2.1. Detection of BoNT/B Using Electrochemiluminescent (ECL) Immunoassay
The gold standard for detection of BoNTs employs the mouse bioassay. The mouse bioassay can detect BoNT/B levels of 25 pg/mL [
13,
20,
23]. However, these assays require about 3–4 days for full confirmation. To improve detection sensitivity and speed, we have previously described the development of high affinity monoclonal antibodies (mAbs), MCS6-27 and BoB92-32, and their use in ELISA detection of BoNT/B [
19,
21]. Both of these mAbs were against the Hc receptor binding domain (E859-E1291) of BoNT/B and were used successfully in electrochemiluminescence (ECL) detection assays in complex food matrices and horse sera [
20]. Limits of detection for BoNT/B in buffer conditions were as low as 13 pg/mL. The ECL assays, like ELISA type immunoassays, take about 4–5 h to complete, but are less sensitive to food matrix effects. In addition, less sample volume is needed (15 μL
vs. 50–100 μL) than an ELISA or animal bioassay.
Mice are highly sensitive to BoNT toxins. The LD
50 for BoNT/B is about 12.5 pg for a 20 g mouse [
20]. In order to determine the biologic half-life of BoNT/B holotoxins in mice, assays need to be able to detect low picogram amounts of BoNT/B in complex matrices, such as sera. To improve the sensitivity of the ECL assay, we tested the use of a rabbit polyclonal anti-BoNT/B antibody coupled with goat anti-Rabbit detector (SULFO-TAG labeled). We improved the limit of detection (LOD) for BoNT/B to 1 ± 0.1 pg/mL with a dynamic range for standard detection from 0.5 pg/mL to 100 ng/mL in buffer conditions (
Figure 1A). Using this assay, we also tested the effect of fresh mouse sera matrix on detection sensitivity. Use of 50%, 75%, or 100% sera had negligible effects on detection sensitivity compared to the buffer matrix (
Figure 1B). We believe the polyclonal rabbit antibodies contained multiple antibodies binding to different epitopes of captured BoNT/B; this, in turn, improved detection sensitivity. An improvement on detection sensitivity was also observed when multiple mAbs were used as detector antibodies in ELISA assays (data not shown).
We used this sensitive ECL assay to determine the biological half-lives of BoNT/B after intravenous (IV) introduction of toxin. Random sets of five mice were treated with 1000 pg BoNT/B holotoxin (about 80 mouse LD
50) via tail vein IV injection. Sera were collected from each set of mice over time and the levels of BoNT/B were determined using the Meso Scale Discovery (MSD) instrument. Sera concentrations of BoNT/B over 3 h were then plotted (
Figure 2). Soon after injection, BoNT/B holotoxin levels declined rapidly within the first 10 min of toxin introduction followed by a slower rate of toxin decline in the bloodstream. This initial phase or alpha half-life (
t1/2α) was determined to be 2.3 min and represented the period of toxin redistribution to tissues, extracellular spaces and uptake by neurons (
Figure 2). The second phase or
t1/2β half-life was determined to be 105 min and likely represented the natural clearance of BoNT from the blood.
Figure 1.
Electrochemiluminescent detection of BoNT/B with a MSD instrument. (A) Diagram of serial 1:5 dilutions of BoNT/B with a range of 10,000 to 0.64 pg/mL detected using an ECL assay using anti-BoNT/B mAb MCS6-27 for capture, and SULFO-TAG-labeled rabbit anti-BoNT/B polyclonal antibody for detection; and (B) the detection of BoNT/B dilution standards in the presence of buffer only or 50%, 75%, or 100% sera were compared. Graph points showed the mean ± SEM of duplicate wells.
Figure 1.
Electrochemiluminescent detection of BoNT/B with a MSD instrument. (A) Diagram of serial 1:5 dilutions of BoNT/B with a range of 10,000 to 0.64 pg/mL detected using an ECL assay using anti-BoNT/B mAb MCS6-27 for capture, and SULFO-TAG-labeled rabbit anti-BoNT/B polyclonal antibody for detection; and (B) the detection of BoNT/B dilution standards in the presence of buffer only or 50%, 75%, or 100% sera were compared. Graph points showed the mean ± SEM of duplicate wells.
Figure 2.
In vivo biological half-live of BoNT/B. Groups of five mice were injected with 1000 pg/mouse of BoNT/B and sera were obtained at 5, 10, 20, 30, 40, 80, 120, and 160 min post-intoxication. The concentration of unknown BoNT/B was determined using the ECL method. Each data point in graph represents the mean ± S.E.M. R2 = 0.8937.
Figure 2.
In vivo biological half-live of BoNT/B. Groups of five mice were injected with 1000 pg/mouse of BoNT/B and sera were obtained at 5, 10, 20, 30, 40, 80, 120, and 160 min post-intoxication. The concentration of unknown BoNT/B was determined using the ECL method. Each data point in graph represents the mean ± S.E.M. R2 = 0.8937.
