Epitope Recognition of a Monoclonal Antibody Raised against a Synthetic Glycerol Phosphate Based Teichoic Acid

Abstract

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.

Figure 1

Generation of the monoclonal antibody against WH7-BSA. (A) Schematic representation of the structure of the synthetic teichoic acid glycoconjugate WH7-BSA. (B) Antigen specificity of the supernatant from the hybridoma cell line producing the WH7.01 mAb. The binding was evaluated by ELISA against the synthetic antigen WH7 (blue) conjugated to BSA (horizontal stripes) or to AdcA (vertical stripes), the unconjugated carrier protein BSA (gray), and the commercially available LTAs from S. aureus (orange), all coated in duplicate with 1 μg well^–1. The concentration of the WH7.01 mAb in the supernatant was 35 μg mL^–1. Bars represent mean data, and the error bars represent the standard errors of the means. Significance was inferred by two tailed unpaired t tests between WH7 conjugates and the carrier protein (**P < 0.01). (C) Opsonophagocytic killing activity of the newly generated WH7.01 mAb against S. aureus MW2 on the left and E. faecalis 12030 on the right. The opsonophagocytic killing activity of the purified monoclonal from the hybridoma cells producing WH7.01 mAb (gray) at different dilutions was evaluated against S. aureus MW2 and E. faecalis 12030. Polyclonal sera raised in a rabbit against the purified LTA from E. faecalis 12030 (black and gray squares) was used as a positive control, and a mAb of the same isotype, IgG1κ, was used as a negative control. Bars represent mean data, and the error bars represent the standard errors of the means. n.s. (not significant), *P < 0.05; ***P < 0.001, all by one-way ANOVA with Dunnett’s multiple comparison to the negative control (monoclonal antibody of the same isotype).

Figure 2

TA-library overview and binding assay results from microarray and ELISA. (A) Overview of the synthetic fragments used for the binding analysis of WH7.01 mAb. (B) TA-microarray results with WH7.01 mAb at 1 μg mL^–1. Compounds WH7, 1 to 7 were immobilized on an epoxide functionalized glass slide in three different concentrations: 30 μM (dark blue), 10 μM (shade blue), and 3 μM (light blue). The average of the triplicate spots was normalized to the highest intensity on the array. (C) ELISA results of WH7.01 mAb at different concentrations against WH7b (light green), 1b (blue), 2b (yellow), 8b (orange), and 9b (red). All biotin derivatives were coated at the same concentration (1 μM) on a streptavidin ELISA plate. Bars represent mean data, and the error bars, the standard errors of the means.

Figure 3

Binding kinetics and kinetic and affinity constants of WH7.01 mAb against WH7b (A) and compounds 1b (B) and 8b (C). Serial dilutions of the analyte, WH7.01 mAb (2000-62.5 nM), were run. The numbers in parentheses represent the standard deviations of k_on and k_off. Results are representative of three independent experiments.

Figure 4

STD-NMR analysis. (A) Assignment of the ¹H NMR spectrum of WH7 in D₂O at RT. H peaks for the intermediate GroP (Int-Gro CH/CH₂), terminal GroP (Term-Gro CH/CH₂OP/HOCH₂), and α-glucose (H1 to 5) are assigned with red, green, and blue, respectively. (B) Off- (top) and on-resonance (bottom) of ¹H-STD-NMR spectra between WH7.01 mAb (2.5 μM) and WH7 (0.5 mM) at 303 K.