Robbins D L, Kenny T, Wutke A, Benisek W
Department of Internal Medicine, University of California School of Medicine, Davis 95616.
J Immunol Methods. 1988 May 25;110(1):111-6. doi: 10.1016/0022-1759(88)90089-0.
In rheumatoid arthritis (RA), the pathogenicity of IgM rheumatoid factor (RF), an autoantibody whose antigen is IgG, is still unclear although RF-IgG complexes appear to be important mediators of immune injury. The polyclonality of RF in RA makes it difficult to characterize certain qualitative properties such as specificity and affinity which may be very important in determining pathogenicity. Monoclonal IgM RF can be used to circumvent this problem. Monoclonal RF secreting cells can be produced via hybridizations with RA B lymphocytes fused with mouse or human myeloma cell lines. Another source of monoclonal RF is the sera of patients with Waldenström's macroglobulinemia (WM). One particular WM IgM RF (Kas) was chosen for our experiments to measure affinities and specificities to eight different monoclonal IgGs (three IgG1s, three IgG3s, one IgG2, and one IgG4). 19 S IgM RF, a pentavalent molecule, was mildly reduced with DTT to make 7 S univalent fragments (7 S IgM RF). 7 S IgM RF was incubated with each of the different IgGs at several different concentrations. These mixtures were allowed to come to equilibrium. An aliquot was then used to determine the amount of free 7 S IgM RF by ELISA. By plotting the reciprocal of the fraction of bound RF versus the reciprocal of the concentration of free antigen at equilibrium, different affinities were determined. The results of these determinations compare favorably with published IgM RF affinities determined by more traditional methods. This method can also be used with proteolytic digest fragments of IgG and short synthetic peptides of the IgG molecule to better locate the antigen binding site. The technique may also help us to determine whether there are select clones of RSC producing RF with different affinities that could complex to a particular type of IgG which, in vivo, could produce greater inflammatory tissue damage. Furthermore, this methodology should be useful in the study of other autoimmune diseases characterized by pathogenic autoantibodies of differing affinities.
在类风湿性关节炎（RA）中，尽管IgM类风湿因子（RF）-IgG复合物似乎是免疫损伤的重要介质，但其作为一种抗原为IgG的自身抗体，其致病性仍不明确。RA中RF的多克隆性使得难以表征某些定性特性，如特异性和亲和力，而这些特性在确定致病性方面可能非常重要。单克隆IgM RF可用于规避这一问题。通过将RA B淋巴细胞与小鼠或人骨髓瘤细胞系融合进行杂交，可产生分泌单克隆RF的细胞。单克隆RF的另一个来源是华氏巨球蛋白血症（WM）患者的血清。我们选择了一种特定的WM IgM RF（Kas）进行实验，以测量其对八种不同单克隆IgG（三种IgG1、三种IgG3、一种IgG2和一种IgG4）的亲和力和特异性。19S IgM RF是一种五价分子，用二硫苏糖醇（DTT）轻度还原以生成7S单价片段（7S IgM RF）。7S IgM RF与每种不同的IgG在几种不同浓度下孵育。使这些混合物达到平衡。然后取一份等分试样通过酶联免疫吸附测定（ELISA）确定游离7S IgM RF的量。通过绘制结合RF分数的倒数与平衡时游离抗原浓度的倒数的关系图，确定不同的亲和力。这些测定结果与通过更传统方法测定的已发表的IgM RF亲和力相比具有优势。该方法也可用于IgG的蛋白水解消化片段和IgG分子的短合成肽，以更好地定位抗原结合位点。该技术还可能帮助我们确定是否存在产生具有不同亲和力的RF的RSC选择克隆，这些克隆可能与特定类型的IgG形成复合物，在体内可能产生更大的炎症组织损伤。此外，这种方法在研究以具有不同亲和力的致病性自身抗体为特征的其他自身免疫性疾病中应该是有用的。