The serum half-life for BoNT/B was comparably faster than to those measured for BoNT/B in rats using radiolabeling [
24]. This could be due to differences in toxin uptake in these two animals. Rats are not as susceptible to BoNT/B intoxication as mice due to differences in toxin receptors [
25,
26].
2.2. Neutralization of BoNT/B with Monoclonal Antibodies
We tested the neutralization potencies of individual and combinations of mAbs against BoNT/B in both the IV and oral mouse models of botulism. Three mAbs, MCS6-27, BoB92-23, and BoB92-32, all specific against the Hc receptor binding domain (amino acids E859-E1291) of BoNT/B were used alone or in combination to treat mice dosed with lethal doses of BoNT/B. These mAbs were first chosen because of their binding activity in BoNT/B capture assays. First, mice were administered different doses of mAbs by IV 30 min before injection with 1000 pg/mouse or about 80 mouse IV LD
50 of BoNT holotoxin. Mice pre-treated with 10 µg of MCS6-27 or 0.4 µg BoB92-23, or 50 µg BoB92-32 prior to BoNT/B injection, were completely protected from death (
Figure 3A–C). Control mice treated with PBS alone died with a median survival time of 6 h. Treatment with 0.4 µg MCS6-27, 0.08 µg BoB92-23, or 2 µg BoB92-32 only led to a slight delay in median survival time to 18, 14, and 14 h, respectively (
Figure 3A–C). In contrast, treatment with 2.5 µg of a combination of these same concentrations of mAbs (from here referred as Combo mAbs: 0.4 µg MCS6-27, 0.08 µg BoB92-23, and 2 µg BoB92-32) conferred complete protection from death (
Figure 3D).
Figure 3.
Monoclonal antibody neutralization of BoNT/B in the intravenous mouse model. The percent survival of mice treated with different doses of MCS6-27 (A); BoB92-23 (B); BoB92-32 (C); or Combo mAbs, containing 0.4 µg MCS6-27, 0.08 µg BoB92-23, and 2 µg BoB92-32 mAbs (D) one hour prior to challenge with BoNT/B were plotted over time. The percent survival of individual mAbs present in the Combo was also plotted for comparison. Control mice were treated with PBS iv instead of mAbs before intoxication.
Figure 3.
Monoclonal antibody neutralization of BoNT/B in the intravenous mouse model. The percent survival of mice treated with different doses of MCS6-27 (A); BoB92-23 (B); BoB92-32 (C); or Combo mAbs, containing 0.4 µg MCS6-27, 0.08 µg BoB92-23, and 2 µg BoB92-32 mAbs (D) one hour prior to challenge with BoNT/B were plotted over time. The percent survival of individual mAbs present in the Combo was also plotted for comparison. Control mice were treated with PBS iv instead of mAbs before intoxication.
Neutralization of BoNT/B with high doses of individual mAbs (such as 10 µg MCS6-27, 0.4 µg of BoB92-23, and 50 µg of 92–32) can fully protect mice from toxin lethality, but as reported by others, a combination of mAbs against different binding sites of BoNT had a synergistic effect on neutralization potential [
27]. A combination of three single mAbs (
Figure 3D) at much lower mAb concentrations can protect better, presumably because mAb binding at multiple toxin sites enables steric hindrance of receptor binding and also presumably by enhancing Fc domain binding and eventually immune clearance of toxins from blood. Previous studies have found that most BoNTs were cleared into the liver and not found associated with non-immune organs [
24,
28].
We wanted to determine the time frames when mAbs can rescue mice from death or delay the time-to-death. This information will help us understand whether there are windows of opportunity for treatment and would also give us clues to the timing of toxin passage in the mouse intestinal tract. Mice were treated IV with 1000 pg of BoNT/B holotoxin. A 12.4 µg dose of Combo mAbs (2 µg 6–27, 0.4 µg 92–23 and 10 µg 92–32) was then administered IV 5, 10, 15, 20, and 40 min post-intoxication. This dose, five times the dose used for complete protection of mice in
Figure 3D, was chosen to ensure complete protection from intoxication. At this dose, toxin that is not neuron or tissue bound would be cleared from the system. Treatment with Combo mAbs 10 min or less after intoxication completely rescued mice from death (
Figure 4). A time-to-death delay was seen when mice were given Combo mAbs 15 min post-intoxication with a median survival time of 60 h. Mice treated 20 min or more post-intoxication showed only a slight delay of time-to-death when compared with PBS-treated mice with median survival times of 14 and 4.5 h, respectively (
Figure 4). This method helps predict the timing of when toxin is absorbed by neurons or tissues. At later time points, lethal amounts of BoNT/B would have been taken up and mAb neutralization would not prolong survival.
