CN111487416A - optrA耐药蛋白双抗夹心ELISA检测试剂盒及检测方法 - Google Patents
optrA耐药蛋白双抗夹心ELISA检测试剂盒及检测方法 Download PDFInfo
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- CN111487416A CN111487416A CN202010542163.8A CN202010542163A CN111487416A CN 111487416 A CN111487416 A CN 111487416A CN 202010542163 A CN202010542163 A CN 202010542163A CN 111487416 A CN111487416 A CN 111487416A
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Abstract
本发明提供一种用于检测细菌中optrA耐药蛋白的双抗夹心ELISA检测试剂盒及其检测方法,包括包被有单克隆抗体5C6的酶标板、检测抗体2F3‑HRP、optrA蛋白。本发明选用5C6作为捕获抗体,2F3‑HRP作为检测抗体研制了optrA双抗夹心ELISA检测试剂盒,并对其进行了方法学***评估,结果显示试剂盒特异性好,灵敏度高,为optrA阳性耐药菌的快速检测提供了一种技术手段。
Description
技术领域
本发明涉及一种用于检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒及方法,属于免疫学分析技术领域。
背景技术
optrA基因由我国学者于2015年从1株临床分离的粪肠球菌09E349中首次发现,并且已被证明还可以介导细菌对氟化和非氟化氯霉素的耐药性。研究表明,optrA属于ABC蛋白家族,是一种新发现的耐药蛋白,既可介导肠球菌对恶唑烷酮类和利胆醇类抗生素的耐药性,还可以介导金黄色葡萄球菌对氯霉素类的耐药性。optrA被确定存在于中国的人源和动物源的肠球菌中,在中国,人源的主要分布于江西、天津、浙江;动物源的主要分布在河南、山东、上海、广东、西藏。自optrA首次在中国报道后,世界范围内多个国家开始检出optrA阳性的肠球菌,并且在部分国家临床和动物来源菌株(如波兰、哥伦比亚)检出的携带optrA基因的质粒与pE349有很高的相似性,这提示可能存在人畜之间的传播。2016年,意大利也发现了携带optrA基因的肠球菌。由于肠球菌的感染已经成为疾病治疗中的主要问题之一,因此耐药基因optrA的出现和蔓延将有望成为临床治疗肠球菌感染的一个重要靶点。
本发明应用的酶联免疫吸附法(ELISA)具有快速、灵敏、特异性好且操作简便等特点,因此建立一种用于检测optrA基因的双抗夹心ELISA检测试剂盒及方法,对其基因检测和方法开发具有重要意义。
发明内容
本发明的目的是提供一种灵敏度高、特异性强、操作简便、检测快速的检测optrA耐药蛋白的双抗夹心酶联免疫试剂盒及检测方法。
为了实现上述目的,本发明采用的技术方案是:
一种用于检测细菌中optrA耐药蛋白的双抗夹心ELISA检测试剂盒,包括:包被有单克隆抗体5C6的酶标板、检测抗体2F3-HRP、optrA蛋白标准品。
在一个实施方案中,所述检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,包括:
PCR扩增目的片段并克隆至表达载体pET28a,将成功构建的重组表达质粒转化至大肠杆菌BL21(DE3)中,IPTG诱导表达,经亲和层析和分子筛层析高度纯化后得到optrA重组蛋白;
上述重组optrA蛋白氨基酸序列为序列1;
