For the last two decades, the relationship between the structure and function of BoNT has been studied intensively. With the complex structure of BoNT/B and its receptors, protein receptor Syt-II 
and its ganglioside receptor GT1b 
, we can combine these data to provide the structure basis of the “double receptor” interaction proposed by Montecucco et al. 
. Bio-functional assays also provided the evidence that both protein receptor SytII and ganglioside co-receptor were necessary for the infective process of BoNT/B 
. This led us to the hypothesis that a combination comprised of a pair of neutralizing antibodies that bound to different receptors binding domains of the toxin would be more effective neutralizers than either member of the pair alone. We generated three mAbs, 8E10, 5G10 and 2F4 which could neutralize the challenge of 20 LD50 of BoNT/B in vivo
. Finely epitope mapping revealed that 8E10 recognized ganglioside binding domain in BoNT/B, 5G10 and 2F4 bound with 2 non-overlapping epitopes surround the SytII binding domain respectively. In addition, we found that the combination between 8E10 (recognizing ganglioside binding domain) and 5G10 or 2F4 (recognizing protein receptor binding domain) could result in a more than 30–60 folds increase in potency compared with that of any the single mAb. However, for the combination of 5G10 and 2F4, which both blocked protein receptor binding sites by non-overlapping epitopes, no synergistic effect was observed. This indicates that double blocking the ganglioside and protein receptor binding domain simultaneously with two mAbs is helpful for the synergistic effects of the two antibodies' combination against BoNT/B.
The combinations of antibodies, which recognize nonoverlapping epitopes, synergistically cooperate in neutralization potency have been reported previously 
. In that study, random combinations between 2 of the 3 neutralizing mAbs significantly prolonged the time to neuroparalysis compared with single mAbs. However, in our study, 2 mAbs, which bind to the same function domain of BoNT/B, had much less synergistic effects than those whose epitopes located in different function domains of BoNT/B. In addition, a BoNT/B-specific triplex antibody combination exhibited cooperative neutralizing effects to the toxin in vivo that were no better than those of the pairs of antibodies (). These findings are different from the conclusion of the previous report. Fine epitope mapping showed that the two mAbs (3D12 and S25) which bind the BoNT/A HCC
(C terminal of Hc) overlap the putative sialoganglioside binding site and cover a large portion of HCC
. The other mAb C25 bound a conformational epitope that consisted of the sequence from the N- and C-terminal subdomains of BoNT/A Hc, which overlapped with a putative inositol phosphate binding site that may be important for attachment to the lipid membrane. This function domain is essential for the binding to anionic lipid in the environment of lipid raft, which is important for the translocation processing of the toxin. 
. Interestingly, the neutralizing potency of the pair of mAbs (C25+3D12), which bind to translocation domain and sialoganglioside binding domain respectively, is 10 times higher than that of S25+3D12, whose epitopes are located at the same function domain. This finding provides evidence that the synergetic effects of mAbs, which recognize different function domains of BoNT/A, are better than the effects of those recognizing the same function domain. Although we didn't observe the cooperative effects between the mAbs binding to SytII binding domain of BoNT/B, we did prove that the combination of two mAbs recognizing different receptors' binding domain in BoNT/B has a synergistic effect. Accordingly, the data indicate that the combination between 2 mAbs recognizing different function domains in BoNTs could be the general principle for the potent synergistic effect.
Neutralizing mAbs binding multiple serotypes of botulinum neurotoxin are rare have been reported previously 
. The cross-reactive mAbs bound to a relatively conserved epitope at the tip of the BoNT HN
. This is a functionally important epitope for intoxication, as mAb binding leads to potent BoNT neutralization. In this study, a neutralizing mAb 8E10 binding the conservative domain on Hc was reported. The structure of Hc from BoNT/B (PDB 2NM1) 
, BoNT/E (PDB 3FFZ) 
and BoNT/F (PDB 3FUQ) 
supports the view that the Hc fold is highly conserved. GT1b binds on a cleft formed by W1266 and Y1267 on one face and E1203, H1253, and F1252 on the other for BoNT/A 
. The structure of Hc from BoNT/B in complex with the trisaccharide sialyllactose, a mimic of GT1b (PDB 1F31), displays a similar binding cavity with corresponding residues W1261, Y1262, and H1240 
. In addition, the crystal structure of Hc from BoNT/F displays a ganglioside-binding pocket with corresponding residues W1250, Y1251, and H1241 
. Amino acid sequence alignment shows that S1264, W1266 and Y1267, conserved among all BoNT serotypes 
, constitute key residues of a lactose-binding motif (H. . .SXWY. . .G) that contribute to the crevice binding with GT1b. The implication is that the GT1b-binding pocket for all these BoNTs is similar. We found in this study that the motif 1259SKWY1262 is the recognizing epitope of 8E10, which could also cross react with the Hc of BoNT A/B/E/F (). This conserved epitope may partially explain how 8E10 could cross-protect the challenge of different serotype of BoNTs in vivo
. Although its synergistic effect with the mAbs blocking protein receptor binding domain in BoNT A, E or F has not been detected, we can predict that 8E10 could act synergistically with the mAbs recognizing protein receptor binding domain against BoNT A, E or F. This study provides a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.