CN112462074B - 一种cd277三种亚型蛋白的鉴定方法及检测试剂盒 - Google Patents
一种cd277三种亚型蛋白的鉴定方法及检测试剂盒 Download PDFInfo
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Abstract
本发明一方面公开了一种CD277亚型蛋白鉴定特异性靶点,CD277亚型蛋白为BTN3A1,BTN3A2和BTN3A3三种亚型,用于检测三种亚型蛋白的特异性靶点氨基酸序列分别是BTN3A1(276-287aa),如SEQ ID NO:1所示;BTN3A2(276-287aa):如SEQ ID NO:2所示;BTN3A3(276-287aa),如SEQ ID NO:3所示,通过三种不同的抗体能够分别识别这三个特异性靶点,从而高效地鉴别CD277三种亚型蛋白。本发明第二方面公开了CD277亚型蛋白ELISA试剂盒及其使用方法。本发明第三方面公开了CD277亚型蛋白TRFIA试剂盒及其使用方法。本发明的鉴定方法及试剂盒的开发和应用填补了三种亚型蛋白鉴定和检测方面的技术空白,同时提供的检测盒及其检测方法有良好的稳定性,操作时间短,成本低。
Description
技术领域
本发明属于医学生物技术研究领域,涉及一种CD277三种亚型蛋白的鉴定方法及检测试剂盒。
背景技术
Butyrophilin(BTN)称为嗜乳脂蛋白,它最初在乳汁中被发现,是乳汁中脂蛋白球的主要成份之一。人的Butyrophilin 3A基因家族位于第6号染色体MHC I类分子基因簇的延伸端,该基因在啮齿类动物中缺失。人的Butyrophilin 3A家族又称为CD277,Butyrophilin 3A蛋白是单次穿膜蛋白,其胞外区结构属于免疫球蛋白超家族,与B7-H4蛋白有结构同源性,包含有一个N端的IgV样结构域和一个近膜端的IgC样结构域。CD277包含三个成员:Butyrophilin 3A1(BTN3A1),Butyrophilin 3A2(BTN3A2)和Butyrophilin 3A3(BTN3A3)。
Butyrophilin 3A家族的三个成员广泛表达于人体绝大部分类型的细胞膜表面。目前国际上对Butyrophilin 3A家族分子在Vγ9Vδ2T细胞上的功能进行了深入的研究。BTN3A1可直接结合靶细胞的磷酸类抗原(phosphoantigens,pAgs),并将信号通过TCR传导,进而活化Vγ9Vδ2T细胞。。
BTN3A1、BTN3A2和BTN3A3的胞外段氨基酸组成同源性高达95%,其中IgV样结构域的同源性更高达99%,因此针对Butyrophilin 3A胞外段的抗体只能够同时识别BTN3A1、BTN3A2和BTN3A3,所以目前的检测技术无法明确具体的BTN3A1、BTN3A2和BTN3A3分子。
因此本领域技术人员致力于研发一种用于鉴定和检测BTN3A1、BTN3A2和BTN3A3的ELISA方法和时间分辨免疫荧光方法及相应的试剂盒,用于填补了这CD277三种亚型蛋白鉴定方面的空白。
发明内容
通过对Butyrophilin 3A家族氨基酸序列分析,在BTN3A1、BTN3A2和BTN3A3三个分子的氨基酸序列中存在一个差异较大的基序(motif),这段基序具有很大的抗原性,能够诱导动物产生特异性抗体。
本发明第一方面通过检测CD277三个蛋白质分子中的276-287aa的特异性靶点来明确其亚型,以此能够协助判断免疫细胞功能和肿瘤细胞增殖状态。
在BTN3A1、BTN3A2和BTN3A3三个分子的氨基酸序列中第276-287位存在一个差异较大且具有独特型的基序(motif),这段基序的结构为EXXXXXXXXXXE(X代表任意氨基酸)。该基序中氨基酸以碱性或酸性氨基酸为主,具有很大的亲水性和抗原性。以此序列合成的肽段能够很快诱导动物产生特异性抗体,并且该特异性抗体能够相应地识别该基序,因此该基序可以作为其特异性鉴定靶点,该靶点分别是BTN3A1(276-287aa),氨基酸序列如SEQID NO:1所述、BTN3A2(276-287aa),氨基酸序列SEQ ID NO:2所示、BTN3A3(276-287aa),氨基酸序列如SEQ ID NO:3所示。
