Site-specific conjugation of metal carbonyl dendrimer to antibody and its use as detection reagent in immunoassay.
We describe here the conjugation of polyclonal goat anti-rabbit antibody to generation 4 polyamidoamine (G4-PAMAM) dendrimers carrying (i) (η(5)-cyclopentadienyl) iron dicarbonyl succinimidato complexes as infrared (IR) probes, (ii) nitroaniline entities as nuclear magnetic resonance (NMR) probes, (iii) acetamide groups for surface neutralization, and (iv) hydrazide-terminated spacer arms for the reaction with aldehyde. To preserve a high binding affinity, the conjugation was performed on the carbohydrate moieties located on the Fc fragment. The resulting conjugates were characterized by Fourier transform-IR, ultraviolet (UV), and high-mass matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. On the basis of relative concentration ratios of IR probes and antibody, an average labeling of 30 IR probes per antibody was reached (i.e., more than twice the value obtained with our previous strategy that generated no spacer arm). Immunoassays revealed that the antibody-dendrimer conjugates retained 55.1% of immunoreactivity on average with respect to underivatized antibody. Finally, the conjugates were used to quantify their antigen by solid-phase carbonyl metallo immunoassay (CMIA). Results showed a significant enhancement of the IR signal, demonstrating the efficiency of the new conjugation strategy and the potential of the new antibody-dendrimer conjugates as universal immunoanalytical reagents.
Fischer-Durand
Nathalie
N
Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, CNRS UMR 7223, 75005 Paris, France.
Salmain
Michèle
M
Rudolf
Bogna
B
Dai
Lili
L
Jugé
Lauriane
L
Guérineau
Vincent
V
Laprévote
Olivier
O
Vessières
Anne
A
Jaouen
Gérard
G
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D056831:Coordination Complexes / Q000737:chemistry*; D050091:Dendrimers / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D007501:Iron / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D009757:Nylons / Q000737:chemistry*; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013056:Spectrophotometry, Ultraviolet
D000906:Antibodies; D056831:Coordination Complexes; D050091:Dendrimers; D009757:Nylons; C520550:PAMAM-G4; D007501:Iron