Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield.
Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immunochemical analysis of these fragments by SDS gel electrophoresis, Western blot and by double immunodiffusion revealed that the solution containing F(ab')2M was free of IgG and of other plasma proteins, whereas that containing F(ab')2B was not. One milligram of either F(ab')2B, F(ab')2M or Fab'B was able to neutralize respectively 20.7 micrograms, 20.2 micrograms and 13.8 micrograms of C.d. terrificus venom.
dos Santos
M C
MC
Fundação Ezequiel Dias, Universidade Federal de Minas Gerais, Brazil.
D'Império Lima
M R
MR
Furtado
G C
GC
Colletto
G M
GM
Kipnis
T L
TL
Dias da Silva
W
W
D000818:Animals; D000918:Antibody Specificity; D000997:Antivenins / Q000302:isolation & purification*; D015153:Blotting, Western; D002210:Caprylates / Q000494:pharmacology; D003435:Crotalid Venoms / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009500:Neutralization Tests
D000997:Antivenins; D002210:Caprylates; D003435:Crotalid Venoms; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G