通过三种不同的抗体识别CD277氨基酸序列中这种独特型基序作为其特异性鉴定靶点,以此来鉴定CD277亚型分子属于哪个具体的BTN3A1、BTN3A2或BTN3A3。
本发明第二方面,提供了一种CD277三种亚型蛋白鉴定的ELISA试剂盒,其包括以下组成:
(1)洗涤液为含0.1%Tween-20的PBS;
(2)加样缓冲液的成分为含1%BSA的PBS,其成分为PBS,1%BSA;
(3)反应终止液为2M H2SO4;
(4)包被在酶标板上的分别是单克隆小鼠抗SEQ ID NO:1抗体、单克隆小鼠抗SEQID NO:2抗体、单克隆小鼠抗SEQ ID NO:3抗体,包被浓度为5ug/ml;
(5)检测抗体分别为生物素化的兔抗SEQ ID NO:1多抗、兔抗SEQ ID NO:2多抗、兔抗SEQ ID NO:3多抗,储存浓度为50ug/ul;
(6)辣根过氧化物酶标记的链亲和霉素,储存浓度为0.5ug/ul;
(7)标准品,重组人BTN3A1、BTN3A2和BTN3A3蛋白,浓度为100ug/ul;
该ELISA试剂盒的使用方法步骤如下:
(1)分别在已包被有单克隆小鼠抗EKKTQFRKKKRE、EITALSSEIESE、EKIALSRETERE抗体的酶标板中加入待测细胞裂解上清样品和倍比稀释的标准品100ul/孔,37℃孵育1h;
(2)弃去样品和标准品,300ul/孔洗涤液洗涤三次;
(3)用加样缓冲液分别稀释检测抗体-生物素化的兔抗EKKTQFRKKKRE、EITALSSEIESE、EKIALSRETERE多抗至浓度为75ug/ml,以100ul/孔加入稀释好的检测抗体,37℃孵育1h;
(4)弃去检测抗体,300ul/孔洗涤液洗涤三次;
(5)用加样缓冲液稀释辣根过氧化物酶标记的链亲和霉素,终浓度为0.5ug/ml,以100ul/孔加入酶标板,37℃避光孵育0.5h;
(6)弃去辣根过氧化物酶标记的链亲和霉素,300ul/孔洗涤液洗涤四次;
(7)加入TMB显色液,室温避光孵育15min;
(8)在酶标仪上读取OD450nm值;
(9)利用统计学绘图软件根据测定的值绘制标准曲线;
(10)通过标准曲线和未知浓度样品的OD450nm值分别计算待测样品中的BTN3A1、BTN3A2和BTN3A3的浓度。
本发明第三方面,提供了一种CD277三种亚型蛋白鉴定的时间分辨免疫荧光试剂盒,又称TRFIA试剂盒,TRFIA试剂盒是通过以下方法实现的:在酶标板中用包被抗体包被,封闭后加入待测样品孵育,洗板,加入检测抗体孵育,再次洗板后加入Eu3+标记的链亲和霉素孵育,然后洗板加入荧光增强液,振荡5分钟后用337nm波长光激发,延时200微秒在615nm波长处检测发射荧光值;通过标准品的浓度和相应的荧光值绘制标准曲线,待检样品中的BTN3A1、BTN3A2和BTN3A3浓度就可以通过它的荧光值并对应标准曲线得到。其包括以下组成:
本发明CD277亚型鉴定时间分辨免疫荧光试剂盒,TRFIA试剂盒包括以下组成:
(1)包被缓冲液为PBS,其成分为140mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mMKH2PO4,pH值为7.4;
(2)封闭液为含1%BSA的PBS,其成分为PBS,1%BSA;
(3)洗涤液为含0.05%Tween-20的Tris-HCl缓冲液,其成分为50mM的Tris-HCl缓冲液(pH 7.8),0.05%Tween-20;
(4)加样缓冲液的成分为含1%BSA的PBS,其成分为PBS,1%BSA;
(5)荧光增强液;
(6)检测缓冲液为50mM的Tris-HCl缓冲液(pH 7.8),其中含有1%BSA,1%bovineglobulin,0.05%Tween 40,二乙烯三胺五乙酸;
