CN112345772A - 一种基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒 - Google Patents
一种基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒 Download PDFInfo
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- CN112345772A CN112345772A CN202011184220.6A CN202011184220A CN112345772A CN 112345772 A CN112345772 A CN 112345772A CN 202011184220 A CN202011184220 A CN 202011184220A CN 112345772 A CN112345772 A CN 112345772A
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- hepcidin
- hepcidin1
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Abstract
本发明公开一种基于单克隆抗体制备的铁调素(HEPCIDIN)的胶乳增强比浊法检测试剂盒,其含有交联混合HEPCIDIN 1‑3单克隆抗体的胶乳微球,所述交联混合HEPCIDIN1‑3单克隆抗体的胶乳微球为将人HEPCIDIN蛋白基因序列切割为三段,分别通过表达载体导入表达菌中进行表达;然后将表达产物纯化和鉴定后免疫小鼠,待免疫达到指定抗体水平后无菌取脾脏,制备杂交瘤细胞;培养筛选杂交瘤,获得三种HEPCIDIN单克隆抗体,然后分别将该三种HEPCIDIN单克隆抗体与聚苯乙烯胶乳微球交联而成。与现有技术相比,本发明自制了三株单克隆抗体交联到胶乳微球上,形成胶乳增强免疫比浊法试剂盒,使用其测定铁调素,可在全自动生化分析仪上使用,成本低廉且自动化高,节省检测时间,提升了铁调素检测的应用价值。
Description
技术领域
本发明涉及一种基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒。
背景技术
抗原通常是由多个抗原决定簇组成的,由一种抗原决定簇刺激机体,由一个B淋巴细胞接受该抗原刺激所产生的抗体称之为单克隆抗体,通常采用杂交瘤技术来制备。杂交瘤抗体技术是在细胞融合技术的基础上,将具有分泌特异性抗体能力的致敏B细胞和具有无限繁殖能力的骨髓瘤细胞融合为B细胞杂交瘤。用具备这种特性的单个杂交瘤细胞培养成细胞群,可制备针对一种抗原表位的特异性抗体即单克隆抗体。
铁调素(HEPCIDIN)是一种25氨基酸肽,具有抗菌肽的特性,是组织对铁的吸收和分配的主要调节者,其表达受机体信号因子以及炎症、低氧等疾病状态的影响。2000年由Krause等和Park等分别从人血清和尿中提纯得到铁调素。研究证实,铁调素基因发生突变引起其体内表达下降将导致严重的铁超载,进而形成血色病。Nemeth等和Lim等的研究表明,通过使铁调素的受体FPN1表达降低并导致其降解,可抑制铁向细胞外的释放。因此目前认为,铁调素是主要的铁调节激素,炎症性贫血的关键调节剂、铁代谢和固有免疫之间联系的桥梁,在机体铁代谢平衡的调节中起关键作用,临床上可作为一个中间指标参考使用。
目前监测铁调素的方法是质谱检测法。该方法检测成本高,仪器专业度要求高,操作过程时间长,样本量有限,难以在临床推广使用。
胶乳增强比浊法是近年来出现的一种较为稳定、准确的均相免疫比浊检测方法。胶乳增强比浊法是在高分子胶乳微球的表面交联抗体,当交联有抗体的微球与抗原结合后,在短时间内会迅速聚集在一起,改变了反应液透光性能(即吸光度)。而且,反应液透光性能的改变与被测抗原的浓度有较强的相关性,在一定范围内可以反映被测抗原的浓度。胶乳增强比浊法是在均相反应体系中进行抗原、抗体反应及结果的测定。抗原、抗体反应后,直接测定反应液的吸光度值,几分钟就能获得结果,成本低且省时省力。此外,该方法可以在大型全自动生化仪上使用,不需要添置额外的设备。胶乳增强比浊法操作步骤的简化也相应地避免了许多人为操作因素和试剂、环境等外界因素的干扰,稳定性和重复性都较好,能较真实地反映被测物质的含量。
