CN116656776B - 一种稳定的醛缩酶测定试剂盒及其制备方法和应用 - Google Patents
一种稳定的醛缩酶测定试剂盒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种稳定的醛缩酶测定试剂盒,由以下成分组成,试剂盒含有试剂R1和试剂R2;试剂R1包含以下组分:缓冲液、NADH、1,6‑二磷酸果糖、磷酸丙糖异构酶、乳酸脱氢酶、甲酸脱氢酶、甲酸、稳定剂、叠氮钠;试剂R2包含以下组分:缓冲液、3‑磷酸甘油脱氢酶、稳定剂、酶激活剂、Proclin 300。该试剂盒是稳定性强、准确度高、抗干扰性强的液体试剂盒。同时还公开了该试剂盒的制备方法和应用,用于非疾病的诊断和治疗目的测定血清中醛缩酶的浓度。
Description
技术领域
本发明涉及生化检测技术领域,特别是涉及一种稳定的醛缩酶测定试剂盒及其制备方法和应用。
背景技术
醛缩酶(aldolase)是一种可以裂解羟醛的酶,有三个不同亚型,分别为aldolaseA(ALDOA)、aldolase B(ALDOB)和aldolase C(ALDOC),其基因染色体定位分别为17q11.2、9q31.1和17q11.2。醛缩酶家族成员在人体组织中的表达具有特异性。研究发现,发育中的胚胎组织及成年人肌肉组织中主要表达ALDOA;成年人肝脏、肾脏组织中多表达ALDOB;在大脑及其他神经组织中,ALDOC的表达则更为丰富。醛缩酶家族主要在糖酵解过程中发挥重要作用,在以葡萄糖为底物的糖酵解过程中,醛缩酶家族参与催化1,6-二磷酸果糖(fructose-1,6-diphosphate,FDP)生成磷酸二羟丙酮(dihydroxyacetone phosphate,DHAP)和3-磷酸甘油醛(3-phosphoglyceraldehyde,G3P)。
血清醛缩酶测定有助于诊断肌肉和肝脏疾病。(1)肌肉疾病:肌营养不良症、多发性肌炎等患者血清醛缩酶活性升高。在肌营养不良症中,以假肥大型、肢带型和远端型的酶活性最高,肩肱型和眼肌型仅轻度升高或正常。通常,醛缩酶活性随年龄增加而递减,在活动期和肌肉萎缩之前酶活力增高最显著,故其测定有助于本病的诊断。心肌梗死患者血清醛缩酶活力升高,一般在胸痛发作24~48h达高峰,其变化规律与AST相同,但比AST改变要早,心绞痛时则正常。(2)肝脏疾病:急性病毒性肝炎醛缩酶活性的升降与ALT相平行。慢性肝炎、肝硬化、阻塞性黄疸仅轻度升高。
另有研究发现,醛缩酶家族成员在恶性肿瘤中的异常表达已被证实与肿瘤患者预后密切相关。ALDOA在胃癌、非小细胞肺癌、前列腺癌、乳腺癌、口腔鳞状上皮细胞癌和甲状腺癌等多种肿瘤组织中均呈现高表达,并可作为一个独立的预后因素,提示患者的不良预后结局。
常用的血清醛缩酶活性测定有比色法、酶偶联法、酶联免疫吸附实验法(ELISA),手动比色法操作复杂,容易出现误差;酶联免疫吸附实验法(ELISA)目前在临床应用广泛,技术成熟,虽然在灵敏度、特异性、试剂价格等方面占有优势,但该技术在检测时间、操作性等方面存在弊端。酶偶联法主要为3-磷酸甘油脱氢酶偶联法,醛缩酶催化1,6-二磷酸果糖生成磷酸二羟丙酮和3-磷酸甘油醛,在NADH存在下磷酸二羟丙酮还原成1,3-二磷酸甘油,于波长340nm监测吸光度下降速率,但是酶偶联法容易受到丙酮酸的干扰,NADH在液体溶液中非常不稳定,容易被氧化成NAD+。
