CN105968196A - G种肠道病毒检测抗体双夹心elisa检测试剂盒 - Google Patents
G种肠道病毒检测抗体双夹心elisa检测试剂盒 Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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Abstract
本发明公开了检测G种肠道病毒病原的双抗体夹心ELISA试剂盒,它采用其碱基序列如序列表SEQ ID NO.1所示的VP1基因,表达的VP1蛋白,免疫BALB/C小鼠后制备的单克隆抗体为捕获抗体,免疫成年健康家兔后制备的多克隆抗体,并标记了辣根过氧化物酶(HRP)。安全性、与RT‑PCR检测结果具有100%符合率。试剂盒4℃保存6个月,检测结果与常规方法无明显差异。
Description
技术领域
本发明属生物技术领域,具体涉及检测G种肠道病毒病原的双抗体夹心ELISA试剂盒。
背景技术
根据病毒的最新分类,小RNA病毒科肠道病毒属包括A、B、C、D、E、F、G、H和J共9个种肠道病毒。这些肠道病毒是引起人类和动物呼吸道和消化道及中枢神经系统疾病的重要病原体。其中A、B、C和D种肠道病毒引起人的感染;E、F和G种肠道病毒引起动物感染如牛的E种和F种肠道病毒感染和猪的G种肠道病毒感染,给人类的健康和畜牧业的健康发展造成了严重危害。羊肠道病毒感染是国内外新发的一种临床上以消化道和呼吸道为主要特征的疫病。国内有关羊肠道病毒感染的检测与诊断尚未见有报道。申请人于2014年从吉林多地爆发的一种临床上以严重腹泻,高发病率和高致死率为主要特征的不明疫病中的山羊体内分离到G种肠道病毒CEV-JL14,经过序列测定与分析发现,该毒株不同于目前报道的所有动物肠道病毒,其核苷酸序列的同源性与绵羊的最高,只有83%,表明所分离的毒株是一种新的肠道病毒。有关该病毒的检测与诊断方法及其防治缺乏研究。
发明内容
本发明目的是提供一种检测G种肠道病毒病原的双抗体夹心ELISA试剂盒。
一种G种肠道病毒抗原捕获抗体,它是采用其碱基序列如序列表SEQ ID NO.1所示的VP1基因,表达VP1蛋白,免疫BALB/C小鼠后制备的单克隆抗体。
一种G种肠道病毒酶标抗体,是采用其碱基序列如序列表SEQ ID NO.1所示的VP1基因,表达VP1蛋白,免疫成年健康家兔后制备的多克隆抗体。
所述的G种肠道病毒VP1基因,可采用PCR技术,以G种肠道病毒基因组cDNA为模板扩增获得,也可人工合成,其编码的氨基酸序列如SEQ ID NO.2所示。
所述的一种G种肠道病毒酶标抗体标记了辣根过氧化物酶。
检测G种肠道病毒病原的双抗体夹心ELISA试剂盒,它的抗原捕获抗体为上述的一种G种肠道病毒抗原捕获抗体,酶标抗体为上述的一种G种肠道病毒酶标抗体;
检测G种肠道病毒病原的双抗体夹心ELISA试剂盒,它是由下述方法制备的:
1)96孔ELISA板:用碳酸盐缓冲液稀释上述的一种G种肠道病毒抗原捕获抗体,包被96孔板,300 ng/孔,4℃,包被过夜;次日甩干,洗涤后,加入含2% BSA的PBST溶液每孔100µL,37℃封闭2h,洗净甩干后塑封;
(2)样品稀释液(PBST pH7.4):NaCl 8.0g;KH2PO4 0.2g;Na2HPO4 ·12H2O 2.9g;KCl0.2g补加二馏水致1000 mL,加入0.5 mL Tween-20,50mL分装;
(3)20倍浓缩洗涤液:NaCl 160.0g;KH2PO4 4g;Na2HPO4 ·12H2O 58g;KCl 4g补加二馏水致1000 ml;加入10 ml Tween-20,50 mL分装;
(4)HRP标记兔源抗VP1 多克隆抗体:1:8000稀释上述的一种G种肠道病毒酶标抗体,无菌分装,每瓶10mL;
(5)终止液:2mol/L H2SO4 浓硫酸44.5 ml,双蒸水355.5 ml,混匀后5mL分装;
