CN105823884A - 检测妊娠特异性糖蛋白3的方法、试剂盒及其制备方法 - Google Patents
检测妊娠特异性糖蛋白3的方法、试剂盒及其制备方法 Download PDFInfo
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Abstract
本发明涉及生物医药检测领域,具体涉及检测PSG3的方法、试剂盒及其制备方法。该试剂盒主要包括标记有示踪标记物或结合于固相载体上的第一抗PSG3抗体的组分A、结合于固相载体上或标记有示踪标记物的第二抗PSG3抗体或PSG3的组分B;所述组分A和组分B中的任意一种标记有示踪标记物,另一种则结合于固相载体上;且第一抗PSG3抗体和第二抗PSG3抗体与PSG3的结合位点不同。利用本发明提供的PSG3检测试剂盒和检测方法,特异性及灵敏度得到了很大的提升,可准确、灵敏、快速地测定样本中的PSG3浓度,借助免疫分析仪,加样方式完全由仪器全自动操作,降低了人为因素对实验结果造成的干扰,大幅度缩短了测试时间,有利于临床上快速的得到诊断结果。
Description
技术领域
本发明涉及生物医药检测领域,具体涉及检测妊娠特异性糖蛋白3的方法、试剂盒及其制备方法。
背景技术
妊娠特异性糖蛋白(PregnancySpecificGlycoprotein,PSG)是一种胎盘多肽,自1971年德国科学家Bohn从人胎盘中分离提纯以来,各国学者对其进行了广泛的研究。发现PSG与早孕并发症、宫内胎儿生长状况及消化道肿瘤疾病关系密切,对临床上相关疾病的诊断具有指示作用。日本学者报道妊娠特异性糖蛋白1(PregnancySpecificGlycoprotein1,PSG1)可在子宫颈癌、子宫体癌、卵巢癌、乳腺癌、淋巴瘤和和胃肠道恶性肿瘤患者的血中测出,进一步研究证实癌细胞浆中存在有PSG1的患者比没有PSG1患者存活时间短。近年来对妊娠特异性糖蛋白3(PregnancySpecificGlycoprotein3,PSG3)的研究受到广泛关注。PSG3是癌胚抗原(CEA)家族的成员,PSG3蛋白全长428个氨基酸,在胚胎发育的过程中同胚胎期免疫耐受的建立以及血管生成有关,同时该指标可应用于消化道肿瘤的诊断。
目前,市场上尚未有检测PSG3相关的试剂盒,使用传统方法进行检测缺乏重复性、准确性,且纯手工加样等系列操作效率低,容易导致实验结果误差大,还易受外部干扰因素影响,不能形成产业化。
发明内容
本发明的目的是解决上述现有技术中的不足,提供检测妊娠特异性糖蛋白3的方法、试剂盒及其制备方法。
本发明是通过以下技术方案实现的:
用于检测妊娠特异性糖蛋白3的试剂盒,所述试剂盒包括组分A和组分B,其中组分A为标记有示踪标记物或结合于固相载体上的第一抗妊娠特异性糖蛋白3抗体,组分B为结合于固相载体上或标记有示踪标记物的第二抗妊娠特异性糖蛋白3抗体或妊娠特异性糖蛋白3;并且,组分A和组分B中的任意一种标记有示踪标记物,另一种则结合于固相载体上;所述第一抗妊娠特异性糖蛋白3抗体和第二抗妊娠特异性糖蛋白3抗体与妊娠特异性糖蛋白3的结合位点不同。
本发明所述的试剂盒,所述示踪标记物为金刚烷、鲁米诺及其衍生物、异鲁米诺及其衍生物、吖啶酯、碱性磷酸酶、稀土元素和辣根过氧化物酶中的至少一种。
本发明所述的试剂盒,所述固相载体为塑料制品、微颗粒或膜载体。
本发明所述的试剂盒,所述固相载体为聚苯乙烯、聚氯乙烯、磁珠、乳胶微球或硝酸纤维膜。
本发明所述的试剂盒,所述标记有示踪标记物或结合于固相载体上的方式可以为直接或间接。
本发明所述的试剂盒,所述间接方式包括通过异硫氰酸荧光素与抗异硫氰酸荧光素抗体体系或通过链霉亲和素与生物素体系进行。
本发明所述的试剂盒,所述试剂盒还包括妊娠特异性糖蛋白3的低点校准品和高点校准品。
用于制备所述试剂盒的方法,所述方法包括:将第一抗妊娠特异性糖蛋白3抗体和第二抗妊娠特异性糖蛋白3抗体中的任意一种直接或间接标记示踪标记物,将另一种直接或间接结合于固相载体上。
