CN1155829C - 以四唑化合物为基础的试剂和测试带 - Google Patents
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Abstract
一种适于测定含有血红蛋白的生物体液,如全血,中的分析物的浓度的试剂。该试剂含有对该分析物具有特异性的脱氢酶、烟酰胺腺嘌呤二核苷酸(NAD)或NAD的衍生物、四唑染料前体、心肌黄酶或其类似物和亚硝酸盐。该试剂导致衡量该分析物的浓度的染色的形成。该亚硝酸盐抑制了由血红蛋白对染料形成的非酶性干扰。优选将该试剂用于干燥带上来测定酮体,如β-羟基丁酸。
Description
技术领域
本发明涉及一种可测定含血红蛋白生物体液中分析物浓度的诊断组合物。该组合物是以四唑染料(tetmazolium dye)前体为基础的,并涉及抑制它们的血红蛋白引起的还原。本发明也涉及以该诊断组合物为基础的测试带。
背景技术
脂肪组织是身体上能量贮藏的最丰富的形式之一。它将被贮藏的脂肪酸释放进入循环系统,并主要由肝脏对其进行代谢作用。在该过程中,消耗了脂肪酸而释放出能量并被身体加以利用。通常,几乎不消耗脂肪,而脂肪酸被完全代谢成二氧化碳和水,并且这种转化不会扰乱身体灵敏的PH平衡。但是,如果例如由于饮食等原因造成身体内糖类数量的不足,则脂肪消耗和脂肪酸的产生会增长到可能有害的程度。除节食者之外,胰岛素依赖性病人也是易患病的,因为他们的糖代谢被削弱了。如果使用过多的脂肪酸来供给身体的能量需要,则会产生大量的乙酰醋酸、丙酮和β-羟基丁酸。这些中间体被称为酮体,而将这种症状称为酮酸中毒。
通常,如果酮体不是过多的话,则它可以被身体重复利用而变成其它形式。因此,一个健康的个体所蓄积的这些分析物是微不足道的。如果在较短的时间内有大量的脂肪被代谢或者如果大部分的能量来源于脂肪,则会产生大量的酮体。过多地产生这些脂肪代谢物会导致某种神经紊乱,如果不及时地纠正该问题。
酮体存在于血液中,如果它超过了界限,则它会通过尿而被排出。采用现代诊断分析仪可以容易地对其进行检测。平均来说,β-羟基丁酸、乙酰酯酸和丙酮的百分数分别为78%、20%和2%。由于丙酮的浓度较低而挥发性较大,因而很少对它进行测定。实际上,通过硝普盐反应来对乙酰酯酸进行定量检测,而采用酶法来确定β-羟基丁酸的数量。乙酰酯酸测试带已用了几十年了。它们是以与醛和酮发生偶合反应的硝普盐离子为基础的。碱性尿样品或血清样品能与硝普盐发生数分钟的反应,并产生紫色。而颜色的强度指示出乙酰酯酸的浓度。但是,由于丙酮对该测试的干扰导致了读数升高。而且,如果病人的酮酸中毒发作康复时,尿和血液中的乙酰酯酸的水平升高,从而使诊断变得困难。
β-羟基丁酸测试更有用之处为监测酮体的浓度。它是以采用β-羟基丁酸对应的脱氢酶在烟酰胺腺嘌呤二核苷酸(NAD)辅助因子的作用下的β-羟基丁酸的氧化为基础的。(严格地说,只有D-β-羟基丁酸天然存在并被氧化,但是为简明起见我们在本说明书及所附的权利要求书中省略了“D”。)在氧化时,产生了NADH,并且它的浓度可以直接用UV分光光度计进行测定。因此,在光谱上的对应的信号的变化与该分析物的浓度成正比。不幸的是,NADH在UV区域发生了过度的激发,因此,这种检测模式仅适于实验室用仪器。另一种监测β-羟基丁酸的方法是通过采用四唑化合物来氧化NADH。
四唑化合物在组织的研究中起到了重要的作用。例如,已将这类化合物用于探测细胞的不需要氧的氧化和还原反应。而且,它们在临床诊断学中被普遍地应用。这些化合物一般为浅色或无色化合物,这些化合物在还原剂的作用下的经过还原反应而产生深色的甲(formazan)。一些还原剂如抗坏血酸、巯基或NADH和NADPH的变体能形成染色。
