HAPTENYLATION OF ANTIBODIES DURING AFFINITY PURIFICATION - A NOVEL AND CONVENIENT PROCEDURE TO OBTAIN LABELED ANTIBODIES FOR QUANTIFICATIONAND DOUBLE-LABELING

Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many hapt ens are available as N-hydroxysuccinimide esters or isothiocyanates. U nfortunately, the hapten group may become covalently attached to or cl ose to the combining site of antibodies, lectins, or other ligand-bind ing proteins during the corresponding of haptenylation. Thus, the inte raction of the corresponding protein with its ligand may become severe ly hampered. To overcome this restriction, we developed a novel proced ure for the haptenylation of polyclonal antibodies that combines purif ication and haptenylation. Haptenylation during adsorption to the affi nity matrix combines two advantages: the antigen binding site is prote cted and the labeling procedure becomes most convenient, as overlabele d proteins and unreacted haptens are easily removed by simple washing. Haptenylation during adsorption to the affinity matrix is a two-phase reaction, which requires different conditions to the conventional pro cedure. To obtain such optimal conditions, stabilities and reactivitie s of N-hydroxysuccinimide esters and isothiocyanate groups were invest igated with a newly developed assay. Based on this information, antibo dies against two recently described calcium-binding proteins, NCS-1 an d NVP-3, were biotinylated or digoxigenylated. The haptenylated antibo dies were successfully applied for biochemical determination and simul taneous immunoenzymatic double labeling of the two proteins.