Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography.
A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30-50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab')2 fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl) A peak containing primarily the F(ab')2 antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.
Saetang
T
T
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
Treamwattana
N
N
Suttijitpaisal
P
P
Ratanabanangkoon
K
K
D000645:Ammonium Sulfate; D000818:Animals; D000997:Antivenins / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange; D003039:Cobra Neurotoxin Proteins / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005590:Fractional Precipitation; D006736:Horses / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000097:blood / Q000302:isolation & purification; D010434:Pepsin A / Q000097:blood
D000997:Antivenins; D003039:Cobra Neurotoxin Proteins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010434:Pepsin A; D000645:Ammonium Sulfate