[一种用于测量人脂肪细胞中瘦素分泌的高灵敏度酶联免疫吸附测定法]

[A highly sensitive enzyme-linked immunosorbent assay for measurement of leptin secretion in human adipocytes].

作者信息

Li Ming, Yin Jin-hua, Zhang Kui, Wu Cong-yuan

机构信息

Department of Endocrinology, Peking Union Medical College Hospital, Peking Union Medical College Hospital, Beijing 100730, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2008 Dec 16;88(46):3293-7.

PMID:19159558

Abstract

OBJECTIVE

To establish a highly sensitive and specific ELISA method for measurement of leptin and further to study the secretion of leptin during human preadipocytes differentiation and effects of troglitazone.

METHODS

Rabbits Balb/c mice were immunized by recombinant human leptin and Balb/c mice were immunized by human leptin so as to produce rabbit anti-human leptin polyclonal antibodies (PAb) and mouse anti-human leptin monoclonal antibodies (MAb). Combination of the PAb as coating antibody, with a carefully paired biotinylated MAb as detector, and the avidin-horseradish peroxidase as the amplifier of detecting signals, a sandwich method, biotin-avidin ELISA (BA-ELISA) was established. Human omental preadipocytes were cultured, introduced to differentiate, and treated with 10 micromol/L troglitazone; the leptin secretion in the supernatant was detected by BA-ELISA. Peripheral blood samples were collected from 114 healthy persons and the serum leptin was detected by BA-ELISA.

RESULTS

The sensitivity of BA-ELISA was 0.03 ng/ml with a working range of 0.05 - 5 ng/ml and a exogenous leptin recovery rate of 97.8%, and the intra- and interassay coefficients of variation (CVs) were less than 7.4% and 9.3% respectively. The assay detected only a single free leptin peak in gel chromatographic fractions from the mixed human sera or adipocytes culture media. The leptin secretion level detected by BA-ELISA showed that the leptin secretion of the preadipocytes increased strongly when the cells differentiated into mature adipocytes. The peak leptin secretion level of the troglitazone treated group was 2 times as that of the control group. The leptin concentration of women was than 7.6 ng/ml, significantly higher than that of the men (3.2 ng/ml, P < 0.001), and the serum leptin level was significantly correlated with body mass index both for men (r = 0.67, P < 0.001) and for women (r = 0.61, P < 0.001).

CONCLUSION

A highly sensitive BA-ELISA specific for free leptin has been developed that is especially suited for the accurate measurement of the rather low leptin levels of clinical blood specimens and for basic research use.

摘要

目的

建立一种高灵敏度、高特异性的用于检测瘦素的酶联免疫吸附测定（ELISA）方法，并进一步研究人前脂肪细胞分化过程中瘦素的分泌情况以及曲格列酮的作用。

方法

用重组人瘦素免疫家兔，用人瘦素免疫Balb/c小鼠，分别制备兔抗人瘦素多克隆抗体（PAb）和小鼠抗人瘦素单克隆抗体（MAb）。以PAb作为包被抗体，精心配对的生物素化MAb作为检测抗体，抗生物素蛋白-辣根过氧化物酶作为检测信号的放大剂，建立夹心型生物素-抗生物素蛋白ELISA（BA-ELISA）法。培养人网膜前脂肪细胞，诱导其分化，并用10 μmol/L曲格列酮处理；采用BA-ELISA法检测上清液中瘦素的分泌量。收集114名健康人的外周血样本，用BA-ELISA法检测血清瘦素水平。

结果

BA-ELISA法的灵敏度为0.03 ng/ml，工作范围为0.05～5 ng/ml，外源性瘦素回收率为97.8%，批内和批间变异系数（CVs）分别小于7.4%和9.3%。该测定法在混合人血清或脂肪细胞培养基的凝胶色谱级分中仅检测到一个单一的游离瘦素峰。BA-ELISA法检测的瘦素分泌水平显示，当脂肪前体细胞分化为成熟脂肪细胞时，瘦素分泌显著增加。曲格列酮处理组的瘦素分泌峰值水平是对照组的2倍。女性的瘦素浓度高于7.6 ng/ml，显著高于男性（3.2 ng/ml，P<0.001），男性（r=0.67，P<0.001）和女性（r=0.61，P<0.001）的血清瘦素水平均与体重指数显著相关。