Purification of fluorescein-labeled specific anti-hemoglobin antibody using cross-linked immunoabsorbent

Abstract

Fluorescein-labeled monospecific anti-hemoglobin antibodies are useful tools for studying hemoglobin expression and somatic point mutation. The yield of monospecific antibodies is extremely low and the purification of these antibodies using hemoglobin immunoabsorbents is further complicated by the fact that hemoglobin ‘leaks’ out from the immunoabsorbent during elution of the antibody with acid medium contaminating the eluted antibody. Two improvements are reported in this paper for the purification of fluorescein-labeled goat anti-mouse hemoglobin antibody specific for DBA/2J hemoglobin that does not react with C57BL/6J mouse hemoglobin: (1) The entire gamma globulin fraction of anti-hemoglobin antiserum was labeled with the fluorochrome before proceeding for the absorption and purification steps (in contrast to the conventional methods where the antibody is first purified followed by the fluorochrome labeling). This modification was expected to yield only the functionally active labeled antibody preparation devoid of molecules that may have undergone denaturation during the labeling procedure. Also, this modification eliminated the need of carrying out the labeling procedure with a small amount of the precious purified antibody thus decreasing the antibody loss. (2) Instead of conventional immunoabsorbents (hemoglobin coupled to CNBr-Sepharose), immunoabsorbents cross-linked with glutaraldehyde were used. The eluted antibody was free of any hemoglobin contamination. The antibody thus purified was found to specifically react with fixed red blood cells from DBA/2J mice but not cells from C57BL/6J mice. The method described in this paper should be of general applicability in the preparation of monospecific antibodies.