(6)底物A:取OPD 200 mg,加双蒸水至1000 mL,10 mL分装;
(7)底物B:0.1 mol/L柠檬酸,0.2 mol/L磷酸氢二纳,0.75 %过氧化氢6.4 mL,加三蒸水至1000 mL,调至pH5.0-5.43,10 mL分装。
本发明提供了检测G种肠道病毒病原的双抗体夹心ELISA试剂盒,它采用其碱基序列如序列表SEQ ID NO.1所示的VP1基因,表达的VP1蛋白,免疫BALB/C小鼠后制备的单克隆抗体为捕获抗体,免疫成年健康家兔后制备的多克隆抗体,并标记了辣根过氧化物酶(HRP)。安全性、与RT-PCR检测结果具有100%符合率。试剂盒4℃保存6个月,检测结果与常规方法无明显差异。
本发明的优点:
1、具有生物安全性。试剂盒所用的抗G种肠道病毒VP1单克隆抗体和多克隆抗体为原核载体表达的重组蛋白诱导BALB/C小鼠和家兔产生,不含G种肠道病毒,因此不存在散毒危险;
2、敏感性高。捕获抗体和HRP标记二抗均具有很高的免疫效价,因此提高了试剂盒的敏感性和特异性。试剂盒检测G种肠道病毒阳性和阴性粪便样品与RT-PCR检测结果具有100%符合率;
3、特异性强。试剂盒与其他病原体无交叉反应,只检出G种肠道病毒抗原,具有很高特异性;
4、稳定性好。试剂盒4℃保存6个月,检测结果与常规方法无明显差异,具有很好的稳定性;
5、快速简便。试剂盒2.0-2.5 h 即可报告结果,使用方便,一般技术人员按说明书即可操作,条件一般实验室均可进行;
6、不影响动物正常生产。待检样品为粪便,采集容易、方便、不影响动物正常生产,容易被基层牛场接受;
7、价格便宜。约3元/头份,HRP标记二抗和抗体制备技术成熟,均可大规模获得。
附图说明
图1 pGEX-4T-1-VP1重组质粒酶切鉴定结果(泳道M:DNA分子marker;泳道1:pGEX-4T-1-VP1 EcoRI/XhoI双酶切;泳道2:pGEX-4T-1-VP1 EcoRI单酶切)
图2重组VP1蛋白的SDS-PAGE检测结果(泳道M:蛋白质分子量marker;泳道1 :pGEX-4T-1-VP1重组菌未诱导总蛋白;泳道2:pGEX-4T-1-VP1重组菌诱导后总蛋白;泳道3:纯化后的重组VP1蛋白)
图3病毒感染细胞IPMA检测结果(A图:未感染的Vero细胞,阴性对照;B图:感染CEV的Vero细胞)
图4试剂盒各组分((1)包被有VP1捕获抗体及预处理的96孔ELISA板;(2)样品稀释液;(3)20倍浓缩洗涤液;(4)HRP标记抗VP1兔源多抗;(5)终止液;(6)底物A;(7)底物B;(8)阳性对照样品;(9)阴性对照样品)。
具体实施方式
实施例1 G种肠道病毒结构蛋白VP1原核表达重组质粒的构建
1、引物设计
根据目标序列的碱基组成,分别设计引物扩增VP1基因,上下游分别含有EcoRI、XhoI酶切位点。引物序列如下:
上游引物:5' TTGAATTCCAGGCTGGGTATGTGACT 3' (EcoRI)
下游引物:5' TCTCGAGTTAATGAAAATCACTGAGGGGTT 3' (XhoI)
2、基因扩增
常规Trizol法提取G种肠道病毒的基因组RNA,采用市售常规反转录试剂盒,按照说明操作获得cDNA,并以其为模板扩增VP1基因,PCR反应体系:
PCR运行条件:94℃预变性3min;94℃变性1.5min、54℃退火40sec、72℃延伸40sec,共35个循环;72℃再延伸6min。反应结束后,取1μL进行0.8%琼脂糖凝胶电泳检测。
3、目标基因与pGEX-4T-1的连接、转化
采用分子生物学领域中的常规酶切、连接等技术,构建重组质粒pGEX-4T-1-VP1,并采用CaCl2法将重组质粒转化大肠杆菌DH5α感受态中。利用含100 µg/mL氨苄霉素的LB培养平板筛选阳性克隆,并进行常规增菌培养,收获菌体,提取质粒,进行EcoRI/XhoI双酶切鉴定,如图1所示。
实施例2 VP1重组蛋白的表达及纯化
