Rogers Mary-Louise, Atmosukarto Ines, Berhanu Degu A, Matusica Dusan, Macardle Peter, Rush Robert A
Centre for Neuroscience, Department of Human Physiology, School of Medicine, Flinders University, South Australia, Australia.
J Neurosci Methods. 2006 Nov 15;158(1):109-20. doi: 10.1016/j.jneumeth.2006.05.022. Epub 2006 Jul 7.
In this study, p75NTREXONIII knockout mice were used as immune-naive hosts to produce functional antibodies to human p75NTR. Three monoclonal antibodies were produced and named MLR1, MLR2 and MLR3, and isotyped as IgG1, IgG2a and IgG2a, respectively. MLR1 and MLR2 bound to human p75NTR with higher affinity than the well-characterized ME20.4 in ELISA and also recognized p75NTR present on neurons in both rat and mouse. MLR1 and MLR2 bound to nerves known to express p75NTR following injection into Balb/C mice but not p75NTREXONIII knockout mice, indicating the antibodies are directed against the ligand binding extracellular region absent in knockout mice. Both MLR1 and MLR2 partially blocked NGF induced cell death in a mouse cell-line that expresses p75NTR but not TrKA. Importantly, intracerebroventricular injections indicated MLR2 was internalized within the cell bodies of mouse basal forebrain neurons, further demonstrating that this antibody is biologically active.
在本研究中，p75NTREXONIII基因敲除小鼠被用作免疫未成熟宿主，以产生针对人p75NTR的功能性抗体。制备了三种单克隆抗体，分别命名为MLR1、MLR2和MLR3，其亚型分别为IgG1、IgG2a和IgG2a。在酶联免疫吸附测定（ELISA）中，MLR1和MLR2与人p75NTR的结合亲和力高于已充分表征的ME20.4，并且还识别大鼠和小鼠神经元上存在的p75NTR。将MLR1和MLR2注射到Balb/C小鼠而非p75NTREXONIII基因敲除小鼠体内后，它们与已知表达p75NTR的神经结合，表明这些抗体针对的是基因敲除小鼠中不存在的配体结合细胞外区域。MLR1和MLR2均部分阻断了在表达p75NTR但不表达TrKA的小鼠细胞系中神经生长因子（NGF）诱导的细胞死亡。重要的是，脑室内注射表明MLR2被内化到小鼠基底前脑神经元的细胞体内，进一步证明该抗体具有生物活性。