在临床诊断学中,已发现这些染色在监测由NAD(P)H的母体化合物,NAD(P)+在不需要氧的反应中形成的NAD(P)H时是没有意义的。该氧化还原反应很快并且对氧不敏感。所得的染色的颜色很深且在水中的溶解度小。
原则上,可将四唑染料前体用于测定全血的酮体和葡萄糖。但是,如果血红蛋白不存在于血液红细胞中,则四唑可被血红蛋白(Fe(II))非酶化还原而形成一种有色的甲。因此,游离的血红蛋白造成了对该测定的严重干扰。事实上,在典型的临床样品中,由于溶血以及所产生的相对于所说分析物而言的大量游离血红蛋白,来自血红蛋白的干扰信号会超过所预期的信号。在高血球比率样品中或当在较高的温度下进行该反应时,特别是其中的血红蛋白氧化反应较快时,这种现象尤其突出。如果采用四唑进行分析,则由于导致游离血红蛋白存在的红细胞的溶血难以避免,因此必须在测试前将红细胞从样品中除去。
可以采用薄膜和过滤器进行过滤、采用化学试剂进行截留或结合采用这两种方法来将红细胞从样品中除去。从全血中分离红细胞的过滤方法成本高并且需要相当大的样品体积。一个采用过滤从全血样品中除去红细胞而进行血酮(β-羟基丁酸)测试的实例是来自GDS诊断学,Elkhart,IN.中的ketosite测试(见临床化学Tietz教科书,第二版,C.Burtis等编辑,W.B.Saunder Co.,Philadelphia,PA,1994,p.974)。在该测试中所用的“测试卡片”具有两层过滤层,它造成了这种卡片的成本较高,并需要大量(25μL)血样品。而且,该血液必须是不能被溶血。
在得自Miles的AmesGlucometer EncoreTM的血液葡萄糖检测带中将过滤和化学截留方法结合使用。该检测带采用一层过滤材料和一种聚集反应助剂(马铃薯凝集素)以消除来自红细胞的干扰。(见Chu等人,欧洲专利申请0638805A2,1995年2月15日公开。)
减少血红蛋白干扰的另一种方法是向系统中引入一种氧化剂以将血红蛋白氧化成高铁血红蛋白。虽然已知氰铁酸盐将血红蛋白转变成高铁血红蛋白,但它们也破坏了预期的产物NADH。
发明内容
本发明提供了一种测定含血红蛋白生物流体中分析物浓度的试剂。该试剂含有:
a)对该分析物具有特异性的脱氢酶,
b)烟酰胺腺嘌呤二核苷酸(NAD)或NAD的衍生物,
c)四唑染料前体
d)心肌黄酶或其类似物,和
e)亚硝酸盐。
特别适合将该试剂涂在一个或多个基片上以形成用于测定分析物的干燥的试剂带。特别优选的试剂带含有:
a)载体层
b)位于该载体层上的含涂料的测试垫板,该涂料含有:
i)对该分析物具有特异性的脱氢酶
ii)烟酰胺腺嘌呤二核苷酸(NAD)或NAD的衍生物,
iii)四唑染料前体,和
iv)心肌黄酶或其类似物
c)位于该测试垫板上的被亚硝酸盐所涂布的吸湿性顶层。
附图说明
图1为本发明测试带的透视图。
图2为本发明的另一种测试带的“部件分解图”。
图3为本发明的又另一种测试带的“部件分解图”。
图4为本发明的酮测定的化学图示描述。
图5的图表显示了以亚硝酸盐作为血红蛋白抑制剂的效果。
具体实施方式
本发明提供了一种通过产生某一浓度的衡量分析物浓度的NADH来测定含血红蛋白的生物体液(如全血)中分析物浓度的试剂。该试剂中含有的亚硝酸盐克服了血红蛋白对NADH浓度测定的干扰。它特别适用于但不限于酮体的测定。
图1描述了本发明的典型的测试带10,它由覆盖在载体14上的测试垫板12组成。该载体可以是塑料-如聚苯乙烯、尼龙或聚酯-或金属薄片或任何一种本技术中已知的其它合适的材料。该测试垫板涂有与该分析物发生反应而导致颜色变化的试剂。该测试垫板优选含有吸湿性材料,如滤纸或聚合物薄膜。但是,由于该反应不需要氧,该测试垫板可以是非吸湿性材料,如塑料薄膜。该试剂含有对该分析物具有特异性的酶、氢负离子转移试剂、四唑染料前体、适宜的酶辅助因子和血红蛋白抑制剂。为更加稳定,该试剂可选择地含有缓冲剂和稳定剂。