检测妊娠特异性糖蛋白3的方法,所述方法包括使用如权利要求8所述的试剂盒,通过免疫学原理检测待测样品中的妊娠特异性糖蛋白3浓度。
本发明所述的方法,其特征在于,所述检测妊娠特异性糖蛋白3的方法为酶联免疫法,其步骤包括:将所述试剂盒的组分A和组分B与待测样品混合,温育,经分离,向沉淀物中加入底物,检测光信号强度;以同样方法测定妊娠特异性糖蛋白3的低点校准品和高点校准品的光信号强度,并获得妊娠特异性糖蛋白3的浓度与光信号强度之间的标准曲线;将检测得到的待测样品的光信号强度与标准曲线对照,获得待测样品中的妊娠特异性糖蛋白3浓度。
本发明所述的方法,所述检测妊娠特异性糖蛋白3的方法为化学发光免疫法,其步骤包括:将所述试剂盒的组分A和组分B与待测样品混合,温育,经分离,向沉淀物中加入发光底物,检测光信号强度;以同样方法测定妊娠特异性糖蛋白3的低点校准品和高点校准品的光信号强度,并获得妊娠特异性糖蛋白3的浓度与光信号强度之间的标准曲线;将检测得到的待测样品的光信号强度与标准曲线对照,获得待测样品中的妊娠特异性糖蛋白3浓度。
本发明所述的方法,所述检测妊娠特异性糖蛋白3的方法为荧光免疫法,其步骤包括:将所述试剂盒的组分A和组分B与待测样品混合,温育,经分离,向沉淀物中加入发光底物,检测光信号强度;以同样方法测定妊娠特异性糖蛋白3的低点校准品和高点校准品的光信号强度,并获得妊娠特异性糖蛋白3的浓度与光信号强度之间的标准曲线;将检测得到的待测样品的光信号强度与标准曲线对照,获得待测样品中的妊娠特异性糖蛋白3浓度。
本发明的有益效果在于:
根据本发明提供的PSG3检测试剂盒,包含以多种标记方式标记示踪标记物或者以多种方式结合于固相载体上的PSG3或PSG3抗体,利用双抗体夹心法或竞争法,可准确、灵敏、快速地测定样本中的PSG3浓度。
使用本发明提供的试剂盒测定PSG3,不需要将待测样本进行稀释处理,可以直接用于检测,从而有利于提高低值样本的检测灵敏度。其中待测样本可为空白管血清、分离胶管血清、促凝剂管血清、EDTA血浆和肝素血浆中的任意一种。
本发明提供的PSG3检测方法,可以采用酶联免疫、化学发光免疫、荧光免疫分析法等多种免疫学检测方法,试剂盒的特异性及灵敏度得到了很大的提升,更好的服务于临床诊断。
本发明提供的PSG3检测方法,使用所提供的试剂盒,借助免疫分析仪,加样方式完全由仪器全自动操作,降低了人为因素对实验结果造成的干扰,大幅度缩短了测试时间,有利于临床上快速的得到诊断结果。
附图说明
图1为本发明重组PSG3的SDS-PAGE检测结果图:
其中,泳道M:蛋白Marker;泳道1:重组PSG3蛋白;
图2为本发明实施例5中层析卡组装方法。
具体实施方式
为更好理解本发明,下面结合实施例及附图对本发明作进一步描述,以下实施例仅是对本发明进行说明而非对其加以限定。
实施例1PSG3抗原的制备
1.构建重组菌:将PSG3的编码基因连接入基因的表达载体pET-30α中,并将构建好的基因表达载体pET-30α-PSG3导入宿主大肠杆菌E.coliJM109(DE3)菌体中,构建重组菌JM109(DE3)/pET-30α-PSG3;其中,编码PSG3的核苷酸序列如SEQIDNO:1所示,PSG3的氨基酸序列如SEQIDNO:3所示。
2.PSG3蛋白溶液的制备
(2-1)将重组PSG3工程菌接种到含有卡那青霉素的LB固体培养基上,37℃恒温培养12~16h,挑取克隆接种于3-5ml的LB培养基中,37℃恒温摇床培养至OD600达到1.5~1.8,转速为220r/min;
(2-2将(2-1)获得的菌液1:100接种到100-200ml的LB培养基中,37℃恒温摇床培养至OD600达到1.5~1.8,转速为220r/min,得到发酵种子液;
(2-3)将配好的LB培养基加到发酵罐中,连接pH探针、溶氧探针进行原位灭菌,灭菌温度为121℃,灭菌时间为20min;
(2-4)将发酵种子液1:100接种于发酵罐中LB培养基上,设置温度为37℃、溶氧为30%、转速为220~270r/min、pH为7.2~7.4条件下发酵;当发酵罐内的OD600达到1.0~1.2时,加入异丙基-β-D-硫代吡喃半乳糖苷至终浓度为1mmol/L,调温度至32℃,诱导表达4~5h,得到发酵液;
