(“丹酰基”);4-二甲胺基偶氮苯-4-磺酸和其卤化物(“dabsyl”);2,4,6-三硝基苯磺酸和其盐(“TNT”);3-苯甲酰基喹啉-2-甲醛;3-(2-呋喃甲酰基(furfoyl))喹啉-2-甲醛;2,4-二硝基氟苯(Sanger's试剂);和茚三酮。特别优选的基团包括:丹酰基、dabsyl、和TNT。
本发明的单克隆或多克隆抗体可以通过本领域技术人员所知的技术进行制备。有许多制备多克隆抗体的技术,包括由B.A.L.Hurn和S.M.Chantler记载的技术,“试剂抗体的制备”,
酶学方法,70:104-142(1980)。制备MAbs的技术首先由G.Kohler和C.Milstein记载,Nature,265:495(1975)。在本发明的免疫测定技术中优选使用MAbs。
为了制备抗特殊标记物的MAbs,首先在接种结合物的小鼠体内制备标记物的致免疫结合物和致免疫载体。在1到300天或更长的时间内进行多次接种。接种可以通过许多途径中的任意一种进行,包括腹腔内、静脉内或皮下注射,每次注射剂量为5-50微克(“μg”)结合物。结合物可以单独注射,或与其它物质结合以增强其致免疫性。在免疫作用期结束时,收集来自脾脏的淋巴细胞,将其与骨髓瘤细胞融合形成杂交瘤。然后将这些杂交瘤分离、培养并测试,以测定哪种杂交瘤对标记物具有所需的特异性和亲和性。然后,根据本领域技术人员所知的技术让来自于这些选定的杂交瘤的MAbs在体外和体内生长。
本发明的杂交瘤包括所谓的Hybrid 1、Hybrid 2、Hybrid 3,通过瑞士或Balb/c小鼠的免疫作用获得。通过这些杂交瘤加工的MAbs,可与PPA本身、与2-羧基丙基(苯基)膦酸(本质上,PPA加一单位的AA)、或与标记的聚合物(PPA-PAA)在低至1ppm的浓度下反应,但是对未标记的聚合物(PAA)仅有少量亲和力或没有亲和力。这些MAbs适用于本发明的免疫测定测定方法中,包括直接夹层、间接夹层和竞争性ELISA测定。优选使用直接夹层ELISA。
在竞争性测定中,首先将抗原吸入到塑料载体上,然后加入游离的(未结合)抗原(通常来自于标准溶液),随后加入抗体,然后孵育溶液。在孵育期结束时,洗去溶液,仅留下附着于结合抗原上的抗体并对其进行检测。剩余抗体的浓度与样品中的半抗原的浓度成反比。
对于非竞争性测定,使用两个不同的抗体,每一个针对抗原上的不同表位,一个抗体将抗原结合于固体基质,第二个用于检测抗原并测定其浓度。在直接夹层中,可检测的标记被直接附着于第二个抗体;而在间接夹层中,使用标记的抗-免疫球蛋白测定第二个抗体本身。
由杂交瘤细胞表达并且对标记物具有所需的特异性和选择性的MAbs(“抗-标记MAbs”)可用于本发明的免疫检测测定方法中。一般地,根据本领域技术人员所知的技术对适当的杂交瘤系进行选择、使用这些杂交瘤系所表达的MAbs作为具体的免疫测定中的反应物并对其进行优化。
在以下的实施例中使用如下缩写。L=升;mL=毫升;μL=微升;g=克,μg=微克;M=摩尔;mM=亳摩尔;rpm=转/分;nm=纳米。KLH=钥孔槭血蓝素;BSA=牛血清白蛋白;OVA=卵白蛋白;AP=碱性磷酸酶。CFA=完全弗氏佐剂;IFA=“不完全”弗氏佐剂。另外,在以下的实施例中使用如下试剂。PBS:13.8mMNaCl+2.7mMKCl,pH7.4。10倍PBS(10L):138mMNaCl(800g)+2.7mMKCl(20g)+4.28mMNaPO4,二碱的(dibasic)7H2O(115g)+1.46mMKPO4(20g),pH7.2。PT(20L):10倍PBS(2L)+5%吐温20(200ml)+水(足量)。PCT(1L):PBS(990ml)+酪蛋白(10g)+5%吐温20(10ml),pH7.2,组织培养液(1L):小牛血清(10%)+L-谷氨酰胺(1%)+2-mercapthanol(6%)+Iscove’s修饰的Dulbecco’s培养液(痕量)。TMB:3,3’,5,5’-四甲基联苯胺(0.4g/L)在酸性缓冲液中的溶液。本段中所有的%均指体积。
实施例1:免疫结合物的制备
A.