将鉴定的阳性重组质粒pGEX-4T-1-VP1转化大肠杆菌BL21(DE3) 感受态细胞后,涂板,37℃培养过夜,挑取单个菌落接种到3 mL含有100 µg/mL氨苄霉素的LB液体培养基中培养过夜,然后取1 mL上述培养物接种到200 mL含有100 µg/mL氨苄霉素的LB液体培养基中37℃振摇培养至对数生长期(OD600=0.6),加入IPTG(终浓度1 mmol/L),20℃诱导培养3 h,经SDS-PAGE检测,获得了重组目标蛋白VP1,如图2所示(泳道2)。离心沉淀菌体, 超声破碎后以1.5M尿素洗涤3次后获得高纯度的重组蛋白,如图2所示(泳道3)。
实施例3抗羊肠道病毒 VP1重组蛋白单克隆抗体和多克隆抗体的制备
1、抗VP1单克隆抗体制备:选择6-8周龄健康BALB/c小鼠以弗氏完全佐剂乳化纯化的VP1重组蛋白,每只小鼠多点皮下注射共100 μg,之后每间隔14d按照同样方法加强免疫一次,同时采集血清检测效价。最后一次加强免疫采用直接腹腔注射100μg纯化的蛋白,3~5天后取免疫小鼠的脾细胞与SP2/0混合于融合管内,以300 g离心10 min,弃去上清,振荡细胞,使两种细胞尽量混合均匀,然后于60 sec内缓慢滴加预热的PEG-4000溶液,再缓慢加入无血清的1640培养基终止融合,静置后再以1 000 r/min离心10 min,弃去上清后加入HAT培养基,使细胞混匀悬浮,于96孔培养板中培养,第14 d换液,并检测筛选分泌抗体的杂交瘤细胞。杂交瘤细胞的筛选采用间接ELISA,用纯化的VP1重组蛋白作为包被抗原包被反应板,加入杂交瘤细胞的培养上清进行ELISA筛选;对ELISA阳性的杂交瘤细胞进一步克隆至所有孔都为阳性。通过3次细胞克隆最终获得杂交瘤细胞株。取高压灭菌的石蜡油0.5 mL,注射到小鼠腹腔,7 d后腹腔注入106个杂交瘤细胞,一周后抽取腹水,并置37℃ 24 h后4℃过夜,然后离心腹水,取上清,即为抗VP1单抗;
按上述操作,经过多次重复性试验,每次均可获得多株合乎标准的杂交瘤细胞株。
2、抗VP1多克隆抗体制备:选择成年健康家兔以弗氏完全佐剂乳化纯化的VP1重组蛋白,每只家兔多点皮下共注射约2mg,之后每间隔14d以弗氏不完全佐剂乳化蛋白多点皮下注射,并检测血清效价。最后一次加强免疫直接采用腹腔注射2mg纯化的蛋白,3~5天内采集血液收集血清。
3、抗VP1多克隆抗体纯化:取高免血清20mL,加入等体积10 mmol/L的PBS缓冲液(pH7.2),混匀后缓慢加入40 mL饱和硫酸铵,边加边搅拌;混合物4℃放置30 min后,以3000rpm离心30 min。沉淀物以20 mL PBS溶解后,缓慢加入10 mL饱和硫酸铵,边加边搅拌,混合物 4℃放置30 min 后,3000 rpm 离心30 min, 如此重复两次。最后沉淀溶于4 mL PBS缓冲液中,4℃透析24 h,蔗糖浓缩后分装-80℃保存。提取的抗CEV VP1抗体过氧化物酶单层细胞试验(IPMA)进行鉴定,结果如图3所示。
实施例4 HRP标记兔源抗VP1 IgG制备
采用郭春祥法对纯化的IgG进行HRP标记:将5 mg HRP溶于H2O,并与0.5 mL的0.1 MNaIO4 溶液混匀,4℃静置30min。然后加入0.5 mL的0.16M乙二醇溶液混匀、室温静置30 min后,加入纯化的IgG 1mL(约10mg),混匀后装入透析袋,以pH9.5的碳酸盐缓冲液4℃透析12-18h。透析后所得溶液加入0.2mL的5 mg/mL的NaBH4溶液,于4℃静置2h。然后采用过硫酸铵沉淀法沉淀HRP标记的IgG,PBS 4℃透析24-48h,蔗糖浓缩后,使用紫外分光光度计测定浓度,分装-80℃保存。
实施例5双抗体夹心ELISA方法的建立
1、反应程序的建立
(1)双抗体夹心ELISA方法的程序