如图2所示,该测试带也可以是顶层16覆盖在测试垫板12上的多层结构。在该结构中,该试剂可在这两层之间被分开。例如,可将血红蛋白抑制剂涂在可选择的顶层16上而将该试剂的剩余部分涂在测试垫板12上。优选顶层16为吸湿性的并用作涂敷层和吸收多余样品的吸收剂层。将样品涂在顶层16,使其经过测试垫板12。可通过测定透过载体层14的颜色的变化,或者如果层14与该反应区域邻接的地方不透明,则可通过观察透过可选择的窗或通孔18的颜色的变化来检测该分析物的浓度。
在图3所示的可选择的实施方案中,隔离物20将顶层16和测试垫板12分开。隔离物20优选为双面具有粘合涂料(没有显示)的非吸湿性塑料薄膜。隔离物20上的槽22给样品提供了毛细通道而使其从开口24流到测试区域26。流动取决于测试垫板12表面和相邻层之间的空气流通,或可选择地通过可选择的出口18。通过可选择的出口/窗口18来监测测定区域26的颜色变化。试剂可以全部在测试垫板12上或者可选择地在测试垫板和一或两层非吸湿性层14和16之间被分开。如果我们称试剂为“涂料”或“盖”在层上,则我们意味着含有试剂将被吸收入该层的可能性,特别是如果它是吸湿性的话。
适于采用本发明进行测定的酶和其对应的分析物为:对乙醇来说是乙醇脱氢酶,对甲醛来说为甲醛脱氢酶,对葡萄糖来说为葡萄糖脱氢酶,对葡萄糖-6-磷酸来说是葡萄糖-6-磷酸脱氢酶,对谷氨酸来说是谷氨酸脱氢酶,对甘油来说是甘油脱氢酶,对于β-羟基丁酸来说是β-羟基丁酸脱氢酶,对于类固醇来说是羟基类固醇脱氢酶,对于L-乳酸来说是L-乳酸盐脱氢酶,对于亮氨酸来是亮氨酸脱氢酶,对于苹果酸来说是苹果酸脱氢酶,对丙酮酸来说是丙酮酸脱氢酶。
需要适宜的酶辅助因子来活化该酶。根据该酶,可用这些辅助因子:β-烟酰胺腺嘌呤二核苷酸(β-NAD)、β-烟酰胺腺嘌呤二核苷酸磷酸盐(β-NADP)、硫代烟酰胺腺嘌呤二核苷酸、硫代烟酰胺腺嘌呤二核苷酸磷酸盐、烟酰安1,N6-亚乙烯基腺嘌呤二核苷酸和烟酰胺1,N6-亚乙烯基腺嘌呤二核苷酸磷酸盐。在酶的作用下,该分析物将该辅助因子还原。
染料-形成过程的下一步骤是从被还原的辅助因子中进行氢化物分离。它可以通过心肌黄酶如硫辛酸脱氢酶,铁氧还蛋白-NADP还原酶,硫辛酰胺脱氢酶,或合成的类似物如吩嗪甲基硫酸盐(PMS)或麦尔多拉蓝。反应动力学和稳定性是选择氢负离子转移试剂或“转移剂”的主要因素。例如,PMS为通用的氢化物转移剂,因为它具有与以下所列的大部分四唑化合物相比较快的反应动力学。但是,它与以酶为基础的氢化物转移剂相比对光的敏感性较高。心黄肌酶更为稳定,并且由于这个原因而作为优选。
将被截留的氢化物转变成四唑化合物(染料前体)以形成有色的甲。最适于这种设计的四唑化合物为:2-(2’苯并噻唑基)-5-苯乙烯基-3-(4’-邻苯二甲酰基肼基)四唑(BSPT)、2-苯并噻唑基-(2)-3,5-二苯基四唑(BTDP)、2,3-二(4-硝基苯基)四唑(DNP)、2,5-二苯基-3-(4-苯乙烯基苯基)四唑(DPSP)、联苯乙烯基氨蓝四唑(distyryl nitroblue tetrazolium)(DS-NBT)、3,3’-[3,3’-二甲氧基-(1,1’-联苯基-4,4’-二基]-双[2-(-4-硝基苯基)-5-苯基(-2H四唑(NBT)、3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H四唑(MTT)、2-苯基-3-(4-羧基苯基)-5-甲基四唑(PCPM)、四唑蓝(TB)、硫代氨基甲酰基氨蓝四唑(TCNBT)、四氨蓝四唑(TNBT)、四唑紫(TV)、2-苯并噻唑噻唑基-3-(4-羧基-2-甲氧基苯基)-5-[4-(2-磺乙基氨甲酰基)苯基]-2H-四唑(WST-4)和2,2’-二苯并噻唑基-5,5’-双[4-二(2-磺乙基)氨甲酰基苯基]-3,3’-(3,3’-二甲氧基-4,4’-亚联苯基)双四唑二钠盐(WST-5)。优选WST-5,因为它易溶于与生物样品最相容的水性介质中。而且,所产生的甲化合物在紫-蓝区域表现出强烈的波谱吸收,因此减少了对来自血红蛋白的背景信号进行修正的需求。
