TY - JOUR
T1 - Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates
AU - Dadachova, E.
AU - Chappell, L. L.
AU - Brechbiel, M. W.
PY - 1999/11
Y1 - 1999/11
N2 - A simple spectrophotometric assay for determination of bifunctional polyazacarboxylate-macrocyclic ligands of different sizes that are conjugated to proteins has been developed for: 12-membered macrocycle DOTA (2-[4- nitrobenzyl]-1, 4, 7, 10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and analogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]- 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), and the large 18-membered macrocycle HEHA (1,4,7,10,13,16- hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''' '-hexaacetic acid). The method is based on titration of the blue-colored 1:1 Pb(II)-Arsenazo III (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 μM, wherein color change occurring upon transchelation of the Pb(II) from the AAIII to the polyazamacrocyclic ligand is monitored at 656 nm. The assay is performed at ambient temperature within 20 min without any interfering interaction between the protein and Pb(II)- AA(III) complex. Thus, this method also provides a ligand-to-protein ratio (L/P ratio) that reflects the effective number of ligands per protein molecule available to radiolabeling. The method is not suitable for 14- membered TETA macrocycle (2-[4-nitrobenzyl]-1, 4, 8, 11- tetraazacyclotetradecane N,N',N'',N'''-tetraacetic acid) because of low stability constant of Pb(II)-TETA complex. The method is rapid, simple and may be customized for other polyazacarboxylate macrocyclic ligands.
AB - A simple spectrophotometric assay for determination of bifunctional polyazacarboxylate-macrocyclic ligands of different sizes that are conjugated to proteins has been developed for: 12-membered macrocycle DOTA (2-[4- nitrobenzyl]-1, 4, 7, 10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and analogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]- 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), and the large 18-membered macrocycle HEHA (1,4,7,10,13,16- hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''' '-hexaacetic acid). The method is based on titration of the blue-colored 1:1 Pb(II)-Arsenazo III (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 μM, wherein color change occurring upon transchelation of the Pb(II) from the AAIII to the polyazamacrocyclic ligand is monitored at 656 nm. The assay is performed at ambient temperature within 20 min without any interfering interaction between the protein and Pb(II)- AA(III) complex. Thus, this method also provides a ligand-to-protein ratio (L/P ratio) that reflects the effective number of ligands per protein molecule available to radiolabeling. The method is not suitable for 14- membered TETA macrocycle (2-[4-nitrobenzyl]-1, 4, 8, 11- tetraazacyclotetradecane N,N',N'',N'''-tetraacetic acid) because of low stability constant of Pb(II)-TETA complex. The method is rapid, simple and may be customized for other polyazacarboxylate macrocyclic ligands.
KW - Antibodies
KW - Conjugates
KW - Polyazamacrocycles
KW - Radioimmunotherapy
KW - Radiolabel ing
KW - Spectrophotometry
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U2 - 10.1016/S0969-8051(99)00054-2
DO - 10.1016/S0969-8051(99)00054-2
M3 - Article
C2 - 10708314
AN - SCOPUS:0033380104
SN - 0969-8051
VL - 26
SP - 977
EP - 982
JO - International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology
JF - International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology
IS - 8
ER -
