Purification and analysis of bispecific tetrameric antibody complexes

Abstract

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 IgG 1 with equimolar amounts of purified mouse IgG 1 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°C, indicating a preference of the tetrameric composition over other immune complex compositions. Size exclusion HPLC also showed that greater then 80% of purified tetramers retained their original dimensions after storage for 1 year at 4°C. thus indicating the long-term stability of tetrameric antibody complexes. When complexes were prepared with a mixture of two different mouse IgG 1 antibodies, bispecific tetramers were obtained that could be separated from monospecific tetramers using DEAE-HPLC. Purified bispecific antibody complexes of mouse IgG 1 anti-CD34 (My10) cross-linked to mouse IgG 1 anti-desferal with F(ab') 2 rat anti-mouse IgG 1 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.