CN111487416B - 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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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系统评估，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，pH 4.4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：WP063854496.1），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镍柱纯化系统纯化表达的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10000 r/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1000 μL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℃，10 min，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100μL，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ATCC6538，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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tagggaacgg ggttgctgac gggtcttgtg atagccgggg agcccgggga tcgtgattgg 300
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Claims (9)
1.一种用于检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒，其特征在于：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；
单克隆抗体5C6的重链可变区的氨基酸序列为序列2；
单克隆抗体5C6的轻链可变区的氨基酸序列为序列3；
单克隆抗体2F3的重链可变区的氨基酸序列为序列4；
单克隆抗体2F3的轻链可变区的氨基酸序列为序列5。
2.根据权利要求1所述的用于检测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2所述的用于检测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。
4.根据权利要求3所述的用于检测optrA耐药蛋白的双抗夹心ELISA检测试剂盒，其特征在于：所述optrA重组蛋白氨基酸序列为序列1。
5.根据权利要求1所述的用于检测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5mg HRP溶于1mL三蒸水中，逐滴缓慢加入0.20mL新配的0.1M NaIO4溶液，4℃避光搅拌25min，活化HRP，颜色由棕色变为绿色；上述溶液装入透析袋中，用1M，pH为4.4的醋酸钠缓冲液透析，4℃过夜，10000r/min，4℃，10min，离心去除沉淀；得到透析后的HRP；
(2)将单克隆抗体5C6抗体用0.2M，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0.16M乙二醇，每mg酶加0.1mL，4℃避光搅拌1h，然后加入透析后的抗体；两者混匀后用0.05M，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0.15M pH 7.4PBS透析过夜；搅拌下逐滴加入等体积饱和硫酸氨，4℃避光搅拌3h；4℃，10000rpm离心15min，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。
6.一种制备权利要求1-5中任一所述的试剂盒的方法，其特征在于：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5μg/mL的抗体包被液，每孔100μL，4℃包被过夜后洗板；然后每孔加入100μL封闭液，37℃、湿度30-40％封闭2h，37℃、湿度30-40％恒温干燥2h；
(2)检测抗体稀释液：0.01M 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：0.01M 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。
7.一种用于检测optrA耐药蛋白的双抗夹心ELISA检测法，其特征在于：采用权利要求1-5中任一所述的试剂盒检测，检测法具体步骤包括：
(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1h；
(2)用PBST洗涤三次，每次3min，200μL/孔，然后甩干反应板；
(3)接着加入检测抗体2F3-HRP，100μL/孔，37℃孵育1h；
(4)用PBST洗涤三次，每次3min，200μL/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100μL，37℃孵育10～30min，在HRP作用下，显色剂发生颜色变化，加入终止液50μL/孔；
(6)测定：用酶标仪检测OD450nm。
8.根据权利要求1所述的试剂盒或权利要求7所述的检测法，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3mol/L硫酸铵；
所述封闭液为2％BSA；
所述洗涤液为含0.1％Tween-20的0.01M pH 7.4PBST溶液。
9.根据权利要求7所述的检测法，其特征在于：所述细菌菌体裂解液的制备方法为：
4℃条件下，3500g离心15min收集菌体，然后使用含150mM NaCl的20mM Tris-HCl重悬菌体，通过超声裂解菌体，超声条件为3000W，10s/10s，15min。
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