Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons

Pellett, Sabine; Tepp, William H.; Johnson, Eric A.; Sesardic, Dorothea

doi:10.1016/j.vascn.2017.04.013

Cited by 15 publications

(6 citation statements)

References 17 publications

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“…In a second step, the large and small subunits are separated by reduction, before the activity of the small subunit in the supernatant is determined by the immunological quantification of neo-epitopes generated on an immobilized substrate in a second well [ 13 ]. A similar approach is taken in cell-based assays, in which the BoNT-A is taken up into the cells of a differentiated human neuronal cell line (SIMA cells) [ 14 ] or neuronal cells derived from hiPSC [ 15 ] and the cleavage of SNAP25 is quantified by a SNAP25 cleavage product-specific ELISA. Both systems suffer from a major limitation as they only work for one specific BoNT subtype.…”

Section: Introductionmentioning

confidence: 99%

Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins

Pathe-Neuschäfer-Rube

Neuschäfer-Rube

Haas

et al. 2018

Toxins

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Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, the current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.

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Section: Introductionmentioning

confidence: 99%

Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins

Pathe-Neuschäfer-Rube

Neuschäfer-Rube

Haas

et al. 2018

Toxins

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“…Whereas future studies could explore if different mixtures of polysialogangliosides can improve the sensitivity of SiMa cells, the focus of this study was to establish the feasibility of the SiMa cell model as a BoNT neutralization test. We focused on the use of ELISA as an end point because it is applicable to high-throughput formats and is a more quantitative method compared to Western Blotting [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Bak

Rajagopal

Stickings

et al. 2017

Toxins

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Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

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“…The expression of six human proteins (POMP, PSMB5, NRF2, XBP1, cMAF and MAFB in MM PCs was analyzed in all samples. Quantitative detection of human protein concentration in PCs was performed by solid-phase sandwich ELISA assay, as previously described [ 37 , 38 ]. POMP (Biobool, Hong Kong), PSMB5 (MyBioSource, San Diego, CA, USA), NRF2 (ThermoFischer Scientific, Waltham, MA, USA), XBP1 (MyBioSource, USA), cMAF (ThermoFischer Scientific, USA) and MAFb (MyBioSource, USA) reagents were applied.…”

Section: Methodsmentioning

confidence: 99%

Prognostic Value of Resistance Proteins in Plasma Cells from Multiple Myeloma Patients Treated with Bortezomib-Based Regimens

Robak

Szemraj

Mikulski

et al. 2021

JCM

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While multiple myeloma (MM) treatment with proteasome inhibitors and other agents yields encouraging results, primary and secondary resistance remains an emerging problem. An important factor in such treatment resistance is the overexpression of several proteins. The present study comprehensively evaluates the expression of POMP, PSMB5, NRF2, XBP1, cMAF and MAFb proteins in plasma cells isolated from the bone marrow of 39 MM patients treated with bortezomib-based regimens using an enzyme-linked immunosorbent assay (ELISA). The proteins were selected on the basis of previous laboratory and clinical studies in bortezomib-treated MM patients. It was found that the expression of the investigated proteins did not significantly differ between bortezomib-sensitive and bortezomib-refractory patients. However, the expression of some proteins correlated with overall survival (OS); this was significantly shorter in patients with higher POMP expression (HR 2.8, 95% CI: 1.1–7.0, p = 0.0277) and longer in those with higher MAFB expression (HR 0.32, 95% CI: 0.13–0.80, p = 0.0147). Our results indicate that a high expression of POMP and MAFB in MM plasma cells may serve as predictors of OS in MM patients treated with bortezomib-based regimens. However, further studies are needed to determine the role of these factors in effective strategies for improving anti-myeloma therapy.

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Assessment of ELISA as endpoint in neuronal cell-based assay for BoNT detection using hiPSC derived neurons

Cited by 15 publications

References 17 publications

Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins

Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Prognostic Value of Resistance Proteins in Plasma Cells from Multiple Myeloma Patients Treated with Bortezomib-Based Regimens

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