(7)包被抗体分别为单克隆小鼠抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3抗体,储存浓度为1ug/ul;
(8)检测抗体分别为生物素化的兔抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3多抗,储存浓度为50ug/ul;
(9)Eu3+标记的链亲和霉素,储存浓度为0.1ug/ul;
(10)标准品,重组人BTN3A1、BTN3A2和BTN3A3蛋白,浓度为100ug/ul;
本发明检测CD277三种亚型蛋白的TRFIA试剂盒的使用步骤如下:
(1)用PBS稀释包被抗体-单克隆小鼠抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3抗体至浓度为4ug/ml,以100ul/孔加入96孔酶标板,4℃过夜;
(2)弃去包被抗体,300ul/孔洗涤液洗涤三次,加入封闭液200ul/孔,37℃孵育1h;
(3)弃去封闭液,300ul/孔洗涤液洗涤三次,加入待测细胞裂解上清样品和倍比稀释的标准品100ul/孔,37℃孵育1h;
(4)弃去样品和标准品,300ul/孔洗涤液洗涤三次;
(5)用加样缓冲液稀释检测抗体-生物素化的兔抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3多抗至浓度为75ug/ml,以100ul/孔加入稀释好的检测抗体,37℃孵育1h;
(6)弃去检测抗体,300ul/孔洗涤液洗涤三次;
(7)用检测缓冲液稀释Eu3+标记的链亲和霉素,终浓度为500ug/ml,以100ul/孔加入酶标板,37℃避光孵育1h;
(8)弃去Eu3+标记的链亲和霉素,300ul/孔洗涤液洗涤四次
(9)加入荧光增强液200ul/孔,室温避光缓慢水平摇动5min;
(10)读板:设定激发光波长为337nm,发射光波长为615nm,延时200微秒测定615nm处的荧光值;
(11)利用统计学绘图软件根据测定的值绘制标准曲线;
(12)通过标准曲线和未知浓度样品的荧光值分别计算待测样品中的BTN3A1、BTN3A2和BTN3A3的浓度。
本试剂盒采用时间分辨免疫荧光方法来分别检测样品中的BTN3A1、BTN3A2和BTN3A3蛋白浓度,其中应用了双抗体夹心法,利用包被抗体单克隆小鼠抗SEQ ID NO:1、SEQID NO:2、SEQ ID NO:3抗体来捕获样品中微量的BTN3A1、BTN3A2和BTN3A3蛋白,然后用生物素化的兔抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3多抗来与被捕获的BTN3A1、BTN3A2和BTN3A3蛋白结合,由于生物素与链亲和霉素能够高亲和力结合,用Eu3+标记的链亲和霉素(Eu3+-labeled streptavidin)来与生物素化的多抗结合,形成三明治样复合结构,样品中的BTN3A1、BTN3A2和BTN3A3就被Eu3+标记上并固定在酶标板中,最后应用337nm波长的激发光激发,在发射光波长为615nm处,测定荧光值;然后根据标准曲线来分别计算BTN3A1、BTN3A2和BTN3A3的浓度。运用时间分辨免疫荧光方法来高灵敏高特异地检测外周血中的BTN3A1、BTN3A2和BTN3A3浓度是该试剂盒的一个最显著特色和创新。
本发明不仅填补了CD277三种亚型蛋白检测领域的空白,同时提供的检测盒及其检测方法有良好的稳定性,操作时间短,成本低。
具体实施方式
本发明中的一些术语与缩写:BTN3A1:嗜乳脂蛋白3A1;BTN3A2:嗜乳脂蛋白3A2;BTN3A3:嗜乳脂蛋白3A3;TRFIA:时间分辨免疫荧光;BSA:牛血清白蛋白;Tween-20:吐温20;Tween-40:吐温40;PBS:磷酸盐缓冲液;TBS:Tris-HCl缓冲液;bovine globulin:牛球蛋白;DTPA:二乙烯三胺五乙酸。
