Immobilization of C3b with retention of functional activity.
We compared eight commercially available, pre-activated affinity chromatography supports for ability to immobilize C3b that would retain functional activity. Pre-activated supports that we studied were: cyanogen bromide activated agarose, N-hydroxysuccinimide activated agarose, Reacti-Gel HW-65, Actigel A aldehyde activated agarose, thiopropyl activated agarose, 1,4-bis(2,3-epoxypropoxy) butane activated agarose, Reacti-Gel GF-2000 and tresyl activated agarose. The amount of C3b immobilized by each support varied from 81% for Actigel A aldehyde activated agarose to only 19% for Reacti-Gel GF-2000. We examined the functional capacity of the C3b immobilized on these various supports to participate in the alternative pathway. Immobilized C3b was mixed with factors D and B of the alternative pathway and examined over time for ability to consume factor B hemolytic activity. C3b immobilized on thiopropyl activated agarose consumed factor B at a rate comparable to unbound fluid phase C3b. C3b immobilized on other supports was less active in participating in factor B consumption. Thus, we have demonstrated the ability to immobilize C3b onto a solid matrix with the immobilized C3b retaining the ability to participate in the alternative pathway. This immobilized C3b can be used to fractionate substances with high C3b binding affinity.
Edens
R E
RE
Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City.
Linhardt
R J
RJ
Weiler
J M
JM
D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods*; D003179:Complement C3b / Q000302:isolation & purification*; D051381:Rats; D012685:Sepharose; D013329:Structure-Activity Relationship
D003179:Complement C3b; D012685:Sepharose