Hyperoxia elevates Cu,Zn-superoxide dismutase of endothelial cells as detected by a sensitive ELISA.

Enzyme Pub Date : 1992-01-01 DOI:10.1159/000468787
S K Das, B L Fanburg
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引用次数: 6

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of bovine Cu,Zn-SOD. Accuracy of the ELISA and specificity of the antibody for cell-free extracts was established by: (1) measurement of antigen levels of bovine endothelial cell extracts reconstituted with pure antigen, and (2) immunoblotting with affinity purified antibody. The ELISA was highly sensitive and 0.05-0.10 ng of pure antigen could be accurately detected, which allowed the measurement of Cu,Zn-SOD in as few as 250 endothelial cells. With utilization of the ELISA for detection, DEAE-cellulose chromatography patterns of endothelial cell Cu,Zn-SOD overlapped those of pure bovine erythrocyte Cu,Zn-SOD. Exposure of cells in culture to 80% O2 for 48 h increased the relative abundance of the Cu,Zn-SOD as measured by the ELISA by 1.8-fold. Thus, endothelial cells in culture respond to hyperoxia by enhanced production of Cu,Zn-SOD protein. The ELISA developed in this study may be useful for assessing other factors that regulate cellular production of Cu,Zn-SOD.

ELISA检测高氧升高内皮细胞铜、锌超氧化物歧化酶。
建立了测定牛铜、锌- sod的酶联免疫吸附法(ELISA)。通过:(1)测定用纯抗原重组的牛内皮细胞提取物的抗原水平,(2)用亲和纯化抗体进行免疫印迹,建立ELISA的准确性和抗体的特异性。ELISA灵敏度高,可准确检测到0.05 ~ 0.10 ng的纯抗原,可检测到250个内皮细胞中Cu、Zn-SOD的含量。利用ELISA法检测内皮细胞Cu、Zn-SOD的deae -纤维素色谱图与纯牛红细胞Cu、Zn-SOD的色谱图重叠。细胞在80% O2环境下培养48 h,通过ELISA测定,Cu,Zn-SOD的相对丰度增加了1.8倍。因此,内皮细胞对高氧的反应是通过增加Cu,Zn-SOD蛋白的产生。本研究开发的酶联免疫吸附试验可用于评估调节细胞中Cu,Zn-SOD生成的其他因素。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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