试剂配方:
1.缓冲液:
包被缓冲液:为磷酸盐缓冲液(PBS),其成分为140mM NaCl,2.7mM KCl,10mMNa2HPO4,1.8mM KH2PO4,pH值为7.4;
洗涤液:为含0.05%Tween-20的Tris-HCl缓冲液(TBS),其成分为50mM的Tris-HCl缓冲液(pH 7.8),0.05%Tween-20;
封闭液:为含1%BSA的PBS,其成分为PBS,1%BSA;
加样缓冲液:为含1%BSA的PBS,其成分为PBS,1%BSA;
检测缓冲液:为50mM的Tris-HCl缓冲液(pH 7.8),其中含有1%BSA,1%bovineglobulin,0.05%Tween 40,DTPA;
2.标准品蛋白:
重组人BTN3A1、BTN3A2和BTN3A3蛋白
4.酶标板:
Nunc八联孔酶标条
实施例1
本发明运用时间分辨免疫荧光方法来检测样品中的BTN3A1、BTN3A2和BTN3A3蛋白浓度,其试剂盒的使用步骤如下:
(1)用PBS稀释包被抗体-单克隆小鼠抗EKKTQFRKKKRE、EITALSSEIESE、EKIALSRETERE抗体至浓度为4ug/ml,以100ul/孔加入96孔酶标板,4℃过夜;
(2)弃去包被抗体,300ul/孔洗涤液洗涤三次,加入封闭液200ul/孔,37℃孵育1h;
(3)弃去封闭液,300ul/孔洗涤液洗涤三次,加入待测细胞裂解上清样品和倍比稀释的标准品100ul/孔,37℃孵育1h;
(4)弃去样品和标准品,300ul/孔洗涤液洗涤三次;
(5)用加样缓冲液稀释检测抗体-生物素化的兔抗EKKTQFRKKKRE、EITALSSEIESE、EKIALSRETERE多抗至浓度为75ug/ml,以100ul/孔加入稀释好的检测抗体,37℃孵育1h;
(6)弃去检测抗体,300ul/孔洗涤液洗涤三次;
(7)用检测缓冲液稀释Eu3+标记的链亲和霉素,终浓度为500ug/ml,以100ul/孔加入酶标板,37℃避光孵育1h;
(8)弃去Eu3+标记的链亲和霉素,300ul/孔洗涤液洗涤四次
(9)加入荧光增强液200ul/孔,室温避光缓慢水平摇动5min;
(10)读板:设定激发光波长为337nm,发射光波长为615nm,延时200微秒测定615nm处的荧光值;
(11)利用统计学绘图软件根据测定的值绘制标准曲线;
(12)通过标准曲线和未知浓度样品的荧光值分别计算待测样品中的BTN3A1、BTN3A2和BTN3A3的浓度。
实施例2
本发明中的另一种运用ELISA方法来检测样品中的BTN3A1、BTN3A2和BTN3A3蛋白浓度,其试剂盒的使用步骤如下:
(1)分别在已包被有克隆小鼠抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3抗体的酶标板中加入待测细胞裂解上清样品和倍比稀释的标准品100ul/孔,37℃孵育1h;
(2)弃去样品和标准品,300ul/孔洗涤液洗涤三次;
(3)用加样缓冲液分别稀释检测抗体-生物素化的兔抗SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3多抗至浓度为75ug/ml,以100ul/孔加入稀释好的检测抗体,37℃孵育1h;
(4)弃去检测抗体,300ul/孔洗涤液洗涤三次;
(5)用加样缓冲液稀释辣根过氧化物酶标记的链亲和霉素,终浓度为0.5ug/ml,以100ul/孔加入酶标板,37℃避光孵育0.5h;
(6)弃去辣根过氧化物酶标记的链亲和霉素,300ul/孔洗涤液洗涤四次;
(7)加入TMB显色液,室温避光孵育15min;
(8)在酶标仪上读取OD450nm值;
(9)利用统计学绘图软件根据测定的值绘制标准曲线;
(10)通过标准曲线和未知浓度样品的OD450nm值分别计算待测样品中的BTN3A1、BTN3A2和BTN3A3的浓度。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
