CN112147333A - 一种基于胃蛋白酶原pgⅱ的免疫分析试剂盒及其制备方法和检测方法 - Google Patents
一种基于胃蛋白酶原pgⅱ的免疫分析试剂盒及其制备方法和检测方法 Download PDFInfo
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Abstract
本发明公开了一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒及其制备方法和检测方法,属于免疫检测分析技术和纳米生物技术领域,在所述试剂盒中,包括集成在试剂条上的反应缓冲液、清洗液、增强液、包被PGⅡ抗体的磁微粒溶液、铕标记的PGⅡ抗体溶液,还有2瓶PGⅡ的冻干校准品。上述试剂盒可以提供一种接近均相的反应体系,检测PGⅡ的含量,缩短检测时间,提高了效率,为临床使用提供便利。
Description
技术领域
本发明属于免疫检测分析技术和纳米生物技术领域,具体涉及一种基于胃蛋白酶原Ⅱ时间分辨荧光免疫分析试剂盒及其制备方法和检测方法。
背景技术
胃蛋白酶原(pepsinogen,PG)是由胃部分泌的参与消化的胃蛋白酶的前体,通常约1%的PG可通过胃黏膜进入血液循环,可分为PGⅠ和PGⅡ两种生化和免疫活性等特征的不同亚群,且细胞来源及组织内分布各不相同。血清PG浓度主要反映胃腺细胞的分泌水平。PGⅠ是检测胃泌酸腺细胞功能的指征,胃酸分泌增多PGⅠ升高,反之则降低;PGⅡ与胃底黏膜病变的相关性较大,其升高与胃底腺管萎缩、胃上皮化生或假幽门腺化生、异型增值有关;PGⅠ/Ⅱ比值进行性降低与胃黏膜萎缩进展相关。联合测定PGⅠ、PGⅡ及PGⅠ/Ⅱ比值可起到胃底腺黏膜“血清学活检”的作用。在做胃镜前后测定PGⅠ和PGⅡ水平,并定期追踪,可有效监控胃黏膜状态和功能。
时间分辨荧光免疫分析(time-resolved fluoroimmunoassay,TRFIA)是一种以镧系元素螯合物为标记物的新兴技术,具有零本底、线性范围宽、稳定性好等特点,灵敏度可达到10-18mol/L,较ELISA、CLIA、ECLIA等的10-9~10-15mol/L更高,TRFIA的主要创新内容在于应用荧光强度大、衰变时间长、分子量小的镧系原子标记免疫分子,而不是常用的大分子的酶或放射性同位素,故建立的免疫方法灵敏度和量程成倍增加,而且没有放射性污染。时间分辨荧光免疫分析技术标记离子的荧光是类线光谱,其特点是激发光波长范围较宽而发射光谱峰范围较窄,这样有利于降低本底的荧光强度,提高检测的分辨率;在时间分辨荧光免疫分的激发光和发射光之间存在较大的Stokes位移,这有利于排除非特异荧光的干扰,从而增强了测量的特异性;标记离子螯合物产生的荧光强度高,寿命长,这有利于消除样品及环境中非特异荧光物质对检测结果的影响;每一次检测都要重复进行1000次,取平均的荧光计数值作为结果,从而提高了检测的准确性。TRFIA是国内外免疫分析的主要发展方向之一。
发明内容
针对现有技术中存在的缺陷,本发明的目的是将TRFIA与磁微粒技术相结合,将所有试剂集成在一个试剂条上,建立PGⅡ-TRFIA方法,该检测可由全自动设备操作,准确度高,单人份检测,使用方便,将此技术应用于胃部病变的研究,可以更准确的鉴别诊断浅表性胃炎、胃粘膜糜烂溃疡、萎缩性胃炎、慢性胃炎、慢性胃扩张、慢性十二指肠炎、十二指肠溃疡等胃部病变,以通过方便的血清学检查进行胃部病变的疗效评估。为胃部病变的诊断和治疗提供一个非常便捷的新手段。