以pH 9.6的碳酸盐缓冲液稀释的抗VP1的单克隆抗体,4℃包被过夜,每孔包被200 ng。以PBST (含0.05%Tween-20的PBS,pH7.2) 洗涤液洗板3次后,加入5%脱脂奶粉37℃封闭60min。以PBST洗涤液洗涤3次后,加入1:5(W/V)预处理样品100 µL,37℃感作60 min,然后以洗涤液洗涤5次,加入HRP标记的二抗,每孔100 μL,37℃感作45 min。以同样方法洗涤后,加入OPD显色试剂,每孔100 μL。室温避光作用15 min后,加入2 mol/L H2SO4 50μL终止反应,测定OD490值。
(2)HRP标记抗VP1 兔源多克隆抗体的最佳孵育量确定
应用直接ELISA方法确定HRP标记抗VP1 抗VP1 兔源多克隆抗体的最佳孵育量。用pH9.6的碳酸盐缓冲液稀释V1抗原,包被0.1µg/孔, 4℃过夜。以PBST(含0.05%Tween-20的PBS,pH7.2)洗涤液洗板3次后,加入5%脱脂奶粉37℃封闭60 min。然后以PBST洗涤液洗涤3次,加入预稀释的HRP标记兔源抗VP1IgG(100×,200×,400×,600×,800×,1000×,2000×,4000×,6000×,8000×,10000×,12000×),37℃感作45 min。以PBST洗板3次后,加入OPD显色试剂,室温避光显色15 min,然后加入2 mol/L H2SO4 50μL终止反应,测定OD490值。选择OD值在0.2左右且阳性OD值/阴性OD值(P/N)比值最大时的一抗和二抗稀释度为最佳工作浓度。最终确定最佳孵育量为8000×稀释。
(3)捕获抗体最佳包被量的确定:
应用双抗体夹心ELISA方法对捕获抗体最佳包被量进行确定。每孔加入以pH9.6的碳酸盐包被缓冲液稀释的100µL的捕获抗体,稀释度分别为1 µg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 30 µg/mL, 40 µg/mL, 50 µg/mL, 60 µg/mL, 80 µg/mL和100 µg/mL,按照前述操作方法进行试验,选择OD值在0.2左右且阳性OD值/阴性OD值(P/N)比值最大时的一抗稀释度为最佳工作浓度。最终捕获抗体最佳包被量为300 ng/孔
(4)封闭液及作用时间的确定:
应用确定的捕获抗体及酶标二抗的最适工作浓度进行双抗体夹心ELISA试验。分别以2%脱脂奶粉、5%脱脂奶粉、1% BSA、2%BSA和5%BSA于37℃分别封闭30 min、1h和2h。显色15min后终止反应。测定各样本OD490值,比较各组的OD490值和P/N值。,确定ELISA反应的最适封条件为2%BSA ,37℃封闭1h。
(5)双抗体夹心ELISA方法的敏感性试验:
判定标准的确定:取40份背景清晰的经RT-PCR鉴定为羊肠道病毒(CEV)病原检测阴性的羊粪样品,按1:10(W/V)将样品以PBS稀释,3000 rpm 离心15 min后,上清液用于双抗体夹心ELISA检测,确定OD490值,以此40份样品的平均OD490加上3倍标准差作为阴性与阳性样品的临界值,以此作为判定标准,即OD490≥OD490(阴性)+3SD阴性=0.109时样品为阳性。
敏感性试验:另取56份未知样品按已建立的双抗体夹心ELISA方法进行试验,判定结果,并对该系列样品进行RT-PCR鉴定,两者试验结果一致,表明所建立的双抗体夹心ELISA方法具有很好的敏感性。
(6)双抗体夹心ELISA方法特异性试验:
与口蹄疫病毒(FMDV)、牛病毒性腹泻病毒(BVDV)、牛细小病毒(BPV)阳性样品进行双抗体夹心ELISA交叉试验检测,结果显示上述样品均为阴性,说明所建立的双抗体夹心ELISA方法与这些病毒无交叉反应,特异性较好。
(7)双抗体夹心ELISA方法的重复性试验
取4块不同批次包被的酶标板,每个稀释度设4个重复,在同一块酶标板上进行批内重复,不同的酶标板之间进行批间重复,测定OD值,计算其变异系数,如果变异系数<10%则说明其重复性和稳定性很好。4个不同批次的酶标板,结果一致。说明检测方法的重复性较好。
