Anti-galactosyl antibodies that react with unmodified agarose: a potential source of artifacts in immunoaffinity chromatography.
Rabbits were immunized with glycolipid and membrane glycoprotein extracts of the promastigotes of Leishmania major partially purified by two-phase extraction with Triton X-114. The resulting antibodies were affinity purified on agarose immunoadsorbents to which a recombinant DNA-produced L. major polypeptide had been coupled. In addition to the expected reaction with the polypeptide, it was found that the affinity-purified antibodies reacted strongly with lipophosphoglycan of L. major amastigotes and to a lesser extent with the lipophosphoglycan of promastigotes. Antibodies with this reactivity could be affinity purified on unmodified agarose and were probably directed against beta-1,3-galactosyl determinants shared between the agarose matrix and Leishmania. In this and other special situations the properties of the support matrix can be exploited to obtain antibodies of defined specificity which may be useful probes for the identification and characterization of carbohydrate structures. This work also points to a potential source of artifacts in affinity chromatography, in which the bead matrix is usually considered as inert. Carbohydrate antigens are ubiquitous, particularly in micro-organisms, and the presence of such antibodies may be more common than previously recognized.
Osborn
A H
AH
Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
Kelleher
M
M
Handman
E
E
Goding
J W
JW
D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D005690:Galactose / Q000276:immunology*; D006028:Glycosphingolipids / Q000276:immunology; D008566:Membranes; D015800:Protozoan Proteins; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012685:Sepharose
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D006028:Glycosphingolipids; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C008290:lipophosphonoglycan; C072094:surface antigen P2, Leishmania; D012685:Sepharose; D005690:Galactose