序列表
<110> 上海交通大学医学院
<120> 一种CD277三种亚型蛋白的鉴定方法及检测试剂盒
<130> 2020
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Lys Lys Thr Gln Phe Arg Lys Lys Lys Arg Glu
1 5 10
<210> 2
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Glu Ile Thr Ala Leu Ser Ser Glu Ile Glu Ser Glu
1 5 10
<210> 3
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Glu Lys Ile Ala Leu Ser Arg Glu Thr Glu Arg Glu
1 5 10
Claims (10)
1.一种制备鉴定人CD277三种亚型蛋白的试剂盒的用途,CD277三种亚型蛋白分别为BTN3A1、BTN3A2、BTN3A3,其特征在于,通过三种亚型蛋白对应的特异性靶点来鉴定CD277三种亚型蛋白,所述特异性靶点是CD277三种亚型蛋白氨基酸序列中的独特型基序,BTN3A1亚型蛋白的特异性靶点氨基酸序列如SEQ ID NO:1所示,BTN3A2亚型蛋白的特异性靶点氨基酸序列如SEQ ID NO:2所示,BTN3A3亚型蛋白的特异性靶点氨基酸序列如SEQ ID NO:3所示。
2.一种基于权利要求1所述特异性靶点鉴定CD277三种亚型蛋白的ELISA试剂盒,其特征在于:所述鉴定CD277亚型蛋白的ELISA试剂盒包括:洗涤液,加样缓冲液,反应终止液,包被在酶标板上的单克隆小鼠抗SEQ ID NO:1抗体、单克隆小鼠抗SEQ ID NO:2抗体、单克隆小鼠抗SEQ ID NO:3抗体,检测抗体,通过辣根过氧化物酶标记的链霉亲和素,重组人BTN3A1、BTN3A2和BTN3A3蛋白标准品和TMB显色液;
其中,所述检测抗体为生物素化的兔抗SEQ ID NO:1多抗、兔抗SEQ ID NO:2多抗、兔抗SEQ ID NO:3多抗。
3.如权利要求2所述的鉴定CD277亚型蛋白的ELISA试剂盒,其特征在于,所述洗涤液为含0.1%Tween-20的PBS;所述加样缓冲液为含1%BSA的PBS;所述反应终止液为2M的H2SO4;所述链霉亲和素重组人BTN3A1、BTN3A2和BTN3A3蛋白标准品浓度为100ug/ul。
4.如权利要求2所述的鉴定CD277亚型蛋白的ELISA试剂盒,其特征在于,单克隆小鼠抗SEQ ID NO:1抗体、单克隆小鼠抗SEQ ID NO:2抗体、单克隆小鼠抗SEQ ID NO:3抗体的包被浓度为5ug/ml。
5.如权利要求2所述的鉴定CD277亚型蛋白的ELISA试剂盒,其特征在于,所述检测抗体为生物素化的兔抗SEQ ID NO:1多抗、兔抗SEQ ID NO:2多抗、兔抗SEQ ID NO:3多抗的储存浓度50ng/ul。
6.如权利要求2所述的鉴定CD277三种亚型蛋白的ELISA试剂盒,其特征在于,所述鉴定CD277三种亚型蛋白的ELISA试剂盒的使用方法包括以下步骤:
A1)分别在已包被有单克隆小鼠抗SEQ ID NO:1抗体、单克隆小鼠抗SEQ ID NO:2抗体、单克隆小鼠抗SEQ ID NO:3抗体的酶标板中加入待测细胞裂解上清样品和倍比稀释的标准品100ul/孔,37℃孵育1h;
A2)弃去样品和标准品,300ul/孔洗涤液洗涤三次;
A3)用加样缓冲液分别稀释检测抗体-生物素化的兔抗SEQ ID NO:1多抗、兔抗SEQ IDNO:2多抗、兔抗SEQ ID NO:3多抗至浓度为75ug/ml,以100ul/孔加入稀释好的检测抗体,37℃孵育1h;
A4)弃去检测抗体,300ul/孔洗涤液洗涤三次;
