RESUMO
Anti-lymphocyte globulin [ALG] is a polyclonal antibody to surface markers of human lymphocyte that is produced in rabbit. ALG is one of the immunosuppressive agents which have been used to prevent organ allograft rejection, treatment of aplastic anemia and some of autoimmune disorders. ALG causes elimination of human lymphocytes from the peripheral blood circulation, regulation of cytotoxic activities and apoptosis. The aim of this study was to produce ALG in country. At the first lymphocytes of 20 healthy people were mixed together after separating with Ficol-Hypaque [D=1.077]. About 4x10[9] lymphocytes were injected to rabbit via marginal vein. Following repetitive immunizations of the animal, ALG was produced and, then its purification was performed using simple, rapid and inexpensive method, ion exchange chromatography. Serum of the immunized rabbit was precipitated with ammonium sulfate in the final concentration of 50%. The precipitant rich in immunoglobulin [ALG] was rewashed with 50% ammonium sulfate, dialyzed against phosphate-buffered saline and applied to ion-exchange chromatography on DEAE-Sepharose 6B. ALG riched fraction was eluted with Tris-Phosphate buffer [pH=8.1] in the first step. Then second fraction containing residual ALG was exited from the column applying stepwise concentrations of 50 mM NaCl. The efficiency of 1/40 dilution of the produced ALG was proved by histocompatibility assays in compare with standard ALG. Also its purification was confirmed by SDS-PAGE in reduced conditions. Hence even 40 times diluted produced ALG can act like standard ALG, so it could be used as positive control in histocompatibility assays, indicating production of ALG is more economical and in the benefit of self sufficiency. In addition purified ALG can be prescribed as immunosuppressive medicine