最后,将血红蛋白抑制剂置于该试剂中以削减在血红蛋白和四唑化合物之间的不期望的染料形成反应。血红蛋白抑制剂的作用是将血红蛋白氧化成不与四唑反应的高铁血红蛋白。令人惊讶的是,亚硝酸盐如亚硝酸钠、亚硝酸钾和它们的衍生物非常有效地抑制了血红蛋白而不破坏NADH。在高温和采用高血球比率的样品时亚硝酸盐也有效。优选亚硝酸钠,因为它具有高水溶性、无毒且成本较低。
虽然可以以湿化学模式如在比色杯中使用本发明的试剂,但在一个优选的实施方案中,本发明是一种用于测定全血中β-羟基丁酸的干燥的带。它由被置于载体和顶层之间的薄膜测试垫板优选为尼龙的测试垫板组成。载体优选是聚酯薄膜。顶层可以是任何一种现有技术中已知的吸湿性材料。优选的材料为购自PorexCorp.of Fairburn,GA的甲基油酰牛磺酸(taurate)钠处理的多孔性聚乙烯。我们将这种材料称为“Porex”。该测试垫板含有一种含β-羟基丁酸脱氢酶、NAD、心肌黄酶和WST-5(以下的表1)的试剂。该Porex顶层含有亚硝酸盐试剂(表2)。
在操作中,使用者将一滴全血涂在该Porex顶层的上表面。当全血或被溶解的血与该Porex发生接触时,亚硝酸钠会再生并与存在的游离血红蛋白发生反应,因此使血红蛋白不妨害该测定。通过毛细管或重力将产生的基本上不含血红蛋白的样品转移到下面的测试垫板中。在该测试垫板上该样品引发了图4所描述的级联反应而得到一种有色的染料,该染料的深度与样品中的β-羟基丁酸成正比并可用光度计对其进行检测。
图5描述了采用含0到15mg/dl的血样品时亚硝酸盐对该系统成色反应的作用。在没有亚硝酸盐存在下,血红蛋白将四唑还原而形成连续增长染料浓度,同时相应地增大光密度。亚硝酸盐通过除去血红蛋白(通过氧化)而将成色限制到只对酮体(如β-羟基丁酸)显色的程度。
以下的实施例显示了本发明的优选的实例,其中该分析物为β-羟基丁酸而酶为β-羟基丁酸脱氢酶。可以容易地对该组合物进行改变而用于前面所列的其它分析物-酶的组合。(例如参见Tietz临床化学教科书,第二版,由C.Burtis等人编辑,W.B.Saunders Co.,Philadelphia,PA,1994,pp976-978和1174-1175。)该实施例并不意味着以任何方式进行限定。
实施例1
将购自Cuno(Meriden,CT,USA)0.8μm的尼龙薄膜浸渍到表1的试剂中,直至饱和。用玻璃棒将多余的试剂轻刮去。将所得的薄膜悬挂于56℃烘箱中干燥10分钟。将Porex(0.6mm厚)浸泡在表2的亚硝酸盐溶液中,接着将其悬挂于100℃烘箱中干燥10小时。最后,在聚酯原料(购自美国ICI,Wilmington,DE的0.4mmMelenex聚酯)和被亚硝酸盐浸渍的Porex之间对该薄膜进行层压。
表1.用于测试垫板的试剂
组分 | 数量 |
水 | 100ml |
三(羟甲基)氨基甲烷(MW 121,Sigma,St.Louis,MO,USA)(通过加入6M的HCL将PH调整至8.5) | 1.2gm |
氯化钠(MW 56.44,Sigma,St.Louis,MO,USA) | 560mg |
氯化镁(MW 203,Sigma,St.Louis,MO,USA) | 2.5gm |
PSSA,聚苯乙烯磺酸,钠盐(MW 70,000,Polysciences,Inc.,Warrington,PA,USA) | 3gm |