(2-5)将发酵液4℃条件下6000r/min离心10min,收集菌体,用适量的0.1M磷酸盐缓冲液重悬菌体,在800bar压力下高压均质破碎,重复破碎2-3次,4℃条件下12000r/min离心10~15min,收集沉淀;重复离心一次,收集沉淀,即可得到重组PSG3蛋白粗品。
3.SDS-PAGE鉴定:对PSG3蛋白进行SDS-PAGE鉴定,相对分子量46kD(图1)。
实施例2PSG3抗体的制备
采用大肠杆菌原核表达PSG3蛋白,表达载体为pET30a。取5-6周龄BALB/c小鼠,利用PSG3全长蛋白作为抗原添加弗氏佐剂免疫制备PSG3抗体。每只小鼠免疫4次,每次间隔2周,每次利用15-30μg抗原蛋白,第一次免疫注射时利用完全佐剂,后三次利用不完全佐剂。细胞融合前三天,从三只脾细胞供体小鼠挑选一只进行静脉注射PSG3抗原做进一步免疫。细胞融合、杂交瘤筛选均依据J.M.Davis的方法(Davisetal1982)。简略过程如下:加强免疫后的小鼠脾细胞和SP2/0骨髓瘤细胞混合,慢慢加入促融合剂50%聚乙二醇0.7ml。37℃培养1min,10mlIMDM培养基稀释,低速离心。然后用40ml含1.25%甲基纤维素、25%胎牛血清、2%HAT(含次黄嘌呤、甲氨蝶呤和胸腺嘧啶核苷)的IMDM培养基重悬。上下颠倒混合,转移到35mm平板中,每板约2ml,于37℃、5%CO2环境中培养。7天后克隆转移入96孔板,加入含15%胎牛血清和2%HT(含次黄嘌呤和胸腺嘧啶核苷)的IMDM培养基再次培养。当细胞融合度达到50%~70%,收集培养基上清,利用ELISA和Westernblot检测是否存在抗体。
实施例3制备PSG3检测的ELISA试剂盒
建立检测PSG3抗原的酶标双抗体夹心法。检测PSG3抗原的酶标双抗体夹心法的建立:首先利用小鼠腹腔生产单克隆抗体,将得到的阳性杂交瘤细胞分别注入小鼠腹腔,使其产生腹水,注射降植烷,每只小鼠0.5ml;注射降植烷1周后,注射杂交瘤细胞,每只小鼠注射106个细胞。7-10天后分别收集腹水,用正辛酸-硫酸铵沉淀法分别提纯抗体,再用过碘酸钠法将抗体分别进行辣根过氧化物酶标记(酶标抗体)。用纯化的单克隆抗体分别包被酶标板,并分别与酶标抗体配对检测PSG3抗原,获得最佳配对的包被抗体P201以及酶标捕获抗体P209。其中包被抗体P201和酶标的捕获抗体P209分别由杂交瘤细胞株P201和P209制备产生。杂交瘤细胞系P201和P209已于2015年11月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号分别为CGMCCNO.11597和CGMCCNO.11598,保藏地址为北京市朝阳区北辰西路一号院3号。
ELISA步骤如下:将PSG3包被抗体P201包被96孔酶标板,100μl/孔,4℃过夜;甩去包被抗体,0.5%PBST洗板3次,300μl/孔;加入2%BSA封闭液,300μl/孔,室温4小时;甩去封闭液,0.5%PBST洗板3次,300μl/孔;酶标板中每孔分别加入PSG3标准蛋白和待测样本,100μl/孔;再加入PSG3酶标抗体P209,100μl/孔,混匀后4℃过夜;甩去反应液体,0.5%PBST洗板5次,300μl/孔;加入TMB显色液,200μl/孔,避光反应15分钟;加入2M硫酸终止反应,100μl/孔,用酶标仪OD450nm处检测吸光度;利用标准蛋白吸光度的值绘制标准曲线,并计算待测样本中PSG3的蛋白浓度。
实施例4制备PSG3检测的化学发光免疫试剂盒
化学发光双抗体夹心法建立。首先利用小鼠腹腔生产单克隆抗体,将得到的阳性杂交瘤细胞分别注入小鼠腹腔,使其产生腹水,注射降植烷,每只小鼠0.5ml;注射降植烷1周后,注射杂交瘤细胞,每只小鼠注射106个细胞。7-10天后分别收集腹水,用正辛酸-硫酸铵沉淀法分别提纯抗体,再用过碘酸钠法将抗体分别进行鲁米诺标记。用纯化的单克隆抗体分别包被磁性微球,并分别与鲁米诺标记抗体配对检测PSG3抗原,获得最佳配对的包被抗体P201以及鲁米诺标记捕获抗体P209。