发明内容
针对HEPCIDIN抗原性较差,HEPCIDIN现有检测方法学时间过长,检测量过小,检测成本高无法在临床推广的问题,本发明提供一种基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,采用融合蛋白增强抗原性,实现胶乳增强比浊检测HEPCIDIN,代替质谱检测方法,提高检测效率,扩大检测样本量。具体技术方案如下:
一种基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,该试剂盒的组分包括交联混合HEPCIDIN1-3单克隆抗体的胶乳微球。所述交联混合HEPCIDIN1-3单克隆抗体的胶乳微球为:将人HEPCIDIN蛋白基因序列切割为三段,分别通过表达载体导入表达菌中进行表达;然后将表达产物纯化和鉴定后免疫小鼠,待免疫达到指定抗体水平后无菌取脾脏,制备杂交瘤细胞;培养筛选杂交瘤,获得三种HEPCIDIN单克隆抗体,然后分别将该三种HEPCIDIN单克隆抗体与聚苯乙烯胶乳微球交联,获得三种交联抗人HEPCIDIN单克隆抗体胶乳微球的混合物,即为交联混合HEPCIDIN1-3单克隆抗体的胶乳微球。所述将人HEPCIDIN蛋白基因序列切割为三段,该三段基因序列之间有交叉,具体如下:
第一段序列为:
ACCAGAGCAAGCTCAAGACCCAGCAGTGGGACAGCCAGACAGACGGCACGATGGCACTGAGCTCCCAGATCTGGGCCGCTTGCCTCCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCG
第二段序列为:
CCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAA
第三段序列为:
AAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAAGTGTGGGATGTGCTGCAAGACGTAGAACCTACCTGCCCTGCCCCCGTCCCCTCCCTTCCTTATTTATTCCTGCTGCCCCAGAACATAGGTCTTGGAATAAAATGGCTGGTTCTTTTGTTTTCCAAA。
前所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,所述交联混合HEPCIDIN1-3单克隆抗体的胶乳微球制备方法包括如下步骤:
S1:重组人HEPCIDIN1-3蛋白的制备
将人HEPCIDIN蛋白基因序列切割为三段,分别通过表达载体导入表达菌中进行表达,获得重组人HEPCIDIN1-3粗制品,将该重组人HEPCIDIN1-3粗制品纯化,获得重组人HEPCIDIN蛋白;
S2:HEPCIDIN1-3单克隆抗体的制备
将步骤S1制备的重组人HEPCIDIN1-3蛋白注射小鼠对其免疫,待免疫达到指定抗体水平后,取小鼠脾脏细胞制备杂交瘤细胞,并培养杂交瘤细胞获得HEPCIDIN1-3单克隆抗体;
S3:交联混合HEPCIDIN1-3单克隆抗体的胶乳微球的制备
将步骤S2制备的HEPCIDIN1-3单克隆抗体与直径100~300nm的聚苯乙烯胶乳微球采用化学交联法交联,获得交联混合HEPCIDIN1-3单克隆抗体的胶乳微球。
其中,步骤S2所述的HEPCIDIN1-3单克隆抗体的制备包括如下子步骤:
S2-1:小鼠免疫
选择6~10周龄,健康、发育良好的雌性小鼠,分别对其注射制备的重组人HEPCIDIN1-3蛋白进行免疫;分别取免疫后的小鼠脾脏,制备脾脏细胞悬液,并培养悬浮细胞,培养的脾细胞供融合用;
S2-2:杂交瘤选择
选择活细胞数大于90%的小鼠骨髓瘤细胞系SP2/0供细胞融合用;
S2-3:细胞融合
将步骤S2-1培养的脾细胞和S2-2准备的小鼠骨髓瘤细胞按比例混合,在PEG作用下使二者融合,形成双核细胞或混核体;
S2-4:筛选克隆培养
用含有次黄嘌呤、氨基喋呤和胸腺嘧啶核苷的培养液对步骤S2-3中融合形成的双核细胞或混核体予以筛选,并进行克隆培养;
S2-5:杂交瘤阳性克隆的克隆化
采用酶联免疫吸附试验对步骤S2-4中筛选克隆培养的细胞进一步进行筛选和克隆化,筛出能稳定生长和有功能特性的阳性克隆杂交瘤细胞;
S2-6:抗HEPCIDIN1-3McAb的制备