发明内容
为了解决上述问题,本发明提供一种稳定的醛缩酶测定试剂盒,该试剂盒是稳定性强、准确度高、抗干扰性强的液体试剂盒。
本发明是通过以下技术方案实现的:
一种稳定的醛缩酶测定试剂盒由以下成分组成,试剂盒含有试剂R1和试剂R2;
试剂R1包含以下组分:
试剂R2包含以下组分:
优选地,试剂R1的pH值为8.0-10.0;试剂R2的pH值为7.0-9.0。
优选地,试剂R1中所述缓冲液为三羟甲基氨基甲烷缓冲液、磷酸盐缓冲液、三乙醇胺缓冲液、甘氨酸缓冲液中的任一种;试剂R2中所述缓冲液为三羟甲基氨基甲烷缓冲液、磷酸盐缓冲液、三乙醇胺缓冲液、甘氨酸缓冲液中的任一种。
优选地,试剂R1中所述的稳定剂由甘露寡糖、牛血清白蛋白和表面活性剂四聚乙二醇单月桂醚组成,且质量比为2-4:3:3-4。
优选地,试剂R2中所述的酶激活剂为ZnSO4·7H2O、ZnCl2的任一种。
优选地,试剂R2中所述的稳定剂为酪蛋白酸钠。
优选地,所述试剂R1与试剂R2的体积比为1~5:1,更优选地,所述试剂R1与试剂R2的体积比为4:1。
上述所述的醛缩酶测定试剂盒的制备方法,包括以下步骤:
(1)试剂R1的配制:取适量水,分别加入R1中的缓冲液、甲酸、1,6-二磷酸果糖(FDP)、稳定剂、叠氮钠,搅匀溶解一个原料后加入下一个原料,用盐酸或氢氧化钠调节到pH值8.0-10.0,再加入磷酸丙糖异构酶、NADH、乳酸脱氢酶、甲酸脱氢酶,搅拌溶解后定容到所需体积;
(2)试剂R2的配制:取适量水,分别加入R2中缓冲液、稳定剂、酶激活剂、Proclin300,搅匀溶解一个原料后加入下一个原料,用盐酸或氢氧化钠调节到pH值7.0-9.0,再加入3-磷酸甘油脱氢酶,搅拌溶解后定容到所需体积。
上述所述的醛缩酶测定试剂盒的应用,用于非疾病的诊断和治疗目的测定血清中醛缩酶的浓度。
本发明醛缩酶测定试剂盒原理为醛缩酶通过分解1,6-二磷酸果糖为磷酸二羟丙酮和3-磷酸-D-甘油醛,3-磷酸-D-甘油醛在磷酸丙糖异构酶的作用下转化为磷酸二羟丙酮,在NADH存在下3-磷酸甘油脱氢酶将磷酸二羟丙酮还原成1,3-二磷酸甘油,于波长340nm监测NADH吸光度下降速率。
本发明检验原理如下:
本发明的有益效果:
1、本发明稳定的醛缩酶测定试剂盒为液体双试剂,无需复溶配制,开瓶可以直接使用。
2、本发明采用NADH辅酶再生,甲酸脱氢酶催化甲酸和NAD+生成CO2和NADH,甲酸和CO2对R1中磷酸丙糖异构酶和乳酸脱氢酶的活性没有影响,有利于提高试剂的稳定性和准确度。
3、本发明试剂R1中添加稳定剂甘露寡糖、牛血清白蛋白和表面活性剂四聚乙二醇单月桂醚,且质量比为2-4:3:3-4,有利于保护R1中磷酸丙糖异构酶、乳酸脱氢酶和甲酸脱氢酶,提高试剂R1的稳定性,表面活性剂四聚乙二醇单月桂醚还有利于消除乳糜干扰。
4、本发明试剂R2中添加稳定剂酪蛋白酸钠,保护3-磷酸甘油脱氢酶,显著提高试剂R2的稳定性。
5、本发明制备的醛缩酶测定试剂稳定性好、准确度高、线性范围广、抗干扰性强,37℃可保存10天,明显优于市售的同类型产品。
附图说明
图1为对比例1与实施例1的线性方程;
图2为对比例1与对比例2的线性方程;
图3为对比例1与对比例7的线性方程;
图4为实施例1、对比例1-6的稳定性测试;
图5为实施例1、对比例1-6的吸光度测试。
