Velardi A, Kubagawa H, Kearney J F
J Immunol. 1984 Oct;133(4):2098-103.
We have used two rat monoclonal antibodies (Mab) (Bet-1 and 331.12), a mouse Mab (AF6-78.25), and an alloantiserum (SJA anti-BAB/14) in two-color immunofluorescence analysis and enzyme-linked immunosorbent assays to examine the expression of IgM allotypes at various stages of mouse B cell development, especially at the pre-B cell stage. In agreement with findings previously reported by others, these antibodies displayed the patterns of allotypic reactivity with IgM+ B cells and plasma cells from the appropriate strains of mice: Bet-1 antibody is specific for a allotype, AF6-78.25 and SJA anti-BAB/14 for b allotype, and 331.12 for both a and b allotypes but not e allotype. These antibodies, however, did not react with isolated mu-heavy chains prepared from secreted molecules or synthesized in situ by normal pre-B cells or pre-B cell-derived hybridomas. The requirement of light chain participation in the expression of IgM allotypic determinant(s) was additionally suggested by analysis of a fetal liver-derived hybridoma (F1-26-11) that has been shown to secrete IgM heteromolecules composed of C57BL/6-derived mu-chain and BALB/c-derived kappa-chains. In contrast to the nonreactivity with C57BL/6 mu-only pre-B cell-derived hybridomas, all three antibodies with specificity for the b allotype (AF6-78.25, SJA anti-BAB/14, and 331.12) reacted with this particular hybridoma. After treating surface IgM molecules on B lymphocytes with papain to cleave Fab fragments, the b allotype reactivity of AF6-78.25 mouse Mab, but not 331.12 rat Mab, disappeared, whereas the a allotype reactivities of two rat Mab (Bet-1, 331.12) were not altered. These results suggest that the IgM allotypic epitopes defined by these antibodies appear to be sterically dependent on the assembly of the whole IgM molecules, and rat Mab 331.12 and mouse Mab AF6-78.25 define two separate b allotype specificities, one on the Fc portion and the other on the Fab portion of the IgM molecules, respectively.
我们使用了两种大鼠单克隆抗体（Mab）（Bet-1和331.12）、一种小鼠Mab（AF6-78.25）和一种同种抗血清（SJA抗BAB/14），通过双色免疫荧光分析和酶联免疫吸附测定，来检测小鼠B细胞发育各个阶段，尤其是前B细胞阶段IgM同种异型的表达。与其他人先前报道的结果一致，这些抗体与来自相应品系小鼠的IgM+B细胞和浆细胞呈现出同种异型反应模式：Bet-1抗体对a同种异型具有特异性，AF6-78.25和SJA抗BAB/14对b同种异型具有特异性，331.12对a和b同种异型均具有特异性，但对e同种异型无特异性。然而，这些抗体与从分泌分子中制备的或由正常前B细胞或前B细胞衍生的杂交瘤原位合成的分离的μ重链不发生反应。对一种源自胎肝的杂交瘤（F1-26-11）的分析进一步表明，轻链参与IgM同种异型决定簇的表达，该杂交瘤已被证明可分泌由C57BL/6来源的μ链和BALB/c来源的κ链组成的IgM异源分子。与对仅含C57BL/6μ链的前B细胞衍生的杂交瘤无反应不同，三种对b同种异型具有特异性的抗体（AF6-78.25、SJA抗BAB/14和331.12）均与这种特定的杂交瘤发生反应。用木瓜蛋白酶处理B淋巴细胞表面的IgM分子以切割Fab片段后，AF6-78.25小鼠Mab的b同种异型反应消失，但331.12大鼠Mab的b同种异型反应未改变，而两种大鼠Mab（Bet-1、331.12）的a同种异型反应性未改变。这些结果表明，这些抗体所定义的IgM同种异型表位似乎在空间上依赖于整个IgM分子的组装，大鼠Mab 331.12和小鼠Mab AF6-78.25分别定义了两种不同的b同种异型特异性，一种在IgM分子的Fc部分，另一种在Fab部分。
