New Insights into the Functional Behavior of Antibodies as Revealed by Binding Studies on an Anti-Uranium Monoclonal Antibody
As part of an ongoing effort to develop immunoassays for chelated uranium(VI) on a hand-held flow fluorimeter, an anti-uranium monoclonal antibody designated as 8A11 was fluorescently labeled using two different strategies. When 8A11 was coupled via reactive lysines to either ALEXATM 488 or Cy5TM, the resulting fluorescent antibody conjugate exhibited positive cooperativity in the presence of its antigen, U(VI) chelated with 2,9-dicarboxy-1,10-phenanthroline (U(VI)-DCP). That is, when one of the two binding sites on the covalently modified 8A11 was occupied with bound antigen, the affinity of the remaining site on the antibody for U(VI)-DCP appeared to increase. Unmodified 8A11 bound U(VI)-DCP with the expected hyperbolic dependence on the concentration of antigen, consistent with independent and equal binding of ligand at both sites. Proteolytic cleavage of the fluorescently conjugated 8A11 to produce the fluorescent monovalent Fab fragment yielded an active preparation that now bound U(VI)-DCP with no evidence of positive cooperativity. Although, in principle, any divalent antibody has the potential to exhibit positive cooperativity in its binding interactions with its antigen, very little literature precedent for this type of behavior exists. Native 8A11 was also noncovalently labeled with highly fluorescent ZENONTM reagents. These reagents are fluorescently-labeled Fab fragments of goat anti-mouse antibodies that bind to the Fc portion of 8A11. These high-affinity, monovalent fluorescent reagents permitted the intact 8A11 mouse antibody to be labeled in situ with no covalent modifications. Incubation of the 8A11 with ZENON 647 produced a fluorescent protein complex that showed an 8-fold higher affinity for U(VI)-DCP than did the free 8A11 alone. Again, very few literature precedents exist for this phenomenon, where agents that bind to the Fc portion of an intact antibody change the affinity of the antibody for the antigen at the structurally distant Fab portion of the molecule. The addition of protein G, a bacterial protein that also binds to the Fc portion of mouse IgG, to the covalently modified 8A11 produced an antibody preparation that showed a lower affinity for U(VI)-DCP than that observed in the absence of protein G. This protein G-dependent decrease in the affinity of 8A11for U(VI)-DCP was dose-dependent. Similarly, U(VI)-DCP was observed to decrease the affinity between 8A11 and protein G, also in a dose-dependent manner. These reciprocal binding effects between protein G and U(VI)-DCP were taken as further evidence that binding to the Fc portion on the intact 8A11 antibody could influence the strength of the interaction at the antigen binding sites on the Fab portions of the protein, and vice versa. These practical, development-driven binding experiments have revealed a fundamental facet of antibody functional behavior that appears to have been largely unnoticed. The binding phenomena described for the first time in this report may have physiological relevance and can be purposefully exploited to improve the sensitivity and utility of selected immunoassays.
- Research Organization:
- Tulane University Health Sciences Center, New Orleans, LA; Xavier University of Louisiana, New Orleans, LA
- Sponsoring Organization:
- USDOE Office of Science (SC)
- OSTI ID:
- 895284
- Report Number(s):
- CONF-NABIR2004-17; TRN: US0700436
- Resource Relation:
- Conference: Annual NABIR PI Meeting, March 15-17, 2004, Warrenton, VA
- Country of Publication:
- United States
- Language:
- English
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