实施例6双抗体夹心ELISA检测试剂盒组装
将建立好的双抗夹心ELISA方法中所用到的试剂盒材料:(1)96孔ELISA板、(2)样品稀释液、(3)20倍浓缩洗涤液、(4)HRP标记兔源抗VP1多克隆抗体、(5)终止液、(6)底物A、(7)底物B、(8)阳性样品、(9)阴性样品,分别用相应的广口瓶加以封装,贴上标签,组装成试剂盒,如图4所示,具体操作如下:
(1)96孔ELISA板:用碳酸盐缓冲液稀释抗VP1鼠源单克隆抗体,包被96孔板,300 ng/孔,4℃,包被过夜;次日甩干,洗涤后,加入含2% BSA的PBST溶液每孔100µL,37℃封闭2h,洗净甩干后塑封。
(2)样品稀释液(PBST pH7.4):NaCl 8.0g;KH2PO4 0.2g;Na2HPO4 ·12H2O 2.9g;KCl 0.2g补加二馏水致1000 mL,加入0.5 mL Tween-20,50mL分装。
(3)20倍浓缩洗涤液:NaCl 160.0g;KH2PO4 4g;Na2HPO4 ·12H2O 58g;KCl 4g补加二馏水致1000 ml;加入10 ml Tween-20,50 mL分装。
(4)HRP标记兔源抗VP1 多克隆抗体:1:8000稀释的HRP标记兔源抗VP1 多克隆抗体,无菌分装,每瓶10mL。
(5)终止液:2mol/L H2SO4 浓硫酸44.5 ml,双蒸水355.5 ml,混匀后5mL分装。
(6)底物A:取OPD 200 mg,加双蒸水至1000 mL,10 mL分装。
(7)底物B:0.1 mol/L柠檬酸,0.2 mol/L磷酸氢二纳,0.75 %过氧化氢6.4 mL,加三蒸水至1000 mL,调至pH5.0-5.43,10 mL分装。
(8)阳性样品:将确定为CEV阳性粪样,以样品稀释液按1:5稀释、离心,上清液经过灭活后,1 mL分装。
(9)阴性样品:将确定为CEV阴性粪样,以样品稀释液按1:5稀释、离心,上清液经过灭活后,1 mL分装。
实施例7双抗体夹心ELISA试剂盒的保质期的确定
将组装好的双抗体夹心ELISA试剂盒4℃放置1个月、2个月、4个月、6个月、九个月测定其特异性和敏感性。结果该试剂盒4℃保存6个月后,检测效果与常规ELISA方法无明显差异,表明该试剂盒保质期至少6个月。
实施例8 人工合成VP1基因
人工合成如序列表SEQ ID NO.1所示的VP1基因,按实施例2方法表达蛋白VP1,用以制备单克隆抗体和多克隆抗体及其检测试剂盒,与实施例1的基因效果相似。
<110> 吉林大学
<120>G种肠道病毒检测抗体双夹心ELISA检测试剂盒
<160> 2
<210> 1
<211> 677
<212> DNA
<213> 人工
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cagaccgcta atatcatctg tttagtggca gcacaaccaa atttttccct gcgagtgtta 120
aaagatcgtc cagacatgga ccagactgcc gctttgcaac taccaccagt gggtgagcag 180
attagagagt ttatgggtga gactgtttct aatgcattga ctgccgccaa caccactgaa 240
agcactcaca atatttcaac gagtgatacc ccagcacttc aagctgccga gacgggggca 300
acgtcgaatg caagtgatga gagtatgctt gagaccagga cggttcttaa ccagaatgga 360
attagggaga catccgttga agccttcttt ggcagatctg gattggccac gattatgacc 420