本发明解决技术问题所采用的技术方案是:一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒,所述试剂盒内包括至少一个试剂架,每个所述的试剂架上包括至少五个试剂条,所述试剂盒内还包括至少二瓶PGⅡ的冻干校准品;所述试剂条上包括磁珠孔、反应缓冲液孔、铕标冻干孔、检测孔、清洗液孔、增强液孔和若干个预留孔;所述磁珠孔内填充包被PGⅡ抗体的磁微粒溶液,所述反应缓冲液孔内填充反应缓冲液,所述铕标冻干孔内填充铕标记的PGⅡ抗体溶液,所述检测孔内填充待检样品,所述清洗液孔内填充清洗液,所述增强液孔内填充增强液。
进一步地,所述试剂盒内包括5~20个试剂架,每个所述的试剂架上设有5个试剂条;所述试剂盒内还包括2瓶PGⅡ的冻干校准品。
进一步地,所述包被PGⅡ抗体的磁微粒溶液经直径为50~5000纳米的活化的磁微粒与PGⅡ抗体连接、洗涤、封闭制备得到;所述铕标记的PGⅡ抗体溶液以Eu3+-N2-[P-异氰酸-苄基]-二乙烯三胺四乙酸钠标记PGⅡ抗体制备得到,所述PGⅡ抗体与所述Eu3+-N2-[P-异氰酸-苄基]-二乙烯三胺四乙酸钠的质量比为5:1。
进一步地,所述反应缓冲液为pH7.8的50mmol/L Tris-HCl含8mmol/L NaCl、0.1%BSA、50μmol/L DTPA、0.1ml/L Tween-80和0.1%NaN3;所述清洗液为含0.48g/L的Tris、12.49g/L的NaCl、1.11g/L的Tween-20,以盐酸调节pH为7.8的缓冲溶液;所述增强液为含体积浓度为3.6‰的冰醋酸、0.5g/L的醋酸钠、0.05g/L的β-萘甲酰三氟丙酮、0.03g/L的三辛基氧化膦和体积浓度为1‰的Triton X-100的混合溶液;所述PGⅡ的冻干校准品为用含2g/L的BSA和1g/L的NaN3的50mmol/L的pH7.8 Tris-HCl的反应缓冲液,将PGⅡ配制成不同浓度的校准品溶液。
一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒的制备方法,所述制备方法包括以下内容:
PGⅡ校准品溶液的制备:取高浓度的PGⅡ抗原溶液用含2g/L的BSA和1g/L的NaN3的50mmol/L的pH为7.8的Tris-HCl反应缓冲液配制成不同浓度的校准品溶液,进行分装,2~8℃保存;
包被PGⅡ抗体的磁微粒溶液的制备:取直径为50~5000nm的四氧化三铁微球用质量浓度为0.5~2%的N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化,室温混匀4小时后,用0.05mol/L的pH为5.0的MES缓冲液清洗1~3次,然后用上述缓冲溶液进行悬浮,制成浓度为100mg/mL的磁微粒悬浮液;取100μL所述磁微粒悬浮液,然后加入1mL的0.05mol/L的pH为5.0的MES缓冲液和50μg的PGⅡ抗体,于25℃混匀温育2小时,然后进行磁性分离,保留磁微粒,用pH为7.2的5%BSA和0.05mol/L的Tris-HCl缓冲液清洗1~3次,用pH为7.2的5%BSA和0.05mol/L的Tris-HCl缓冲液于25℃下封闭30分钟,用含有质量浓度为0.5%BSA和质量浓度为0.1%的NaN3的0.05mol/L的pH为7.2的Tris-HCl缓冲液清洗1~3次,用含有质量浓度为0.5%BSA和质量浓度为0.1%的NaN3的0.05mol/L的pH为7.2的Tris-HCl缓冲液重悬,分装,置于2~8℃下保存;
铕标记的PGⅡ抗体溶液的制备:取PGⅡ抗体1mg,经PD-10转换缓冲条件,洗脱液为含0.155mol/L NaCl的50mmol/L的pH为8.5的Na2CO3-NaHCO3缓冲液,获得浓缩至2g/L的PGⅡ抗体溶液;取所述PGⅡ抗体溶液500μL加入0.2mg的异硫氰酸苄基二亚乙基三胺四乙酸铕钠,25℃下振荡反应20小时,将反应液转移到预先用pH为7.8的浓度为80mmol/L的Tris缓冲液平衡的Sephadex G-50柱上进行层析,收集蛋白峰,稀释,进行分装,真空冷冻干燥,置于2~8℃保存;
