Inhibition by human sera of botulinum neurotoxin-A binding to synaptosomes: A new assay for blocking and non-blocking antibodies

The mouse protection assay (MPA), which is an in vivo assay, is currently the most widely used method for monitoring blocking antibodies (Abs) in botulinum neurotoxin (BoNT)-treated patients. In recent studies we found that a number of the regions on the heavy (H) subunit of BoNT/A that bind blocking mouse Abs coincided, or overlapped, with the regions that bind to mouse synaptosomes (snps). This suggested that blocking anti-BoNT/A Abs would be expected to inhibit BoNT/A binding to snps. In the present work, we analyzed sera from 58 cervical dystonia (CD) patients who had been treated with BOTOX® (a preparation of BoNT/A serotype) for blocking Abs by MPA and by their abilities to inhibit in vitro the binding of 125I-labeled active BoNT/A or inactive toxin (toxoid) to mouse brain snps. With active 125I-labeled BoNT/A-snps binding, the MPA-positive sera (n = 30) displayed inhibition levels that were distinctly higher (mean = 21.1 ± 5.8) than those obtained with MPA-negative sera (n = 28) (mean = −1.3 ± 3.9; p < 0.0001) or control sera (n = 19) (mean = −3.4 ± 2.8; p < 0.0001). Similarly, inhibition levels by MPA-positive sera of 125I-labeled toxoid snp-binding (mean = 48.6 ± 8.7) were distinctly higher than inhibition by MPA-negative sera (mean = 10.0 ± 7.6; p < 0.0001) or control sera (mean = 1.8 ± 6.9; p < 0.0001). Thus, using labeled active toxin or toxoid, the inhibition assay correlated very well with the MPA. The inhibitory activity of the non-protective sera generally correlated with the duration of survival after toxin challenge (correlation coefficients of inhibition: active toxin = 0.445; p = 0.0167; inactive toxoid = 0.774; p < 0.0001). It is concluded that the snp-inhibition assay reported here is reliable, reproducible and correlates very well with the MPA. It requires much less serum (0.75% of the amount needed for the MPA) and is considerably less costly than the MPA. With either 125I-labeled active toxin or toxoid, it is possible to distinguish CD sera that have blocking Abs from those that lack such Abs. Since the results with the toxoid were as discriminating as those of the active toxin, it would not even be necessary to use active toxin in these assays.