将PSG3包被抗体P201包被磁性微球,微球粒径0.5~3μm,包被浓度16μg抗体/1mg磁性微球,室温包被2h;磁分离包被完的磁性微球并用0.05M的BBST0.5%BSA封闭液封闭磁性微球30min;0.05M的BBST洗涤3次封闭完的磁性微球;0.01MPBS缓冲液重选磁性微球待用。
试剂盒检测操作步骤:将包被完成的磁性微球、待测样本或PSG3标准蛋白、酶标记抗体P209同时加入反应杯,37°孵育10min,通过磁铁分离出夹心偶合物,0.01MPBS缓冲液洗涤3次,加入鲁米诺发光底物,用光电倍增管检测发光值;利用标准蛋白的发光值及浓度绘制标准曲线,并计算待测样本中PSG3的含量。
实施例5制备PSG3检测的荧光免疫试剂盒
PSG3荧光免疫层析试剂盒体系建立。首先利用小鼠腹腔生产单克隆抗体,将得到的阳性杂交瘤细胞分别注入小鼠腹腔,使其产生腹水,注射降植烷,每只小鼠0.5ml;注射降植烷1周后,注射杂交瘤细胞,每只小鼠注射106个细胞。7-10天后分别收集腹水,用正辛酸-硫酸铵沉淀法分别提纯抗体,再用过碘酸钠法将抗体分别进行稀土元素标记。用纯化的单克隆抗体分别包被乳胶微球,并分别与稀土元素标记抗体配对检测PSG3抗原,获得最佳配对的包被抗体P201以及稀土元素捕获抗体P209。。
将PSG3包被抗体P201包被乳胶微球,微球粒径80~200nm,包被浓度60μg抗体/1mg乳胶微球,室温或37°包被2h;离心分离包被完的磁性微球并用0.2M的BBST洗涤3次,0.5%BSA封闭液封闭磁性微球30min;0.05M的BBST洗涤3次封闭完的磁性微球;0.01MPBS缓冲液重选磁性微球待用。
通过喷金划膜仪器将包被有P201抗体的微球喷涂于玻璃纤维制成的结合垫上,喷涂完毕烘烤干燥;将P209抗体和羊抗鼠抗体按照一定间距分别喷涂于硝酸纤维薄膜上,喷涂完毕干燥形成两条抗体线,喷涂P209抗体的抗体线作为T线(Test线),喷涂羊抗鼠抗体的抗体线作为C线(control线);将玻璃纤维制成的样品垫、喷涂了乳胶微球的结合垫、硝酸纤维薄膜、吸水纸按照图2所示依次粘贴于带粘胶的聚氯乙烯(PVC)背板上,形成检测试剂条,再将试剂条装入卡壳内。
荧光免疫层析实验操作步骤。将待测样本或标准蛋白由卡条样本孔滴入,50μl/条;室温孵育层析15min,然后将卡条插入荧光层析检测仪器中读取C\T线荧光值并计算T线与C线荧光值的比值;通过标准蛋白的T线和C线荧光比值及对应浓度绘制标准曲线,将样本的T\C线比值带入曲线获得待测样本中PSG3蛋白含量。
以上所述实施方式仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明的权利要求书确定的保护范围内。
序列表
<110>中国医学科学院肿瘤医院;必欧瀚生物技术(合肥)有限公司
<120>检测妊娠特异性糖蛋白3的方法、试剂盒及其制备方法
<130>3
<160>3
<170>PatentInversion3.3
<210>1
<211>1182
<212>DNA
<213>人工序列
<400>1
caagtcacgattgaagccgagccaaccaaagtttccaaggggaaggacgttcttctactt60
gtccacaatttgccccagaatcttgctggctacatctggtacaaagggcaaatgaaggac120
ctctaccattacattacatcatacgtagtagatggtcaaataattatatatgggcctgca180
tacagtggacgagaaacagtatattccaatgcatccctgctgatccagaatgtcacccgg240
gaggacgcaggatcctacaccttacacatcgtaaagcgaggtgatgggactagaggagaa300
actggacatttcaccttcaccttatacctggagactcccaagccctccatctccagcagc360
