Cyclic tetramolecular complexes of monoclonal antibodies: A new type of cross‐linking reagent

Abstract

A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies, when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1, yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody, stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens, and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine.

Use of tetrameric antibody complexes to stain cells for flow cytometry

“…Increasing the ratio of anti-TNP to Leu-3a resulted in a higher inten-DISCUSSION sity of the specific fluorescence by increasing the ratio of Tetrameric antibody complexes in which two mouse bispecific complexes to monospecific Leu-3Aeu-3a com-monoclonal antibodies are cross-linked to each other via plexes. Although still stronger fluorescence was obtained Fc-specific rat antibodies have previously been used for at ratios of 9 and 19, ratios of 2 to 4 also gave satisfactory the immunological cross-linking of cells and for the imfluorescence intensities and did not require unpractically munoenzymatical detection of cell-surface antigens (6). high anti-TNP and TNP-BPE concentrations.…”

“…Three metric analyses in combination with FITC, conjugated populations of cells are clearly distinguishable; one pop-antibodies. ulation is negative for both antigens, one population is Since immune complex formation is completed within reactive exclusively with Leu-3a-FITC antibodies, and seconds, the complexes can be used immediately after the third population is reactive exclusively with the mixing the components (6). The inherent specificity of Leu-2a-anti-BPEBPE complexes.…”

“…The mouse hybridoma cell line producing IgGl antibodies specific for the hapten trinitrophenol (TNP) was a gift from Dr. G. Kohler (Max Planck Inst., Tubingen, FRG). The preparation of rat monoclonal antibodies specific for mouse IgG, (CLB-mIgG1) has been described previously (6).…”

“…Immune complexes were then generated by the addition of rat antibodies specific for mouse IgGl (6) at a concentration equimolar to the total mouse antibody concentration. Leu-3a and Leu-2a concentrations were 2 pglml.…”

“…These limitations practically exclude direct labeling techniques for the screening of newly developed monoclonal antibodies. We recently reported an immunological technique for the labeling of antibodies that circumvents many of these potential disadvantages of covalent Iabeling procedures (6). In this technique mouse IgGl monoclonal antibodies are noncovalently crosslinked to mouse IgGl monoclonal antibodies with specificity for the label via a selected rat monocIona1 antibody specific for the Fc-portion of mouse IgG1.…”