Gangliosides noncovalently bound to DEAE-Sephadex: application to purification of anti-ganglioside antibodies.
A simple, rapid, effective, and inexpensive method for the purification of ligands having high affinity for gangliosides has been developed. DEAE-Sephadex has a high capacity for binding gangliosides (approx 1/1.6, w/w). The gangliosides, bound to the support by electrostatic and hydrophobic interactions, showed a high resistance, in an aqueous environment, to being detached by eluants commonly employed to desorb ligands (i.e., low or high pH or chaotropic agent solutions) or by nonionic detergent solutions as well as by organic solvents. The DEAE-Sephadex-ganglioside complex was assayed as an immunoadsorbent for purifying anti-GM1 ganglioside antibodies from serum of an immunized rabbit. The specific activity of the purified antibodies was 200- to 400-fold higher, and the recovery of the anti-ganglioside activity was above 50%, with respect to the untreated antiserum. The preparation of the complex and the purification of the antibodies can be done in less than 5 h. The glycolipids from the complex can be recovered by elution with organic solvents containing salt or volatile base solutions, and reused. In principle, this method can be adapted for other anionic amphipathic receptor molecules to purify ligands which bind to them.
Rodriguez
P E
PE
Departamento de Quimica Biologica, CIQUIBIC, Facultad de Ciencias Quimicas, CONICET, Universidad Nacional de Cordoba, Argentina.
Cumar
F A
FA
D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003637:DEAE-Dextran; D004797:Enzyme-Linked Immunosorbent Assay; D005677:G(M1) Ganglioside / Q000276:immunology; D005732:Gangliosides / Q000032:analysis*; D007202:Indicators and Reagents; D011817:Rabbits / Q000276:immunology; D012710:Serum Albumin, Bovine / Q000276:immunology; D012997:Solvents
D000906:Antibodies; D005732:Gangliosides; D007202:Indicators and Reagents; D012997:Solvents; D012710:Serum Albumin, Bovine; D005677:G(M1) Ganglioside; D003637:DEAE-Dextran