CN110501489A - 一种结核免疫组化试剂盒在结核病病变组织诊断中的应用 - Google Patents
一种结核免疫组化试剂盒在结核病病变组织诊断中的应用 Download PDFInfo
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Abstract
本发明公开了一种结核免疫组化试剂盒在结核病病变组织(包括肺内和肺外结核病变组织)诊断中的应用,属于结核病诊断领域。本发明采用一种结核抗原特异性适配体核酸“抗体”,采用生物素、或荧光、化学发光等标记,检测结核病变组织中存在的结核分枝杆菌特异抗原(ManLAM抗原),该抗原可存在于菌体及其分泌到周围空间;通过临床样本免疫组化证实,该结核抗原特异性适配体“抗体”在结核感染组织着色高,且背景着色低。所述结核病为结核分枝杆菌或耐药结核分枝杆菌导致的结核病(包括肺内和肺外结核病),以及所有人畜共患的结核病。本发明提供了一种结核免疫组化试剂盒在结核病病变组织,为结核病变组织的组织病理学确诊提供了新的工具。
Description
技术领域
本发明涉及结核病诊断领域,提供了一种结核免疫组化试剂盒在结核病(包括肺内和肺外结核病)病变组织诊断中的应用,为结核病以及人畜共患结核病病变组织的组织病理学确诊提供了新的工具。
背景技术
结核病目前仍是严重危害人类健康的慢性传染病,结核分枝杆菌所致结核病是人畜共患的结核病。全球目前有近三分之一的人感染结核分枝杆菌,据WHO《2018全球结核报告》估计2017年新发结核病例1000万例,因结核病死亡人数达130万,超过其它传染病死亡人数的总和。
早期以及明确的诊断对结核治疗、预后以及传播的控制有着极其重要的意义。目前临床上应用的结核病诊断技术主要有结核菌素(PPD)试验、抗酸染色、血清学抗体检测、IFN-γ的ELISpot检测及基因芯片等方法,在临床应用中均存在或多或少的局限性,如,假阳性率高、操作繁琐等,尤其是这些方法不能作为结核病确诊依据;而能作为结核病确诊依据的痰细菌培养和PCR方法:培养同样操作繁琐,且耗时过长,因而往往无法指导临床治疗,另外由于各种原因目前我国结核培养阳性率偏低,影响结核的及时准确诊断;PCR方法相对费时较短,但存在费用高,且不能区分既往已愈感染和新发活动性结核。因此,提高结核病确诊率有重要意义。根据《中华人民共和国卫生行业标准—肺结核诊断》(WS288-2017)标准,肺组织免疫病理学检查阳性也可作为结核病确诊依据。因此开发结核病病变组织组织病理学检测方法对于提高结核病确诊率有重要助益。
脂糖是结核分枝杆菌胞壁的主要成分,大约30%结核分枝杆菌基因参与了脂类的合成和代谢。甘露糖修饰的脂阿拉伯甘露聚糖(Mannosylated lipoarabinomannan,ManLAM)是主要存在于致病的人结核分枝杆菌胞壁中的脂糖。研究表明活结核分枝杆菌不断释放ManLAM,因此ManLAM不仅表达于菌体,也表达于其周围空间,分布空间广,因此适于作为结核病变组织(包括肺内和肺外结核病变组织)免疫组化检测靶标,能提高显色信号强度和范围。目前尚无靶向结核菌表面糖脂的免疫组化方法报道。
发明内容
基于此,本发明的目的是一种结核免疫组化试剂盒在结核病(包括肺内和肺外结核病)病变组织诊断中的应用,为结核病(包括肺内和肺外结核)病变组织的组织病理学确诊提供新的工具。
具体的技术方案如下:
本发明的一个目的是提供一种用于结核分枝杆菌检测的组织切片染色的试剂盒,包括DAB染色液、石碳酸复红染色液、苏木素染色液、适配体“抗体”。
本发明提供结核分枝杆菌检测的组织切片染色方法,包括如下步骤:
1)将待检测组织切片与适配体“抗体”溶液反应,得到一抗结合切片;
2)将所述一抗结合切片与二抗(Streptavidin-HRP)溶液反应,得到二抗结合切片;
3)用DAB染色液对所述二抗结合切片进行染色,得到DAB染色切片(免疫组化染色);
4)[可选步骤]用石碳酸复红染色液对所述DAB染色切片染色,得到石碳酸复红染色切片(抗酸染色);
5)用苏木素染色液对所述石碳酸复红染色切片染色,得到染色切片,实现组织切片染色(复染);
所述适配体“抗体”为以结核特异性糖脂抗原为靶标设计而成。