aacttataccccagggaggacatggaggctgtgagcttaacctgtgatcctgagactccg420
gacgcaagctacctgtggtggatgaatggtcagagcctccctatgactcacagcttgcag480
ttgtccaaaaacaaaaggaccctctttctatttggtgtcacaaagtacactgcaggaccc540
tatgaatgtgaaatacggaacccagtgagtgccagccgcagtgacccagtcaccctgaat600
ctcctcccgaagctgcccaagccctacatcaccatcaacaacttaaaccccagggagaat660
aaggatgtcttagccttcacctgtgaacctaagagtgagaactacacctacatttggtgg720
ctaaatggtcagagcctcccggtcagtcccagggtaaagcgacccattgaaaacaggatc780
ctcattctacccagtgtcacgagaaatgaaacaggaccctatcaatgtgaaatacaggac840
cgatatggtggcatccgcagttacccagtcaccctgaatgtcctctatggtccagacctc900
cccagaatttacccttcattcacctattaccattcaggagaaaacctctacttgtcctgc960
ttcgcggactctaacccaccagcagaatattcttggacaattaatgggaagtttcagcta1020
tcaggacaaaagctctttatcccccagattactacaaagcatagcgggctctatgcttgc1080
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gctccttcaggaacaggacatcttcctggccttaatccatta1182
<210>2
<211>1194
<212>DNA
<213>人工序列
<400>2
catatgcaagtcacgattgaagccgagccaaccaaagtttccaaggggaaggacgttctt60
ctacttgtccacaatttgccccagaatcttgctggctacatctggtacaaagggcaaatg120
aaggacctctaccattacattacatcatacgtagtagatggtcaaataattatatatggg180
cctgcatacagtggacgagaaacagtatattccaatgcatccctgctgatccagaatgtc240
acccgggaggacgcaggatcctacaccttacacatcgtaaagcgaggtgatgggactaga300
ggagaaactggacatttcaccttcaccttatacctggagactcccaagccctccatctcc360
agcagcaacttataccccagggaggacatggaggctgtgagcttaacctgtgatcctgag420
actccggacgcaagctacctgtggtggatgaatggtcagagcctccctatgactcacagc480
ttgcagttgtccaaaaacaaaaggaccctctttctatttggtgtcacaaagtacactgca540
ggaccctatgaatgtgaaatacggaacccagtgagtgccagccgcagtgacccagtcacc600
ctgaatctcctcccgaagctgcccaagccctacatcaccatcaacaacttaaaccccagg660
gagaataaggatgtcttagccttcacctgtgaacctaagagtgagaactacacctacatt720
tggtggctaaatggtcagagcctcccggtcagtcccagggtaaagcgacccattgaaaac780