增强液的制备:体积浓度为3.6‰的冰醋酸,0.5g/L的醋酸钠,0.05g/L的β-萘甲酰三氟丙酮,0.03g/L的三辛基氧化膦和体积浓度为1‰的Triton X-100的混合溶液。
反应缓冲液为pH7.8的50mmol/L Tris-HCl含8mmol/L NaCl、0.1%BSA、50μmol/LDTPA、0.1ml/L Tween-80和0.1%NaN3;
清洗液为含0.48g/L的Tris,12.49g/L的NaCl,1.11g/L的Tween-20,以盐酸调节pH为7.8的缓冲溶液。
一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒的检测方法,所述检测方法主要包括以下步骤:
1)分别取PGⅡ的冻干校准品溶液和待测样品,与包被PGⅡ抗体的磁微粒溶液、反应缓冲液稀释的铕标记的PGⅡ抗体溶液混合,进行孵育,孵育后进行磁性分离,然后用清洗液进行清洗,再加入增强液;
2)采用时间分辨荧光免疫分析仪分别测定加增强液后的溶液中铕的荧光值,获得所述PGⅡ的冻干校准品溶液和待测样品的荧光值,根据PGⅡ校准品溶液中各PGⅡ的浓度和其各自对应的荧光值绘制标准曲线,然后将待测样品的荧光值代入对应的PGⅡ标准曲线中,便可得到所述待测样品中PGⅡ的含量。
本发明的有益效果是:与现有技术相比,本发明提供的技术方案具有以下优点:
(1)本发明提供一种检测PGⅡ的时间分辨荧光免疫分析试剂盒,反应缓冲液、清洗液、增强液、包被PGⅡ抗体的磁微粒溶液、铕标记的PGⅡ抗体溶液都集成在一个试剂条上,全自动操作,简便易行。24min内即可检测得到血清中PGⅡ的含量,检测结果准确可靠。除了拥有TRFIA技术的灵敏度高、储存时间长、无放射性污染、测量范围宽等诸多优点外,还可通过免疫磁微粒的富集作用以及磁微粒在液体中充分扩散使得结合表面积扩大的特性,大大缩短反应时间,提高检测灵敏度,同时磁微粒与抗体通过化学基团定向连接,大大减少了抗原的用量以及显著提高检测的精密度,同时实现自动化,克服了传统微孔板式酶联免疫法(ELISA)技术需要样本积累到一定数量才能检测,实现了样本的即时检测,效率高;
(2)该试剂盒是以纳米磁微粒为载体的高效时间分辨荧光技术:磁微粒与PGⅡ抗体的自由氨基连接,形成包被有PGⅡ抗体的免疫磁微粒,该免疫磁微粒依靠其超大的比表面积可在短时间内结合大量PGⅡ,加入Eu标记的PGⅡ抗体进行示踪,通过磁分离,用增强液将Eu从复合物上解离下来后,与增强液中的螯合剂螯合形成一种胶态分子团,分子团在紫外光的激发下能发出很强的发射波长的荧光,信号增强了百万倍,可灵敏的检测血清中抗PGⅡ的含量。
附图说明
图1为本发明提供的免疫分析试剂盒的检测原理示意图。
图2为本发明提供的免疫分析试剂盒中PGⅡ标准品的双对数标准曲线图。
图3为本发明提供的集成5人份试剂条的试剂架的结构示意图。
图4为本发明提供的试剂条的结构示意图。
其中,1-试剂条;2-试剂架;3、4、6、10、15-预留孔;5-磁珠孔;7-反应缓冲液孔;8-铕标冻干孔;9-检测孔;11~13-清洗液孔;14-增强液孔。
具体实施方式
下面通过具体实施案例来进一步说明本发明。但这些实例仅用于说明本发明而不用于限制本发明的范围。
如图3和4所示,一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒,所述试剂盒内包括5~20个试剂架,每个所述的试剂架上设有5个试剂条;所述试剂盒内还包括2瓶PGⅡ的冻干校准品。所述试剂条上包括磁珠孔、反应缓冲液孔、铕标冻干孔、检测孔、清洗液孔、增强液孔和若干个预留孔;所述磁珠孔内填充包被PGⅡ抗体的磁微粒溶液,所述反应缓冲液孔内填充反应缓冲液,所述铕标冻干孔内填充铕标记的PGⅡ抗体溶液,所述检测孔内填充待检样品,所述清洗液孔内填充清洗液,所述增强液孔内填充增强液。
