CN111487417B - Mcr-1耐药蛋白双抗夹心elisa检测试剂盒及检测方法 - Google Patents
Mcr-1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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- CN111487417B CN111487417B CN202010542174.6A CN202010542174A CN111487417B CN 111487417 B CN111487417 B CN 111487417B CN 202010542174 A CN202010542174 A CN 202010542174A CN 111487417 B CN111487417 B CN 111487417B
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Abstract
本发明提供一种用于检测细菌中MCR‑1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4E5的酶标板、检测抗体5B3‑HRP、MCR‑1蛋白标准品。本发明选用4E5作为捕获抗体，5B3‑HRP作为检测抗体研制了MCR‑1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MCR‑1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MCR-1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
近年来，由于多重耐药菌株的出现，粘菌素在人类医学中被重新引入，以治疗与健康相关的感染，从而导致大肠埃希菌和沙门菌对黏菌素的耐药日趋严重。2015年，华南农业大学药理课题组于上海一猪场首次发现一株质粒介导的可转移耐药基因MCR-1，且报道在人和动物和食物中均检测出质粒介导的粘菌素耐药基因MCR-1，并证实该基因可通过接合型质粒在不同菌属间传播，且速度快。这与之前大家所认知的染色体介导粘菌素耐药所不同，从分子机制上解释了目前国内外多粘菌素耐药性迅速升高的原因。MCR-1基因的发现，预示着抗生素的最后一道防线---多粘菌素已然遭到威胁，同时对动物和人类的健康也造成了严重的威胁。
目前，现有的耐药基因检测方法中，一般通过传统的微生物培养法、药敏试验法、聚合酶链反应（PCR）技术、基因芯片技术等方法来判定微生物是否对某一种抗生素具有耐药性，但前两种方法费时且操作复杂，不能满足现场检测的需求。而后两种检测方法操作繁琐，需要专业仪器且昂贵等缺点，不适合临床大规模推广应用。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MCR-1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
在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MCR-1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MCR-1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4E5的酶标板、检测抗体5B3-HRP、MCR-1蛋白标准品。
在一个实施方案中，所述检测MCR-1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MCR-1重组蛋白；
上述重组MCR-1蛋白氨基酸序列为序列1。
在一个实施方案中，所述MCR-1单克隆抗体4E5，5B3为采用重组MCR-1蛋白免疫小鼠，经融合、克隆、筛选，得到的；
所述单克隆抗体4E5的重链可变区的氨基酸序列为序列2；
所述单克隆抗体4E5的轻链可变区的氨基酸序列为序列3；
所述单克隆抗体5B3的重链可变区的氨基酸序列为序列4；
所述单克隆抗体5B3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5B3-HRP的制备方法包括如下步骤：
（1）称取5 mg HRP溶于1mL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5B3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HRP-5B3。
在一个实施方案中，所述检测细菌中MCR-1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4E5的酶标板的制备：将单克隆抗体4E5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5B3-HRP按照1:30000比例稀释后备用；
4）样品稀释液的制备：0.01 M 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MCR-1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MCR-1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4E5(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（0.01M PB，pH 7.4）和阳性对照孔（MCR-1蛋白，4 ng/mL），100 μL/孔，37℃孵育1h；（3）接着加入检测抗体5B3-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 pH 7.4 PBST溶液。
本发明的有益效果是：本发明制备了MCR-1单克隆抗体并建立了双抗夹心ELISA方法，该方法检测MCR-1蛋白LOD为0.5ng/mL，与大肠杆菌、肺炎克雷伯氏菌、志贺菌、鼠伤寒沙门氏菌无交叉，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 MCR-1蛋白双抗体夹心ELISA标准曲线；
横坐标为不同稀释浓度的MCR-1蛋白标准品，纵坐标为相应的OD450nm吸光值。
图2 MCR-1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.MCR-1耐药蛋白的双抗夹心ELISA检测试剂盒的组成
本发明的双抗夹心ELISA 试剂盒包括包被有单克隆抗体4E5的酶标板、检测抗体5B3-HRP、MCR-1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.MCR-1耐药蛋白的双抗夹心ELISA检测试剂盒的制备
1）制备MCR-1蛋白
A、MCR-1基因的合成
从Genebank中获取MCR-1基因的氨基酸序列（Accession：WP 049589868.1），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50秒。
C、MCR-1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℃，200rpm培养至OD600达到0.6~0.8时，加入终浓度为0.5mM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150mMNaCl）重悬菌体，3000 W，10 s/10s，15 min超声裂解菌体。采用Ni-NTA镍柱纯化系统纯化表达的MCR-1重组蛋白，用来制备鼠单克隆抗体。
测序结果：重组MCR-1蛋白时所用氨基酸序列如序列表中的序列1所示。
2）MCR-1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MCR-1蛋白）溶液，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，，免疫剂量为100 μg/只。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和灵敏度。
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MCR-1阳性大肠杆菌、肺炎克雷伯氏菌、志贺菌、鼠伤寒沙门氏菌裂解液包被的酶标板检测细胞的交叉反应，挑选所有MCR-1阳性菌株均有反应的强阳性孔进行性亚克隆。其中MCR-1阳性菌的鉴定方法参照：Liu Y Y , et al. Emergence ofplasmid-mediated colistin resistance mechanism MCR-1 in animals and humanbeings in China: a microbiological and molecular biological study[J]. LancetInfectious Diseases, 2016, 16:161-168.
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醋酸钠缓冲液（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4E5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；5B3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MCR-1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5B3检测抗体用0.2M，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5B3-HRP检测抗体。
F、包被有4E5单克隆抗体的酶标板的制备
将4E5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. MCR-1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9.5的碳酸盐缓冲液包被捕获抗体4E5 (3 ng/mL)，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3min。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MCR-1蛋白(每孔5 ng/mL)，100 μL/孔，37℃孵育1 h；
（3）接着加入检测抗体5B3-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）MCR-1蛋白的线性范围
以MCR-1蛋白标准品作系列稀释至0.5~16 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MCR-1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MCR-1阳性的大肠杆菌、肺炎克雷伯氏菌、志贺菌、鼠伤寒沙门氏菌，以及大肠杆菌ATCC25922，大肠杆菌ATCC35150，肺炎克雷伯氏菌ATCC 10031，志贺菌CICC 21534，鼠伤寒沙门氏菌ATCC 13311进行特异性检测。
4.结果
1）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MCR-1，检出限为0.5 ng/mL，R2=0.997，方程为Y=2.419+(0.23-2.419)/(1+(x/1.066)1.7)。
（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MCR-1阳性的大肠杆菌、肺炎克雷伯氏菌、志贺菌、鼠伤寒沙门氏菌（图2）。结果表明，与MCR-1阳性的大肠杆菌、肺炎克雷伯氏菌、志贺菌、鼠伤寒沙门氏菌进行特异性检测均有交叉反应，与标准菌株大肠杆菌ATCC 25922，大肠杆菌ATCC 35150，肺炎克雷伯氏菌ATCC10031，志贺菌CICC 21534，鼠伤寒沙门氏菌ATCC 13311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> MCR-1耐药蛋白双抗夹心ELISA检测试剂盒及检测方法
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<150> 2020101803879
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Claims (8)
1.一种用于检测细菌中MCR-1耐药蛋白的双抗夹心ELISA检测试剂盒，其特征在于：包被有单克隆抗体4E5的酶标板、检测抗体5B3-HRP、MCR-1蛋白标准品；
所述的试剂盒的方法，包括如下步骤：
(1)包被有单克隆抗体4E5的酶标板的制备：将单克隆抗体4E5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5B3-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；
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。
2.根据权利要求1所述的用于检测细菌中MCR-1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4E5和检测抗体5B3为采用重组MCR-1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所述的用于检测细菌中MCR-1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4E5的重链可变区的氨基酸序列为序列2；
所述单克隆抗体4E5的轻链可变区的氨基酸序列为序列3；
所述单克隆抗体5B3的重链可变区的氨基酸序列为序列4；
所述单克隆抗体5B3的轻链可变区的氨基酸序列为序列5。
4.根据权利要求2所述的用于检测细菌中MCR-1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MCR-1重组蛋白。
5.根据权利要求4所述的用于检测细菌中MCR-1耐药蛋白的双抗夹心ELISA检测试剂盒，其特征在于：MCR-1重组蛋白的氨基酸序列为序列1。
6.根据权利要求1所述的用于检测细菌中MCR-1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5B3-HRP的制备方法步骤包括：
(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的醋酸钠缓冲液中4℃过夜透析，然后10000r/min，4℃，离心10min，去除沉淀；得到透析后的HRP；
(2)将单克隆抗体5B3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 HRP加0.1mL所述乙二醇，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5B3-HRP，使用酶标记物稀释液1:30000倍稀释后备用。
7.根据权利要求1所述的用于检测细菌中MCR-1耐药蛋白的双抗夹心ELISA检测试剂盒，其特征在于：所述试剂盒的检测包括如下步骤：
(1)向包被有单克隆抗体4E5的酶标板中加入细菌菌体裂解液和MCR-1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5B3-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7所述的用于检测细菌中MCR-1耐药蛋白的双抗夹心ELISA检测试剂盒，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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