aggatcctcattctacccagtgtcacgagaaatgaaacaggaccctatcaatgtgaaata840
caggaccgatatggtggcatccgcagttacccagtcaccctgaatgtcctctatggtcca900
gacctccccagaatttacccttcattcacctattaccattcaggagaaaacctctacttg960
tcctgcttcgcggactctaacccaccagcagaatattcttggacaattaatgggaagttt1020
cagctatcaggacaaaagctctttatcccccagattactacaaagcatagcgggctctat1080
gcttgctctgttcgtaactcagccactggcatggaaagctccaaatccatgacagtcaaa1140
gtctctgctccttcaggaacaggacatcttcctggccttaatccattactcgag1194
<210>3
<211>395
<212>PRT
<213>人工序列
<400>3
MetGlnValThrIleGluAlaGluProThrLysValSerLysGlyLys
151015
AspValLeuLeuLeuValHisAsnLeuProGlnAsnLeuAlaGlyTyr
202530
IleTrpTyrLysGlyGlnMetLysAspLeuTyrHisTyrIleThrSer
354045
TyrValValAspGlyGlnIleIleIleTyrGlyProAlaTyrSerGly
505560
ArgGluThrValTyrSerAsnAlaSerLeuLeuIleGlnAsnValThr
65707580
ArgGluAspAlaGlySerTyrThrLeuHisIleValLysArgGlyAsp
859095
GlyThrArgGlyGluThrGlyHisPheThrPheThrLeuTyrLeuGlu
100105110
ThrProLysProSerIleSerSerSerAsnLeuTyrProArgGluAsp
115120125
MetGluAlaValSerLeuThrCysAspProGluThrProAspAlaSer
130135140
TyrLeuTrpTrpMetAsnGlyGlnSerLeuProMetThrHisSerLeu
145150155160
GlnLeuSerLysAsnLysArgThrLeuPheLeuPheGlyValThrLys
165170175
TyrThrAlaGlyProTyrGluCysGluIleArgAsnProValSerAla
180185190
SerArgSerAspProValThrLeuAsnLeuLeuProLysLeuProLys
195200205
ProTyrIleThrIleAsnAsnLeuAsnProArgGluAsnLysAspVal
210215220
LeuAlaPheThrCysGluProLysSerGluAsnTyrThrTyrIleTrp
225230235240
TrpLeuAsnGlyGlnSerLeuProValSerProArgValLysArgPro
245250255
IleGluAsnArgIleLeuIleLeuProSerValThrArgAsnGluThr
260265270
GlyProTyrGlnCysGluIleGlnAspArgTyrGlyGlyIleArgSer
275280285
TyrProValThrLeuAsnValLeuTyrGlyProAspLeuProArgIle
290295300
TyrProSerPheThrTyrTyrHisSerGlyGluAsnLeuTyrLeuSer
305310315320
CysPheAlaAspSerAsnProProAlaGluTyrSerTrpThrIleAsn