选择6-8周龄的健康小鼠,先于小鼠腹腔注射降植烷,7~10d后于每只小鼠腹腔注射步骤S2-5中筛选的阳性克隆的杂交瘤细胞,待小鼠腹部明显膨大到一定程度时,抽取并收集腹水,离心后,取上清分装;
S2-7:腹水的纯化
采用辛酸-硫酸铵法将步骤S2-6中获得的腹水上清进行粗纯化,然后进行亲和层析进一步纯化腹水,获得腹水纯化产物1-3;
S2-8:HEPCIDIN1-3McAb鉴定
以步骤S2-7中获得腹水纯化产物1-3分别作为第一抗体,使用步骤S1中制备的重组人HEPCIDIN1-3蛋白作为抗原,同时使用羊抗小鼠IgG作为第二抗体,对应好抗原和抗体分别孵育,做Western Blot鉴定小鼠抗人HEPCIDIN蛋白;同时检查克隆细胞的染色体数目,验证HEPCIDIN1-3单克隆抗体制备成功。
作为优选的技术方案的,步骤S2-1中,对小鼠注射制备的重组人HEPCIDIN1-3蛋白进行免疫,具体操作为:第一次采用腹腔注射,注射量为0.2mL/鼠;此后间隔2周重复注射1次;在取小鼠脾脏细胞进行融合的前3d用同样剂量静脉注射加强免疫一次;所述培养悬浮细胞,具体操作为:采用完全培养液悬浮细胞,并加入rHulL-2、rHulL-6、PWM和HEPCIDIN1-3,使其终浓度分别为50U/mL、500U/mL、1200ug/mL和500ug/mL;分别置于5%CO2,37℃温箱中培养5d后,收获细胞供融合用。
作为优选的技术方案的,步骤S2-2中所述小鼠骨髓瘤细胞选择标准为:在倒置显微镜下观察为圆形明亮,排列整齐,形态完整,密度0.1×106~1×106个/ml。
作为优选的技术方案的,步骤S2-3中所述骨髓瘤细胞与小鼠脾细胞混合的比例为1:5。
作为优选的技术方案的,步骤S2-6中所述注射降植烷为0.3mL/鼠;每只小鼠注射阳性克隆的杂交瘤细胞为1.0×108个。
前述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其组分包括试剂R1、试剂R2及HEPCIDIN校准品,具体地,
试剂R1包括:Tris-HCl缓冲液10~100mM/L,PEG6000 10~50g/L,BSA1~9g/L,NaCl 1~10g/L,MgCl2 0.1~1.0g/L,NaN3 0.1~1.0g/L,EDTA 0.1~0.5g/L;
试剂R2包括:Tris-HCl缓冲液10~50mM/L,BSA 1~9g/L,NaCl 1~10g/L,交联混合HEPCIDIN1-3单克隆抗体的胶乳微球5~20%,TX-100 0.1~1.0%,NaN3 0.1~1.0g/L,EDTA 0.1~0.5g/L;
HEPCIDIN校准品包括:Tris-HCl缓冲液10~50mM/L,BSA 1~9g/L,NaCl5~10g/L,rHEPCIDIN1-3 10~200mg/L,NaN 0.1~0.5g/L(V/V),EDTA0.1~0.5g/L。
本发明的有益效果是:
本发明试剂盒,自制了三株单克隆抗体,交联到胶乳微球上,形成胶乳增强免疫比浊法试剂盒。相比质谱法,使用本发明试剂盒测定铁调素,可在全自动生化分析仪上使用,成本低廉且自动化高,节省检测时间,具有非常好的实用及经济价值。
附图说明
图1为重组HEPCIDIN蛋白Western Blot鉴定结果;
图2为三株HEPCIDIN单克隆抗体Western Blot鉴定结果;
图3为本发明试剂盒与质谱检测方法学相关性曲线图;
图4为本发明试剂盒线性范围验证图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合实施例,对本发明的技术方案进行清楚、完整地描述。实施例1为重组人HEPCIDIN蛋白的制备,实施例2为HEPCIDIN1-3单克隆抗体的制备,实施例3为一种铁调素胶乳增强免疫比浊法试剂盒及其使用方法,实施例4为效果例,铁调素胶乳增强免疫比浊法试剂盒的评价。
实施例1
本实施例为重组人HEPCIDIN蛋白的制备,具体包括如下步骤:
(1)人HEPCIDIN蛋白基因的切割
人HEPCIDIN完整基因序列如下:
ACCAGAGCAAGCTCAAGACCCAGCAGTGGGACAGCCAGACAGACGGCACGATGGCACTGAGCTCCCAGATCTGGGCCGCTTGCCTCCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAAGTGTGGGATGTGCTGCAAGACGTAGAACCTACCTGCCCTGCCCCCGTCCCCTCCCTTCCTTATTTATTCCTGCTGCCCCAGAACATAGGTCTTGGAATAAAATGGCTGGTTCTTTTGTTTTCCAAA
人为切割第一段序列为:
ACCAGAGCAAGCTCAAGACCCAGCAGTGGGACAGCCAGACAGACGGCACGATGGCACTGAGCTCCCAGATCTGGGCCGCTTGCCTCCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCG
人为切割第二段序列为:
CCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAA
人为切割第三段序列为:
AAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAAGTGTGGGATGTGCTGCAAGACGTAGAACCTACCTGCCCTGCCCCCGTCCCCTCCCTTCCTTATTTATTCCTGCTGCCCCAGAACATAGGTCTTGGAATAAAATGGCTGGTTCTTTTGTTTTCCAAA
(2)重组人HEPCIDIN1-3蛋白的表达:
三段基因分别送至华大基因进行合成,收到工程菌后于含氨苄青霉素100μg/mL的LB培养基中37℃摇菌2h复苏工程菌活性,后在LB培养基(含氨苄青霉素100μg/mL)中放大培养8h后,测OD值达到1.0时,加入IPTG,32℃诱导表达(终浓度10μg/mL)8h,收集细菌,破碎后获得上清和沉淀,即为带HIS标签融合蛋白的目的蛋白rHEPCIDIN1-3。
(3)重组人HEPCIDIN1-3粗制品的制备:
加入质量体积比1:2的PBS重悬沉淀,-20℃与4℃反复冻融沉淀3次,4℃超声裂解细菌沉淀,功率40W,工作2s,间隔1s,超声破碎10min,重复3次,于4℃,12000r/min离心30min分别取上清、沉淀,得到粗制蛋白。
(4)重组人HEPCIDIN1-3粗制品纯化:
亲和层析:将粗制蛋白过滤处理后过HIS亲和层析柱,用Elution buffer(50mMTris-Cl 30mM还原性谷胱甘肽pH 8.6)梯度洗脱,收集rHEPCIDIN峰;
脱盐、缓冲液置换:将第一步纯化的rHEPCIDIN过脱盐柱,用缓冲液(50mM Tris-ClpH10.0)置换,准备下一步离子交换层析;
离子交换层析:分别用Loading buffer(50mM Tris-Cl pH7.0)平衡好柱体,上样后用Elution buffer(50mM Tris-Cl 1M NaCl pH7.0)梯度洗脱,收集rHEPCIDIN峰;
分子筛层析:离子交换层析收集到的rHEPCIDIN过分子筛层析柱,Elution buffer(50mM Na2HPO4 0.15M NaCl pH7.0)收集rHEPCIDIN峰;
取收集rHEPCIDIN做Wersten Blot验证:使用abcam公司HEPCIDIN抗体(EPR18074)为第一抗体,使用abcam公司山羊抗兔IgG(HRP标记)为第二抗体做WB,结果如图1所示,图中泳道M为蛋白Marker 26610,泳道1为空菌对照,泳道2为重组人HEPCIDIN-1纯化后样本,泳道3为重组人HEPCIDIN-2纯化后样本,泳道4为重组人HEPCIDIN-3纯化后样本,沉淀与上清样本在约17kD处均有阳性条带,表明纯化后所得rHEPCIDIN为铁调素,即为制备的重组人HEPCIDIN1-3蛋白。
然后加入20%甘油无菌分装,于-80℃保存备用。
实施例2
本实施例为HEPCIDIN1-3单克隆抗体的制备,具体包括如下步骤:
S2-1:小鼠免疫
选用6~10周龄,健康、发育良好的雌性BALb/c小鼠进行注射制备的重组人HEPCIDIN1-3蛋白免疫。第一次腹腔注射量为0.2mL/鼠,间隔2周重复注射1次,在融合前3d用同样剂量静脉注射加强免疫一次,使免疫抗体水平达到1:64。取免疫后的小鼠脾脏,制备脾细胞悬液,用完全培养液悬浮细胞,加入rHulL-2、rHulL-6、PWM和HEPCIDIN1-3,使其终浓度分别为50U/mL、500U/mL、1200ug/mL和500ug/mL,置5%CO2,37℃温箱中培养5d后,收获细胞供融合用。
