CN110161241A - 一种快速检测生物组织切片cosmc基因突变试剂盒 - Google Patents
一种快速检测生物组织切片cosmc基因突变试剂盒 Download PDFInfo
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Abstract
本发明及生物医学领域，具体涉及一种快速检测生物组织切片COSMC基因突变试剂盒。该试剂盒内含：1)荧光(红色)凝集素HAA浓缩液1管和2)荧光(绿色)凝集素PNA浓缩液1管、3)细胞核荧光染色剂DAPI1管。4)凝集素稀释液1瓶、5)TBS缓冲液1瓶、6)抗原修复液1瓶、7)封片剂1瓶、8)阳性对照病理切片1塑料盒(五张)、9)说明书和10)质检报告各一份。此外，试剂盒的盒体和盒内带下凹位的试剂架均为硬纸材料。本试剂盒通过荧光免疫凝集素直标双染色法，同时检测组织切片中CD175抗原和CD176抗原的表达情况，HAA阳性同时PNA阴性者，即表明COSMC基因突变和T‑合酶活性丧失。该产品能迅速确定生物组织切片COSMC基因突变和T‑合酶失活情况，对于研究T‑合酶生物学意义以及与人类疾病的关系都有重要的意义。
Description
技术领域
本发明涉及生物医学领域，具体涉及一种快速检测生物组织切片COSMC 基因突变试剂盒，COSMC是T-合酶(T-synthase)的分子伴侣，T-合酶又叫半乳糖基转移酶(core1β3-galactosyltransferase)，COSMC基因突变意味着T-合酶活性的丧失，诱发肿瘤的发生和转移。
背景技术
T-合酶是合成核心1(core1)结构的唯一糖基转移酶，在分子伴侣COSMC 的协助下，将半乳糖(Galactose)添加到CD175抗原(GalNAcα1-Ser/Thr) 糖链上形成CD176抗原。用示意图可以简单表述为：CD175+UDP-Galactose T-合酶(COSMC) CD176抗原。在人体和其他脊椎动物中，T-合酶失活将导致机体细胞内堆积大量的CD175抗原，但是仅有肿瘤糖蛋白CD175抗原的过量表达，并不代表COSMC基因突变及T-合酶活性丧失，相反有时CD175表达增多时，往往伴有T-合酶活性异常升高。当只有CD175抗原过量表达而没有CD176抗原同时表达时，才能确认COSMC基因突变和T-合酶丧失活性。T-合酶的活性正常与否与癌症发生和转移密切相关。目前，已有一些检测新鲜组织细胞裂解液T-合酶活性的方法，原文的标题是Anovel fluorescent assayfor T-synthase activity，文献发表在2010年的《Glycobiology》volume21(3) p352-362上，该方法只能用于新鲜的组织细胞样品，检测方法比较繁琐，且不能用于病理组织切片的快速检测。
发明内容:
有鉴于此，本发明研制一种快速检测生物组织切片COSMC基因突变试剂盒。该试剂盒包括：1)1管标记荧光的抗CD175抗原的凝聚素HAA(蜗牛凝集素，Helixaspersa)。2)1管标记荧光的能与CD176抗原特异性结合的凝聚素PNA(花生凝集素，Peanut lectin)。3)1管细胞核荧光染色剂DAPI。 4)1瓶荧光凝聚素稀释液。5)1瓶TBS缓冲液。6)1瓶抗原修复液。7)1瓶封片剂。8)1塑料盒(五张)阳性对照病理切片、9)一份说明书和10) 一份质检报告。本试剂盒用荧光凝集素双染色法，同时检测病理组织切片 CD175和CD176抗原，HAA阳性同时PNA阴性的组织细胞即表明COSMC 基因突变和T-合酶失活(见下图)；而PNA阳性和HAA与PNA双阳性者则表明T-合酶活性依然存在，甚至T-合酶活性增高。
