Conceived and designed the experiments: SK CGR JL TJS JDM JRB. Performed the experiments: SK JB. Analyzed the data: SK CGR JL JB TJS JDM LAS JLP JRB. Contributed reagents/materials/analysis tools: CGR JL TJS JDM LAS. Wrote the paper: SK.
The authors have declared that no competing interests exist.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.
Botulinum neurotoxins (BoNTs) are protein neurotoxins produced by some species of the genus
Previously, we reported the development of an assay for BoNT detection and serotype differentiation termed the Endopep-MS method
As previously reported, Endopep-MS is highly effective in identifying BoNT/A, /B, /E, and /F, the four serotypes that commonly affect humans, in clinical samples. This method uses an antibody affinity concentration/purification step prior to reacting with the substrate
Because the antibodies used in the affinity concentration/purification step are serotype-specific, in order to test for the presence of four serotypes, a sample must either be split into four aliquots or the same sample must be extracted four times, once with each serotype-specific antibody. Neither of these steps is ideal, since they negatively impact the assay in terms of sample volume requirements or total analysis time used, as each extraction step is approximately 1 hr. Therefore, the use of a single antibody which can extract all four serotypes of BoNT at once would be beneficial in an assay to rapidly detect BoNT. Here, we report the use of a unique high-affinity monoclonal antibody, 4E17.1, in the Endopep-MS assay for detection of all available subtypes of BoNT/A, /B, /E, and /F. This multi-specific cross reactive antibody allows for the simultaneous detection of BoNT/A, /B, /E,and /F by Endopep-MS without requiring any additional time for multiple toxin extractions, and it produces limits of detection which are similar to those obtained with serotype-specific monoclonal antibodies.
Studies using mice were conducted at USAMRIID, whose ethics committee specifically approved this study. Studies were performed under an approved IACUC protocol in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals, and adhere to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
Botulinum neurotoxin is very toxic and therefore requires appropriate safety measures. All neurotoxins were handled in a level 2 biosafety cabinet equipped with HEPA filters. Commercially purified complex toxins BoNT/A1, BoNT/A2, BoNT/B1, BoNT/C, BoNT/E3, protBoNT/F, and BoNT/G were purchased from Metabiologics (Madison, WI). Tetanus toxin (TeNT) was purchased from List Biological Laboratories, Inc. (Campbell, CA). Tetanus toxin antibody (TetE3) was purchased from Abcam (Cambridge, MA). Polyclonal rabbit IgGs for BoNT/E and BoNT/F were purchased from Metabiologics. Monoclonal antibodies CR2, RAZ1, and B12.1 were provided by Professor James Marks of the University of California, San Francisco. Dynabeads® Protein G were purchased from Invitrogen (Carlsbad, CA) at 1.3 g/cm3 in phosphate buffered saline (PBS), pH 7.4, containing 0.1% Tween®-20 and 0.02% sodium azide. Sequencing grade modified trypsin at 0.5 mg/mL in 50 mM acetic acid was purchased from Promega (Madison, WI). All chemicals were from Sigma-Aldrich (St. Louis, MO) except where indicated. Peptide substrates were synthesized by Los Alamos National Laboratory (Los Alamos, NM,
Peptide | Sequence | |
BoNT/A substrate | Biotin-KGSNRTRIDQGN |
2878.7 |
BoNT/A NT product | Biotin-KGSNRTRIDQGNQ | 1699.9 |
BoNT/A CT product | RATRXLGGK-Biotin | 1197.8 |
BoNT/B substrate | LSELDDRADALQAGAS |
4024.7 |
BoNT/B NT product | LSELDDRADALQAGASQ | 1759.9 |
BoNT/B CT product | FETSAAKLKRKYWWKNLK | 2283.4 |
BoNT/E substrate | IIGNLRHMALDMGNEIDTQNRQID |
4041.1 |
BoNT/E NT product | IIGNLRHMALDMGNEIDTQNRQIDR | 2923.6 |
BoNT/E CT product | IMEKADSNKT | 1136.6 |
BoNT/D and /F substrate | LQQTQAQVDEVVDIMRVNVDKVLERD |
4497.5 |
BoNT/D NT product | LQQTQAQVDEVVDIMRVNVDKVLERDQK | 3296.9 |
BoNT/D CT product | LSELDDRADAL | 1217.7 |
BoNT/F NT product | LQQTQAQVDEVVDIMRVNVDKVLERDQ | 3167.8 |
BoNT/F CT product | KLSELDDRADAL | 1345.2 |
The observed
Monoclonal antibody (mAb) 4E17.1 was first selected and engineered as a single chain fragment of variable domain (scFv) antibody using yeast display technology. The original antibody gene was selected from material of human volunteers immunized with pentavalent BoNT toxoid using toxin of the BoNT/E3 subtype
As discussed in greater detail in work by Garcia-Rodriguez, et.al.
