CN117405884A - 一种羊全血布鲁氏菌抗体检测试剂盒 - Google Patents
一种羊全血布鲁氏菌抗体检测试剂盒 Download PDFInfo
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Abstract
本发明提供了一种羊全血布鲁氏菌检测试剂盒。所述试剂盒中包括三棱针、塑料滴管、离心管、血液稀释液以及包含荧光标记的羊全血布鲁氏菌单克隆抗体，所述羊全血布鲁氏菌单克隆抗体重链可变区序列如SEQ ID NO.1所示，轻链可变区序列如SEQ ID NO.2所示。本试验标记的荧光抗体具有特异性和检测下线，其灵敏度可高达100％。表明所建立的羊全血布鲁氏菌荧光抗体检测方法具有较高的灵敏度，能满足高通量，快速检测的要求，具有潜在推广应用价值。
Description
技术领域
本发明涉及布鲁氏菌检测技术领域，更具体地，涉及一种羊全血布鲁氏菌抗体检测试剂盒。
背景技术
布鲁氏菌病是由布鲁氏菌引起的人畜共患的慢性传染病。其特点是生殖器官、胎膜及多种器官组织发炎、坏死和肉芽肿的形成，引起流产、不孕、睾丸及关节炎等症状。多种动物对本病均有不同程度的易感性，自然感染以羊、牛和猪常见。
布鲁氏菌病的快速诊断对于布鲁氏菌病的早发现，早治疗，以及布鲁氏菌病大样本的筛查都具有重要的意义，然而传统布鲁氏菌病的诊断以病原分离和血清学检验为主，无论是病原分离还是血清学检验，其特异性和敏感性都不高，容易造成漏诊。因此，开发一种新的检测方法，快速、敏感、特异、准确地检测羊全血布鲁氏菌对该疾病预防和治疗具有重要的意义。
发明内容
针对现有技术所存在的技术问题，本发明提供了一种羊全血布鲁氏菌抗体检测试剂盒。本研究首次以布鲁氏菌omp19蛋白为免疫原，并制备得到羊全血布鲁氏菌单克隆抗体，并基于该单克隆抗体建立了羊全血布鲁氏菌的直接免疫荧光检测方法。
具体而言，本发明首先是提供了一种羊全血布鲁氏菌检测试剂盒，其特征在于，所述试剂盒中包含荧光标记的羊全血布鲁氏菌单克隆抗体，所述羊全血布鲁氏菌单克隆抗体重链可变区序列如SEQ ID NO.1所示，轻链可变区序列如SEQ ID NO.2所示。
优选地，所述羊全血布鲁氏菌单克隆抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示，所述羊全血布鲁氏菌单克隆抗体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ IDNO.6-8；
进一步地，所述试剂盒还包括如下组分：三棱针、塑料滴管、离心管、血液稀释液；
进一步优选地，所述血液稀释液的配方为每100mL无菌水中含有枸橼酸钠二水0.8g，枸橼酸一水0.055g，葡萄糖2.05g，氯化钠0.42g；
进一步地，本发明还提供了一种荧光标记的羊全血布鲁氏菌单克隆抗体，采用荧光基团标记所述羊全血布鲁氏菌单克隆抗体，其中所述羊全血布鲁氏菌单克隆抗体的轻链CDR4-6分别如SEQ ID NO.6-8所示，所述单克隆抗体的重链CDR1-3分别如SEQ DI NO.3-5所示。
优选地，本发明提供的所述荧光标记为FITC、FAM、Rhodamine B、TAMRA、Cy3、Cy5等荧光基团标记。
优选地，本发明提供的所述荧光标记为FITC。
优选地，本发明提供的所述荧光抗体的最适工作浓度确定为1:240。
进一步地，本发明还提供了一种非诊断目的的羊全血布鲁氏菌的直接免疫荧光检测方法，该方法包括采用所述试剂盒中荧光标记的单克隆抗体接触待检样本，孵育结束后，在荧光显微镜下观察结果或酶标仪读取荧光值。
进一步地，本发明还提供了上述荧光标记的单克隆抗体在制备检测羊全血布鲁氏菌检测试剂盒中的应用。
本发明的优点如下：本发明提供的羊全血布鲁氏菌单克隆抗体是针对布鲁氏菌omp19蛋白的特异性抗体，其与羊炭疽细菌、羊破伤风杆菌、羊李氏杆菌和羔羊大肠杆菌均不发生交叉反应。经鉴定，所述单克隆抗体具备良好的检测特异性和检测下线，且在实际样本检测中的灵敏度可高达100％。表明所建立的羊全血布鲁氏菌荧光抗体检测方法具有较高的灵敏度，能满足高通量，快速检测的要求，具有潜在推广应用价值。
