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.
Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City.
R E
RE
Edens
R J
RJ
Linhardt
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JM
Weiler
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