Crude culture supernatants representing various BoNT/A, /B, /D, /E, and /F subtypes (
Toxin | Strain | Accession # | Amino acid sequence | KD (pM) |
BoNT/A1 | Hall | AF488749 | 1.83 | |
BoNT/A2 | CDC 1436 | EF028393 | 7.98 | |
BoNT/A3 | Loch Maree | DQ185900 | 4.5 | |
BoNT/A4 | Strain 657 | EU341407 | ND | |
BoNT/A5 | H0 4402 065 | EU679004 | ND | |
BoNT/B1 | Okra | AB232927 | 43760 | |
BoNT/B2 | 213B (ATCC 7949) | EF028395 | 41330 | |
BoNT/B3 | CDC 795 | EF028400 | ND | |
BoNT/B4 | Eklund 17B | EF051570 | 16.52 | |
BoNT/B5 | Strain 657 | EF033130 | 45210 | |
BoNT/B6 | Osaka05 | AB302852 | ND | |
BoNT/C | Stockholm | X62389 | NB | |
BoNT/D | CB-16 | S49407 | NB | |
BoNT/E1 | German sprats (Hazen 35396) | AB082519 | 730 | |
BoNT/E2 | CDC 5247 | EF028404 | 205 | |
BoNT/E3 | Alaska E43 | EF028403 | 241 | |
BoNT/E4 | BL5262 | AB088207 | 260 | |
BoNT/E5 | LCL155 | AB037704 | ND | |
BoNT/E6 | K35 | AM695752 | ND | |
BoNT/F1 | Langeland | GU213203 | 65080 | |
BoNT/F2 | Strain 84 | Y13631 | ND | |
BoNT/F3 | CDC 54086 | GU213218 | ND | |
BoNT/F4 | CDC 54078 | GU213214 | ND | |
BoNT/F5 | CDC 54074 | GU213211 | ND | |
BoNT/F6 | Eklund 202F | M92906 | ND | |
BoNT/F7 | Sullivan | HM746656 | ND | |
BoNT/G | 113/30 | X74162 | ND | |
TeNT | CN3911 (Harvard) | X06214 | ND |
Residues that play an important role in 4E17.1 binding are bolded and underlined. Dissociation rates (KD) in pM of BoNT with mAb 4E17.1 are also listed. NB indicates no binding was observed and ND indicates that the KD was not determined. Each BoNT is also identified by strain tested where appropriate. Equilibrium dissociation constant (KD) were measured by flow fluorimetry in a KinExA
For toxin titrations, samples were diluted 2-fold in gelatin-phosphate buffer (0.2% gelatin, 0.4% sodium phosphate), pH 6.2. Female Crl∶CD-1 mice, 16–22 g on receipt (Charles River Laboratories, Raleigh, NC) were injected i.p. with total volumes of 0.1 mL diluted toxin. Mice were observed for 5 days, survivors were tallied, and the results were analyzed using probit analysis (SPSS, Chicago, IL). The final LD50/mL of the preparation was calculated by dividing the initial dilution used in the assays by the probit result. Titrations were done in duplicate and averaged to obtain the working titers for each toxin. Duplicate titrations were also done on commercially obtained toxins (Metabiologics, Madison, WI; Wako Biologicals, Richmond, VA) using their stated toxicity as the initial dilution, and the titers were averaged to obtain their working titer.
Monoclonal antibody 4E17.1 was immobilized and crosslinked to the Dynabeads® Protein G as described in the manufacturer's protocol using 40 µg of antibody diluted into 500 µL of PBS for every 100 µ L of Dynabeads® Protein G. Cross-linked IgG-coated Dynabeads® were stored in PBS-Tween buffer (PBS with 0.05% Tween®-20) at 4°C for up to 12 weeks. For the BoNT extraction assay, an aliquot of 20 µL of antibody-coated beads was mixed for 1 hr with a solution of 5–100 µL of each culture supernatant, which is mixed with 495 µL of phosphate buffered saline with 0.01% Tween (PBST) buffer. After mixing for 1 hr with constant agitation at room temperature, the beads were washed twice in 1 mL each of PBST and then washed once in 100 µL of water. For limit of detection tests, BoNT at varying levels was spiked into an aliquot of 500 µL of PBST. Negative controls consisted of PBST with no spiked toxin extracted using the above protocol. For TeNT work, 5 µg of TeNT was spiked into 50 µL of PBST with the remainder of the extraction protocol as above. For simultaneous multiple serotype BoNT detection, 100 mLD50 of BoNT/A, /B, /E, and /F were spiked into 500 µL of serum mixed with 50 µL of 10× PBST.
The reaction was performed as previously described
A 2 µL aliquot of each reaction supernatant was mixed with 18 µL of matrix solution consisting of alpha-cyano-4-hydroxy cinnamic acid (CHCA) at 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA), and 1 mM ammonium citrate. A 0.5 µL aliquot of the resulting milieu was pipeted onto a 192-spot matrix-assisted laser desorption/ionization (MALDI) plate (Applied Biosystems, Framingham, MA). Mass spectra of each spot were obtained by scanning from 1100 to 4800
Following bead extraction of spiked TeNT from PBST buffer, the beads were reconstituted in 10 µL of 50 mM ammonium bicarbonate, pH = 7.5 and 2 µL of stock trypsin. 2 µL of 500 mM ammonium bicarbonate, pH = 7.5 and 2 µL of stock trypsin were also added to 20 µL of the extraction supernatant. These mixtures were digested overnight at 37°C. Following digestion, 1 µL of 10% TFA was added to both mixtures. The supernatant was then removed from the beads for analysis. 5 µL of this supernatant and the extraction supernatant mixture were analyzed by LC-MS/MS for amino acid sequence as reported by Kalb, et.al.
Monoclonal antibody (mAb) 4E17.1 interacts with residues Y750, Y753, E756, and E757 within the translocation domain of BoNT
The peptide cleavage products indicating BoNT/A are
After determining that mAb 4E17.1 could be used successfully to extract and detect all subtypes tested of BoNT/A via Endopep-MS, we investigated the use of this antibody to detect BoNT/B as well. The sequences of BoNT/B subtypes within the 4E17.1 epitope are listed in
The peptide cleavage products indicating BoNT/B are
Because the amino acid sequences of all BoNT/E subtypes contain all four amino acids necessary for optimum 4E17.1 binding, it was expected that the 4E17.1 antibody should be able to bind and extract all BoNT/E subtypes. Beads coated with 4E17.1 were therefore used to extract BoNT/E subtypes from culture supernatant media.
The peptide cleavage products indicating BoNT/E are
The amino acid sequences of the 7 BoNT/F subtypes contain Y750, Y753, and E757, three of the four amino acids determined to be important in 4E17.1 binding. The BoNT/B subtypes also contain these three amino acids, and it has already been shown that the BoNT/B subtypes recognize 4E17.1, so it was expected that the four subtypes of BoNT/F would also recognize 4E17.1.
The peptide cleavage products indicating BoNT/F are
BoNT/C and /D both contain Y750 and Y753, but not E756 or E757 (
The peptide cleavage products indicating BoNT/D are
Additionally, some of this toxin activity can be explained by non-specific binding of the toxin to the beads. This is illustrated in
Tetanus toxin (TeNT) contains Y750 and Y753, but not E756 or E757 (
There were several peptides which originated from TeNT in the digest of the beads which used anti-TeNT for extraction, but these peptides were not present in the digested beads which used anti-4E17.1 for extraction (data not shown). Furthermore, a digest of the supernatant consisting of material left behind after the extraction of TeNT with 4E17.1 coated beads demonstrated that TeNT remained behind in solution following extraction with 4E17.1 coated beads (data not shown).
Monoclonal antibody 4E17.1 has been shown to bind separately multiple subtypes of BoNT/A, /B, /E, and /F; therefore, it was assumed that this antibody could bind multiple subtypes of BoNT/A, /B, /E, and /F simultaneously. Beads coated with 4E17.1 antibody were used to extract BoNT/A, /B, /E, and /F spiked into a single sample. The beads were then added to reaction buffer with the four peptide substrates used to detect BoNT/A, /B, /E, and /F.
Cleavage products indicating those four BoNTs are marked.
Although the toxins were present in an equimolar ratio in this case, we also experimented with each individual toxin present at levels up to 1000 times the others. Those results (data not shown) indicate that we can still detect all four toxins in that case. Additionally, serotype specific antibodies were also used to extract BoNT/A, /B, /E, and /F spiked into serum, and those results are depicted in
Under natural conditions, it is unlikely that a single sample would contain four serotypes of BoNT; however, there are some cases when a single sample contains more than one serotype. Several bivalent strains exist, known as A2b, Ba4, Af, and Bf, in which the bacterium produces more than one serotype of BoNT under certain conditions.
Peaks at
The Endopep-MS assay detects BoNT via its activity, and it uses antibody extraction to isolate and concentrate the toxin prior to analysis. Some antibodies inhibit the enzymatic activity of the toxin and therefore increase the limit of detection (LOD) of BoNT. We therefore wanted to determine the LOD of BoNT/A, BoNT/B, BoNT/E, and BoNT/F using mAb 4E17.1 for extraction of those toxins from buffer. Using commercially-available, mouse-titered BoNT/A, /B, /E, and /F, we determined that the LOD of those toxins spiked into 0.5 mL of PBST buffer are 1 mLD50 for BoNT/A, 0.05 mLD50 of BoNT/B, 0.1 mLD50 of BoNT/E, and 0.05 mLD50 of BoNT/F. These LOD are equivalent to those obtained with other serotype-specific antibodies used for toxin extraction, with the exception of BoNT/A, and the LOD in serum and milk are the same as in PBST buffer. The LOD of BoNT/A using a combination of the RAZ1 and CR2 antibodies engineered for high affinity to BoNT/A is 0.5 mLD50.
The different serotypes of BoNT are defined by their ability to be neutralized by specific antitoxins; therefore, it is unusual for one antibody to bind multiple serotypes of botulinum neurotoxin. Monoclonal antibody 4E17.1 binds the translocation domain of BoNT, and residues Y750, Y753, E756, and E757 of BoNT/A play important roles in antibody binding. These residues are fairly well conserved among all of the known botulinum neurotoxins; hence, a single monoclonal antibody could bind most of the known botulinum neurotoxins at the same epitopic location.
All currently known subtypes of BoNT/A contain residues Y750, Y753, E756, and E757. BoNT/A1-/A4 were tested for their ability to bind monoclonal antibody 4E17.1. To test for the presence or absence of the extracted toxin on the antibody-coated beads, we used Endopep-MS. BoNT/A1-/A4 were all efficiently extracted from buffer with antibody 4E17.1, and the beads from those extractions all demonstrated the presence of active BoNT/A, meaning that all four of those BoNT/A subtypes bind the 4E17.1 antibody. Although BoNT/A5 was unavailable for testing, it is expected that this serotype will also effectively interact with 4E17.1 on the basis of its amino acid sequence (Y750, Y753, E756, and E757)
Most subtypes of BoNT/B contain three of the four key amino acids in 4E17.1 binding; the exception to this is BoNT/B4, which contains all four amino acids. Five of the currently known subtypes of BoNT/B were available for testing, and all interacted with the 4E17.1 antibody. This interaction indicates that E756 is perhaps not as critical for binding as the other amino acids. E756 is a lysine in most of the BoNT/B serotypes, and a mutation from glutamic acid to lysine is not a conserved mutation. This non-conserved mutation may be responsible for the higher KD associated with BoNT/B in general for this antibody (
All currently known subtypes of BoNT/E contain all four key amino acids in 4E17.1 binding. Subtypes BoNT/E1–E4 were available for testing, and all four interacted with 4E17.1. Subtypes BoNT/E5 and /E6 were unavailable for testing, but based on the amino acid homology
BoNT/C, /D, /G, and TeNT all contain Y750 and Y753, two of the amino acids known to be important in 4E17.1 binding. However, upon testing, it was determined that these toxins do not efficiently interact with the 4E17.1 antibody. Hence, these data show that a minimum of three of the four critical amino acids, Y750, Y753, and E757, are required for efficient binding. Furthermore, BoNT/G contains three of the four key amino acids in 4E17.1 binding including Y750, Y753 and E756, but it does not contain E757 and BoNT/G does not interact with 4E17.1. These additional data show that E756 is not critical for 4E17.1 interactions but that Y750, Y753, and especially E757 are required for efficient binding.
Endopep-MS was developed to detect the presence of BoNT in a clinical or food sample. An antibody affinity concentration/purification step prior to reaction with the substrate is critical to the success of the method when testing complex matrices such as serum or stool extracts. Prior to the discovery of the 4E17.1 antibody, a sample needed to either be split into 4 aliquots or extracted 4 separate times to test for the presence of four serotypes of BoNT. Either option negatively affected the Endopep-MS assay in terms of either sample volume requirements or time as each extraction step is approximately 1 hr. Therefore, the development of a single antibody which can be used to extract four serotypes of BoNT simultaneously, especially the four serotypes commonly involved in human botulism, is beneficial in the Endopep-MS assay to detect BoNT and would be beneficial in other assays which use antibody-affinity for BoNT detection.
We have tested the 4E17.1 antibody with spiked clinical and food samples such as serum, stool extracts, and milk and found that this antibody is particularly effective with serum and milk. For stool extracts with a severe protease composition, we have had recent success with 2M sodium chloride washes to remove excessive proteases from the antibody-coated beads (manuscript in preparation). In the presence of such extreme conditions, BoNT/B and /F in particular unfortunately are removed from the antibody-coated beads, due to the higher dissociation rates
The opinions, interpretations and recommendations are those of the authors and are not necessarily those of the Centers for Disease Control and Prevention or the US Army.