Korn A, Rajabi Z, Wassum B, Ruiner W, Nixdorff K
Department of Microbiology, University of Darmstadt, Germany.
Infect Immun. 1995 Jul;63(7):2697-705. doi: 10.1128/iai.63.7.2697-2705.1995.
Monoclonal antibodies (MAb) to lipopolysaccharide (LPS) and to the major outer membrane protein OmpA from Proteus mirabilis were generated and used to monitor the kinetics of uptake in macrophages of LPS as well as LPS bound to OmpA. Uptake was measured by a modified enzyme-linked immunosorbent assay (ELISA) in a microtiter culture system. The MAb were of various immunoglobulin G subclasses and showed strong reactivities with their antigens. Four hybridoma clones recognizing LPS and three recognizing OmpA from P. mirabilis 19 were selected for the present study on the basis of reactions in ELISA and Western blot (immunoblot) analyses. In the uptake assay, it was possible to differentiate between antigen on the cell surface and antigen which had been internalized. Uptake of LPS by macrophages was relatively rapid during the first 4 h of culture and then progressed more slowly over the remaining 24-h observation period. The level of detection of LPS in this assay system was in the nanogram range. When macrophages were pulsed with LPS for 30 min and subsequently washed to remove antigen not bound to the cells, the amount of LPS detectable on the macrophage surface decreased progressively for 3 h after the pulse, which indicated internalization of the antigen. Thereafter, LPS rose to an increased level on the cell surface. The rate of uptake of LPS was more rapid when it was in complex with OmpA. When the fate of OmpA was monitored in the same LPS-protein complexes by use of MAb to OmpA in a pulse experiment, the level of protein measured on the cell surface decreased after an initial rise, which again indicated internalization, but the protein did not reappear on the cell surface in a form detectable with the MAb. Compared with the LPS monitoring system, detection of OmpA associated with macrophages was weak, although the MAb to OmpA reacted strongly with the protein in the ELISA and Western blot analyses.
制备了针对奇异变形杆菌脂多糖（LPS）和主要外膜蛋白OmpA的单克隆抗体（MAb），并用于监测巨噬细胞对LPS以及与OmpA结合的LPS的摄取动力学。在微量培养系统中，通过改良的酶联免疫吸附测定（ELISA）来测量摄取情况。这些单克隆抗体属于不同的免疫球蛋白G亚类，并且与其抗原表现出强烈的反应性。基于ELISA和蛋白质印迹（免疫印迹）分析中的反应，选择了四个识别LPS的杂交瘤克隆和三个识别奇异变形杆菌19的OmpA的杂交瘤克隆用于本研究。在摄取试验中，能够区分细胞表面的抗原和已内化的抗原。巨噬细胞对LPS的摄取在培养的最初4小时内相对较快，然后在剩余的24小时观察期内进展较慢。该测定系统中LPS的检测水平在纳克范围内。当巨噬细胞用LPS脉冲处理30分钟，随后洗涤以去除未与细胞结合的抗原时，脉冲后3小时内巨噬细胞表面可检测到的LPS量逐渐减少，这表明抗原发生了内化。此后，LPS在细胞表面上升至更高水平。当LPS与OmpA形成复合物时，其摄取速率更快。在脉冲实验中，通过使用针对OmpA的单克隆抗体在相同的LPS-蛋白质复合物中监测OmpA的命运时，细胞表面测量的蛋白质水平在最初升高后下降，这再次表明发生了内化，但该蛋白质没有以单克隆抗体可检测的形式重新出现在细胞表面。与LPS监测系统相比，尽管针对OmpA的单克隆抗体在ELISA和蛋白质印迹分析中与该蛋白质反应强烈，但与巨噬细胞相关的OmpA的检测较弱。
