In this study, the optimal F:P ratio, ~1.6 C 5, is in agreement with previous estimates but developed using a new minimization metric. highest F:P ratio) in antibody-antigen binding. An optimal F:P ratio that minimized both inactivation and unlabeled antibody was calculated. This procedure can be utilized to prepare functional, labeled antibody Oxtriphylline reagents with defined activity and can aid in quantitative applications in which the stoichiometry and functionality of the labeled antibody is critical. Keywords: antibody, avidity, fluorophore:protein ratio, kinetic ELISA, global fitting, optimal labeling Introduction The coupling of fluorescent moieties to antibodies to produce labeled antibody reagents, first reported by Coons and collaborators over 60 years ago, has become a routine and important process in the biological sciences and medicine [1; 2]. Often, a succinimidyl-ester functional group is attached to a fluorophore core and this functionality confers reaction specificity with main amines to form fluorophore-antibody conjugates. The presence of multiple main amines, especially Oxtriphylline main amines in the antibody active site, can result in fluorophore conjugation that changes antigen binding characteristics and in the extreme, completely inactivates the antibody [3; 4]. Steric hindrance and the absence of additional reactive sites around the fluorophore are presumed to limit the degree of antibody modification by the conjugation reaction. Furthermore, as commercial protein labeling kits state, antibodies react with fluorophores at different rates Oxtriphylline and retain biological activity at different degrees of fluorophore labeling (FluoReporter FITC Protein Labeling Kit, Molecular Probes, Invitrogen). Thus, protocols may inadvertently recommend a suboptimal fluorophore to protein ratio for the specific coupling reaction of interest [5; 6; 7]. Moreover, the coupling reaction results in a populace of antibodies using a distribution in labeling where the quantity of fluorescence molecules per antibody is usually variable and best described by the labeling distribution [8; 9]. Finally, there is a limit to the number of fluorescence molecules that can be attached to an antibody. The presence of multiple fluorophores in close proximity can decrease fluorescence POLDS via quenching mechanisms; increased labeling may produce a reagent that is Oxtriphylline dimmer then one with less labeling [6; 7; 10; 11; 12; 13; 14]. Previous optimization studies recognized problems related to under and over antibody labeling including decreases in fluorescence due to too few or many fluorophores, non specific staining, and loss of antibody-antigen specificity [8; 9; 15; 16; 17; 18; 19]. To further understand the role of derivitization in antibody function, an anti-hemaglutinin (HA) monoclonal antibody (Fc125) coupled to fluorescein was evaluated. A microplate kinetic ELISA assay was used to quantitatively evaluate antibody-antigen binding [20; 21; 22; 23; 24; 25]. A Michaelis-Menten model was used to evaluate ELISA rate data as a function of antibody concentration. One strategy to avoid deleterious effects is usually to reduce the level of labeling. Decreasing the imply quantity of fluorophore molecules per antibody is usually hypothesized to decrease the number of antibodies using a deleteriously high number of fluorophores, but may create a significant proportion of unlabeled antibodies. Analysis is usually developed here to optimally Oxtriphylline label an antibody sample that takes into consideration these trade-offs. This analysis may be useful in evaluating other antibody conjugations. Materials and Methods Antibody and Antigen Preparation Fc125 anti-HA monoclonal antibodies were prepared from ascites by precipitation with 60% saturated ammonium sulfate followed by affinity purification using a solid-phase protein A adsorbent (UltraLink immobilized protein A, Pierce). FluoReporter FITC Protein Labeling Kit (Molecular Probes) was used to label Fc125. The amount of FITC labeled dye (Component A) was varied (reaction volume 1, 3, and 10 L) and the corresponding fluorophore:protein (F:P) ratio, based on A280 and A494 absorption readings, was calculated according to the labeling kit instructions including the recommended correction factors for the absorbance of the dye at 280 nm (1.9, 3.7, 7.4, respectively). Influenza computer virus (strain A2/Japan/305/57) was obtained from Charles River Laboratories. The computer virus was cultivated in specific pathogen free (SPF) chicken eggs and purified by centrifugation in a sucrose gradient. Viral envelope protein was extracted by mixing 1 ml viral suspension (2 mg protein / ml) with 1 ml 15% n-octyl–D-glucopyranoside (Calbiochem) in PBS (final detergent concentration, 7.5%) and incubating at 23 C for 30 min. [26]. The suspension was centrifuged at 20,000 g for 60 min. to remove.