在一个实施方案中,所述optrA单克隆抗体5C6,2F3为采用重组optrA蛋白免疫小鼠,经融合、克隆、筛选,得到的;
所述单克隆抗体5C6的重链可变区的氨基酸序列为序列2;
所述单克隆抗体5C6的轻链可变区的氨基酸序列为序列3;
所述单克隆抗体2F3的重链可变区的氨基酸序列为序列4;
所述单克隆抗体2F3的轻链可变区的氨基酸序列为序列5。
在一个实施方案中,所述检测抗体2F3-HRP的制备方法包括如下步骤:
(1)称取5 mg HRP溶于1 mL三蒸水中,逐滴缓慢加入0.20 mL新配的0.1 M NaIO4溶液,4℃避光搅拌25 min,活化HRP,颜色由棕色变为绿色;上述溶液装入透析袋中,用1 M,pH4.4的醋酸钠缓冲液中4℃过夜透析,然后10000 r/min,4℃,离心10 min,去除沉淀;得到透析后的HRP;
(2)将单克隆抗体2F3用0.2 M,pH为9.5的碳酸缓冲液4℃透析过夜,得到透析后的抗体;
(3)将透析后的HRP加入0.16 M乙二醇(每mg酶加0.1 mL),4℃避光搅拌1 h,然后加入透析后的抗体;两者混匀后用0.05 M,pH为9.5的碳酸缓冲液4℃透析过夜,得到HRP-抗体混合液;
(4)对0.15 M pH 7.4 PBS透析过夜;搅拌下逐滴加入等体积饱和硫酸氨,4℃避光搅拌3 h;4℃,10000 rpm离心15 min,弃上清;PBS 溶解沉淀,得到经辣根过氧化物酶标记的检测抗体2F3-HRP。
在一个实施方案中,所述检测细菌中optrA耐药蛋白的双抗夹心ELISA检测试剂盒,还包括如下步骤:
1)包被有单克隆抗体5C6的酶标板的制备:将单克隆抗体5C6用碳酸盐缓冲液稀释成浓度为5 μg/mL的抗体包被液,每孔100 μL,4℃包被过夜后洗板;然后每孔加入100 μL封闭液,37℃、湿度30-40%封闭2 h,37℃、湿度30-40%恒温干燥2 h;
2)检测抗体稀释液:0.01 M pH 7.4的PBST溶液;
3)检测抗体工作液的制备:检测抗体2F3-HRP按照 1:30000比例稀释后备用;
4)样品稀释液的制备:0.01M pH7.4的PBS溶液。
5)底物A液的制备:含0.08%过氧化脲、0.025%PEG-2000、3.58%十二水合磷酸氢二钠、0.96%一水合柠檬酸水溶液,调pH值至7.4;
6)底物B液的制备:含1.03%一水合柠檬酸、0.04% TMB、0.0008%硫代硫酸钠、0.1%光稳定剂292、3% DMF水溶液,调pH值至5.0。
本发明的第二个目的在于提供一种optrA蛋白双抗夹心ELISA检测方法,该方法操作简便,检测快速,特异性强,灵敏度高。
具体的说,一种optrA蛋白双抗夹心ELISA检测法,采用以下步骤:(1)以pH为9.5的Na2CO3溶液包被捕获抗体5C6(2 ng/mL),酶标板中每孔加入100 μL,4℃过夜,使其与酶标板紧密结合;(2)次日,弃去孔内溶液,用洗涤液(PBST)洗板3次,每次3 min;每孔加入100 μL2%BSA进行封闭,37℃温育2 h;封闭结束后向酶标板孔内加入细菌裂解液液,并同时设置阴性对照孔(0.01 M PB,pH 7.4)和阳性对照孔(optrA蛋白,4 ng/mL),100 μL/孔,37℃孵育1 h;(3)接着加入检测抗体2F3-HRP,100 μL/孔,37℃孵育1 h;(4)最后加入的显色剂TMB溶液(现用现配),每孔100 μL,37℃孵育10 min。在HRP作用下,显色剂发生颜色变化,加入终止液50 μL/孔;(5)测定:用酶标仪检测OD450nm。
在一个实施方案中,所述试剂盒中还包括终止液、封闭液和洗涤液;
具体的所述终止液为3 mol/L硫酸铵;
所述封闭液为2% BSA;
所述洗涤液为含0.1% Tween-20的0.01 M pH7.4 PBST溶液。
