Ni-IMAC Purification of IgG3 Murine Monoclonal Antibody Against Trypanosoma Cruzi Glycoprotein Extract

ALEANZI, M.; OGGERO EBERHARDT, M.; BOLLATI FOGOLÍN M. R.; MARCIPAR, A.; VIJAYALAKSHMI, M.

International Journal of Bio-Chromatography

Hardwood Academic Publishers

Lugar: Amsterdam; Año: 1998 vol. 4 p. 85 - 94

1068-0659

Monoclonal IgG3 from mouse ascitic fluid was purified on iminodiacetate-Sepharose 6B column coupled either with Ni(II). Both metal chelate adsorbents bound the antibody. Separation was performed by elution with imidazole followed by EDTA. Under starting conditions assayed, 0,1 M phosphate buffer, pH 7.5, 1 M NaC1, antibody was strongly retained and a significantly pure fraction was eluted with EDTA at neutral pH. The yield of antibody activity was found to be between 75% and 90% in this last fraction, depending on the nature of bound metal and the amount of sample load on column. The purity of immunoglobulin fractions was analyzed by SDS-PAGE and was determined to be 80% in eluates from Ni(II) and 90% in eluates from Zn(II) columns. By increasing sample load, antibody was recovered in two main fractions from Zn(II) columns, both of different degree of purity, and consequently an actual decrease of yield in the purest antibody fraction was obtained. This chromatographic pattern was not observed on Ni(II) columns which could be loaded up to 30 mg/ml of gel matrix without change in the elution profile thus showing the possibility of application of this technique for large scale purification. Prefractionation of ascitic fluid with ammonium sulfate followed by chromatography on immobilized Ni(II) gels, increased purity of antibody obtained in EDTA peak to 90% with a recovery of 70%.