具体实施方式
以下是本发明的具体实施例结合附图说明,对本发明的技术方案作进一步的描述,但本发明并不限于这些实施例。本发明试剂盒测定血清中醛缩酶的测试条件为:方法:速率法;主/副波长:340nm/405nm;温度:37℃;校正类型:线性;校准方法:两点定标;反应方向:向下。
具体操作如表1所示:
表1醛缩酶测定试剂操作步骤
样本要求:
1.不溶血血清。
2.样本稳定性:标本2~8℃保存可稳定3天,-20℃保存可稳定2周。
实施例1
试剂R1包含以下组分:
pH 8.5
试剂R2包含以下组分:
pH 8.3。
制备方法为:(1)试剂R1的配制:取适量水,分别加入R1中的三羟甲基氨基甲烷、甲酸、1,6-二磷酸果糖、甘露寡糖、牛血清白蛋白、四聚乙二醇单月桂醚、叠氮钠,搅匀溶解一个原料后加入下一个原料,用盐酸或氢氧化钠调节到pH值8.5,再加入磷酸丙糖异构酶、NADH、乳酸脱氢酶、甲酸脱氢酶,搅拌溶解后定容到所需体积。
(2)试剂R2的配制:取适量水,分别加入R2中三羟甲基氨基甲烷、酪蛋白酸钠、ZnSO4·7H2O、Proclin 300,搅匀溶解一个原料后加入下一个原料,用盐酸或氢氧化钠调节到pH值8.3,再加入3-磷酸甘油脱氢酶,搅拌溶解后定容到所需体积。
实施例2
试剂R1包含以下组分:
pH 8.5
试剂R2包含以下组分:
pH 8.5。
制备方法同实施例1。
实施例3
试剂R1包含以下组分:
pH 8.5
试剂R2包含以下组分:
pH 8.8。
制备方法同实施例1。
对比例1
醛缩酶ELISA试剂盒。
对比例2
试剂R1包含以下组分:
pH 8.5
试剂R2包含以下组分:
三羟甲基氨基甲烷缓冲液 100mmol/L
3-磷酸甘油脱氢酶 30KU/L
Proclin 300 1ml/L
pH 8.3
制备方法同实施例1(其他不同的成分不添加)。
对比例3
与实施例1中醛缩酶测定试剂盒的区别仅在于试剂R1不含甘露寡糖,其它与实施例1相同。
对比例4
与实施例1中醛缩酶测定试剂盒的区别仅在于试剂R1不含牛血清白蛋白,其它与实施例1相同。
对比例5
与实施例1中醛缩酶测定试剂盒的区别仅在于试剂R1不含四聚乙二醇单月桂醚,其它与实施例1相同。
对比例6
与实施例1中醛缩酶测定试剂盒的区别仅在于试剂R2不含酪蛋白酸钠,其它与实施例1相同。
对比例7
与实施例1中醛缩酶测定试剂盒的区别仅在于试剂R2不含ZnSO4·7H2O,其它与实施例1相同。
性能验证
试验一相关性试验
实验方案为:实施例1、对比例1、对比例2和对比例7,同时检测了40个临床血清样本,对两组检测结果进行相关性分析,计算相关系数r;以对比例1检测结果作为靶值,分别计算40对数据的相对偏差(Bias%)。要求r不小于0.990,相对偏差不超过±10%。
表2相关性对比实验结果
表3对比例1分别与实施例1、对比例2和对比例7的相关系数
相关系数r | |
实施例1和对比例1 | 0.9982 |
对比例2和对比例1 | 0.8185 |
对比例7和对比例1 | 0.9855 |
由表3和图1的可以看出,实施例1和对比例1的试剂盒血清样本测试偏差最大值为2.98%,试剂的相关系数为0.9982,实施例1和对比例1的检测结果非常接近,因此本发明提供的实施例1检测试剂准确度较高;由表3和图2可以看出,对比例2和对比例1检验结果偏差较大,相关系数为0.8185,说明本发明试剂盒准确度优于对比例2。对比例7的检测结果与对比例1相关性提高,但由于ZnSO4·7H2O的缺失,有些样本还是偏差较大,说明ZnSO4·7H2O的添加促进3-磷酸甘油脱氢酶发挥作用,有利于进一步提高试剂的准确度。
试验二线性相关试验
取醛缩酶高值样本为200U/L,并进行稀释,配制6个不同浓度的样本,依次为200U/L、100U/L、50U/L、25U/L、12.5U/L、0U/L浓度的样本,每个浓度水平各样本分别测定三次,分别取其平均值。用实施例1的试剂进行检测。检测结果如表4所示:
表4线性相关验证实验检测结果
理论浓度(U/L) | 实测浓度(U/L) |
0.00 | 1.20 |
12.50 | 11.80 |
25.00 | 26.40 |
50.00 | 49.20 |
100.00 | 103.10 |
200.00 | 198.50 |
相关系数r | 0.9995 |
由表4可以看出,本发明实施例1随稀释浓度呈线性变化,线性相关系数达到0.9995,大于0.990,说明实施例1线性范围较广,能够符合临床病例样本的要求,对于临床检验有重要意义。
试验三稳定性试验
对实施例1和对比例1、2、3、4、5、6提供的醛缩酶测定试剂进行稳定性试验,实验方案为:对实施例1和对比例1、2、3、4、5、6提供的试剂,一起放入37℃水浴箱中,每天检测靶值为16.0±2.3U/L的质控品和水,并监测质控品测定值和空白吸光度的变化。
表5试剂热稳定性验证结果(质控品)
表6试剂热稳定性验证结果(空白吸光度)
时间(天) | 实施例1 | 对比例1 | 对比例2 | 对比例3 | 对比例4 | 对比例5 | 对比例6 |
1 | 1.416 | 1.411 | 1.440 | 1.464 | 1.371 | 1.334 | 1.489 |
2 | 1.452 | 1.484 | 1.238 | 1.409 | 1.330 | 1.310 | 1.450 |
3 | 1.446 | 1.459 | 1.182 | 1.345 | 1.244 | 1.228 | 1.185 |
4 | 1.408 | 1.443 | 1.128 | 1.319 | 1.184 | 1.201 | 1.156 |
5 | 1.430 | 1.422 | 1.083 | 1.253 | 1.105 | 1.168 | 1.118 |
6 | 1.422 | 1.450 | 1.062 | 1.109 | 1.014 | 1.146 | 1.109 |
7 | 1.445 | 1.409 | 1.058 | 0.996 | 0.985 | 1.076 | 1.046 |
8 | 1.404 | 1.410 | 0.930 | 0.885 | 0.973 | 0.955 | 1.001 |
9 | 1.476 | 1.432 | 0.925 | 0.846 | 0.930 | 0.940 | 0.996 |
10 | 1.480 | 1.495 | 0.918 | 0.828 | 0.916 | 0.914 | 0.917 |