藏花精(Crotein)(Croda Inc.Parsippany,NJ,USA) | 3gm |
草氨酸,钠盐(MW 111.03,Aldrich Chemicals,Milwaukee,WI,USA) | 250mg |
Tetronic 1307(BASF Corporation,Mount Olive,New Jersey,USA) | 2gm |
蔗糖(MW 342.30,Aldrich chemicals,Milwaukee,WO,USA) | 5gm |
NAD(MW 663.4,N-7004,Sigma,St.Louis,MO,USA) | 450mg |
D-3-羟基丁酸脱氢酶(来源:假单胞菌属SP.HBD-301,125U/mg,Toyobo,日本) | 50,000U |
心肌黄酶(来源:B.stearothermophilus,New,1033u/mg,Toyobo,日本) | 340890U |
WST-5(MW1331.37,Dojindo,日本) | 1.8gm |
表2.亚硝酸盐试剂
组分 | 数量 |
10mM磷酸盐缓冲液,PH7.4,(P-3813,Sigma,St.Louis,MO,USA) | 70ml |
乙醇 | 30ml |
亚硝酸钠(MW69,Aldrich Chemicals,Milwaukee,WI,USA) | 5mg |
聚乙烯吡咯烷(MW 40,000,Sigma,St.Louis,MO,USA | 200mg |
草氨酸,钠盐(MW 111.03,Aldrich Chemicals,Milwaukee,WI,USA) | 500mg |
Claims (10)
1.一种用于测定含有血红蛋白的生物体液中的分析物浓度的试剂,含有:
a)对该分析物具有特异性的脱氢酶,
b)烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷酸的衍生物,
c)四唑染料前体
d)心肌黄酶或其类似物,和
e)亚硝酸盐。
2.权利要求1的试剂,其中该分析物为β-羟基丁酸,而该酶为β-羟基丁酸脱氢酶。
3.一种用于测定含有血红蛋白的生物体液中的分析物的存在及其数量的染色试剂带,含有一个载体层,在该载体层上有含权利要求1的试剂涂料层的测试垫板。
4.权利要求3的试剂带,进一步含有覆盖在该测试垫板上的吸湿性顶层。
5.一种测定含有血红蛋白的生物体液中的分析物的存在及其数量的染料试剂带,含有一个载体层,在该载体层上有一测试垫板和覆盖在该测试垫板上的顶层,其中权利要求1的试剂的a)-d)组分在该测试垫板上,而该试剂的e)组分在该载体层和/或顶层上。
6.权利要求5的试剂带,进一步含有位于顶层和测试垫板之间的隔离物和槽,从而在该层和垫板之间提供毛细通道。
7.权利要求5的试剂带,其中该分析物为β-羟基丁酸,而该酶为β-羟基丁酸脱氢酶。
8.权利要求5的试剂带,其中该分析物为葡萄糖,而该酶为葡萄糖脱氢酶。
9.权利要求5的试剂带,其中该四唑染料前体为2,2’-二苯并噻唑基-5,5’-双[4-二(2-磺乙基)氨基甲酰基苯基]-3,3’-(3,3’-二甲氧基-4,4’-亚联苯基)双四唑二钠盐。
10.一种用于检测含有血红蛋白的生物体液中的分析物的存在及其数量的染料试剂测试带,含有:
a)载体层
b)位于该载体层上的含涂料层的测试垫板,该涂料层含有:
i)对该分析物具有特异性的脱氢酶
ii)烟酰胺腺嘌呤二核苷酸或烟酰胺腺嘌呤二核苷酸的衍生物,
iii)四唑染料前体,和
iv)心肌黄酶或其类似物
c)位于该测试垫板上的被亚硝酸盐所涂布的吸湿性顶层。
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