CN114460190A - 一种β2-微球蛋白含量测定方法 - Google Patents
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Abstract
本发明公开了一种β2‑微球蛋白含量测定方法,步骤一:制备供试品溶液;取样品1ml,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,用0.45μm滤膜过滤,即得;步骤二:制备对照品溶液;取β2‑微球蛋白对照品适量,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,制得的溶液中每1ml含β2‑微球蛋白10μg;步骤三:按照高效液相色谱法测定;本发明采用高效液相色谱法进行测定,可以快速便捷的测定患者的尿液和腹膜透出液中β2‑微球蛋白结果,对于监测患者体内β2‑微球蛋白具有重要意义,同时对于人工肾研发具有重要意义。
Description
技术领域
本发明属于医药技术领域,具体涉及一种β2-微球蛋白含量测定方法。
背景技术
β2-微球蛋白(β2-MG)是人类白细胞抗原(HLA)Ⅰ类抗原的轻链,是由淋巴细胞、血小板、多形核白细胞产生的一种低分子量血清球蛋白,存在于成熟红细胞和胎盘滋养层细胞外的各种细胞表面。β2-微球蛋白极易通过肾小球滤过膜,几乎全部被近曲小管细胞重吸收和降解,不再回流入血。健康人β2-微球蛋白的合成速度和细胞膜释放量非常恒定,故体内β2-微球蛋白保持稳定。
检测血或尿中的β2-MG浓度为临床肾功能测定、肾移植成活、糖尿病肾病、重金属镉、汞中毒、某些恶性肿瘤、病毒感染、自身免疫性疾病的临床诊断提供较早、可靠和灵敏的指标,同时也为鉴别蛋白尿性质、肾小球或肾小管病变、尿路感染部位等提供依据。唾液中β2-微球蛋白检测可为诊断干燥综合征及某些口腔疾病提供依据,干燥综合征患者血清和唾液β2微球蛋白含量明显升高,且唾液β2微球蛋白含量较血清中的高。
目前β2-微球蛋白的检测方法,均为免疫法,包括放射免疫分析法、酶联免疫吸附法、时间分辨荧光免疫分析法、免疫透射法、免疫散射比浊法、胶乳免疫散射比浊法、乳胶增强免疫比浊法等,其试剂盒需要使用配套型号的全自助生化分析仪,局限性较强,现急需一种可使用通用仪器进行检测的方法。
本发明中提供了一种高效液相法实现了对β2-微球蛋白的检测,可采用本发明中的方法对尿毒症患者的尿液及腹膜透析患者的透出液进行测定。
发明内容
本发明的目的在于提供一种β2-微球蛋白含量测定方法,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:一种β2-微球蛋白含量测定方法,步骤一:制备供试品溶液;取样品1ml,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,用0.45μm滤膜过滤,即得;
步骤二:制备对照品溶液;取β2-微球蛋白对照品适量,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,制得的溶液中每1ml含β2-微球蛋白10μg;
步骤三:按照高效液相色谱法测定,精密量取供试品溶液和对照品溶液,分别注入液相色谱仪,记录色谱图,按外标法计算供试品溶液中β2-微球蛋白含量。
优选的,高效液相色谱法测定的色谱条件为:用十八烷基硅烷键合硅胶为填充剂;流动相:A相:0.1%三氟乙酸-水,B相:0.09%三氟乙酸-乙腈(A:B=65~85:35~15);检测波长为210~230nm;流速为每分钟0.5~1.0ml;柱温为40~55℃。
优选的,系统适用性为取对照品溶液进样,主峰的理论塔板数应大于2000。
优选的,步骤一和步骤二中进行室温放置时,需要保持量瓶密封。
优选的,所述高效液相色谱法测定中,进样体积为20μl。
与现有技术相比,本发明的有益效果是:本发明采用高效液相色谱法进行测定,可以快速便捷的测定患者的尿液和腹膜透出液中β2-微球蛋白结果,对于监测患者体内β2-微球蛋白具有重要意义,同时对于人工肾研发具有重要意义。
附图说明
图1为本发明的对照品溶液图谱示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明方法主要的研究过程如下:
系统适用性
对照品溶液:取β2-微球蛋白对照品适量,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,制得的溶液中每1ml含β2-微球蛋白10μg。
取对照品溶液进样,结果表明,β2-微球蛋白色谱峰,峰型对称,理论塔板数大于2000。
专属性
对照品溶液:取β2-微球蛋白对照品适量,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,制得的溶液中每1ml含β2-微球蛋白10μg。
尿素溶液:取6mol/L尿素溶液,即得。
小分子毒素混合溶液:取乳酸钠对照品、肌酸对照品、尿酸对照品、肌酐对照品、苯乙酸对照品、马尿酸对照品、5-羟甲基糠醛对照品、3-吲哚乙酸对照品、对甲苯酚对照品各适量,加流动相溶解并定量稀释制成每1ml中约含乳酸钠400μg、肌酸4μg、尿酸20μg、肌酐4μg、苯乙酸20μg、马尿酸20μg、5-羟甲基糠醛20μg、3-吲哚乙酸20μg、对甲苯酚10μg的溶液。
取上述三种溶液分别进样,结果表明,尿素溶液和小分子毒素混合溶液,在本色谱条件下均不出峰,不干扰β2-微球蛋白的检测。
重复性
配制对照品溶液6份,分别进样,结果见下表。
由上表可知,6次结果的重复性良好。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (5)
1.一种β2-微球蛋白含量测定方法,其特征在于:步骤一:制备供试品溶液;取样品1ml,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,用0.45μm滤膜过滤,即得;
步骤二:制备对照品溶液;取β2-微球蛋白对照品适量,置100ml量瓶中,加入6mol/L尿素溶液至刻度,室温放置1小时后,制得的溶液中每1ml含β2-微球蛋白10μg;
步骤三:按照高效液相色谱法测定,精密量取供试品溶液和对照品溶液,分别注入液相色谱仪,记录色谱图,按外标法计算供试品溶液中β2-微球蛋白含量。
2.根据权利要求1所述的一种β2-微球蛋白含量测定方法,其特征在于:高效液相色谱法测定的色谱条件为:用十八烷基硅烷键合硅胶为填充剂;流动相:A相:0.1%三氟乙酸-水,B相:0.09%三氟乙酸-乙腈(A:B=65~85:35~15);检测波长为210~230nm;流速为每分钟0.5~1.0ml;柱温为40~55℃。
3.根据权利要求1所述的一种β2-微球蛋白含量测定方法,其特征在于:系统适用性为取对照品溶液进样,主峰的理论塔板数应大于2000。
4.根据权利要求1所述的一种β2-微球蛋白含量测定方法,其特征在于:步骤一和步骤二中进行室温放置时,需要保持量瓶密封。
5.根据权利要求1所述的一种β2-微球蛋白含量测定方法,其特征在于:所述高效液相色谱法测定中,进样体积为20μl。
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