由表5、6和图4、5可以看出,与对比例1相似,本发明提供的实施例1试剂与对比例1类似,在37℃水浴条件下10天内基本无变化,稳定性较好;而对比例2、3、4、5、6试剂在37℃水浴条件下10天内水空白吸光度降低,样本值降低,实施例1的试剂盒稳定性优于对比例2、3、4、5、6试剂盒的稳定性。说明本发明通过添加辅酶再生的甲酸脱氢酶和甲酸,使氧化的NAD+又生成NADH,提高R1中NADH的稳定性,而甘露寡糖、牛血清白蛋白、四聚乙二醇单月桂醚的同时加入,可以协同提高试剂R1中磷酸丙糖异构酶、乳酸脱氢酶和甲酸脱氢酶的稳定性,酪蛋白酸钠的加入提高试剂R2中3-磷酸甘油脱氢酶的稳定性。
试验四 抗干扰试验
对实施例1和对比例2、5提供的醛缩酶测定试剂盒进行抗干扰性实验,实验方案为:取靶值为36±3.4U/L的醛缩酶质控品,分成25等份,加入干扰物质丙酮酸钠和乳糜,使其在血清中的浓度达到表7的要求,然后分别采用实施例1和对比例2、5所得的试剂,测定质控品中醛缩酶的含量,其中相对偏差(%)=(干扰样本的测定均值-对照样本的测定均值)/对照样本的测定均值×100%。
表7试剂抗干扰性能结果比较
由表7可以看出,在上述浓度干扰物质存在时,本发明试剂盒的检测结果相对偏差小,没有受到明显干扰,而对比例2和对比例5试剂相对偏差较大,干扰效果明显,对比例2虽然添加等量的乳酸脱氢酶,但没添加稳定剂的情况下,乳酸脱氢酶不稳定,并不能有效消除丙酮酸钠的干扰。对比例2和对比例5试剂在表面活性剂四聚乙二醇单月桂醚缺失的情况下,容易受乳糜微粒的干扰,说明本发明的试剂盒抗干扰能力优于对比例试剂,各种保护剂的添加增强酶的活性和试剂的抗干扰性能。
文中所描述的具体实施例仅仅是对本发明精神作举例说明。本发明所属技术领域的技术人员可以对所描述的具体实施例做各种修改或补充或采用类似的方式替代,但并不会偏离本发明的精神或者超越所附权利要求书所定义的范围。
Claims (2)
1.一种稳定的醛缩酶测定试剂盒,其特征在于,试剂盒含有试剂R1和试剂R2;
试剂R1包含以下组分:
三羟甲基氨基甲烷缓冲液 100mmol/L
NADH 3g/L
1,6-二磷酸果糖 15g/L
磷酸丙糖异构酶 20KU/L
乳酸脱氢酶 20KU/L
甲酸脱氢酶 30KU/L
甲酸 1ml/L
甘露寡糖 2g/L
牛血清白蛋白 3g/L
四聚乙二醇单月桂醚 3g/L
叠氮钠 1g/L
pH 8.5;
试剂R2包含以下组分:
三羟甲基氨基甲烷缓冲液 100mmol/L
3-磷酸甘油脱氢酶 40KU/L
酪蛋白酸钠 2g/L
ZnSO4·7H2O 0.005g/L
Proclin 300 1ml/L
pH 8.3。
2.一种权利要求1所述的一种稳定的醛缩酶测定试剂盒的制备方法,其特征在于,包括以下步骤:
(1)试剂R1的配制:取适量水,分别加入R1中的三羟甲基氨基甲烷缓冲液、甲酸、1,6-二磷酸果糖、甘露寡糖、牛血清白蛋白、四聚乙二醇单月桂醚和叠氮钠,搅匀溶解一个原料后加入下一个原料,用盐酸或氢氧化钠调节到 pH 值8.5,再加入磷酸丙糖异构酶、NADH、乳酸脱氢酶和甲酸脱氢酶,搅拌溶解后定容到所需体积;
(2)试剂R2的配制:取适量水,分别加入R2中三羟甲基氨基甲烷缓冲液、酪蛋白酸钠、ZnSO4·7H2O和Proclin 300,搅匀溶解一个原料后加入下一个原料,用盐酸或氢氧化钠调节到pH值8.3,再加入3-磷酸甘油脱氢酶,搅拌溶解后定容到所需体积。
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