Purification of Gnathostoma spinigerum larval antigens by monoclonal antibody affinity chromatography.
An immunoaffinity column was prepared by coupling a partially purified Gnathostoma spinigerum-specific IgG1, MAb SK-6C4 (5 mg/ml) to CNBr-activated Sepharose 4B. Ten milliliters of approximately 0.3 mg/ml of crude soluble G. spinigerum larval antigens (GsAL3) were loaded onto the affinity column at a flow rate of about 5 ml/hour. Elution of the bound antigens was accomplished using 50 mM diethylamine-HCI containing 0.15 M NaCL, pH 11.5. The average amount of eluted antigens obtained by one passage of crude GsAL3 (1-4 mg) through 4 to 8 ml of column matrix was 143 micrograms (range, 67 - 414 micrograms). The minimal amount of purified GsAL3 detectable by ELISA using MAb SK - 6C4 (100 micrograms/ml) was 50 ng/ml. The SK - 6C4 affinity-purified GsAL3 was found to be relatively pure and immunologically specific as determined by SDS-PAGE and Western blotting, respectively.
Rojekittikhun
W
W
Department of Immunology and Parasitology, Yamagata University School of Medicine, Japan.
Yamashita
T
T
Saito
S
S
Watanabe
T
T
Azuma
T
T
Sendo
F
F
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000947:Antigens, Helminth / Q000302:isolation & purification*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006039:Gnathostoma / Q000276:immunology*; D007814:Larva / Q000276:immunology
D000911:Antibodies, Monoclonal; D000947:Antigens, Helminth