附图说明
图1为羊全血布鲁氏菌单克隆抗体对布鲁氏菌检测灵敏度分析。
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明，以使本领域的技术人员更加清楚地理解本发明。
以下各实施例，仅用于说明本发明，并不用来限制本发明的范围。基于本发明中的具体实施例，本领域普通技术人员在没有做出创造性劳动的情况下，所获得的其他所有实施例，都属于本发明的保护范围。
在本发明实施例中，若无特殊说明，所有原料组分均为本领域技术人员熟知的市售产品；在本发明实施例中，若未具体指明，所用的技术手段均为本领域技术人员所熟知的常规手段。
实施例1羊全血布鲁氏菌单克隆抗体
1.1杂交瘤细胞系制备
以100μg/mL布鲁氏菌omp19蛋白(Genbank：AAB06277.1)作为免疫抗原对8只6周龄雌性BALB/c小鼠进行免疫注射，每次皮下注射200μL，间隔时间1周。经连续三次间隔免疫后，对小鼠断尾采血，用间接ELISA方法监测血清抗体效价，并选择效价最高的免疫小鼠，用纯的抗原液尾静脉加强免疫，3天后无菌采取小鼠脾细胞与SP2/0细胞融合，两种细胞之比为1:10，将培养板放置37℃5％的CO2培养箱中培养。经6天的连续培养后，取上清进行抗体检测。
1.2杂交瘤细胞系筛选
用间接ELISA方法检测杂交瘤细胞(长至孔底的1/2以上)分泌抗体的情况，以筛选出阳性细胞株。具体方法为取杂交瘤细胞培养上清100μL加入前日包被好的酶标板孔中，同时设立阳性、阴性对照和空白对照。判定标准为OD490值阳性孔与对照孔比值≥2.1判定为阳性。阴性孔3d后再检测一次，如仍为阴性则弃之。
1.3单克隆抗体免疫球蛋白重链和轻链类型的鉴定
取杂交瘤细胞株培养上清，采用IgG抗体检测试剂盒(上海抚生实业有限公司)测定抗体重链、轻链类型。
经鉴定，本发明所述羊全血布鲁氏菌单克隆抗体的重链可变区序列如SEQ IDNO.1所示，轻链可变区序列如SEQ ID NO.2所示。所述羊全血布鲁氏菌单克隆抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示，所述羊全血布鲁氏菌单克隆抗体体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ ID NO.6-8。
1.4单克隆抗体的特性鉴定
单抗效价的测定
用间接ELISA方法测定单克隆抗体的效价。提纯的羊全血布鲁氏菌作为包被抗原，将羊全血布鲁氏菌单克隆抗体以1:10开始倍比稀释，同时以同等稀释度的注射SP2/0细胞的小鼠腹水作为阴性对照。以平行稀释的单抗OD490值/对照OD490≥2.1的最高稀释倍数为单抗的效价。结果显示：三免后小鼠的血清效价均在106以上，达到融合要求。
与其他细菌的交叉反应
用间接ELISA方法测定单克隆抗体与羊炭疽细菌、羊破伤风杆菌、羊李氏杆菌和羔羊大肠杆菌的交叉反应。分别将上述细菌作为包被抗原，与所述单抗作用，以阴性腹水对照。结果表明：本发明制备的羊全血布鲁氏菌单克隆抗体是针对布鲁氏菌的特异性抗体，与羊炭疽细菌、羊破伤风杆菌、羊李氏杆菌和羔羊大肠杆菌均不发生交叉反应，结果见表1，证明此所述单抗对羊全血布鲁氏菌具有良好的特异性。
表1羊全血布鲁氏菌单克隆抗体特异性分析
实施例2单克隆抗体荧光学检测方法
2.1荧光抗体的制备
取一定量的纯单抗溶液，在0.5mol/L pH9.2碳酸盐缓冲液透析过夜，按荧光素与蛋白质质量比1:50称取适量FITC，加入二甲亚砜(DMSO)，使终浓度为5mg FITC/1mL DMSO；将单抗溶液置于电磁搅拌器上，轻轻搅拌，以不起沫为准。按上述比例将FITC-DMSO溶液逐滴加入单抗溶液中，磁力搅拌器4℃避光搅拌24h；加完后，随时用试纸测定搅拌液的pH值，若低于9.0，则应以碳酸钠溶液调整。置4℃搅拌24h即可。用Sephadex G25柱过滤标记的荧光抗体，以去除游离荧光素。
2.2抗体工作浓度的确定
将荧光抗体自1:15～1:960倍比稀释，分别用各稀释度对羊布鲁氏菌感染甲状腺上皮细胞做染色。以能清晰显示特异荧光(+++)、且无非特异染色的最高稀释度为该荧光抗体的工作浓度。
