Epitope characterization of sero-specific monoclonal antibody to Clostridium botulinum neurotoxin type A

Abstract

Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis.

FIG. 1.

Reactivity of MAbs developed against the H_C50 binding domain of BoNT/A. Anti-H_C50/A MAb reactivity assessed by ELISA, as described previously.⁽³²⁾ One clone, F90G5-3, elicited strong reactivity with whole BoNT/A and was chosen for further characterization.

FIG. 2.

Reactivity and specificity of MAb F90G5-3. (A) ELISA demonstrating concentration-specific reactivity of F90G5-3 with H_C50/A and H_CN25/A; no reactivity is observed with H_CC25/A. (B) Lower limit of detection ELISA illustrating that F90G5-3 can detect as low as 8 ng H_C50/A. (C) Western immunoblot depicting specific reactivity of F90G5-3 with denatured H_C50/A (lane 5) and H_CN25/A (lane 3). No reactivity is observed with irrelevant antigen, BSA (lane 1), H_CC25/A (lane 2), H_C50/B (lane 6), or H_C50/E (lane 7). Molecular mass markers (kDa), 194, 128, 87, 39, 32, 17, 0.6 (lane 4). (D) Competitive ELISA demonstrating that pre-incubation with H_CN25/A (▪) decreases F90G5-3 reactivity with whole H_C50/A, whereas pre-incubation with H_CC25/A (▴) or irrelevant protein (BSA, ●) does not decrease reactivity.

FIG. 3.

Analysis of the H_C50/A epitope bound by MAb F90G5-3. (A) Pepscan analysis: pin peptide epitope mapping of MAb F90G5-3. Reactivity of F90G5-3 (▾) and negative control (▴) with overlapping synthetic 15-mer peptides spanning the entire amino acid sequence of H_C50/A. F90G5-3 binds specifically to one linear epitope, ₉₈₅WTLQDTQEIKQRVVF₉₉₉, which is found in both H_C50/A and H_CN25/A. (B) Sequence alignment of the residues in BoNT/B and BoNT/E corresponding to residues W985–F999 in BoNT/A, demonstrating the primary amino acid sequence diversity in this region. *, identical residues; :, conserved residues; ., semi-conserved residues. Residues unique to BoNT/A are represented in blocks. (C) Schematic of crystal structure of BoNT/A (reproduced from Lacy and associates⁽¹²⁾ created with Raswin software). Enzymatic (light chain) is represented in yellow, H_N50 translocation domain represented in green, H_CN25 in blue, and H_CC25 in magenta. The F90G5-3 epitope, residues W985–F999, is highlighted in red. BoNT/A GT1b ganglioside binding residues identified by Rummel and colleagues⁽¹⁶⁾ are depicted in orange space. Residues identified to interact with the neutralizing antibodies CR1 and AR2⁽⁴⁰⁾ are depicted in pink and cyan space fill.

FIG. 4.

Reactivity and functional activity of MAb F90G5-3 with whole BoNT. (A) End-point titration of F90G5-3 against BoNT/A, BoNT/B, and BoNT/E assessed via ELISA demonstrating specificity for BoNT/A. Reactivity with BoNT/A (▪), BONT/B (▴), or BONT/E (▾) is depicted. (B) Functional activity of MAb F90G5-3 assessed via MPA for BoNT/A neutralization. Although there is not complete neutralization of BoNT/A, delayed time to death is significant (p<0.005). F90G5-3 treated mice (▾); mock-treated mice (▴).