A5)用加样缓冲液稀释辣根过氧化物酶标记的链霉亲和素,终浓度为0.5ug/ml,以100ul/孔加入酶标板,37℃避光孵育0.5h;
A6)弃去辣根过氧化物酶标记的链霉亲和素,300ul/孔洗涤液洗涤四次;
A7)加入TMB显色液,室温避光孵育15min;
A8)在酶标仪上读取OD450nm值;
A9)利用统计学绘图软件根据测定的值绘制标准曲线;
A10)通过标准曲线和待测样品的OD450nm值分别计算待测样品中CD277三种亚型蛋白BTN3A1、BTN3A2和BTN3A3的浓度。
7.一种基于权利要求1所述特异性靶点鉴定CD277三种亚型蛋白的时间分辨免疫荧光试剂盒,其特征在于:所述时间分辨免疫荧光试剂盒又称为TRFIA试剂盒,所述TRFIA试剂盒包括:包被缓冲液,封闭液,洗涤液,加样缓冲液,荧光增强液,检测缓冲液,包被抗体,检测抗体,Eu3+标记的链霉亲和素和重组人BTN3A1、BTN3A2和BTN3A3蛋白标准品;
其中,所述包被抗体对应三种亚型蛋白分别为单克隆小鼠抗SEQ ID NO:1抗体、单克隆小鼠抗SEQ ID NO:2抗体、单克隆小鼠抗SEQ ID NO:3抗体;
所述检测抗体对应三种亚型蛋白分别为生物素化的兔抗SEQ ID NO:1多抗、兔抗SEQID NO:2多抗、兔抗SEQ ID NO:3多抗。
8.如权利要求7所述的鉴定CD277亚型蛋白的TRFIA试剂盒,其特征在于,所述包被抗体的储存浓度为1ug/ul。
9.如权利要求7所述的鉴定CD277亚型蛋白的TRFIA试剂盒,其特征在于,所述检测抗体的储存浓度50ng/ul。
10.如权利要求7所述的试剂盒,其特征在于,所述试剂盒的使用方法包括以下步骤:
B1)用包被缓冲液稀释包被抗体-单克隆小鼠抗SEQ ID NO:1抗体、单克隆小鼠抗SEQID NO:2抗体、单克隆小鼠抗SEQ ID NO:3抗体至浓度为4ug/ml,以100ul/孔加入96孔酶标板,4℃过夜,包被缓冲液为PBS,其成分为140mM NaCl,2.7mM KCl,10mM Na2HPO4,1.8mMKH2PO4,pH值为7.4;
B2)弃去包被抗体,300ul/孔洗涤液洗涤三次,加入封闭液200ul/孔,37℃孵育1h,洗涤液为含0.05%Tween-20的Tris-HCl缓冲液,其成分为50mM的Tris-HCl缓冲液(pH 7.8),0.05%Tween-20,封闭液为含1%BSA的PBS;
B3)弃去封闭液,300ul/孔洗涤液洗涤三次,加入待测细胞裂解上清样品和倍比稀释的标准品100ul/孔,37℃孵育1h;
B4)弃去样品和标准品,300ul/孔洗涤液洗涤三次;
B5)用加样缓冲液稀释检测抗体-生物素化的兔抗EKKTQFRKKKRE、EITALSSEIESE、EKIALSRETERE多抗至浓度为75ug/ml,以100ul/孔加入稀释好的检测抗体,37℃孵育1h,加样缓冲液的成分为含1%BSA的PBS;
B6)弃去检测抗体,300ul/孔洗涤液洗涤三次;
B7)用检测缓冲液稀释Eu3+标记的链霉亲和素,终浓度为500ug/ml,以100ul/孔加入酶标板,37℃避光孵育1h,检测缓冲液为50mM的Tris-HCl缓冲液,pH=7.8,其中含有1%BSA,1%bovine globulin,0.05%Tween 40,二乙烯三胺五乙酸,Eu3+标记的链霉亲和素的储存浓度为0.1ug/ul;
B8)弃去Eu3+标记的链霉亲和素,300ul/孔洗涤液洗涤四次
B9)加入荧光增强液200ul/孔,室温避光缓慢水平摇动5min;
B10)读板:设定激发光波长为337nm,发射光波长为615nm,延时200微秒测定615nm处的荧光值;
B11)利用统计学绘图软件根据测定的值绘制标准曲线;
B12)通过标准曲线和未知浓度样品的荧光值分别计算待测样品中的BTN3A1、BTN3A2和BTN3A3的浓度。
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