S2-2:杂交瘤选择
选用小鼠骨髓瘤细胞系SP2/0。复苏的瘤细胞需在含10%新鲜小牛血清的RPMI-1640培养液中,置5%CO237℃温箱培养,每2d换培养液一次,3d传代一次。在倒置显微镜下挑选观察为圆形明亮,排列整齐,形态完整,密度适宜(0.1×106~1×106个/ml)。经台盼蓝染色,活细胞数应大于90%时供细胞融合用。
S2-3:细胞培养和融合
加入小鼠腹腔巨噬细胞作为饲养细胞,调整细胞浓度为1×105/mL,免疫亲代脾细胞与骨髓瘤细胞在PEG作用下,膜先行融合形成双核细胞或混核体:
将准备好的骨髓瘤细胞与小鼠脾细胞按1:5比例混合,加入20mL RPMI-1640液,1500rpm×5min离心,弃上清,用手指轻轻击打离心管底部,使沉淀细胞分散,将离心管置37℃水浴中,吸取1mL37℃预温的50%PEG缓缓滴入离心管内,1min内加完。37℃静置1min,滴加完全培养液(37℃预温)1mL,1min内加完,再加RPM1-1640液到50mL使PEC作用终止。800r/min×10min离心,弃上清,将沉淀细胞轻轻悬浮于所需容积的HAT培养液中,接种24孔或96孔培养板置37℃5%CO2温箱中培养。
S2-4:筛选克隆培养
经PEC处理后的两种亲本细胞,应用含有次黄嘌呤、氨基喋呤和胸腺嘧啶核苷(HAT)培养液予以筛选出来克隆培养,融合后每3d换一次HAT培养液,每次吸出培养孔旧液1/2换入新液,连续2周(14天)。5d后未融合及自身融合的细胞逐渐死亡,1周左右可出现克隆,并不断增殖,当集落大于3mm或布满孔底1/2时,即取上清作抗体活性检测,同时补加新鲜HAT培养液。
S2-5:杂交瘤阳性克隆的克隆化
杂交瘤细胞在HAT培养液中生长形成克隆后,其中仅少数是分泌预定特异性抗HEPCIDIN1-3的McAb的细胞,用酶联免疫吸附试验(ELISA)进行筛选和克隆化。初筛出能分泌与预定抗原起反应的McAb杂交瘤细胞,再进一步从中筛选出有预定特异性的杂交瘤细胞,然后选出可供实际应用,具有能稳定生长和有功能特性的细胞克隆。采用软琼脂法筛选分离后将选出的杂交瘤细胞放入96孔板中培养。
S2-6:抗HEPCIDIN1-3McAb的制备
采取体内诱生法,制备腹水。
选择6-8周龄健康小鼠,先于小鼠腹腔注射0.3mL降植烷,7~10d后于每只小鼠腹腔注射阳性克隆的杂交瘤细胞1.0×108个,正常饲养。待小鼠腹部明显膨大,用9号针头抽取腹水或用16号针头放腹水,可反复收集数次。腹水经12000rpm离心10min后,取上清分装。
S2-7:腹水的纯化
采用辛酸-硫酸铵法进行粗纯化后进行亲和层析:腹水4℃,12000rpm离心15min,腹水上清滤纸过滤去除脂质和大的颗粒沉淀,滤液用4倍体积的60mM醋酸缓冲液(pH4.0)稀释后,用1M NaOH调pH至4.5逐滴加入辛酸(终浓度为25μl/ml稀释腹水),待溶解后再加入另一滴,室温搅拌30min,然后4℃静置2h以上,使其充分沉淀。12000r/min,4℃,30min,收集上清。上清液经普通滤纸过滤1次,加入1/10体积的pH7.4的0.1M10*PBS用1M NaOH调至7.4,冰浴至4℃。根据每ml上述混合液加0.277g固体硫酸铵,再搅拌30min,继续静置过夜。再13000r/min,4℃离心30min,弃上清,沉淀溶解于结合缓冲液中。结合缓冲液洗柱子,流速为1ml/min,上样,结合缓冲液冲洗柱子,直到流出液中没有杂质,用10个柱床体积洗脱液进行洗脱。纯化产物可以利用Hi Trap Desalting交换缓冲液。
S2-8:HEPCIDIN1-3McAb鉴定
对产生抗体的细胞克隆,检查染色体数目,约80~100条。
取腹水纯化产物1-3作为第一抗体,使用上述制备的rHEPCIDIN1-3为抗原,使用abcam公司羊抗小鼠IgG(HRP标记,ab7063)为第二抗体,对应好抗原和抗体分别孵育,做WB,结果如图2所示,图中泳道M为蛋白Marker26610,泳道1为BSA对照,泳道2为HEPCIDIN单克隆抗体1,泳道3为HEPCIDIN单克隆抗体2,泳道4为HEPCIDIN单克隆抗体3,泳道5为abcam公司HEPCIDIN单抗对照,图中可见在17kD处泳道2-5均有阳性条带产生。因为rHEPCIDIN1-3已经商品化单抗验证,且使用abcam公司商品化单克隆抗体验证rHEPCIDIN同样出现阳性条带,表明单克隆抗体制备成功。
