以人CD14合成肽为免疫抗原制备抗CD14单克隆抗体。

Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

作者信息

Maleki Leili Aghebati, Shanehbandi Dariush, Majidi Jafar, Yusefi Mehdi, Abdolalizadeh Jalal, Orangi Mona, Baradaran Behzad

机构信息

Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

出版信息

Hum Antibodies. 2013;22(3-4):67-71. doi: 10.3233/HAB-140272.

DOI:10.3233/HAB-140272

PMID:24961864

Abstract

BACKGROUND

CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock.

OBJECTIVE

The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes.

METHODS

To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb.

RESULTS

Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14.

CONCLUSIONS

The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.

摘要

背景

CD14是一种主要在外周血单核细胞、树突状细胞和巨噬细胞上表达的髓系分化抗原。它是对革兰氏阴性菌炎症反应、氧化爆发和脓毒性休克的关键调节因子。

目的

本研究旨在制备并鉴定用于单核细胞检测和诊断的抗CD14单克隆抗体。

方法

为制备抗CD14蛋白的单克隆抗体，用两种与钥孔血蓝蛋白（KLH）偶联的CD14肽免疫小鼠。然后通过杂交瘤技术将免疫小鼠的脾细胞与SP2/0细胞融合。融合细胞在选择性培养基中培养，并通过有限稀释法克隆。选择所需克隆，用酶联免疫吸附测定（ELISA）筛选杂交瘤细胞的上清液中的抗体。通过层析法纯化单克隆抗体，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。最后，采用免疫印迹法和流式细胞术探究该单克隆抗体的特异性。