结果显示：当对荧光抗体做1:30稀释时，阴性对照细胞出现少量的非特异性荧光；为保证荧光抗体的特异性荧光亮度，并且消除与正常细胞的非特异性反应，将荧光抗体的最是工作浓度确定为1:240。具体检测结果见表2。
表2羊全血布鲁氏菌荧光抗体最适工作浓度的测定
2.3荧光抗体的最低检测线
在96孔微量培养板中加入依次加入浓度为16.0μg/mL、8.0μg/mL、4.0μg/mL、2.0μg/mL、1.0μg/mL、0.5μg/mL、0.25μg/mL、0.125μg/mL的羊全血布鲁氏菌，每个浓度的细菌样品分别做8个孔。放入37℃细胞培养箱内培养48h，甩去板内培养液，PBS洗液洗涤3次，250μL/孔，每次3min。每孔加入200μL-20℃预冷80％的丙酮液，-20℃冰箱内过夜固定过夜。弃丙酮，同上洗涤。加入羊全血布鲁氏菌荧光抗体(以PBS稀释至工作浓度，并加入0.01％的伊文思蓝)，100μL/孔，37℃孵育60min，PBS洗涤3次。最后用三蒸水洗1min，于酶标仪下记录，激发波长460nm，发射波长520nm。
结果显示：经荧光检测分析可知，如图1所示，本发明提供荧光标记的单克隆抗体能很好地实现对羊全血布鲁氏菌的检测，且其在0.125-16μg/mL之间存在线性相关，可实现高灵敏地检测羊全血布鲁氏菌。
2.4实际样本检测
取已知羊全血布鲁氏菌感染的阳性样本80份，阴性样本120份。分别采用标准的虎红平板凝集试验和本发明羊全血布鲁氏菌荧光抗体进行检测，结果如表3所示。
表3实际样本检测结果
结果显示：经本方法羊全血布鲁氏菌检测的灵敏度为100％，表明所建立的羊全血布鲁氏菌荧光抗体检测方法具有较高的灵敏度，能满足高通量，快速检测的要求，具有潜在推广应用价值。
在此有必要指出的是，以上实施例仅限于对本发明的技术方案做进一步的阐述和说明，并不是对本发明的技术方案的进一步的限制，本发明的方法仅为较佳的实施方案，并非用于限定本发明的保护范围。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种羊全血布鲁氏菌检测试剂盒，其包括三棱针、塑料滴管、离心管、血液稀释液，其特征在于，所述试剂盒中还包括包含荧光标记的羊全血布鲁氏菌单克隆抗体，所述羊全血布鲁氏菌单克隆抗体重链可变区序列如SEQ ID NO.1所示，轻链可变区序列如SEQ ID NO.2所示。
2.如权利要求1所述的试剂盒，其特征在于，所述羊全血布鲁氏菌单克隆抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示。
3.如权利要求1所述的试剂盒，其特征在于，所述羊全血布鲁氏菌单克隆抗体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ ID NO.6-8。
4.如权利要求1所述的试剂盒，其特征在于，所述荧光标记为FITC、FAM、Rhodamine B、TAMRA、Cy3、Cy5荧光基团标记。
5.如权利要求4所述的试剂盒，其特征在于，所述荧光标记为FITC荧光基团标记。
6.一种荧光标记的羊全血布鲁氏菌单克隆抗体，其特征在于，所述羊全血布鲁氏菌单克隆抗体重链可变区序列如SEQ ID NO.1所示，轻链可变区序列如SEQ ID NO.2所示。
7.如权利要求6所述的单克隆抗体，其特征在于，所述羊全血布鲁氏菌单克隆抗体重链可变区包括CDR1、CDR2和CDR3，其序列分别对应于SEQ ID NO.3-5所示。
8.如权利要求6所述的单克隆抗体，其特征在于，所述羊全血布鲁氏菌单克隆抗体轻链可变区包括CDR4、CDR5和CDR6，其序列分别对应于SEQ ID NO.6-8。
9.权利要求6-8任一项所述的单克隆抗体在制备检测羊全血布鲁氏菌试剂盒中的应用。
10.一种非诊断目的的羊全血布鲁氏菌的直接免疫荧光检测方法，其特征在于，所述方法中采用权利要求1-5任一项试剂盒中的单克隆抗体接触待检样本，孵育结束后，在荧光显微镜下观察结果或酶标仪读取荧光值。
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