高灵敏酶联免疫分析法测定人脂肪细胞瘦素分泌

目的 建立高灵敏、特异的酶联免疫分析法测定人脂肪细胞瘦素的分泌.方法 制备兔抗人瘦素多克隆抗体和鼠抗人瘦素单克隆抗体,建立生物素一亲和素放大酶联免疫分析方法(BAELISA),观测原代培养的人网膜前脂肪细胞分化过程中瘦素的分泌及曲格列酮对其影响.结果 BA-ELISA方法灵敏度可达0.03 ng/ml,曲线工作范围0.05～5 ng/ml,批内批间变异系数分别小于7.4%和9.3%,平均回收率97.8%.测定经柱层析分离的混合人血清或脂肪细胞培养上清,仅显示单峰游离瘦素.观测到人网膜前脂肪细胞随诱导分化成熟而瘦素分泌量递增;10 umol/L曲格列酮可使瘦素的分泌峰值增加1倍.健康成人空腹血清瘦素水平女性显著高于男性(均值:7.6 ng/ml vs 3.2 ng/ml,P<0.001),与体重指数均有较强的相关性(r=0.61、0.67;P<0.001).结论 建立了高灵敏的BA-ELISA测定游离瘦素水平,尤其适于瘦素浓度低的临床血样本测定及基础研究之用.

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

Objective To establish a highly sensitive and specific ELISA method for measurement of leptin and further to study the secretion of leptin during human preadipecytes difierentiation and effects of troglitazone.Methods Rabbits Balb/c mice were immunized by recombinant human leptin and Balb/e 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 difierentiate,and treated with 10μmoL/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.s/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 adipecytes culture media.The leptin secretion level detect 5ed bv BA-ELISA showed that the leptin secretion of the preadipocytes increased strongly when the cells difierentiated into mature adipocytes.The Deak leptin secretion level of the tmglitazone treated group waft 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 ns/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 forwomen(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 1eptin levels of clinical blood specimens and for basic research use.