Haptenylation of antibodies during affinity purification: a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling.
Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during the process of haptenylation. Thus, the interaction of the corresponding protein with its ligand may become severely hampered. To overcome this restriction, we developed a novel procedure for the haptenylation of polyclonal antibodies that combines purification and haptenylation. Haptenylation during adsorption to the affinity matrix combines two advantages: the antigen binding site is protected and the labeling procedure becomes most convenient, as overlabeled 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 procedure. To obtain such optimal conditions, stabilities and reactivities of N-hydroxysuccinimide esters and isothiocyanate groups were investigated with a newly developed assay. Based on this information, antibodies against two recently described calcium-binding proteins, NCS-1 and NVP-3, were biotinylated or digoxigenylated. The haptenylated antibodies were successfully applied for biochemical determination and simultaneous immunoenzymatic double labeling of the two proteins.
Pitt
J C
JC
Zentrum für Molekulare Neurobiologie, Institut für Neurale Signalverarbeitung, Universität Hamburg, Germany.
Lindemeier
J
J
Habbes
H W
HW
Veh
R W
RW
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000042:Absorption; D000327:Adsorption; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D001666:Binding Sites, Antibody; D001710:Biotin / Q000031:analogs & derivatives; D019921:Biotinylation; D001923:Brain Chemistry; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000276:immunology*; D002846:Chromatography, Affinity; D004076:Digoxigenin; D004797:Enzyme-Linked Immunosorbent Assay; D016650:Fluorescein-5-isothiocyanate; D006241:Haptens; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000276:immunology*; D051596:Neurocalcin; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins / Q000276:immunology; D013388:Succinimides
D000906:Antibodies; D002135:Calcium-Binding Proteins; D006241:Haptens; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; D013388:Succinimides; C496690:Vsnl1 protein, rat; C033788:biotinyl N-hydroxysuccinimide ester; C101464:frequenin calcium sensor proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D001710:Biotin; D016650:Fluorescein-5-isothiocyanate; D004076:Digoxigenin