Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation

Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

doi:10.1016/j.jchromb.2011.06.003

Cited by 12 publications

(9 citation statements)

References 22 publications

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“…In the untreated sample with 2-mercaptoethanol, the toxin maintained its molecular mass of 150 kDa, whereas, when the sample was incubated with the reducing agent, there was a 100 kDa band and another 50 kDa band. Since 2-mercaptoethanol is a reducing agent and dissociates bonds made by disulfide bridges [ 50 ], the observed profile was expected, and the literature shows similar results obtained under the same conditions [ 51 , 52 ].…”

Section: Discussionmentioning

confidence: 57%

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Sartori

Costa

Macarini

et al. 2021

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60 Co. Methods: Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results: Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin

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Section: Discussionmentioning

confidence: 57%

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Sartori

Costa

Macarini

et al. 2021

J. Venom. Anim. Toxins incl. Trop. Dis

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“…4 Despite having achieved their goal, they were unable to remove the putative S-layer protein and four other contaminating proteins. Stojicévićet al 1 reported the use of interaction hydrophobic chromatography (HIC), chelating chromatography (CC), and immobilized metal ion affinity chromatography (IMAC) for the purification of the Ttx. The best results were obtained when a first tangential ultrafiltration step was used together with HIC (Y% ∼ 50−80%), similar to those obtained with the traditional method.…”

Section: Resultsmentioning

confidence: 99%

“…The use of the tetanus toxin (Tt) as the target protein for the screening process is not recommended because, as a biohazard, special equipment and infrastructure would be required. The Ttx, used to produce tetanus vaccines, is prepared by inactivation of the Tt with formaldehyde . Traditional Ttx purification is achieved by a two-step salting out process followed by ultrafiltration. − Although this low-cost methodology has been used for many decades by manufacturers of most of the antitetanus vaccines, it is a laborious process that gives low yields.…”

Section: Introductionmentioning

confidence: 99%

“…The Ttx, used to produce tetanus vaccines, is prepared by inactivation of the Tt with formaldehyde. 1 Traditional Ttx purification is achieved by a two-step salting out process followed by ultrafiltration. 2−4 Although this low-cost methodology has been used for many decades by manufacturers of most of the antitetanus vaccines, it is a laborious process that gives low yields.…”

Section: Introductionmentioning

confidence: 99%

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Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification

et al. 2021

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Peptide ligands are widely used in protein purification by affinity chromatography. Here, we applied a fully automated two-stage library screening method that avoids false positive peptidyl-bead selection and applied it to tetanus toxoid purification. The first library screening was performed using only sulforhodamine (a fluorescent dye), and fluorescent beads were isolated automatically by flow cytometry and discarded. A second screening was then performed with the rest of the library, using the target protein (tetanus toxoid)–rhodamine conjugate. This time, fluorescent beads were isolated, and peptide sequences were identified by matrix-assisted laser desorption/ionization tandem mass spectrometry. Those appearing with greater frequency were synthesized and immobilized on agarose to evaluate a range of chromatographic purification conditions. The affinity matrix PTx1-agarose (Ac-Leu-Arg-Val-Tyr-His-Gly-Gly-Ala-Gly-Lys-agarose) showed the best performance when 20 mM sodium phosphate, 0.05% Tween 20, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. A pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with the PTx1 matrix, and 96% adsorption was achieved, with a K d of 9.18 ± 0.07 nmol/L and a q m of 1.31 ± 0.029 μmol Ttx/mL matrix. Next, a Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a band, identified by electrospray ionization mass spectrometry as the Ttx, that appeared only in the elution fraction, where an S-layer protein was also detected.

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“…TeNT is inactivated by formaldehyde to produce the toxoid [89]. Subsequently, the toxoid is further purified from the detoxified cultures by methods such as ammonium sulphate precipitation [92], ultrafiltration [93], tangential flow filtration [94,95], or chromatography [96].…”

Section: Clostridium Tetanimentioning

confidence: 99%

Vaccine Production to Protect Animals Against Pathogenic Clostridia

Zaragoza

Orellana

Moonen³

et al. 2019

Toxins

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Clostridium is a broad genus of anaerobic, spore-forming, rod-shaped, Gram-positive bacteria that can be found in different environments all around the world. The genus includes human and animal pathogens that produce potent exotoxins that cause rapid and potentially fatal diseases responsible for countless human casualties and billion-dollar annual loss to the agricultural sector. Diseases include botulism, tetanus, enterotoxemia, gas gangrene, necrotic enteritis, pseudomembranous colitis, blackleg, and black disease, which are caused by pathogenic Clostridium. Due to their ability to sporulate, they cannot be eradicated from the environment. As such, immunization with toxoid or bacterin-toxoid vaccines is the only protective method against infection. Toxins recovered from Clostridium cultures are inactivated to form toxoids, which are then formulated into multivalent vaccines. This review discusses the toxins, diseases, and toxoid production processes of the most common pathogenic Clostridium species, including Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Clostridium chauvoei, Clostridium septicum, Clostridium novyi and Clostridium hemolyticum.

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Tetanus toxoid purification: Chromatographic procedures as an alternative to ammonium-sulphate precipitation

Cited by 12 publications

References 22 publications

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification

Vaccine Production to Protect Animals Against Pathogenic Clostridia

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