实施例3
本实施例为一种铁调素胶乳增强免疫比浊法试剂盒及其使用方法,具体如下:
本实施中所述铁调素胶乳增强免疫比浊法试剂盒包括试剂R1、试剂R2和OC校准品,具体组分如下:
(1)试剂R1中含有:
Tris-HCl缓冲液 ················50mM/L,
PEG6000····················20g/L,
BSA······················5g/L,
NaCl ·····················9g/L,
MgCl2·····················0.5g/L,
NaN3 ·····················0.1g/L,
EDTA ·····················0.5g/L;
(2)试剂R2中含有:
Tris-HCl缓冲液················50mM/L,
BSA ·····················5g/L,
NaCl·····················9g/L,
交联球····················5%,
TX-100····················0.5%,
NaN3·····················0.1g/L,
EDTA·····················0.5g/L;
(3)HEPCIDIN校准品中含有:
Tris-HCl缓冲液···············20mM/L,
BSA ····················5g/L,
NaCl····················9g/L,
rHEPCIDIN1-3················90mg/L,
NaN3····················0.1g/L,
EDTA····················0.5g/L。
本实施还提供一种使用上述试剂盒的测定方法,具体为:
分析方法:两点终点法;
反应方向:上升反应;
校准方式:Logit-Log(5P);
测定波长:570nm;
测定温度:37℃;
样本:试剂R1:试剂R2=20:210:70(μL)
测试步骤:吸取20μL样本,加入210μL试剂R1,37℃孵育5min,加入70μL试剂R2,1min后读取吸光值A1,3min后读取吸光值A2,计算ΔA。
定标方法:5点定标,采用日立7180全自动生化分析仪(或其他品牌型号),进行检测,设置校准品浓度分别为:5.625、11.25、22.5、45、90ng/ml(mg/L)。
依据浓度和ΔA值测算样本中HEPCIDIN含量。
实施例4效果例
(1)相关性验证
利用实施例3配方配制试剂,与质谱法进行对照检测,检测50份临床血清样本,检测结果如下表1,获得了本发明试剂盒与质谱法相关性曲线,如图3所示,通过检测结果显示,本案试剂与质谱法的线性相关曲线为y=0.8027x+2.7608,相关系数R2=0.8152,说明两者较大的相关性。
表1本发明试剂盒与质谱检测相关性比对值
(2)线性范围验证
使用重组HEPCIDIN1-3纯化品和生理盐水配制成浓度100mg/L、50mg/L、25mg/L、12.5mg/L、6.25mg/L和0mg/L(生理盐水对照)的测试品,使用本发明试剂盒测定各测试品浓度,以稀释浓度为自变量,以测定结果为因变量求出线性回归方程,计算测定结果的相对偏差。结果显示测定结果与稀释浓度之间的线性回归方程为y=1.0163x+0.1231,如图4所示。相关系数R2=0.997,说明线性关系良好,线性范围可达100mg/L。
表2本发明试剂盒线性范围验证
(3)准确度及精密度验证
取高值血清质控和低值血清质控各一份,使用所述试剂盒检测6次,取均值,与质控靶值进行比对。结果表明检测值较靶值相对偏差较小,CV较小,准确度及精密度较高。见表3。
表3所述试剂盒准确度验证结果
测定值1 | 测定值2 | 测定值3 | 测定值4 | 测定值5 | 测定值6 | 测定均值 | 血清靶值 | 相对偏差 | CV |
94.23 | 94.58 | 93.55 | 96.34 | 91.27 | 97.23 | 94.53 | 95.44 | -0.95% | 2.04% |