所述包被PGⅡ抗体的磁微粒溶液经直径为50~5000纳米的活化的磁微粒与PGⅡ抗体连接、洗涤、封闭制备得到;所述铕标记的PGⅡ抗体溶液以Eu3+-N2-[P-异氰酸-苄基]-二乙烯三胺四乙酸钠标记PGⅡ抗体制备得到,所述PGⅡ抗体与所述Eu3+-N2-[P-异氰酸-苄基]-二乙烯三胺四乙酸钠的质量比为5:1。
所述反应缓冲液为pH7.8的50mmol/L Tris-HCl含8mmol/L NaCl、0.1%BSA、50μmol/L DTPA、0.1ml/L Tween-80和0.1%NaN3;所述清洗液为含0.48g/L的Tris、12.49g/L的NaCl、1.11g/L的Tween-20,以盐酸调节pH为7.8的缓冲溶液;所述增强液为含体积浓度为3.6‰的冰醋酸、0.5g/L的醋酸钠、0.05g/L的β-萘甲酰三氟丙酮、0.03g/L的三辛基氧化膦和体积浓度为1‰的Triton X-100的混合溶液;所述PGⅡ的冻干校准品为用含2g/L的BSA和1g/L的NaN3的50mmol/L的pH7.8 Tris-HCl的反应缓冲液,将PGⅡ配制成不同浓度的校准品溶液。
一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒的制备方法,所述制备方法包括以下内容:
PGⅡ校准品溶液的制备:取高浓度的PGⅡ抗原溶液用含2g/L的BSA和1g/L的NaN3的50mmol/L的pH为7.8的Tris-HCl反应缓冲液配制成不同浓度的校准品溶液,进行分装,2~8℃保存;
包被PGⅡ抗体的磁微粒溶液的制备:取直径为50~5000nm的四氧化三铁微球用质量浓度为0.5~2%的N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化,室温混匀4小时后,用0.05mol/L的pH为5.0的MES缓冲液清洗1~3次,然后用上述缓冲溶液进行悬浮,制成浓度为100mg/mL的磁微粒悬浮液;取100μL所述磁微粒悬浮液,然后加入1mL的0.05mol/L的pH为5.0的MES缓冲液和50μg的PGⅡ抗体,于25℃混匀温育2小时,然后进行磁性分离,保留磁微粒,用pH为7.2的5%BSA和0.05mol/L的Tris-HCl缓冲液清洗1~3次,用pH为7.2的5%BSA和0.05mol/L的Tris-HCl缓冲液于25℃下封闭30分钟,用含有质量浓度为0.5%BSA和质量浓度为0.1%的NaN3的0.05mol/L的pH为7.2的Tris-HCl缓冲液清洗1~3次,用含有质量浓度为0.5%BSA和质量浓度为0.1%的NaN3的0.05mol/L的pH为7.2的Tris-HCl缓冲液重悬,分装,置于2~8℃下保存;
铕标记的PGⅡ抗体溶液的制备:取PGⅡ抗体1mg,经PD-10转换缓冲条件,洗脱液为含0.155mol/L NaCl的50mmol/L的pH为8.5的Na2CO3-NaHCO3缓冲液,获得浓缩至2g/L的PGⅡ抗体溶液;取所述PGⅡ抗体溶液500μL加入0.2mg的异硫氰酸苄基二亚乙基三胺四乙酸铕钠,25℃下振荡反应20小时,将反应液转移到预先用pH为7.8的浓度为80mmol/L的Tris缓冲液平衡的Sephadex G-50柱上进行层析,收集蛋白峰,稀释,进行分装,真空冷冻干燥,置于2~8℃保存;
增强液的制备:体积浓度为3.6‰的冰醋酸,0.5g/L的醋酸钠,0.05g/L的β-萘甲酰三氟丙酮,0.03g/L的三辛基氧化膦和体积浓度为1‰的Triton X-100的混合溶液。
