Lansdorp P M, Thomas T E
Terry Fox Laboratory, Cancer Control Agency, Vancouver, British Columbia, Canada.
Mol Immunol. 1990 Jul;27(7):659-66. doi: 10.1016/0161-5890(90)90009-o.
In order to study the type and yield of immune complexes obtained by the mixing of purified F(ab')2 fragments of rat monoclonal antibodies specific for mouse IgG1 with equimolar amounts of purified mouse IgG1 size exclusion HPLC of the reaction mixture was performed. Immune complexes eluted as a single peak at a position compatible with a tetrameric antibody complex configuration. The yield of tetramers could be increased by incubation of the antibody mixture for several hours at 37 degrees C, indicating a preference of the tetrameric composition over other immune complex compositions. Size exclusion HPLC also showed that greater than 80% of purified tetramers retained their original dimensions after storage for 1 year at 4 degrees C, thus indicating the long-term stability of tetrameric antibody complexes. When complexes were prepared with a mixture of two different mouse IgG1 antibodies, bispecific tetramers were obtained that could be separated from monospecific tetramers using DEAE-HPLC. Purified bispecific antibody complexes of mouse IgG1 anti-CD34 (My10) cross-linked to mouse IgG1 anti-desferal with F(ab')2 rat anti-mouse IgG1 were useful for the purification of cells expressing CD34 from human bone marrow. For this purpose cells were labelled with the antibody complexes, selectively adsorbed onto columns containing desferal coated glass beads and then selectively eluted by treatment with dithiothreitol resulting in reductive cleavage of the disulfide bonds of the F(ab')2 fragments. This relatively simple cell fractionation technique illustrates the unique cross-linking properties of bispecific tetrameric antibody complexes. The procedure appears useful for further studies of hemopoietic cells and bone marrow transplantation.
为了研究通过将对小鼠IgG1具有特异性的大鼠单克隆抗体的纯化F(ab')2片段与等摩尔量的纯化小鼠IgG1混合所获得的免疫复合物的类型和产量，对反应混合物进行了尺寸排阻高效液相色谱分析。免疫复合物以与四聚体抗体复合物构型相符的位置作为单峰洗脱。通过在37℃下将抗体混合物孵育数小时，四聚体的产量可以增加，这表明四聚体组成比其他免疫复合物组成更受青睐。尺寸排阻高效液相色谱还显示，超过80%的纯化四聚体在4℃储存1年后仍保持其原始尺寸，因此表明四聚体抗体复合物具有长期稳定性。当用两种不同的小鼠IgG1抗体混合物制备复合物时，获得了双特异性四聚体，其可以使用DEAE-高效液相色谱与单特异性四聚体分离。用F(ab')2大鼠抗小鼠IgG1交联的小鼠IgG1抗CD34（My10）与小鼠IgG1抗去铁胺的纯化双特异性抗体复合物可用于从人骨髓中纯化表达CD34的细胞。为此，用抗体复合物标记细胞，选择性吸附到含有包被去铁胺的玻璃珠的柱上，然后通过用二硫苏糖醇处理进行选择性洗脱，导致F(ab')2片段的二硫键还原裂解。这种相对简单的细胞分级分离技术说明了双特异性四聚体抗体复合物独特的交联特性。该方法似乎对造血细胞和骨髓移植的进一步研究有用。