Figure 4.
Neutralization of BoNT/B after intravenous intoxication. The percent survival of groups of 10 mice challenged iv with 1000 pg of BoNT/B holotoxin followed by treatment with PBS or 12.4 µg of Combo mAbs at 10, 15, and 20 min post-intoxication were plotted over time.
Figure 4.
Neutralization of BoNT/B after intravenous intoxication. The percent survival of groups of 10 mice challenged iv with 1000 pg of BoNT/B holotoxin followed by treatment with PBS or 12.4 µg of Combo mAbs at 10, 15, and 20 min post-intoxication were plotted over time.
Both the half-life data and the post-IV intoxication mAb neutralization results indicate that once BoNT/B enters the blood stream, the window of time to neutralize toxins is short. The remaining BoNT/B that is not absorbed into neurons in the sera is slowly cleared. This remaining toxin reservoir is the target of current antitoxin therapies. For most foodborne cases of botulism, equine antitoxin therapy is recommended as early as possible after toxin confirmation. Positive treatment outcomes are directly correlated with early administration of antitoxins [
1,
14].
Compared to systemic intoxication, relatively large amounts of BoNT must be present to cause disease after oral ingestion [
11,
18]. Most of the BoNTs are likely degraded in the intestinal tract or shed as waste, with very little actually absorbed by the animal. Previous research showed that the BoNT/A complex was 17 times more toxic than BoNT/A holotoxin in the oral route of intoxication. This is likely due to protection from degradation of the botulinum toxin in the acidic environment and neurotoxin-associated proteins (NAP) mediated entry [
10,
11]. Although BoNT/A and BoNT/B complex have similar neurotoxin-associated protein (NAP) compositions, BoNT/B has been observed to form larger complexes than BoNT/A complexes in native gels [
29]. BoNT/B complexes are also about 100× more toxic than BoNT/A complexes in oral intoxications of mice.
In this study, we tested the neutralization of BoNT/B complex in the oral mouse model of intoxication. Mice were treated with 0.06 µg or about three oral LD
50 of BoNT/B complex by gavage, followed by IV injection with 12.4 µg of Combo mAbs at (
Figure 5). Intravenous injection of Combo mAb within 8 h conferred complete protection (defined as no lethality among treated mice). Treatment with the same dose of Combo mAb at 10 h post-intoxication reduced mortality to 20% and mice treated with Combo mAbs at 12 h had 80% lethality with a median survival of 32 h (
Figure 5). Mice that survive toxin or antibody treatment will recover completely over time. Control mice treated with PBS at 6 h post-intoxication had a median survival of 26 h.
Figure 5.
Neutralization of BoNT/B after oral intoxication. The percent survival of groups of 10 mice administered 0.06 µg or about three mouse oral LD50 of BoNT/B complex by gavage followed by rescue with 12.4 µg of Combo mAbs by IV at 8, 10, 12 h post-intoxication were plotted over time. Control mice were injected with PBS IV at 6 h post-intoxication instead of Combo mAbs.
Figure 5.
Neutralization of BoNT/B after oral intoxication. The percent survival of groups of 10 mice administered 0.06 µg or about three mouse oral LD50 of BoNT/B complex by gavage followed by rescue with 12.4 µg of Combo mAbs by IV at 8, 10, 12 h post-intoxication were plotted over time. Control mice were injected with PBS IV at 6 h post-intoxication instead of Combo mAbs.
Orally-ingested BoNT/B would have to reach the blood stream or lymph to cause disease, thus one can predict the timing of when toxins reach those destinations by when protective antibodies cease to protect animals from intoxication. The timing of protection suggests that BoNT/B likely reaches the bloodstream at about 10 h after oral ingestion of BoNT/B complex. This is in sharp contrast to the much shorter oral protection window exhibited by mAb Combos against BoNT/A with BoNT/A complex transit through the intestinal tract and exit into the bloodstream estimated at about 7 h after oral ingestion [
10,
22,
30]. The time between ingestion of toxin to the detection of BoNTs in the blood stream represented the time for toxin transit through the stomach, the intestinal tract, and receptor-mediated translocation through the intestinal epithelial cells into the blood stream and/or lymph [
10]. Thus, it appears that the BoNT/B complex protected toxin better from intestinal degradation [
31] but also slowed intestinal tract transit when compared with BoNT/A complex. It is not clear yet whether complex size or unique properties of the BoNT/B NAPs contributed to the increased toxicity of BoNT/B complex. Once orally-ingested BoNT reaches the blood stream, it would be quickly absorbed, following the systemic biologic half-life pattern (
Figure 2). For both systemic and oral mouse antibody neutralization models, no mAb rescue was observed when mAbs were administered after visible symptoms of botulism (such as slowness, limping,
etc.) became apparent.