反应缓冲液为pH7.8的50mmol/L Tris-HCl含8mmol/L NaCl、0.1%BSA、50μmol/LDTPA、0.1ml/L Tween-80和0.1%NaN3;
清洗液为含0.48g/L的Tris,12.49g/L的NaCl,1.11g/L的Tween-20,以盐酸调节pH为7.8的缓冲溶液。
一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒的检测方法,所述检测方法主要包括以下步骤:
1)校准品溶液定标后,将待测血清取100ul,与所述包被PGⅡ抗体的50ul磁微粒溶液、反应缓冲液稀释的铕标记的PGⅡ抗体溶液混合,反应12分钟,进行磁性分离,清洗液清洗3次,然后加入所述增强液;
2)全自动时间分辨荧光免疫分析仪分别测定加增强液后的溶液中铕的荧光值,然后根据标准曲线,将待测样品的对应荧光值代入对应的标准曲线中,计算得到待测样品中PGⅡ的含量。
检测过程中的反应原理如图1所示,将PGⅡ抗体包被在磁珠上,铕标记PGⅡ抗体,如果样本中存在PGⅡ,则形成磁珠-PGⅡ抗体-PGⅡ-铕标记PGⅡ抗体复合物,分离后加入增强液解离铕离子,采用时间分辨荧光仪检测荧光值,荧光值的强弱与样本中PGⅡ含量成正比,根据PGⅡ的冻干校准品荧光值绘制标准曲线,计算样本中PGⅡ的含量。
检测结果如下:胃蛋白酶原Ⅱ的双对数标准曲线见图2,血清样本测试结果见表1,该检测方法具有较好的灵敏度,且实现了荧光检测的自动化。
表1血清样本测试结果
以上实施方式仅用于说明本发明,而并非对本发明的限制,有关技术领域的普通技术人员,在不脱离本发明的精神和范围的情况下,还可以做出各种变化和变型,因此所有等同的技术方案也属于本发明的范畴,本发明的专利保护范围应由权利要求限定。
Claims (6)
1.一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒,其特征在于:所述试剂盒内包括至少一个试剂架,每个所述的试剂架上包括至少五个试剂条,所述试剂盒内还包括至少二瓶PGⅡ的冻干校准品;所述试剂条上包括磁珠孔、反应缓冲液孔、铕标冻干孔、检测孔、清洗液孔、增强液孔和若干个预留孔;所述磁珠孔内填充包被PGⅡ抗体的磁微粒溶液,所述反应缓冲液孔内填充反应缓冲液,所述铕标冻干孔内填充铕标记的PGⅡ抗体溶液,所述检测孔内填充待检样品,所述清洗液孔内填充清洗液,所述增强液孔内填充增强液。
2.如权利要求1所述的一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒,其特征在于:所述试剂盒内包括5~20个试剂架,每个所述的试剂架上设有5个试剂条;所述试剂盒内还包括2瓶PGⅡ的冻干校准品。
3.如权利要求1所述的一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒,其特征在于:所述包被PGⅡ抗体的磁微粒溶液经直径为50~5000纳米的活化的磁微粒与PGⅡ抗体连接、洗涤、封闭制备得到;
所述铕标记的PGⅡ抗体溶液以Eu3+-N2-[P-异氰酸-苄基]-二乙烯三胺四乙酸钠标记PGⅡ抗体制备得到。
4.如权利要求1所述的一种基于胃蛋白酶原PGⅡ的免疫分析试剂盒,其特征在于:所述反应缓冲液为pH7.8的50mmol/L Tris-HCl含8mmol/L NaCl、0.1%BSA、50μmol/L DTPA、0.1ml/L Tween-80和0.1%NaN3;
所述清洗液为含0.48g/L的Tris、12.49g/L的NaCl、1.11g/L的Tween-20,以盐酸调节pH为7.8的缓冲溶液;
所述增强液为含体积浓度为3.6‰的冰醋酸、0.5g/L的醋酸钠、0.05g/L的β-萘甲酰三氟丙酮、0.03g/L的三辛基氧化膦和体积浓度为1‰的Triton X-100的混合溶液;