TNT-酶结合物(DNP-AP)的制备
TNT-酶结合物通过将TNT衍生物(二硝基苯磺酸)以各种(w/w)比例加入到碱性磷酸酶中进行制备。
B.
PAP-PAA-蛋白结合物的制备
将苯膦酸(PAA)修饰的聚合物(其中PAA用作链转移剂)的丙烯酸部分用各种摩尔比的碳二亚胺激活,然后将这些激活的聚合物以各种(w/w)比例加入到载体蛋白(选于:KLH,BSA,OVA或AP)中,产生适当的PPA-PAA-蛋白结合物。未反应的半抗原通过用PBS充分透析去除。为了进行比较,用类似方法制备PPA-蛋白结合物。
实施例2:Mabs的制备
根据如下的免疫作用方法,对于各组的5只小鼠用3个月的时期注射一种实施例1B的免疫结合物。
在出生大约2个月后,将5只瑞士小鼠放血(尾静脉放血),合并血液测定血清。大约2周后,将0.1ml抗原(免疫结合物)在0.1mlCFA的盐水溶液中乳化的混合物注射入各小鼠的腹腔内。随后以2周为间隔,将0.1ml抗原的盐水溶液用0.1mlIFA乳化并注射入小鼠的腹腔内,共进行大约3个月。以1个月的间隔、以及在免疫作用期结束时取血样,以监测抗体水平。
到免疫作用期结束时,从脾脏获得致敏的淋巴细胞并将其与骨髓瘤细胞融合以形成杂交瘤细胞。可使用任何一种大量的骨髓瘤细胞系,但是优选的细胞系为P3×63Ag8.653。这些融合方法是本领域技术人员已知的;例如,Kearney等所记载的
J.Immunol.,123:1548(1979)。简单地讲,将淋巴细胞与骨髓瘤细胞在聚乙二醇的存在下结合一段时间、培养、检测对抗原各部分的特异性。
实施例3:检测Mab特性
使用以各种末端基团标记的聚合物进行竞争性测定。
TNT-CYS-聚合物(标记的聚合物)通过将半胱氨酸修饰的聚合物的氨基末端与TNBS反应进行制备。未反应的TNBS通过用Amicron微型浓缩器过滤除去。这种半胱氨酸修饰的聚合物(其中的半胱氨酸用作链转移剂)由McCallum等记载,参见前。
将Immulon-2培养皿用纯化的抗TNT MAb在0.1M碳酸盐缓冲液(pH9.6)中的5μg/mL的溶液包被,每孔100μL,将培养皿在4℃下孵育过夜或在37℃下孵育1小时。孵育后,将培养皿用PT冲洗3次,旋转180°,再用PT冲洗3次。此刻,加入125μL/孔PCT,于室温下将培养皿在设置在120rpm的培养皿摇动器上孵育1小时。孵育后,将培养皿用PT冲洗3次,旋转180°,再用PT冲洗3次。制备TNT-CYS-聚合物、CYS-PAA、PPA-PAA、和Acusol的不同浓度的抑制溶液,将各种抑制溶液和DNP-AP的1∶75的稀释液同时加入孔中。然后于室温下将培养皿在设置在120rpm的培养皿摇动器上孵育1小时。孵育后,将培养皿用PT冲洗3次,旋转180°,再用PT冲洗3次。然后向各孔中加入100μL对硝基苯基磷酸盐(PNPP)的硫酸二乙醇胺缓冲液的稀释液。然后于室温下将培养皿在设置在120rpm的培养皿摇动器上另外孵育15分钟。最后,将培养皿在设置在405nm的荧光计上读数。
图1显示该实验的结果。图解数据清楚地证明TNT-CYS-PAA在0.1到10μg/ml的浓度范围下易于检测,而相同浓度下的CYSPAA、PPA-PAA、和Acusol445N反应微小。而且,图1的数据显示,至少在检测的浓度范围内,在反应性和聚合物浓度之间存在着线性关系。
Claims (6)
1.一种鉴别和定量水溶液系统中的聚合物的方法,其中所述聚合物通过链聚合生产,且其中所述方法包括以下步骤:
a)获得所述水溶液系统的样品,其中所述系统中至少一定百分数的所述全部聚合物为可检测聚合物,所述各种可检测聚合物具有单一的可检测末端,所述末端选自由链转移剂、链引发剂、链引发剂片段和连接于链转移剂的基团组成的组,