本发明的有益效果是:本发明制备了optrA单克隆抗体并建立了双抗夹心ELISA方法,本发明具有检测方法简单,灵敏度高,检测optrA蛋白LOD为1 ng/mL,与屎肠球菌、链球菌、金黄色葡萄球菌、松鼠葡萄球菌、副溶血弧菌无交叉,说明具有良好的特异性,为试剂盒方法开发提供了科学依据。
附图说明
图1 optrA蛋白双抗体夹心ELISA标准曲线;
横坐标为不同稀释浓度的optrA蛋白标准品,纵坐标为相应的OD450nm吸光值。
图2 optrA与其他菌属的交叉反应。
具体实施方案
为了使本发明的目的和技术方案更加清楚,下面对本发明的优选实施案例进行详细描述。
1.optrA耐药蛋白的双抗夹心ELISA检测试剂盒的组成
本发明双抗夹心ELISA检测试剂盒,包括包被有单克隆抗体5C6的酶标板、检测抗体2F3-HRP、optrA蛋白标准品、包被缓冲液、检测抗体稀释液、检测抗体工作液、样本稀释液、底物A液、底物B液、显色液、终止液、封闭液、洗涤液。
2.optrA耐药蛋白的双抗夹心ELISA检测试剂盒的制备
A、optrA基因的合成
从Genebank中获取optrA基因的氨基酸序列(Accession Number:WP 063854496.1),委托南京金斯瑞生物科技有限公司合成优化后的基因序列。
B、载体的构建
通过PCR扩增目的片段并克隆至表达载体pET28a,PCR反应扩增条件:95℃预变性5分钟;然后95℃变性40秒,58℃退火30秒,72℃延伸40秒,共进行25个循环;72℃延伸2分钟。
C、optrA重组蛋白的表达与纯化
构建完成的重组质粒转入感受态细胞转化入大肠杆菌BL21(DE3)宿主菌株中,挑取单菌落接种至含有50 μg/mL卡那霉素的LB培养基中,37℃,200 rpm培养至OD600在0.6~0.8范围时,加入终浓度为0.5 mM的IPTG,25℃条件下进行诱导表达。
4℃条件下,3200 g离心15 min收集菌体;然后使用20 mM Tris-HCl(含150 mMNaCl)重悬菌体,通过超声裂解菌体。采用Ni-NTA镍柱纯化***纯化表达的optrA重组蛋白,用来制备鼠单克隆抗体。
测序结果:重组optrA蛋白时所用氨基酸序列如序列表中的序列1所示。
2)optrA单克隆抗体的制备
A、免疫实验动物
取步骤2制备的免疫原(重组optrA蛋白)溶液,胺100μg/只,将免疫原用无菌生理盐水稀释至1mg/mL,首次免疫加入等量的弗氏完全佐剂,完全乳化后,采用颈背部皮下、多点注射的方式免疫8只小鼠。共免疫6次,每次免疫间隔时间均为2周,具体免疫程序见表1。
表1. 单克隆抗体(小鼠)的免疫程序
B、抗血清的筛选
四免一周后,对小鼠眼眶采血,室温放置2 h,4000 rpm离心10 min后取血清检测;抗血清筛选先采用间接ELISA方阵滴定法确定包被原、抗体的最佳工作浓度,再采用间接竞争ELISA方法检测抗体的特异性。
C、杂交瘤细胞株的融合与筛选
按照常规方法进行,取免疫小鼠的脾细胞与处于对数生长期的小鼠骨髓瘤细胞(SP2/0)混合,然后用50%PEG进行免疫融合,用HAT培养基悬浮均匀,再加入适量的饲养细胞,培养于96孔培养板,于37℃,5%CO2培养箱中培养,5天后用HAT培养基半换液,9天时候进行全换液。
细胞融合后,待细胞长到培养孔面积的1/4时,吸出杂交瘤细胞上清液,并采用重组MCR-1蛋白包被酶标板及间接竞争ELISA方法筛选阳性、效价高的培养孔,得到的阳性孔用中国农业大学提供的optrA屎肠球菌、链球菌、金黄色葡萄球菌、松鼠葡萄球菌、副溶血弧菌裂解液包被的酶标板检测细胞的交叉反应,挑选所有optrA阳性菌株均有反应的强阳性孔进行性亚克隆。其中optrA阳性菌的鉴定方法参照:赵丽青. 广东某猪场噁唑烷酮类耐药基因optrA的流行特点[D].华南农业大学,2016.
