CN112114157B - 基于细胞代替荧光编码微球检测hcg的方法 - Google Patents
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Abstract
本发明公开一种基于细胞代替荧光编码微球检测HCG的方法。首先,选用细胞,先将其用4%的多聚甲醛在37度下固定,然后加1‑(3‑二甲氨基丙基)‑3‑乙基碳二亚胺盐酸盐(EDC)将HCG的包被抗体在PBS中孵育连在细胞表面,然后在有HCG抗原的作用下在加入标记荧光的标记抗体可以形成三明治免疫夹心结构,在荧光通道下可见细胞膜一层荧光,实现了HCG的高效、快捷、灵敏度高、特异性强的检测。
Description
技术领域
本发明属于生物技术领域,具体为一种基于细胞代替荧光编码微球检测HCG的方法。
背景技术
人绒毛膜促性腺激素(HCG)是一类具有促性腺发育的蛋白类激素,生物活性与黄体生成素十分接近,其水平含量均在女性体内不断变化。而HCG的检查不仅对女性早期妊娠诊断具有突出的临床意义,还与妊娠相关疾病的诊断、治疗及预后具有较为密切的关系。
在女性相关疾病中HCG的检测有关于宫外孕,宫外孕中最常见的疾病类型即为输卵管妊娠,妊娠妇女体内的滋养层细胞较正常妊娠具有明显的差异,孕期越长,HCG的分泌量越少,所以可将HCG作为诊断宫外孕的有效指标之一。部分生理性或病理性妊娠的孕妇,可表现出类甲亢样的临床表现或经检测其判断甲亢的各项指标数值较为异常,而由于妊娠引起的甲亢主要是由于孕期HCG异常分泌引起的THS抑制所致。还有关于胎儿监测中的HCG检测,例如胎儿宫内发育迟缓以及胎儿窘迫症。还有HCG在男性患者中的变化,有相关文献报道在患膀胱癌的男性患者中HCG较正常人有明显的变化;另外HCG对不孕症精子的影响,因HCG与黄体生成素的结构及功能较为相似,所以其具有激发睾酮由间质细胞产生,提高并存进生精功能的作用。所以HCG在临床中的检测具有非常重要的意义。
传统的HCG检测方法一般采用试纸条,检测试剂应用免疫层析双抗体夹心法原理,制成HCG检测试纸,可在3分钟内测定尿液标本中的HCG。这种方法可以说是非常迅速,灵敏度和特异性好。然而我们看到HCG还和其它疾病息息相关,并不只是检测早孕这么简单,所以想要定量的检测需要更灵敏的方法和环境友好的方法。
发明内容
本发明克服现有技术的不足和空白,提出一种基于细胞代替荧光编码微球检测HCG的方法。
本发明的技术方案:基于细胞代替荧光编码微球检测HCG的方法,包括如下步骤:
(1)细胞固定,孵育HCG包被抗体到细胞膜;
(2)加入HCG抗原及标记了FITC的标记抗体,形成三明治夹心结构,在激发光490nm,发射光525nm下用荧光倒置显微镜可见细胞膜有一层绿色荧光;
(3)在流式细胞仪下FITC通道收集荧光强度。
进一步,细胞选取乳腺癌细胞、K562、Hela或HEK293。
进一步,用于检测HCG时所标记抗体的方法是用EDC活化羧基,细胞膜蛋白和抗体氨基和羧基共价连接,连接包括以下步骤:
首先,细胞培养,在传代的时候铺12孔板104个,隔两天固定,4%的多聚甲醛在37度固定30分钟;
然后,在黑暗条件下加EDC,其终浓度0.1M以及包被抗体,其终浓度0.13mg/ml,在PBS中反应。
进一步,用于检测检测HCG时包被抗体和细胞孵育的最适时间是30分钟。
进一步,检测的方法是用荧光倒置显微镜和流式细胞仪在FITC通道下进行定性和定量检测。
有益效果:该方法具有良好的特异性、灵敏度以及抗干扰能力,为建立灵敏而特异的HCG检测方法提供了新方向,并且用细胞代替荧光编码微球实现了生物安全,环境友好的一大优势。并且我们将细胞表达的颜色作为区分实现多检。表现如下:
1)本发明中,设计了一种由基于细胞代替荧光编码微球的免疫夹心的检测方法。该方法利用细胞表面的蛋白通过EDC的作用和HCG的包被抗体结合,在有目标物和HCG的标记抗体形成三明治的夹心结构,在标记抗体接上FITC标记的标记抗体进行检测。先通过荧光倒置显微镜进行定性的观察,再通过流式细胞仪定量的计算荧光强度进行检测的分析。该方法最大的创新性是通过环境友好的细胞代替荧光编码微球实现抗原的检测,不仅仅检测HCG,当细胞连接其他疾病的待检物的包被抗体是就可以检测其它的疾病。
