Bakillah A, Zhou Z, Luchoomun J, Hussain M M
Department of Pathology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
Lipids. 1997 Oct;32(10):1113-8. doi: 10.1007/s11745-997-0143-8.
We have standardized simple but sensitive enzyme-linked immunoassays to understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibodies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts of low density lipoproteins to obtain a standard curve. Conditioned media were added to other wells in parallel, and the amount of apoB was quantitated from a linear regression curve. To standardize conditions for the measurement of intracellular apoB, cells were homogenized and solubilized with different concentrations of taurocholate. We found that 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2, Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular levels of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were positively correlated (r2 = 0.81, P = 0.0009) in HepG2 cells. Furthermore, a positive correlation (r2 = 0.88, P < 0.0001) was found between intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and apoB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but not the lipid-free apoB, was tightly controlled.
我们已经标准化了简单但灵敏的酶联免疫测定法，以了解载脂蛋白B（apoB）的细胞内水平与分泌率之间的关系。这些测定法基于市售抗体，且对人apoB具有特异性。一种单克隆抗体1D1固定在微量滴定板孔上，并与不同量的低密度脂蛋白孵育以获得标准曲线。将条件培养基平行添加到其他孔中，并根据线性回归曲线对apoB的量进行定量。为了标准化细胞内apoB测量的条件，用不同浓度的牛磺胆酸盐对细胞进行匀浆和溶解。我们发现0.5%的牛磺胆酸盐足以溶解HepG2、Caco-2和McA-RH7777细胞中的所有apoB。接下来，在存在牛磺胆酸盐的情况下制备标准曲线，并用于测定不同细胞系中apoB的细胞内水平。在HepG2细胞中，apoB100的细胞内水平（pmol/mg细胞蛋白）与分泌率（pmol/mg/h）呈正相关（r2 = 0.81，P = 0.0009）。此外，在稳定转染的McA-RH7777细胞中，细胞内和分泌的apoB42之间存在正相关（r2 = 0.88，P < 0.0001）。相比之下，在稳定转染的细胞中，对于部分与脂蛋白相关或完全与脂蛋白无关分泌的人apoB28和apoB18，未观察到相关性。这些研究表明，与脂质相关的apoB的分泌率受到严格控制，而与脂质无关的apoB则不然。