将数次亚克隆建株后的杂交瘤细胞扩大培养,收集上清液用间接ELISA测定效价,冻存;并取8-10周龄Balb/c小鼠腹腔注射0.3 mL/只含1.3x106个细胞的细胞悬液。6天后观察小鼠,当小鼠腹部膨大时,抽取腹水,每隔2天观察小鼠,及时抽取腹水;将抽取的腹水10000r/min离心5 min,收集上清,分装保存于-20℃冰箱。
D、腹水中抗体的纯化
将5 mL腹水在10000 r/min、4℃条件下离心5 min,收集上清液,然后加入20 mL倍体的0.06 mM醋酸盐缓冲液(pH 4.0)进行稀释,再使用0.2 M NaOH调节pH至4.5左右;加入1000μL正辛酸缓慢加入正辛酸并搅拌30 min,4℃静置1 h;将上述液体在6000 r/min、4℃条件下离心30 min,收集上清并过滤;加入2.6 mL的PBS缓冲液进行稀释(加入的量应为上述滤液的10%);加入等体积的饱和硫酸铵,并搅拌30 min,4℃静置1 h;弃上清,加入适量PBS缓冲液重悬后装入透析袋,置于0.02 mM PBS缓冲液中与4℃透析24-48 h并适时换液,收集透析袋内液体,-20℃保存,得到单克隆抗体。
测序结果:本发明5C6单克隆抗体的重链和轻链可变区的氨基酸序列分别如序列表中序列2和序列3所示,2F3单克隆抗体的重链和轻链可变区的氨基酸序列分别如序列表中序列4和序列5所示。
E、经辣根过氧化物酶标记的optrA检测抗体的制备方法
(1)称取5 mg HRP(辣根过氧化物酶,购自Sigma)溶于1 mL三蒸水中,逐滴缓慢加入0.20 mL新配的0.1 M NaIO4溶液,4℃避光搅拌25 min,活化HRP,颜色由棕色变为绿色。上述溶液装入透析袋中,用1 M,pH为4.4的醋酸钠缓冲液透析,4℃过夜。10000 r/min,4℃,10min,离心去除沉淀。得到透析后的HRP。
(2)将2F3检测抗体用0.2 M,pH为9.5的碳酸缓冲液4℃透析过夜。观察是否有沉淀,并分析沉淀性状,10000 r/min,4℃,10 min,离心去除沉淀,得到透析后的抗体。
(3)将透析后的HRP加入0.16 M乙二醇(每mg酶加0.1 mL),4℃避光搅拌1 h,然后加入透析后的抗体;两者混匀后用0.05 M,pH为9.5的碳酸缓冲液4℃透析过夜,得到HRP-抗体混合液;
(4)将上述溶液装入透析袋中,对0.15 M pH 7.4 PBS透析过夜。搅拌下逐滴加入等体积饱和硫酸氨,4℃避光搅拌3 h。4℃,10000 rpm离心15 min,弃上清。PBS 溶解沉淀,得到经辣根过氧化物酶标记的2F3-HRP检测抗体。
F、包被有5C6单克隆抗体的酶标板的制备
将5C6单克隆抗体用碳酸盐缓冲液稀释成浓度为5 μg/mL的抗体包被液,每孔100 μL,4℃包被过夜后洗板;然后每孔加入100 μL封闭液,37℃、湿度30-40%封闭2 h,37℃、湿度30-40%恒温干燥2 h。
3.optrA双抗夹心Elisa检测方法建立
A、双抗体夹心ELISA检测法测定步骤
(1)以pH为9.4的Na2CO3,、NaHCO3包被捕获抗体5C6(2 ng/mL),作为捕获抗体包被酶标板,在96孔酶标板中每孔加入100 μL,4℃包被过夜,使其与酶标板紧密结合;
(2)次日,弃去孔内溶液,用洗涤液 (PBST)洗板3次,每次3 min。每孔再加100 μL的2%牛血清蛋白作为封闭液,37℃温育2 h。封闭结束后,弃去孔内溶液,洗板3次,在酶标板孔内加入待测蛋白提取物溶液(以待测样品与PBS缓冲液的体积比为稀释倍数)和optrA蛋白(每孔4 ng/mL),100 μL/孔,37℃孵育1 h;
(3)接着加入检测抗体2F3-HRP,100 μL/孔,37℃孵育1 h;
(4)最后在形成的复合物中加入临时配制的显色剂TMB溶液,每孔100 μL,37℃孵育10~30 min。在HRP作用下,显色剂发生颜色变化,加入终止液50 μL/孔;