b)将该样品与特异性结合到所述可检测末端的抗体一起孵育;并且
c)检测并测量所述抗体与所述系统中可检测末端的结合程度,其中所
述结合表示所述可检测聚合物的浓度,并允许测量水溶液系统中各种
聚合物的浓度,
其中所述链转移剂选自:硫醇、次膦酸、膦酸、亚磺酸、氨基硫醇、或其盐。
2.权利要求1的方法,进一步包括制备适当的抗可检测末端的单克隆或多克隆抗体的步骤。
3.权利要求1的方法,其中的可检测末端包括引发剂或引发剂片段,所述引发剂或引发剂片段选自:过氧酯、二烷基过氧化物、二芳基过氧化物、氢过氧化物、和偶氮化合物。
4.权利要求1的方法,其中的可检测末端包括连接于链转移剂上的可与胺反应的基团,且其中的链转移剂包括氨基硫醇。
5.权利要求4的方法,其中的可与胺反应的基团选自:1-(二甲胺氨基)-5-萘磺酸和其卤化物;4-二甲胺基偶氮苯-4-磺酸和其卤化物;2,4,6-三硝基苯磺酸和其盐;3-苯甲酰基喹啉-2-甲醛;3-(2-呋喃甲酰基)喹啉-2-甲醛;2,4-二氟硝基苯;和茚三酮。
6.权利要求1的方法,其中使用酶连免疫吸附测定来检测并测量单克隆或多克隆抗体的结合程度。
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- 1997-01-17 CN CNB971022720A patent/CN1144045C/zh not_active Expired - Lifetime
- 1997-01-17 SG SG1997000107A patent/SG60035A1/en unknown
- 1997-01-17 PL PL97318010A patent/PL318010A1/xx unknown
- 1997-01-17 JP JP01768397A patent/JP3776543B2/ja not_active Expired - Lifetime
- 1997-04-03 TW TW086104286A patent/TW454088B/zh not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
TW454088B (en) | 2001-09-11 |
AU1016397A (en) | 1997-07-24 |
CN1165298A (zh) | 1997-11-19 |
MX9700286A (es) | 1997-07-31 |
DE69712805T2 (de) | 2002-12-19 |
NO970167L (no) | 1997-07-21 |
JPH09218199A (ja) | 1997-08-19 |
US6197522B1 (en) | 2001-03-06 |
AU705491B2 (en) | 1999-05-20 |
ZA97248B (en) | 1997-07-18 |
PL318010A1 (en) | 1997-07-21 |
CA2195288A1 (en) | 1997-07-19 |
EP0785431B1 (en) | 2002-05-29 |
SG60035A1 (en) | 1999-02-22 |
NO970167D0 (no) | 1997-01-15 |
BR9700705A (pt) | 1998-08-18 |
EP0785431A1 (en) | 1997-07-23 |
DE69712805D1 (de) | 2002-07-04 |
JP3776543B2 (ja) | 2006-05-17 |
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