ttggcagcag gtgatgtgaa gacccagtgg accatcaatt tcaatgagtt cgttcagctt 480
agagccaagt tagatttgtt cacttaccta cgatttgaca ttgagttcac tttcgtggcg 540
acatccacga aaaagggtaa gtacaattct gagcccatcc agttacaact gatgtacgtc 600
cccccgggcg ccactcygcc gacggatcaa gacacctatc agtggcagac tgcggcaaac 660
ccctcagtga ttttcat 677
<210> 2
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<212> DNA
<213> 人工
<400> 2
qagyvtgwyq tnmvippefp qtaniiclva aqpnfslrvl kdrpdmdqta alqlppvgeq 60
irefmgetvs naltaantte sthnistsdt palqaaetga tsnasdesml etrtvlnqng 120
iretsveaff grsglatimt laagdvktqw tinfnefvql rakldlftyl rfdieftfva 180
tstkkgkyns epiqlqlmyv ppgatxptdq dtyqwqtaan psvif 225
Claims (6)
1. 一种G种肠道病毒抗原捕获抗体,其特征在于:它是采用其碱基序列如序列表SEQID NO.1所示的VP1基因,表达VP1蛋白,免疫BALB/C小鼠后制备的单克隆抗体。
2. 一种G种肠道病毒酶标抗体,其特征在于:它是采用其碱基序列如序列表SEQ IDNO.1所示的VP1基因,表达VP1蛋白,免疫成年健康家兔后制备的多克隆抗体。
3.根据权利要求1或2所述的一种G种肠道病毒酶标抗体,其特征在于:所述的G种肠道病毒VP1基因为人工合成。
4.根据权利要求2所述的一种G种肠道病毒酶标抗体,其特征在于:标记了辣根过氧化物酶。
5.检测G种肠道病毒病原的双抗体夹心ELISA试剂盒,其特征在于:它是由抗原捕获抗体为权利要求1所述的一种G种肠道病毒抗原捕获抗体,酶标抗体为为权利要求1所述的一种G种肠道病毒酶标抗体。
6.检测G种肠道病毒病原的双抗体夹心ELISA试剂盒,其特征在于:它是由下述方法制备的:
(1)96孔ELISA板:用碳酸盐缓冲液稀释为权利要求1所述的一种G种肠道病毒抗原捕获抗体,包被96孔板,300 ng/孔,4℃,包被过夜;次日甩干,洗涤后,加入含2% BSA的PBST溶液每孔100µL,37℃封闭2h,洗净甩干后塑封;
(2)样品稀释液(PBST pH7.4):NaCl 8.0g;KH2PO4 0.2g;Na2HPO4 ·12H2O 2.9g;KCl0.2g补加二馏水致1000 mL,加入0.5 mL Tween-20,50mL分装;
(3)20倍浓缩洗涤液:NaCl 160.0g;KH2PO4 4g;Na2HPO4 ·12H2O 58g;KCl 4g补加二馏水致1000 ml;加入10 ml Tween-20,50 mL分装;
(4)HRP标记兔源抗VP1 多克隆抗体:1:8000稀释为权利要求2所述的一种G种肠道病毒酶标抗体,无菌分装,每瓶10mL;
(5)终止液:2mol/L H2SO4 浓硫酸44.5 ml,双蒸水355.5 ml,混匀后5mL分装;
(6)底物A:取OPD 200 mg,加双蒸水至1000 mL,10 mL分装;
(7)底物B:0.1 mol/L柠檬酸,0.2 mol/L磷酸氢二纳,0.75 %过氧化氢6.4 mL,加三蒸水至1000 mL,调至pH5.0-5.43,10 mL分装。
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