(5)测定:用酶标仪检测OD450nm。
B、方法的建立
(1)线性范围
以optrA蛋白标准品作系列稀释至1~32 ng/mL,用上述建立的双抗体夹心ELISA方法进行检测,进行3次重复,以optrA蛋白标准品质量浓度(ng/mL)为横坐标,OD450值为纵坐标制作标准曲线,用origin8.0(OriginLab Corp, Northampton, MA, USA) 软件中的四参数拟合竞争标准曲线,确定线性范围与检测限LOD(LOD是空白的平均吸收值加3倍的空白吸收值的标准偏差对应的抗原浓度)。
(2) 特异性验证
以最佳配对的抗体以及最佳浓度来进行双抗体夹心ELISA检测特异性,分别用optrA阳性的屎肠球菌、链球菌、金黄色葡萄球菌、松鼠葡萄球菌、副溶血弧菌,以及屎肠球菌CICC10840,链球菌ATCC 13813,金黄色葡萄球菌ATCC 6538,松鼠葡萄球菌CICC 23480,副溶血弧菌ATCC 17802进行特异性检测。
2)ELISA检测方法建立
(1)标准曲线
通过测试数据拟合曲线表明(图1),本方法对optrA,检出限为1 ng/mL,R2= 0.983,方程为Y=2.462+(0.306-2.462)/(1+(x/2.378) 1.840)。
(2)特异性的检测
以最佳配对的抗体以及最佳浓度来进行双抗体夹心ELISA检测特异性.其分别与带有optrA屎肠球菌、链球菌、金黄色葡萄球菌、松鼠葡萄球菌(图2)。结果表明,与optrA阳性的屎肠球菌、链球菌、金黄色葡萄球菌、松鼠葡萄球菌、副溶血弧菌进行特异性检测均有交叉反应,与标准菌株屎肠球菌CICC 10840,链球菌ATCC 13813,金黄色葡萄球菌ATCC 6538,松鼠葡萄球菌CICC 23480副溶血弧菌ATCC 17802均无交叉反应,说明特异性良好。
序列表
<110> 北京维德维康生物技术有限公司
<120> optrA耐药蛋白双抗夹心ELISA检测试剂盒及检测方法
<141> 2020-06-15
<150> 2020101802999
<151> 2020-03-16
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Claims (10)
1.一种用于检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,包括:包被有单克隆抗体5C6的酶标板、检测抗体2F3-HRP、optrA蛋白标准品。
2.根据权利要求1所述的检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,其特征在于:
所述捕获抗体5C6和检测抗体2F3为采用optrA重组蛋白免疫小鼠,经融合、克隆、筛选,得到的。
3.根据权利要求1或2所述的检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,其特征在于:
所述单克隆抗体5C6的重链可变区的氨基酸序列为序列2;
所述单克隆抗体5C6的轻链可变区的氨基酸序列为序列3;
所述单克隆抗体2F3的重链可变区的氨基酸序列为序列4;
所述单克隆抗体2F3的轻链可变区的氨基酸序列为序列5。
4.根据权利要求2所述的检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,其特征在于:
PCR扩增目的片段并克隆至表达载体pET28a,将成功构建的重组表达质粒转化至大肠杆菌BL21(DE3)中,IPTG诱导表达,经亲和层析和分子筛层析高度纯化后得到optrA、重组蛋白。
5.根据权利要求4所述的检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,其特征在于,重组optrA蛋白氨基酸序列为序列1。
