ISOLATION AND PURIFICATION OF POLYCLONAL IgG ANTIBODIES FROM BOVINE SERUM BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

摘要

Three different high-performance liquid chromatographic (HPLC) techniques, i.e. affinity, ion exchange, and gel filtration chromatography, have been used to purify polyclonal antibodies from bovine serum. Polyclonal antibodies were obtained from animals infected with bovine leukaemia virus, which was confirmed by AGID and ELISA tests. Precipitation of the antibodies by ammonium sulphate prior to HPLC made possible to purify the antibodies in one chromatographic step. However, if the highest purity isrequired and separation IgG_1 from IgG_2, it should be used one and/or two additional columns. In sodium dodecyl sulphate polyacrylamide gel electrophoresis, the purest preparation revealed only one band without tailing or any additional extra peaks. Attempts were also made to purify the antibodies without prior ammonium sulphate precipitation. But the best results in immunoglobulins preparation were obtained in a combination of affinity, ion-exchange and gel filtration chromatography, and sample preparation by ammonium sulphate precipitation and delipidation by n-hexane. These preparations are comparable in purity to those commercially available immunoglobulin standards. The rapid HPLC techniques were found to be very useful for the purification of polyclonal antibodies on a preparative scale, where sample loading of up to 25 mg of serum protein could be fully resolved in satisfactory yields.