本发明的另一个目的是提供该试剂盒用于结核病变组织(包括肺内和肺外结核病变组织)免疫组化诊断的应用。
本发明的有益效果:
目前结核病实验室诊断手段存在各种缺陷,导致结核病确诊率低。大多数结核诊断仅仅依据临床症状,结合影像学检测、T-SPOT等检测结果,进行临床诊断,依据《中华人民共和国卫生行业标准—肺结核诊断》(WS288-2017)标准,采用组织病理学方法确诊,可提高结核病确诊率。本发明公开的一种结核抗原特异性结核免疫组化试剂盒能有效用于结核病变组织(包括肺内和肺外结核病变组织)的组织病理学诊断,所检测的结核抗原表达于菌体及其周围空间,分布空间广,提高了显色信号强度和范围;通过临床样本免疫组化证实,该结核抗原特异性适配体“抗体”在结核感染组织着色高,且背景着色低。能够提高结核病的诊断确诊率,并能提高结核病预防和控制的有效性,从而为更好的预防和控制结核病打下基础。
附图说明
图1.肠结核病变组织切片免疫组化染色+抗酸染色+复染与抗酸染色+复染两方法比较。黑色箭头所示为抗酸染色为阳性区域,右图可见抗酸染色与免疫组化染色重叠。
图2.适配体“抗体”免疫组化检测效果与其他抗体免疫组化检测效果比较验证。
图3.适配体“抗体”免疫组化方法用于临床病理组织切片检测。
具体实施方式
以下结合附图和具体实施例来说明本发明。
如无特殊说明,以下实施例中所使用的实际均来源于市售,操作方法均为现有常规操作方法。
实施例1试剂盒组成
1.PBS配方(浓度为0.01M、pH值为7.4):2.9g Na2HPO4·12H2O,0.3g NaH2PO4·2H2O及9g NaCl溶于1L蒸馏水中,调节pH值为7.4。
2.PBST配方:在上述PBS基础上,加终浓度为0.025%的Tween-20混匀。
3.柠檬酸钠缓冲液(10mM柠檬酸钠,0.05%Tween-20,pH 6.0)。
4.适配体“抗体”(300nM):由SELEX筛选得到,其核苷酸序列如SEQ ID No.1所示,以ddH2O调整终浓度至300nM。
5.Streptavidin-HRP:按1:500稀释使用。
6.DAB染色液:先配置20×DAB染色母液:将0.1g二氨基联苯胺(3,3’-diaminobenzidine,DAB)倒入10mL蒸馏水中,得到混合液,向混合液中加入3-5滴10M HCl至混合液变为浅棕色则DAB溶解完全,分装,保存于-20℃。
7.石碳酸复红染色液:珠海贝索商品化/先配置10mL碱性复红乙醇饱和液(将1g碱性复红溶于10mL无水乙醇中)和配置90mL 5%石碳酸溶液(将5g苯酚溶于蒸馏水),再将这两种溶液混合即为石炭酸复红液。
8.分化液:在2mL蒸馏水中加入3mL浓盐酸和95mL无水乙醇,混匀即为分化液。
9.苏木素染色液:将5g苏木素溶于50mL无水乙醇,得到苏木素无水乙醇液;再将44g硫酸铝钾放入1L蒸馏水中加热溶解,得到硫酸铝钾溶液;再将苏木素无水乙醇液和硫酸铝钾溶液混合,得到混合液;等混合液沸腾后搅拌冷却至91℃左右,缓慢加入2.5g氧化汞,溶解完毕后置于冷水中冷却,得到反应产物,次日过滤反应产物,收集滤液,再向滤液中加4g柠檬酸,搅匀即为苏木素染色液。
实施例2组织切片的制作(以石蜡包埋为例)
所有切片组织均取自武汉市金银潭医院(武汉市医疗救治中心)2018年1月~2018年8月住院手术治疗患者。
所有标本均经病理证实,切片标本取自受累部位,所有标本均来自均取自武汉市金银潭医院(武汉市医疗救治中心)病理科。所有组织标本均常规10%中性福尔马林固定,石蜡包埋,蜡块经筛选无明显缺陷,置于室温储存备用。
制作成组织切片主要流程为:
(1)将组织(厚度<3mm)块用10%中性福尔马林过夜固定。
(2)以自来水冲洗切片5min,去除多余福尔马林。
(3)分别将切片浸入70%乙醇(5min)、80%乙醇(5min)、95%乙醇(5min)、100%乙醇(5min×3次)中,以脱除组织中水分。
(4)将脱水后的切片浸入二甲苯(xylene)中,20min×2次。
(5)将组织浸入预先在55℃的恒温烤箱中加热融化的石蜡中,5分钟×3次,再将组织块包埋入石蜡块中。
(6)切片机对组织块进行切片(5-8μm),将连续切片分别漂在凉水中,使其自然展开,再将切片转移至45℃温水中展片2分钟左右,待展开后将其贴在经过防脱片处理的载玻片上晾干。
(8)将切片置于37℃的环境下过夜烤片,用60℃15min烤片。
(9)将做好的组织切片保存于切片盒,置于室温备用。
实施例3免疫组化染色+抗酸染色+苏木素复染:
(1)石蜡切片脱蜡复水:石蜡切片放60度烘箱热烘30分钟。二甲苯I、II脱蜡5分钟,放入10%、95%、90%、80%、70%的甲醇中各2分钟,再放入蒸馏水中5分钟。
(2)加100μL 3%H2O2室温孵育5-10分钟,以消除内源性过氧化物酶的活性。
(3)抗原修复:烧杯中加柠檬酸钠缓冲液(10mM柠檬酸钠,0.05%Tween-20,pH6.0)放入水浴锅加热至沸腾。将切片放入载片架放入烧杯隔水煮片30分钟。将烧杯从水浴锅中取出,自然冷却到室温。
(4)PBS(1mL)清洗切片两次。
(5)10%正常山羊血清(0.5%PBS稀释)封闭,室温孵育30分钟。
(6)去除液体,用纸巾吸走多余液体。加稀释好的一抗100μL(适配体“抗体”:300nmole/Rv2645:1:300)在切片上,用封口膜覆盖放湿盒37度1小时孵育。
(7)0.025%PBST清洗3次,每次5分钟。每次清洗时,用纸巾吸走多余液体。
(8)滴加适量标记二抗工作液(Streptavidin-HRP:1:500),37度0.5小时。
(9)0.025%PBST清洗3次,每次5分钟。每次清洗时,用纸巾吸走多余液体。
(10)DAB显色剂显色约30秒,自来水充分冲洗。
(11)石碳酸染液10分钟,3%盐酸乙醇分化30秒。
(12)苏木精复染30秒(0063201,珠海贝索生物)。1%盐酸乙醇分化3秒。
(13)ddH2O浸泡2分钟。
(14)自来水充分冲洗,脱水,透明,中性树脂胶封片。
(15)莱卡显微镜扫描切片,组织相应部位出现棕黄色作为阳性表现。
结果表明ManLAM阳性主要表达于病变部位(肉芽肿、坏死)的细菌菌体和细菌菌体周围空间,因抗原分布更广,因而较仅检测菌体的抗酸染色,着色更明显(图1),提高了检测率。
实施例4适配体“抗体”免疫组化检测效果与其他抗体免疫组化检测效果比较:我们采用ManLAM适配体“抗体”,同时采用对照抗体:Rv2645抗体(Anti-Rv2645)(Zhang XL*etal.J Infect,2015),CFP10-ESAT6抗体(Anti-CE)(Zhang XL*et al.J Infect,2014),进行组织免疫荧光检测,做平行比较。
(1)石蜡切片脱蜡复水:石蜡切片放60度烘箱热烘30分钟。二甲苯I、II脱蜡5分钟,放入10%、95%、90%、80%、70%的甲醇中各2分钟,再放入蒸馏水中5分钟。
(2)加100μL 3%H2O2室温孵育5-10分钟,以消除内源性过氧化物酶的活性。
(3)抗原修复:烧杯中加柠檬酸钠缓冲液(10mM柠檬酸钠,0.05%Tween-20,pH6.0)放入水浴锅加热至沸腾。将切片放入载片架放入烧杯隔水煮片30分钟。将烧杯从水浴锅中取出,自然冷却到室温。
(4)PBS(1mL)清洗切片两次。
(5)10%正常山羊血清(0.5%PBS稀释)封闭,室温孵育30分钟。
(6)去除液体,用纸巾吸走多余液体。加稀释好的一抗100μL(适配体“抗体”:300nmole/Rv2645等抗体:1:300)在切片上,用封口膜覆盖放湿盒37度1小时孵育。
(7)0.025%PBST清洗3次,每次5分钟。每次清洗时,用纸巾吸走多余液体。
(8)滴加适量标记二抗工作液(Streptavidin-HRP:1:500;HRP标记的羊抗兔二抗:1:2000),37度0.5小时。
(9)0.025%PBST清洗3次,每次5分钟。每次清洗时,用纸巾吸走多余液体。
(10)DAB显色剂显色约30秒,自来水充分冲洗。
(11)苏木精复染30秒(0063201,珠海贝索生物)。1%盐酸乙醇分化3秒。
(12)ddH2O浸泡2分钟。
(13)自来水充分冲洗,脱水,透明,中性树脂胶封片。
(14)荧光显微镜观察切片,组织相应部位出现棕黄色作为阳性表现。