6.根据权利要求1所述的检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒,其特征在于,所述检测抗体2F3-HRP制备步骤包括:
(1)称取5 mg HRP溶于1 mL三蒸水中,逐滴缓慢加入0.20 mL新配的0.1 M NaIO4溶液,4℃避光搅拌25 min,活化HRP,颜色由棕色变为绿色;上述溶液装入透析袋中,用1 M,pH为4.4的醋酸钠缓冲液透析,4℃过夜,10000 r/min,4℃,10 min,离心去除沉淀;得到透析后的HRP;
(2)将单克隆抗体5C6抗体用0.2 M,pH为9.5的碳酸缓冲液4℃透析过夜,得到透析后的抗体;
(3)将透析后的HRP加入0.16 M乙二醇(每mg酶加0.1 mL),4℃避光搅拌1 h,然后加入透析后的抗体;两者混匀后用0.05 M,pH为9.5的碳酸缓冲液4℃透析过夜,得到HRP-抗体混合液;
(4)对0.15 M pH 7.4 PBS透析过夜;搅拌下逐滴加入等体积饱和硫酸氨,4℃避光搅拌3 h;4℃,10000 rpm离心15 min,弃上清;PBS溶解沉淀,得到经辣根过氧化物酶标记的检测抗体2F3-HRP,使用酶标记物稀释液1:30000倍稀释后备用。
7.一种制备权利要求1-6中任一所述的试剂盒的方法,包括如下步骤:
1)包被有单克隆抗体5C6的酶标板的制备:将单克隆抗体5C6用碳酸盐缓冲液稀释成浓度为5 μg/mL的抗体包被液,每孔100 μL,4℃包被过夜后洗板;然后每孔加入100 μL封闭液,37℃、湿度30-40%封闭2 h,37℃、湿度30-40%恒温干燥2 h;
2)检测抗体稀释液:0.01 M pH7.4的PBST溶液;
3)检测抗体工作液的制备:检测抗体2F3-HRP按照 1:30000比例稀释后备用;
4)样品稀释液的制备:0.01 M pH 7.4的PBS溶液;
5)底物A液的制备:含0.08%过氧化脲、0.025%PEG-2000、3.58%十二水合磷酸氢二钠、0.96%一水合柠檬酸水溶液,调pH值至7.4;
6)底物B液的制备:含1.03%一水合柠檬酸、0.04% TMB、0.0008%硫代硫酸钠、0.1%光稳定剂292、3% DMF水溶液,调pH值至5.0。
8.一种用于检测optrA耐药蛋白的双抗夹心ELISA检测法,其特征在于,
(1)向包被有单克隆抗体5C6的酶标板中加入细菌菌体裂解液和optrA蛋白稀释液,100μL/孔,37℃孵育1 h;
(2)用PBST洗涤三次,每次3 min,200 μL/孔,然后甩干反应板;
(3)接着加入检测抗体2F3-HRP,100 μL/孔,37℃孵育1 h;
(4)用PBST洗涤三次,每次3 min,200 μL/孔,然后甩干反应板;
(5)最后在形成的复合物中加入临时配制的显色剂TMB溶液,每孔100 μL,37℃孵育10~30 min,在HRP作用下,显色剂发生颜色变化,加入终止液50 μL/孔;
(6)测定:用酶标仪检测OD450nm。
9.根据权利要求1所述的试剂盒或权利要求8所述的方法,其特征在于,所述试剂盒中还包括终止液、封闭液和洗涤液;
具体的所述终止液为3 mol/L硫酸铵;
所述封闭液为2% BSA;
所述洗涤液为含0.1% Tween-20的0.01 M pH 7.4 PBST溶液。
10.根据权利要求8所述的方法,其特征在于,所述细菌菌体裂解液的制备方法为:
4℃条件下,3500 g离心15 min收集菌体,然后使用20 mM Tris-HCl(含150 mM NaCl)重悬菌体,通过超声裂解菌体,超声条件为3000 W,10 s/10s,15 min。
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