所述PGⅡ的冻干校准品为用含2g/L的BSA和1g/L的NaN3的50mmol/L的pH7.8 Tris-HCl的反应缓冲液,将PGⅡ配制成不同浓度的校准品溶液。
5.一种如权利要求1至4中任意一项所述的基于胃蛋白酶原PGⅡ的免疫分析试剂盒的制备方法,其特征在于,所述制备方法包括以下内容:
PGⅡ校准品溶液的制备:取高浓度的PGⅡ抗原溶液用含2g/L的BSA和1g/L的NaN3的50mmol/L的pH为7.8的Tris-HCl反应缓冲液配制成不同浓度的校准品溶液,进行分装,2~8℃保存;
包被PGⅡ抗体的磁微粒溶液的制备:取直径为50~5000nm的四氧化三铁微球用质量浓度为0.5~2%的N-羟基琥珀酰亚胺(NHS)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺(EDC)活化,室温混匀4小时后,用0.05mol/L的pH为5.0的MES缓冲液清洗1~3次,然后用上述缓冲溶液进行悬浮,制成浓度为100mg/mL的磁微粒悬浮液;取100μL所述磁微粒悬浮液,然后加入1mL的0.05mol/L的pH为5.0的MES缓冲液和50μg的PGⅡ抗体,于25℃混匀温育2小时,然后进行磁性分离,保留磁微粒,用pH为7.2的5%BSA和0.05mol/L的Tris-HCl缓冲液清洗1~3次,用pH为7.2的5%BSA和0.05mol/L的Tris-HCl缓冲液于25℃下封闭30分钟,用含有质量浓度为0.5%BSA和质量浓度为0.1%的NaN3的0.05mol/L的pH为7.2的Tris-HCl缓冲液清洗1~3次,用含有质量浓度为0.5%BSA和质量浓度为0.1%的NaN3的0.05mol/L的pH为7.2的Tris-HCl缓冲液重悬,分装,置于2~8℃下保存;
铕标记的PGⅡ抗体溶液的制备:取PGⅡ抗体1mg,经PD-10转换缓冲条件,洗脱液为含0.155mol/L NaCl的50mmol/L的pH为8.5的Na2CO3-NaHCO3缓冲液,获得浓缩至2g/L的PGⅡ抗体溶液;取所述PGⅡ抗体溶液500μL加入0.2mg的异硫氰酸苄基二亚乙基三胺四乙酸铕钠,25℃下振荡反应20小时,将反应液转移到预先用pH为7.8的浓度为80mmol/L的Tris缓冲液平衡的Sephadex G-50柱上进行层析,收集蛋白峰,稀释,进行分装,真空冷冻干燥,置于2~8℃保存;
增强液的制备:体积浓度为3.6‰的冰醋酸,0.5g/L的醋酸钠,0.05g/L的β-萘甲酰三氟丙酮,0.03g/L的三辛基氧化膦和体积浓度为1‰的Triton X-100的混合溶液。
反应缓冲液为pH7.8的50mmol/L Tris-HCl含8mmol/L NaCl、0.1%BSA、50μmol/LDTPA、0.1ml/L Tween-80和0.1%NaN3;
清洗液为含0.48g/L的Tris,12.49g/L的NaCl,1.11g/L的Tween-20,以盐酸调节pH为7.8的缓冲溶液。
6.一种如权利要求1至4中任意一项所述的基于胃蛋白酶原PGⅡ的免疫分析试剂盒的检测方法,其特征在于,所述检测方法主要包括以下步骤:
1)分别取PGⅡ的冻干校准品溶液和待测样品,与包被PGⅡ抗体的磁微粒溶液、反应缓冲液稀释的铕标记的PGⅡ抗体溶液混合,进行孵育,孵育后进行磁性分离,然后用清洗液进行清洗,再加入增强液;
2)采用时间分辨荧光免疫分析仪分别测定加增强液后的溶液中铕的荧光值,获得所述PGⅡ的冻干校准品溶液和待测样品的荧光值,根据PGⅡ校准品溶液中各PGⅡ的浓度和其各自对应的荧光值绘制标准曲线,然后将待测样品的荧光值代入对应的PGⅡ标准曲线中,便可得到所述待测样品中PGⅡ的含量。
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