Purification Of Immunoglobulin A From Bovine Colostrum

Bovine colostrum contains a considerable amount of IgG, sIgA and IgM, which are 10-100 times higher than those in regular milk. The sIgA in colostrum has immune homology with sIgA in breast milk, which has functions of anti-bacterial, anti-inflammatory, anti-virus and others. Therefore, purification of bovine colostrum sIgA has very important practical significance.This paper studied the separation and purification of immunoglobulin A from bovine colostrum sample (containing 81-84% IgG and 16-19% sIgA+IgM), which was conducted with derosination and salting-out prior. It included two main processes: separation of secretory immunoglobulin A and immunoglobulin M mixture (sIgA+IgM) from the sample by anion-exchange chromatography, followed by separation of sIgA from sIgA+IgM mixture by gel filtration chromatography.Firstly, a preliminary separation of three immunoglobulins was done through anion-exchange chromatography, to remove most of IgG and obtain a sIgA+IgM mixture product (containing a small amount of IgG). DEAE-650M anion-exchange matrix was used to study about the separation of different immunoglobulins through breakthrough curve experiments. A discussion of the effects of the buffer pH and ionic strength was made. In a 40.2 mL of DEAE-650M anion-exchange column, a purity of 56.69% of slgA+IgM product was obtained at the optimal chromatographic operation conditions of pH 7.3,0.15 M sodium phosphate by loading 1.24 mg/mL DEAE-650M ainon-exchange matrix with a feeding concentration of 50 mg/mL. And all the dynamic adsorption capacity of different immunoglobulins decreased as the buffer ionic strength increased. And 3# alkali resin was used to study about the effects of different buffer systems, buffer pH and ionic strength by chromatographic experiments. In a 32.8 mL of 3# alkali resin column, the separation of IgG and sIgA+IgM was much better in soudium phosphate buffer system than that in tris-HCl buffer system. The slgA+IgM product with the purity of 91.24% and the recovery of 47.00% was obtained at the optimal chromatographic operation condition, pH 7.0, 0.03 M sodium phosphate, by loading 1.52 mg/mL 3# alkali resin with a feeding concentration of 50 mg/mL. The sIgA+IgM product solution separated from 3# alkali resin chromatography at the optimal condition was dialyzed against in de-ionized water for desalination, and freeze-dried. As a result,0.0010 g powder sample/mL sIgA+IgM solution was obtained. In a 36.8 mL of HW-55F gel filtration column, at the condition of pH 7.0,0.03 M sodium phosphate by loading 1.35 mg powder sample /mL HW-55F gel matrix with a feeding concentration of 10mg/mL, sIgA and IgM could be effectively separated and the sIgA product with a purity of 96.88% probably was obtained.