Novel hydrophobic interaction chromatography matrix for specific isolation and simple elution of immunoglobulins (A, G, and M) from porcine serum.
A new, highly acetylated agarose matrix (HA-Sepharose) was synthesized and used as a hydrophobic interaction chromatography (HIC) medium to specifically isolate immunoglobulins (Igs) from porcine serum. Recovery of Igs was in a single step and under mild conditions. HA-Sepharose adsorption was studied in terms of salt, gel acetylation time, flow rate, and protein concentration on the loading buffer. At 0.5 M Na2SO4, control with unmodified Sepharose retained a small fraction (0.70 mg/mL of matrix) of serum albumin. On the contrary HA-Sepharose retained primary Igs (IgA, IgG, and 53% of IgM) as revealed by sodium dodecyl sulphate 10% polyacrylamide gel electrophoresis (SDS-PAGE), quantitative radial immunodiffusion and immunodetection. At a flow rate of 1 mL/min, the HA-Sepharose column capacity (3.9 mg/mL of matrix) was similar to the reported capacity for the commercial thiophilic T-gel. However, HA-Sepharose showed higher recovery of IgA and IgM than the T-gel in the same salt conditions, clearly an advantage in terms of immunoglobulin recovery strategies. Acetylation changed the matrix adsorption from albumin to immunoglobulins; thus, the highly acetylated gel rendered recoveries of Igs from unprocessed porcine serum practically free of albumin.
Ramos-Clamont
Gabriela
G
Centro de Investigacion en Alimentacion y Desarrollo A.C. Coordinacion de Ciencia de los Alimentos, Apartado Postal 1735, Hermosillo, Sonora 83000, Mexico.
del Carmen Candia-Plata
Maria
M
Zamudio
Roberto Guzman
RG
Vazquez-Moreno
Luz
L
D000327:Adsorption; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D057927:Hydrophobic and Hydrophilic Interactions; D015151:Immunoblotting; D007070:Immunoglobulin A / Q000097:blood / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000097:blood / Q000302:isolation & purification; D007136:Immunoglobulins / Q000097:blood* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012685:Sepharose / Q000737:chemistry; D013552:Swine
D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007136:Immunoglobulins; D012685:Sepharose