Complexing of biologically active or functional compounds and methods of preparing and utilizing same.

摘要

Complexes containing a biologically active or functional compound or compounds are prepared by attaching or fixing a first compound, which may be a biologically active or functional compound, to a solid substrate under conditions such that the active site of said first compound is protected, thereupon derivatizing or combining said first compound with a derivatizing or modifying compound while said first compound is attached to said solid substrate. The resulting complex or complexed first compound is then released from attachment to said solid substrate to yield the complex, said complex comprising said derivatizing compound attached to said first compound, said first compound, upon release from the solid substrate having available its functional or biologically active site, For example, alkaline phosphatase is attached to a solid substrate, such as dextran beads, preferably through a ligand, such as a competitive inhibitor for alkaline phosphatase, e.g. para-aminobenzyl-phosphonic acid (p-ABPA), The dextran beads would be first treated with (p-ABPA) and the alkaline phosphatase brought into contact with the thus-treated dextran beads. The competitive inhibitor p-ABPA serves as a ligand for attachment of the alkaline phosphatase to the dextran beads while at the same time occupying and protecting the active site of the alkaline phosphatase. Thereupon, the thus-attached alkaline phosphatase is derivatized or modified, such as by biotinylation. Upon biotinylation of the alkaline phosphatase, the resulting biotinylated alkaline phosphatase is eluted or removed from attachment to the p-ABPA treated solid substrate or dextran beads to which it was fixed, yielding a biotinylated alkaline phosphatase with the active or functional site of the alkaline phosphatase intact. The complex, while still attached to the dextran beads, could be further treated with a second compound or component which might also, if desired, provide an active or functional site, such as by contact with avidin, which would bind to the modifying or derivatizing moiety, i.e. biotin, of the biotinylated alkaline phosphatase and the thus-produced avidin-biotinylated alkaline phosphatase complex then eluted. This complex, such as a complex comprising avidin-biotinylated alkaline phosphatase, could be employed as a detector in a detection system for the identification and/or location of another compound, such as a biotinylated compound, e.g. a biotinylated nucleotide or biotinylated DNA.