本发明的有益效果如下：本发明通过荧光凝聚素双染色法的检测方法，快速判定病变组织COSMC基因突变的存在与否，目前已证实在消化道组织中，发生COSMC基因突变的病变部位，其癌变率明显增高，例如结肠息肉伴有COSMC基因突变则癌变风险增大。因此，结肠镜检查时如发现息肉应作COSMC基因突变的快速检测，有利于对病人采取及时有效的治疗措施。
附图说明
图1标示1为棕色塑料管，用于凝聚素和细胞核荧光染料DAPI。标示2 为白色试剂瓶，凝聚素稀释液、TBS缓冲液和抗原修复液均用此瓶。标示3为棕色玻璃试剂瓶，用于封片剂。标示4为长方形无色塑料盒，内装病理切片。图2标示5为试剂盒内的纸质带下凹位的试剂架，标示10为试剂盒的硬质盒体。图3标示6安放棕色塑料试剂管，标示7安放白色塑料试剂瓶，标示8 安放棕色玻璃试剂瓶。标示9安放病理切片无色塑料盒。
具体实施方式
通过下面给出的本发明的具体实施例可以进一步清楚的了解本发明，但它们不是对本发明的限定。
实施例1
荧光凝聚素双染色法操作比较简单，符合快速诊断的要求。
对于石蜡切片，首先需要脱蜡水化和抗原修复；此后，石蜡切片和冰冻切片方法步骤相同，故一同描述。
1、荧光凝集素染色：首先用免疫组化笔圈好待检组织切片，将HAA和 PNA荧光凝集素原液按1：500的比例加入到凝集素稀释液中充分混匀，然后滴加凝集素混合液到载玻片上，4℃孵化20-30分钟。
2、细胞核染色：将载玻片从冰箱中取出，放入缓冲液TBS中洗3次，每次3-5分钟，擦干组织周围的PBS后加上细胞核蓝色荧光染料DAPI，室温下5分钟。
3、封片观察：用缓冲液TBS洗3次，每次3-5分钟，然后滴加防荧光退色封片剂，覆上盖玻片，在荧光显微镜下进行观察。
Claims (5)
1.一种快速检测生物组织切片COSMC基因突变试剂盒。该试剂盒包括纸质盒体和带下凹位的试剂架：内装两管凝集素和一管细胞核荧光染液，三瓶缓冲液和一瓶封片液，一个五片装的病理切片塑料盒，一份说明书和一份质检报告。其特征在于：2管凝集素HAA和PNA浓缩液均已标记不同颜色的荧光，4瓶试剂分别为凝集素稀释液、TBS缓冲液、抗原修复液和封片剂，五片装的病理切片塑料盒为长方形，以及说明书和质检报告各一份，本试剂盒内的下凹位共8个，除说明书和质检报告，全部放入各自的下凹位。采用荧光免疫凝集素直标双染色法，同时检测组织切片中CD175抗原和CD176抗原的表达情况，一步完成免疫凝集素-抗原反应，快速获得结果。若HAA阳性同时PNA阴性者，即表明COSMC基因突变和T-合酶活性丧失。
2.根据权利要求1所述的试剂盒，其特征在于：盒体和下凹位均是硬纸材料。
3.两管凝集素和一管细胞核荧光染料均为棕色塑料管身。其中，凝集素HAA用红色管帽，凝集素PNA用绿色管帽，细胞核荧光染料用蓝色管帽，管帽颜色即为荧光显微镜下各自呈现的颜色。
4.凝集素稀释液、TBS缓冲液、抗原修复液均为白色塑料瓶，封片剂则为棕色玻璃瓶。
5.根据权利要求1所述的荧光凝集素双染色试剂盒，其特征在于，所述的所有试剂瓶(管)和病理切片塑料盒的大小与所对应的下凹槽大小相匹配。
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|---|---|---|---|---|
| CN116539396A (zh) * | 2023-02-20 | 2023-08-04 | 深圳裕康医学检验实验室 | 基于直标荧光与多重免疫荧光的多癌种染色方法 |
| CN116539396B (zh) * | 2023-02-20 | 2024-04-16 | 深圳裕康医学检验实验室 | 基于直标荧光与多重免疫荧光的多癌种染色方法 |
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