6.19 | 7.19 | 8.12 | 8.03 | 7.99 | 7.45 | 7.50 | 7.32 | 2.39% | 8.98% |
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的。此外,应当理解,虽然本说明书按照实施方式加以描述,但并非只包含一个技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (10)
1.一种基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:该试剂盒的组分包括交联混合HEPCIDIN1-3单克隆抗体的胶乳微球。
2.根据权利要求1所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:所述交联混合HEPCIDIN1-3单克隆抗体的胶乳微球为:
将人HEPCIDIN蛋白基因序列切割为三段,分别通过表达载体导入表达菌中进行表达;然后将表达产物纯化和鉴定后免疫小鼠,待免疫达到指定抗体水平后无菌取脾脏,制备杂交瘤细胞;培养筛选杂交瘤,获得三种HEPCIDIN单克隆抗体,然后分别将该三种HEPCIDIN单克隆抗体与聚苯乙烯胶乳微球交联,获得三种交联抗人HEPCIDIN单克隆抗体胶乳微球的混合物,即为交联混合HEPCIDIN1-3单克隆抗体的胶乳微球。
3.根据权利要求2所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:所述将人HEPCIDIN蛋白基因序列切割为三段,该三段基因序列之间有交叉,具体如下:
第一段序列为:
ACCAGAGCAAGCTCAAGACCCAGCAGTGGGACAGCCAGACAGACGGCACGATGGCACTGAGCTCCCAGATCTGGGCCGCTTGCCTCCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCG
第二段序列为:
CCTGCTCCTCCTCCTCCTCGCCAGCCTGACCAGTGGCTCTGTTTTCCCACAACAGACGGGACAACTTGCAGAGCTGCAACCCCAGGACAGAGCTGGAGCCAGGGCCAGCTGGATGCCCATGTTCCAGAGGCGAAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAA
第三段序列为:
AAGGAGGCGAGACACCCACTTCCCCATCTGCATTTTCTGCTGCGGCTGCTGTCATCGATCAAAGTGTGGGATGTGCTGCAAGACGTAGAACCTACCTGCCCTGCCCCCGTCCCCTCCCTTCCTTATTTATTCCTGCTGCCCCAGAACATAGGTCTTGGAATAAAATGGCTGGTTCTTTTGTTTTCCAAA。
4.根据权利要求2所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:所述交联混合HEPCIDIN1-3单克隆抗体的胶乳微球制备方法包括如下步骤:
S1: 重组人HEPCIDIN1-3蛋白的制备
将人HEPCIDIN蛋白基因序列切割为三段,分别通过表达载体导入表达菌中进行表达,获得重组人HEPCIDIN1-3粗制品,将该重组人HEPCIDIN1-3粗制品纯化,获得重组人HEPCIDIN1-3蛋白;
S2: HEPCIDIN1-3单克隆抗体的制备
将步骤S1制备的重组人HEPCIDIN1-3蛋白注射小鼠对其免疫,待免疫达到指定抗体水平后,取小鼠脾脏细胞制备杂交瘤细胞,并培养杂交瘤细胞获得HEPCIDIN1-3单克隆抗体;
S3:交联混合HEPCIDIN1-3单克隆抗体的胶乳微球的制备
将步骤S2制备的HEPCIDIN1-3单克隆抗体与直径100~300nm的聚苯乙烯胶乳微球采用化学交联法交联,获得交联混合HEPCIDIN1-3单克隆抗体的胶乳微球。
5.根据权利要求4所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:步骤S2所述 HEPCIDIN1-3单克隆抗体的制备包括如下子步骤:
S2-1:小鼠免疫
