Jyonouchi H, Voss R M, Good R A
Department of Pediatrics, University of South Florida, St. Petersburg 33701.
Cell Immunol. 1988 Apr 15;113(1):158-74. doi: 10.1016/0008-8749(88)90014-7.
Humoral factors in serum of young NZB mice enhance maturation of B-lymphocyte precursors in vitro. A blot ELISA assay identified autoantibodies against the serum factors. NZB-SFs (designated NZB-SF alpha, pI 3.5-4.0, and NZB-SF beta, pI 7.8) were purified by sequential steps. Both had a molecular weight (MW) of approximately 23,000 in SDS-PAGE. NZB mice develop autoantibodies against NZB-SFs by 2 months of age; titers increased progressively with age. Non-autoimmune-prone mice did not produce autoantibodies against NZB-SFs. We then developed two hybridoma clones, IIC1C1 and IIC1M4, which produce monoclonal autoantibodies against NZB-SF alpha and NZB-SF beta, respectively. Both IgM autoantibodies could be affinity purified with a column of CNBr-Sepharose 4B gel conjugated with anti-mouse IgM antibody. Neither IIC1C1 nor IIC1M4 abolished bioactivity of recombinant mouse IL-1 alpha, human IL-1, mouse, rat, or human IL-2, mouse IL-3, or colony-stimulating factor. Neither antibody reacted to recombinant mouse IL-1 alpha, IL-4, TNF alpha, or IFN gamma in blot ELISA assays. Monoclonal autoantibodies IIC1C1 and IIC1M4 were used to purify NZB-SFs. SDS-PAGE of the affinity-purified NZB-SFs revealed bands of 23 and 60 kDa, and proteins extracted from the bands were reactive to our monoclonal autoantibodies.
幼年新西兰黑鼠血清中的体液因子可在体外增强B淋巴细胞前体的成熟。一种斑点ELISA检测法鉴定出了针对血清因子的自身抗体。通过一系列步骤纯化了NZB-SFs（命名为NZB-SFα，pI 3.5 - 4.0，以及NZB-SFβ，pI 7.8）。在SDS-PAGE中，二者的分子量（MW）均约为23,000。新西兰黑鼠在2月龄时会产生针对NZB-SFs的自身抗体；抗体滴度随年龄增长而逐渐升高。不易发生自身免疫的小鼠不会产生针对NZB-SFs的自身抗体。随后我们获得了两个杂交瘤克隆，IIC1C1和IIC1M4，它们分别产生针对NZB-SFα和NZB-SFβ的单克隆自身抗体。两种IgM自身抗体都可用与抗小鼠IgM抗体偶联的CNBr - Sepharose 4B凝胶柱进行亲和纯化。IIC1C1和IIC1M4均未消除重组小鼠IL-1α、人IL-1、小鼠、大鼠或人IL-2、小鼠IL-3或集落刺激因子的生物活性。在斑点ELISA检测中，这两种抗体均不与重组小鼠IL-1α、IL-4、TNFα或IFNγ反应。单克隆自身抗体IIC1C1和IIC1M4用于纯化NZB-SFs。亲和纯化的NZB-SFs的SDS-PAGE显示出23 kDa和60 kDa的条带，从这些条带中提取的蛋白质与我们的单克隆自身抗体发生反应。
