基于金磁微粒的免疫磁性细胞分选方法的建立
发布时间：2018-10-23 11:58
【摘要】：磁性细胞分选(Magnetic-activated cell separation, MACS)技术已经成为一项快速发展的生物技术,它是基于抗原抗体特异性反应原理,并结合磁性材料特有的超顺磁性来完成细胞分选的。德国美天旎(?)(Miltenyi);纳米级葡聚糖磁性粒子和美国戴诺(?)(Dynal)微米级聚苯乙烯高分子磁性微球在磁性细胞分选领域的应用已经得到了国际认可,而我国此项技术非常落后,只能依赖进口产品。本课题旨在以具有自主知识产权的GoldMag(?)金磁微粒为载体,结合“链霉亲和素(Streptavidin, SA)-生物素(Biotin)"的高度放大效应,基于抗原抗体特异性结合的原理建立免疫磁性细胞分选技术。 1.以金磁微粒为载体,制备链亲和素磁性复合微粒(SA-GoldMag)。分别通过物理方法以及化学方法将链霉亲和素偶联于金磁微粒表面。在化学方法中首先以聚丙烯酸(polyacrylic acid, PAA)对金磁微粒表面进行修饰并在1－(3－二甲氨基丙基)－3－乙基碳二亚胺盐酸盐(1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide, EDC)的作用下进行SA偶联形成SA-PAA-GoldMag;分别对物理以及化学方法偶联所得的SA-GoldMag进行筛选及评价。结果显示：30nm金磁微粒经物理方法偶联所得SA-GoldMag以及50nm金磁微粒经化学方法偶联所得SA-PAA-GoldMag均可使用于下游的细胞分选,二者对SA的偶联量分别为22.14μg/mg和13.54μg/mg,且SA活性高、稳定性好。 2.以制备得到的SA-GoldMag为载体建立磁性细胞分选体系,对外周血淋巴细胞的表面CD4+的细胞进行分选；并对细胞分选过程中所用的缓冲液、抗体用量、孵育时间以及磁珠用量进行优化；对免疫磁性细胞分选方法分选所得的CD4+细胞进行评价。结果显示：在含5%BSA、2m M EDTA的PBS缓冲液作用下,细胞与磁珠始终呈现良好的分散性；在0.5μg抗体、10min(4℃)抗体-细胞孵育时间、15min(4℃)磁珠-细胞孵育时间以及65μg50nm SA-PAA-GoldMag为最适用量的条件下分选CD4+细胞得率可达90.4%,纯度为91.72%；分选前后细胞存活率分别为(97.3+0.9)%和(96.4+1.3)%,细胞状态没有受到明显影响,且WST结果显示磁珠对细胞没有明显的毒性作用,不影响细胞的正常培养。
[Abstract]:Magnetic cell sorting (Magnetic-activated cell separation, MACS) has become a rapidly developing biological technique, which is based on the antigen-antibody specific reaction principle and combined with the magnetic material specific superparamagnetism to complete cell sorting. The application of) (Miltenyi); nanometer-grade dextran magnetic particles and?) (Dynal) micron polystyrene magnetic microspheres in magnetic cell separation has been internationally recognized in Germany, but this technology is very backward in China. We can only rely on imported products. The purpose of this paper is to use GoldMag (?) Combining the high amplification effect of "streptavidin (Streptavidin, SA)-biotin (Biotin)", an immunomagnetic cell sorting technique was established based on the principle of specific binding of antigen and antibody. 1. Chain affinity magnetic composite particles (SA-GoldMag) were prepared with gold magnetic particles as the carrier. Streptavidin was coupled to the surface of gold magnetic particles by physical and chemical methods respectively. In the chemical method, the surface of gold particles was first modified with polyacrylic acid (polyacrylic acid, PAA) and coupled with 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide-3-ethylcarbodiimide (EDC) in the presence of 1- (3- dimethylaminopropyl) -3-ethylcarbodiimide (EDC) to form SA-PAA-GoldMag; pairs. The SA-GoldMag obtained by physical and chemical methods were screened and evaluated. The results showed that SA-GoldMag obtained by physical coupling of 30nm gold magnetic particles and SA-PAA-GoldMag obtained by chemical coupling of 50nm gold magnetic particles could be used in downstream cell sorting. The coupling amounts of SA to SA were 22.14 渭 g/mg and 13.54 渭 g / mg, respectively, and the SA activity was high. Good stability. 2. The magnetic cell sorting system was established by using the prepared SA-GoldMag as the carrier, the CD4 cells on the surface of peripheral blood lymphocytes were sorted, and the buffer solution, antibody dosage, incubation time and the amount of magnetic beads used in the cell sorting process were optimized. The CD4 cells obtained from immunomagnetic cell sorting were evaluated. The results showed that under the action of PBS buffer containing 5 BSA-2m M EDTA, the cells and magnetic beads always showed good dispersion. Under the conditions of 0.5 渭 g antibody, 10min (4 鈩，
本文编号：2289164
[Abstract]:Magnetic cell sorting (Magnetic-activated cell separation, MACS) has become a rapidly developing biological technique, which is based on the antigen-antibody specific reaction principle and combined with the magnetic material specific superparamagnetism to complete cell sorting. The application of) (Miltenyi); nanometer-grade dextran magnetic particles and?) (Dynal) micron polystyrene magnetic microspheres in magnetic cell separation has been internationally recognized in Germany, but this technology is very backward in China. We can only rely on imported products. The purpose of this paper is to use GoldMag (?) Combining the high amplification effect of "streptavidin (Streptavidin, SA)-biotin (Biotin)", an immunomagnetic cell sorting technique was established based on the principle of specific binding of antigen and antibody. 1. Chain affinity magnetic composite particles (SA-GoldMag) were prepared with gold magnetic particles as the carrier. Streptavidin was coupled to the surface of gold magnetic particles by physical and chemical methods respectively. In the chemical method, the surface of gold particles was first modified with polyacrylic acid (polyacrylic acid, PAA) and coupled with 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide-3-ethylcarbodiimide (EDC) in the presence of 1- (3- dimethylaminopropyl) -3-ethylcarbodiimide (EDC) to form SA-PAA-GoldMag; pairs. The SA-GoldMag obtained by physical and chemical methods were screened and evaluated. The results showed that SA-GoldMag obtained by physical coupling of 30nm gold magnetic particles and SA-PAA-GoldMag obtained by chemical coupling of 50nm gold magnetic particles could be used in downstream cell sorting. The coupling amounts of SA to SA were 22.14 渭 g/mg and 13.54 渭 g / mg, respectively, and the SA activity was high. Good stability. 2. The magnetic cell sorting system was established by using the prepared SA-GoldMag as the carrier, the CD4 cells on the surface of peripheral blood lymphocytes were sorted, and the buffer solution, antibody dosage, incubation time and the amount of magnetic beads used in the cell sorting process were optimized. The CD4 cells obtained from immunomagnetic cell sorting were evaluated. The results showed that under the action of PBS buffer containing 5 BSA-2m M EDTA, the cells and magnetic beads always showed good dispersion. Under the conditions of 0.5 渭 g antibody, 10min (4 鈩，
本文编号：2289164
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