High-throughput homogeneous immunoassay readily identifies monoclonal antibody to serovariant clostridial neurotoxins.

High-throughput screening can create the potential ability to screen large numbers of monoclonal antibodies (mAb) in a short time period. A major bottleneck in the hybridoma method for mAb development has historically been the inability to sift through large numbers of hybridoma culture supernatants to identify clones secreting mAbs of the desired specificity. Herein, we develop a homogeneous fluorometric microvolume assay technology (FMAT) and compare it to conventional ELISA screening techniques for monoclonal antibody against soluble protein toxin fragments of the Clostridium botulinum types A, B and E neurotoxin (BoNT) proteins. In total 8,744 hybridoma clones were screened to identify 29 stable hybridomas to neurotoxin binding domain; six of these would have been missed by ELISA alone. Screening of hybridoma supernatants on days 1 and 4 following cloning from semi-solid HAT agarose reveals that FMAT provides a reliable method for screening hybridoma clones to purified protein toxins. The homogeneous FMAT utilizes far less reagent (antigen and hybridoma supernatant) allowing for simultaneous screening against multiple serovariant antigens early in the hybridoma cloning cycle. This reduces costs for reagents and labour by lowering numbers of clones being maintained with undesired specificity. Furthermore, this assay easily accommodates replicate screening which facilitates identification of cross-reactivity to neurotoxin serotypes, thus readily identifying mAb to serovariant antigens. These findings have broad application in accelerating mAb development to serovariant cell-surface or bead bound targets without arraying devices. In summary, FMAT provides a reliable method for the screening of mAbs against C. botulinum neurotoxins.

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

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.