结果显示:相较于其他抗体:Rv2645抗体(Anti-Rv2645)、CFP10-ESAT6抗体(Anti-CE),适配体“抗体”能更好的区分正常肺组织和结核感染肺组织,且荧光强度更强(图2)。因此结核分枝杆菌ManLAM抗原(病理组织可见光免疫组化)诊断试剂盒较其他结核抗体具有更好的检测效果。
实施例5适配体“抗体”免疫组化方法用于临床病理组织切片检测我们采用结核分枝杆菌ManLAM抗原(病理组织可见光免疫组化)诊断试剂盒,以及抗酸染色方法,对临床病理组织切片样本进行平行比较。检测方法如上述。所有检测结果经ImageJ普通软件(下载网址:https://imagej.nih.gov/ij/)(Varghese F,et al.PLoS One,2014;H Score:BakerGM,et al.Cancer Manag Res,2015;H Score计算公式为:H Score=5×强阳性像素点占总像素百分数+4×阳性像素点占总像素百分数+弱阳性像素点占总像素百分数),得出免疫组化评分(H Score),按H Score分析,该试剂盒正常值参考值(Cutoff值)范围为0-60;阳性值为大于60。
结果显示:结核分枝杆菌ManLAM抗原诊断试剂盒能很好地检测结核感染,菌量多与少不影响检测结果,且该试剂盒由于检测结核杆菌分泌的抗原,因此显色反应强,更易于检测,这表明该试剂盒明显优于目前临床结核病理检测所用的抗酸染色方法。
结果分以下四种情况:
一、抗酸染色阳性的组织切片样本,本试剂盒检测亦呈阳性;显示本试剂盒与抗酸染色具有一致性(图3A)。
二、抗酸染色阴性的组织切片样本,本试剂盒检测亦呈阴性;显示本试剂盒具有特异性(见图3B)。
三、抗酸染色阴性的组织切片样本,本试剂盒检测呈阳性;显示本试剂盒较抗酸染色具有更高的敏感性(图3C)。
四、对非结核性疾病的组织切片样本(占位性病变,抗酸染色-AFS呈阴性),本试剂盒检测呈阴性,显示本试剂盒较抗酸染色具有很高的特异性(图3D)。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
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gcggaattca acagtccgag ccttctgatg gagagatgga gagtgagaga gagggtcaat 60
gcgtcatagg atcccgc 77
Claims (6)
1.一种结核免疫组化试剂盒在结核病病变(肺内和肺外病变组织)组织诊断中的应用。
2.根据权利要求1所述的应用,一种用于结核分枝杆菌检测的组织切片染色的试剂盒,其特征在于包括核酸适配体(子),其可用生物素、荧光或化学发光染料等标记。
3.根据权利要求1和2所述适配体“抗体”,其特征在于所述适配体“抗体”为以结核特异性糖脂抗原为靶标设计而成,其核苷酸序列如SEQ ID No.1所示。
4.根据权利要求1所述的应用,一种用于结核分枝杆菌检测的组织切片染色的试剂盒,其特征在于包括生物素、荧光或化学发光染料等标记核酸适配体(子),洗涤液,底物溶液等。
5.根据权利要求1-4所述的应用,其免疫组化染色方法,包括如下步骤:
1)将待检测组织切片与生物素标记的适配体“抗体”溶液反应,得到一抗结合切片;
2)将所述一抗结合切片与二抗(Streptavidin-HRP)溶液反应,得到二抗结合切片;
3)用DAB染色液对所述二抗结合切片进行染色,得到DAB染色切片(免疫组化染色);
4)[可选步骤]用石碳酸复红染色液对所述DAB染色切片染色,得到石碳酸复红染色切片(抗酸染色);
5)用苏木素染色液对所述石碳酸复红染色切片染色,得到染色切片,实现组织切片染色(复染);
所述待检测组织切片为石蜡包埋或冷冻切片等待检测组织切片。
6.根据权利要求1-4所述的应用,其荧光染色方法检测方法,包括如下步骤:
1)将待检测组织切片与荧光染料(如AF488)标记的适配体“抗体”溶液反应,得到荧光结合切片;
2)用DAPI染料染细胞核(可选);
3)用荧光显微镜观察结果,阳性为绿色荧光
所述待检测组织切片为石蜡包埋或冷冻切片等待检测组织切片。
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