325330335
GlyLysPheGlnLeuSerGlyGlnLysLeuPheIleProGlnIleThr
340345350
ThrLysHisSerGlyLeuTyrAlaCysSerValArgAsnSerAlaThr
355360365
GlyMetGluSerSerLysSerMetThrValLysValSerAlaProSer
370375380
GlyThrGlyHisLeuProGlyLeuAsnProLeu
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Claims (12)
1.用于检测妊娠特异性糖蛋白3的试剂盒,其特征在于:所述试剂盒包括组分A和组分B,其中组分A为标记有示踪标记物或结合于固相载体上的第一抗妊娠特异性糖蛋白3抗体,组分B为结合于固相载体上或标记有示踪标记物的第二抗妊娠特异性糖蛋白3抗体或妊娠特异性糖蛋白3;并且,组分A和组分B中的任意一种标记有示踪标记物,另一种则结合于固相载体上;所述第一抗妊娠特异性糖蛋白3抗体和第二抗妊娠特异性糖蛋白3抗体与妊娠特异性糖蛋白3的结合位点不同。
2.根据权利要求1所述的试剂盒,其特征在于:所述示踪标记物为金刚烷、鲁米诺及其衍生物、异鲁米诺及其衍生物、吖啶酯、碱性磷酸酶、稀土元素和辣根过氧化物酶中的至少一种。
3.根据权利要求1所述的试剂盒,其特征在于:所述固相载体为塑料制品、微颗粒或膜载体。
4.根据权利要求4所述的试剂盒,其特征在于:所述固相载体为聚苯乙烯、聚氯乙烯、磁珠、乳胶微球或硝酸纤维膜。
5.根据权利要求1所述的试剂盒,其特征在于:所述标记有示踪标记物或结合于固相载体上的方式可以为直接或间接。
6.根据权利要求5所述的试剂盒,其特征在于:所述间接方式包括通过异硫氰酸荧光素与抗异硫氰酸荧光素抗体体系或通过链霉亲和素与生物素体系进行。
7.根据权利要求1-6中任意一项所述的试剂盒,其特征在于,所述试剂盒还包括妊娠特异性糖蛋白3的低点校准品和高点校准品。
8.用于制备如权利要求7所述的试剂盒的方法,所述方法包括:将第一抗妊娠特异性糖蛋白3抗体和第二抗妊娠特异性糖蛋白3抗体中的任意一种直接或间接标记示踪标记物,将另一种直接或间接结合于固相载体上。
9.检测妊娠特异性糖蛋白3的方法,其特征在于,所述方法包括使用如权利要求7所述的试剂盒,通过免疫学原理检测待测样品中的妊娠特异性糖蛋白3浓度。
10.根据权利要求9所述的方法,其特征在于,所述检测妊娠特异性糖蛋白3的方法为酶联免疫法,其步骤包括:将所述试剂盒的组分A和组分B与待测样品混合,温育,经分离,向沉淀物中加入底物,检测光信号强度;以同样方法测定妊娠特异性糖蛋白3的低点校准品和高点校准品的光信号强度,并获得妊娠特异性糖蛋白3的浓度与光信号强度之间的标准曲线;将检测得到的待测样品的光信号强度与标准曲线对照,获得待测样品中的妊娠特异性糖蛋白3浓度。
11.根据权利要求9所述的方法,其特征在于,所述检测妊娠特异性糖蛋白3的方法为化学发光免疫法,其步骤包括:将所述试剂盒的组分A和组分B与待测样品混合,温育,经分离,向沉淀物中加入发光底物,检测光信号强度;以同样方法测定妊娠特异性糖蛋白3的低点校准品和高点校准品的光信号强度,并获得妊娠特异性糖蛋白3的浓度与光信号强度之间的标准曲线;将检测得到的待测样品的光信号强度与标准曲线对照,获得待测样品中的妊娠特异性糖蛋白3浓度。
12.根据权利要求9所述的方法,其特征在于,所述检测妊娠特异性糖蛋白3的方法为荧光免疫法,其步骤包括:将所述试剂盒的组分A和组分B与待测样品混合,温育,经分离,向沉淀物中加入发光底物,检测光信号强度;以同样方法测定妊娠特异性糖蛋白3的低点校准品和高点校准品的光信号强度,并获得妊娠特异性糖蛋白3的浓度与光信号强度之间的标准曲线;将检测得到的待测样品的光信号强度与标准曲线对照,获得待测样品中的妊娠特异性糖蛋白3浓度。
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