2)首先,先将细胞固定,然后在EDC的作用下实现包被抗体和细胞膜蛋白的连接,在有HCG和标记抗体时形成三明治的免疫夹心结构,在流式细胞仪中实现了HCG的高效、快捷、灵敏度高、特异性强的检测。
附图说明
图1反应原理图;
图2标记抗体荧光标记选择图;
图3包被抗体与细胞连接方法图;
图4最适包被抗体标记细胞膜时间荧光倒置显微镜表征图;
图5流式细胞仪荧光强度图。
具体实施方式
下面的实施案例中将对本发明作进一步的阐述,但本发明不限于此。
本发明的具体步骤如下:图1为本发明的反应原理图。
实施例1:基于细胞代替荧光编码微球检测HCG的方法,具体步骤如下:
(1)MCF-7细胞的固定
12孔板铺板每孔铺104个细胞,隔两天长满每孔加1ml 4%多聚甲醛在37度固定30min,洗掉固定液加PBS。
(2)细胞膜连接包被抗体
加入EDC(终浓度0.1M)和包被抗体(0.13mg/ml)在500ulPBS反应。
(3)加入HCG和标记抗体
加入HCG和标记抗体进行抗原抗体特异性结合反应。
(4)实验优化及数据处理。
分别对整个流程选用的细胞活性、荧光染料标记抗体、抗体连接细胞方法进行筛选,对EDC用量、反应时间、温度等参数进行优化,最终得到该方法的最佳条件。
(1)可行性实验:为了在实验可以顺利进行,需要探究出适合标记抗体的荧光:
a)TRITC在547nm的激发光572nm的发射光发红光,自己标记抗体,但是在检测过程中发现染料是脂溶性的容易进细胞,影响结果。
b)选用公司标记好的FITC,在激发光490nm,发射光525nm下发绿光,这个在孵育30min时效果最好,如图2所示。
(2)包被抗体与细胞连接方法图
如图3所示,将细胞和包被抗体孵育,通过EDC活化羧基细胞膜蛋白和抗体的氨基羧基共价结合,图3加EDC组连接的更好。
(3)最适包被抗体标记细胞膜时间荧光倒置显微镜表征图
为了实验结果有更好的灵敏度与稳定性,包被抗体与细胞膜的蛋白结合的时间进行了优化,发现30min的时候标记的最好,进细胞的最少,所以检测的时候这个时间最好
(4)流式细胞仪荧光强度图
如图5所示,用流式细胞仪定量的进行HCG的检测,通过荧光强度的变化。
实施例2:选取K562细胞,其他步骤同实施例1。
实施例3:选取Hela细胞,其他步骤同实施例1。
实施例4:选取HEK293细胞,其他步骤同实施例1。
Claims (5)
1.基于细胞代替荧光编码微球检测HCG的方法,其特征在于,包括如下步骤:
(1)细胞固定,孵育HCG包被抗体到细胞膜;
(2)加入HCG抗原及标记了FITC的标记抗体,形成三明治夹心结构,在激发光490nm,发射光525nm下用荧光倒置显微镜可见细胞膜有一层绿色荧光;
(3)在流式细胞仪下FITC通道收集荧光强度。
2.根据权利要求1所述的基于细胞代替荧光编码微球检测HCG的方法,其特征在于,细胞选取乳腺癌细胞、K562、Hela或HEK293。
3.根据权利要求1或2所述的基于细胞代替荧光编码微球检测HCG的方法,其特征在于,用于检测HCG时所包被抗体的方法,包括以下步骤:
首先,细胞培养,在传代的时候铺12孔板104个,隔两天固定,4%的多聚甲醛在37度固定30分钟;
然后,在黑暗条件下加EDC,其终浓度0.1M以及包被抗体,其终浓度0.13mg/ml,在PBS中反应。
4.根据权利要求1所述的基于细胞代替荧光编码微球检测HCG的方法,其特征在于,用于检测HCG时包被抗体和细胞孵育的最适时间是30分钟。
5.根据权利要求1所述的基于细胞代替荧光编码微球检测HCG的方法,其特征在于,检测的方法是用荧光倒置显微镜和流式细胞仪在FITC通道下进行定性和定量检测。
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