An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies.
An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.
Aithal
H N
HN
Department of Medicine, University of Chicago, IL 60637.
Knigge
K M
KM
Kartha
S
S
Czyzewski
E A
EA
Toback
F G
FG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D011817:Rabbits
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases