Yang Tangbin, Zhong Ping, Qu Lina, Wang Chunyan, Yuan Yanhong
Lab of Space Cellular & Molecular Biology, Institute of Space Medico-Engineering, Beijing, China.
J Immunol Methods. 2006 Jun 30;313(1-2):20-8. doi: 10.1016/j.jim.2005.12.013. Epub 2006 Mar 29.
2, 4-Dinitrophenyl (DNP) is a widely used hapten in molecular biology and immunoassay fields. Considering that 2, 4-dintrophenylhydrazine (DNPH) could be used as DNA probe and bind with protein carbonyl to form a stable 2 4-dinitrophenyl (DNP) hydrazone product, on which the level of oxidative stress could be validated with a sensitive noncompetitive ELISA, we prepared DNP-aminocaproic acid and NHS-aminocaproic acid-dinitrobenzene and the conjugates between DNP and carrier proteins such as bovine thyroglobulin (BTG) and bovine serum albumin (BSA). High titer antibody producing spleen cells were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, twenty stable murine monoclonal antibodies (MAbs) producing cell lines to DNP were generated. The donor mouse produced antiserum with a high titer of 1/1,280,000. Five MAbs were selected for further characterization as class and subclass. After four successive limiting dilutions, antibodies were produced by five clones with high affinities ranging from 10(10) to 10(11) M(-1). These clones were found to be of IgG(1) subclass with kappa and lambda light chain. Competitive ELISA and SPR-based sensing system for the detection of DNPH are both used to confirm the specificity of MAb (4D(9)A(9)C(2)C(2)).
2,4-二硝基苯基（DNP）是分子生物学和免疫测定领域广泛使用的半抗原。鉴于2,4-二硝基苯肼（DNPH）可作为DNA探针并与蛋白质羰基结合形成稳定的2,4-二硝基苯基（DNP）腙产物，可通过灵敏的非竞争性酶联免疫吸附测定法验证氧化应激水平，我们制备了DNP-氨基己酸和NHS-氨基己酸-二硝基苯以及DNP与载体蛋白如牛甲状腺球蛋白（BTG）和牛血清白蛋白（BSA）之间的缀合物。去除产生高效价抗体的脾细胞，并与源自SP2/0的骨髓瘤细胞融合。采用传统免疫方案，产生了20个针对DNP的稳定小鼠单克隆抗体（MAb）产生细胞系。供体小鼠产生了效价高达1/1,280,000的抗血清。选择5个MAb进行进一步的类别和亚类鉴定。经过连续4次有限稀释后，5个克隆产生了亲和力范围为1×10¹⁰至1×10¹¹ M⁻¹的高亲和力抗体。发现这些克隆属于IgG(1)亚类，轻链为κ链和λ链。用于检测DNPH的竞争性酶联免疫吸附测定法和基于表面等离子体共振的传感系统均用于确认单克隆抗体（4D(9)A(9)C(2)C(2)）的特异性。