选择6~10周龄,健康、发育良好的雌性小鼠,分别对其注射制备的重组人HEPCIDIN1-3蛋白进行免疫;分别取免疫后的小鼠脾脏,制备脾脏细胞悬液,并培养悬浮细胞,培养的脾细胞供融合用;
S2-2:杂交瘤选择
选择活细胞数大于90%的小鼠骨髓瘤细胞系SP2/0供细胞融合用;
S2-3:细胞融合
将步骤S2-1培养的脾细胞和S2-2准备的小鼠骨髓瘤细胞按比例混合,在PEG作用下使二者融合,形成双核细胞或混核体;
S2-4:筛选克隆培养
用含有次黄嘌呤、氨基喋呤和胸腺嘧啶核苷的培养液对步骤S2-3中融合形成的双核细胞或混核体予以筛选,并进行克隆培养;
S2-5:杂交瘤阳性克隆的克隆化
采用酶联免疫吸附试验对步骤S2-4中筛选克隆培养的细胞进一步进行筛选和克隆化,筛出能稳定生长和有功能特性的阳性克隆杂交瘤细胞;
S2-6:抗HEPCIDIN1-3McAb的制备
选择6-8周龄的健康小鼠,先于小鼠腹腔注射降植烷,7~10d后于每只小鼠腹腔注射步骤S2-5中筛选的阳性克隆的杂交瘤细胞,待小鼠腹部明显膨大到一定程度时,抽取并收集腹水,离心后,取上清分装;
S2-7:腹水的纯化
采用辛酸-硫酸铵法将步骤S2-6中获得的腹水上清进行粗纯化,然后进行亲和层析进一步纯化腹水,获得腹水纯化产物1-3;
S2-8:HEPCIDIN1-3McAb鉴定
以步骤S2-7中获得腹水纯化产物1-3分别作为第一抗体,使用步骤S1中制备的重组人HEPCIDIN1-3蛋白作为抗原,同时使用羊抗小鼠IgG作为第二抗体,对应好抗原和抗体分别孵育,做Western Blot 鉴定小鼠抗人HEPCIDIN蛋白;同时检查克隆细胞的染色体数目,验证HEPCIDIN1-3单克隆抗体制备成功。
6.根据权利要求5所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:步骤S2-1中
对小鼠注射制备的重组人HEPCIDIN1-3蛋白进行免疫,具体操作为:第一次采用腹腔注射,注射量为0.2mL/鼠;此后间隔2周重复注射1次;在取小鼠脾脏细胞进行融合的前3d用同样剂量静脉注射加强免疫一次;
所述培养悬浮细胞,具体操作为:采用完全培养液悬浮细胞,并加入rHulL-2、rHulL-6、PWM和HEPCIDIN1-3,使其终浓度分别为50U/mL、500 U/mL、1200 ug/mL和500 ug/mL;分别置于5% CO2,37℃温箱中培养5d后,收获细胞供融合用。
7.根据权利要求5所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:步骤S2-2中所述小鼠骨髓瘤细胞选择标准为:在倒置显微镜下观察为圆形明亮,排列整齐,形态完整,密度0.1×106~1×106个/ml。
8.根据权利要求5所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:步骤S2-3中所述骨髓瘤细胞与小鼠脾细胞混合的比例为1:5。
9.根据权利要求5所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:步骤S2-6中所述注射降植烷为0.3mL/鼠;每只小鼠注射阳性克隆的杂交瘤细胞为1.0×108个。
10.根据权利要求1所述的基于单克隆抗体制备的铁调素的胶乳增强比浊法检测试剂盒,其特征在于:该试剂盒的组分包括试剂R1、试剂R2及HEPCIDIN校准品,具体地,
试剂R1包括:Tris-HCl缓冲液 10~100mM/L,PEG6000 10~50g/L,BSA 1~9g/L,NaCl 1~10g/L,MgCl2 0.1~1.0g/L,NaN3 0.1~1.0g/L,EDTA 0.1~0.5g/L;
试剂R2包括:Tris-HCl缓冲液 10~50mM/L,BSA 1~9g/L,NaCl 1~10g/L,交联混合HEPCIDIN1-3单克隆抗体的胶乳微球 5~20%,TX-100 0.1~1.0%,NaN3 0.1~1.0g/L,EDTA0.1~0.5g/L;
HEPCIDIN校准品包括:Tris-HCl缓冲液 10~50mM/L,BSA 1~9g/L,NaCl 5~10g/L,rHEPCIDIN1-3 10~200mg/L,NaN3 0.1~0.5g/L,EDTA 0.1~0.5g/L。
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