摘要
目的建立较为稳定的定量检测尿液水通道蛋白-2(AQP2)的酶联免疫吸附测定(ELISA)法。方法人工合成AQP2蛋白C-末端的CELHSPQSLPRGSKA多肽片段,并与KLH联接,制备兔抗AQP2多肽片段的多克隆抗体,用辣根过氧化物酶标记部分抗AQP2抗体IgG。建立检测充血性心力衰竭模型大鼠尿液AQP2的双抗体夹心ELISA法。结果双抗体夹心ELISA法成功建立,灵敏度为15.625 pmol/ml,批内及批间变异系数分别为4.65%和14.05%;用该法定量检测左冠状动脉结扎术后不同梗死面积充血性心力衰竭模型大鼠的尿AQP2浓度,其结果与Western blotting半定量检测结果一致。结论双抗体夹心ELISA法可以较好地定量检测充血性心力衰竭模型大鼠的尿AQP2蛋白浓度且较Western blotting更加简便易行,便于临床推广使用。
Objective To develop an efficient and stable enzyme-linked immunosorbent assay (ELISA) for detecting urinary aquaporin-2 (AQP2) water channel protein. Methods Rat AQP2 C-terminal peptides (CELHSPQSLPRGSKA) were synthesized and linked to KLH to prepare rabbit anti-AQP2 polyclonal antibodies, and IgG of the antibodies were labeled with horseradish peroxidase (HRP). Rat models of congestive heart failure (CHF) was established by ligation of the left coronary artery, in which both direct and sandwich ELISA for urinary AQP2 detection were tested. Results Double antibody sandwich ELISA was able to detect urinary AQP2 as low as 15.625 pmol/ml with intra-and inter-assay coefficients of variance (CVs) of 4.65% and 14.05% respectively. Urinary AQP2 concentration determined by this assay showed significant positive relation to that by Western blot analysis in CHF rats. Conclusion Double antibody sandwich ELISA was successfully established to detect urine AQP2 in CHF rats, which is more efficient and simpler than Western blot analysis.
出处
《第一军医大学学报》
CSCD
北大核心
2002年第6期486-489,共4页
Journal of First Military Medical University
基金
广东省自然科学基金(970355)
