Mouse Anti-Digoxin Recombinant Antibody (clone 26-10); Fab Fragment (CAT#: PFBC-032)

Figure 1 Specificity profiles for sFv and 26-10 Fab species.

Microtiter plates were coated first with affinity-purified goat antimouse Fab antibody, followed by sFv or 26-10 Fab in 1% horse serum/0.01 M sodium acetate, pH 5.5. Then ¹²⁵I-labeled digoxin (50,000 cpm) having specific activity of 1800, μCi/,μg (Cambridge Diagnostics; 1 Ci = 37 GBq) was added in the presence of a series of glycoside concentrations. The inhibition of radioligand binding by each of seven cardiac glycosides was plotted and relative affinities for each digoxin analogue were calculated (Table 2). The sFv inhibition curves have been displaced to lower glycoside concentrations than corresponding 26-10 Fab curves, because the concentration of active binding sites on the plate was less for sFv than for 26-10 Fab. When 0.25 M urea was added to the sFv in 0.01 M sodium acetate (pH 5.5), more active sFv bound to the goat anti-mouse Fab on the plate. Hence, the sFv specificity profile shifted toward higher glycoside concentrations, closer to the position of that for 26-10 Fab.

Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotný, J., Margolies, M. N., ... & Crea, R. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences, 85(16), 5879-5883.

Figure 2 Specificity analysis

Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotný, J., Margolies, M. N., ... & Crea, R. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences, 85(16), 5879-5883.

Figure 3 Analysis of digoxin binding affinity.

(A) sFv binding isotherm and Sips plot (Inset). (B) 26-10 Fab binding isotherm and Sips plot (Inset). Binding isotherms display data plotted as the concentration of digoxin bound versus the log of the unbound digoxin concentration, and the dissociation constant corresponds to the ligand concentration at 50% saturation. Sips plots (Inset) present the data in linear form, with the same abscissa as the binding isotherm but with the ordinate representing log[r/(n - r)] (defined below). The average intrinsic association constant (Kₐ) was calculated from the modified Sips equation, log[r/(n - r)] = a log C - a log Kₐ, where r equals mol of digoxin bound per mol of antibody at an unbound digoxin concentration equal to C; n is mol of digoxin bound at saturation of the antibody binding site, and a is an index of heterogeneity, which describes the distribution of association constants about the average intrinsic association constant, K8. Least-squares linear regression analysis of the data indicated correlation coefficients for the lines obtained were 0.96 for sFv and 0.99 for 26-10 Fab. Equilibrium binding was conducted in solution, as follows. Aliquots (100 ,ul) of [3H]digoxin at a series of concentrations (10⁻⁶-10⁻¹⁰ M) in 0.01 M sodium acetate (pH 5.5) with 1% bovine serum albumin were added to 26-10 Fab or sFv (100 μl) at a fixed concentration in 0.01 M sodium acetate, pH 5.5/0.5 M urea/1% bovine serum albumin. After 2-3 hr of incubation at room temperature, the protein was precipitated by the successive addition of goat anti-mouse Fab serum, the IgG fraction of rabbit anti-goat IgG, and protein A-Sepharose. After 2 hr on ice, bound and free [³H]digoxin were separated by vacuum filtration of samples, and radioligand bound to the protfin entrapped on glass fiber filters was measured by scintillation counting.

Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotný, J., Margolies, M. N., ... & Crea, R. (1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences, 85(16), 5879-5883.