Oriented Functionalization of Magnetic Beads with <i>in Vivo</i> Biotinylated Nanobodies for Rapid MALDI-TOF MS Ultrasensitive Quantitation of Microcystins in Biological Samples

Pírez-Schirmer, Macarena; Brena, Beatríz M.; González‐Sapienza, Gualberto

doi:10.1021/acs.analchem.9b01596

Cited by 13 publications

(11 citation statements)

References 25 publications

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“…Otra posible limitación de los inmunoensayos se debe a que, además de las variantes de MCs presentes, podrían detectar metabolitos de detoxificación (conjugados MCs con cisteína y glutatión), que presentan mucho menor toxicidad que las correspondientes toxinas libres (Schmidt, et al, 2014;Li, et al, 2014). Para confirmar y cuantificar la presencia de diferentes variantes de MCs y distinguirlas de sus metabolitos, se utilizó un nuevo método ultrasensible de MALDI-TOF cuantitativo, originalmente validado para aguas y sueros de animales (Pírez-Schirmer, et al, 2019).…”

Section: Discussionunclassified

“…Como primer paso hacia estos objetivos, en este trabajo se presentan los resultados del análisis de peces provenientes de un bioensayo subcrónico de exposición (18 días) en Astraloheros facetus (nv: Castañetas), utilizando una floración productora de MCs en concentraciones frecuentemente encontradas en nuestros cuerpos de agua en temporada estival. La concentración de MCs en los peces expuestos fue analizada por dos métodos inmunoquímicos, recientemente desarrollados en el país, que involucran la utilización de anticuerpos monoclonales recombinantes de llama (nanobodies): ELISA (Pírez-Schirmer, et al, 2017) y MALDI-TOF cuantitativo con pre-concentración en partículas magnéticas (Pírez-Schirmer, et al, 2019). Además se realizaron cortes histológicos de los peces para el estudio de los daños provocados por la exposición (histopatología).…”

Section: Figura 1 Estructura General De Las Microcistinasunclassified

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Nuevas metodologías para el análisis de microcistinas en peces. Estudio de Astraloheros Facetus expuestos in vitro.

Baharian

Letamendía

Pírez

et al. 2020

INNOTEC

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Section: Discussionunclassified

Section: Figura 1 Estructura General De Las Microcistinasunclassified

Nuevas metodologías para el análisis de microcistinas en peces. Estudio de Astraloheros Facetus expuestos in vitro.

Baharian

Letamendía

Pírez

et al. 2020

INNOTEC

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“…The ELISA developed with this Nb turned out to be highly sensitive and selective for the analysis of the water samples [ 45 ]. In a subsequent application, the Nb was used as an immuno-concentration element for the quantitative analysis of MCs captured on magnetic beads, functionalized with NbA2.3, and pre-loaded with a MC-analog internal standard prior to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) [ 46 ]. This methodology, named Nb-QMALDI MS, is very sensitive and allows for the determination of multiple chemical MC-variants simultaneously by MALDI-TOF MS, without previous chromatographic separation.…”

Section: Introductionmentioning

confidence: 99%

Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody

Badagian

Schirmer

Pérez-Parada

et al. 2023

Toxins

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The development of simple, reliable, and cost-effective methods is critically important to study the spatial and temporal variation of microcystins (MCs) in the food chain. Nanobodies (Nbs), antigen binding fragments from camelid antibodies, present valuable features for analytical applications. Their small antigen binding site offers a focused recognition of small analytes, reducing spurious cross-reactivity and matrix effects. A high affinity and broad cross-reactivity anti-MCs-Nb, from a llama antibody library, was validated in enzyme linked immunosorbent assay (ELISA), and bound to magnetic particles with an internal standard for pre-concentration in quantitative-matrix-assisted laser desorption ionization-time of flight mass spectrometry (Nb-QMALDI MS). Both methods are easy and fast; ELISA provides a global result, while Nb-QMALDI MS allows for the quantification of individual congeners and showed excellent performance in the fish muscle extracts. The ELISA assay range was 1.8–29 ng/g and for Nb-QMALDI, it was 0.29–29 ng/g fish ww. Fifty-five fish from a MC-containing dam were analyzed by both methods. The correlation ELISA/sum of the MC congeners by Nb-QMALDI-MS was very high (r Spearman = 0.9645, p < 0.0001). Using ROC curves, ELISA cut-off limits were defined to accurately predict the sum of MCs by Nb-QMALDI-MS (100% sensitivity; ≥89% specificity). Both methods were shown to be simple and efficient for screening MCs in fish muscle to prioritize samples for confirmatory methods.

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“…Nbs are more amenable to faster engineering and cheaper production, and exhibit enhanced tissue penetration and lower immunogenicity [31]. The superior stability, high affinity, high target specificity, and fast clearance of Nbs offer special advantages compared to conventional antibodies in application of noninvasive diagnostic imaging and tumor therapy [31][32][33]. Nb is able to mediate the endocytosis of specific receptors, such as EGFR, in tumor cells, promoting Nb as tumor-targeting ligands for drug nanocarrier designed in targeted cancer therapy and diagnosis [34][35][36].…”

Section: Introductionmentioning

confidence: 99%

Targeted nanobody complex enhanced photodynamic therapy for lung cancer by overcoming tumor microenvironment

Zhang

Liu

et al. 2020

Cancer Cell Int

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Background To investigate the efficacy of a PLGA-based nanobody complex in photodynamic therapy (PDT) and NIR-II imaging in A549 tumor hypoxic model. Method IR1048-MZ was firstly synthesized by conjugating a nitro imidazole group to IR1048. IR1048-MZ and Cat were then encapsulated in PLGA-SH solution. Anti-EGFR-Nanobody was also expressed and purified, and finally Anti-EGFR-Nanobody@PLGA-IR1048MZ-Cat (Nb@IC-NPs) nanobody complex was obtained based on the formation of desulfide bond between PLGA-SH and Anti-EGFR-Nanobody. Size distribution and morphology were characterized by TEM and DLS. Spectrum of Nb@IC-NPs towards NTR was measured by UV and fluorescence, while the particle’s selective response was studied using fluorescence. The uptake of Nb@IC-NPs in A549 cells was observed by flow cytometry and CLSM. In the meantime, its’ catalytic ability that decomposes H2O2 both extra-and intra-cellular was observed by fluorescence and CLSM. In vitro photodynamic toxicity of Nb@IC-NPs was examined by MTT, Live/Dead Cell Staining, Flow Cytometry and Apoptosis Assay. Tumor-bearing model was constructed to observe a semi-quantitative fluorescent distribution and the possibility of NIR-II fluorescence/photoacoustic (PA) imaging. Effect of Nb@IC-NPs on enhancing A549 tumor hypoxia and expression profile of HIF-1α was investigated in the presence of NIR. An A549 tumor metastasis model was also constructed to confirm the complex’ potential to destroy primary tumor, inhibit lung metastasis, and prolong mice’ survival. Lastly, impact of Nb@IC-NPs on mice’ main organs and blood indices was observed. Results Nb@IC-NPs was successfully fabricated with good homogeneity. The fluorescent absorbance of Nb@IC-NPs showed a linear relationship with the concentration of NTR, and a higher concentration of NTR corresponded to a stronger photoacoustic signal. In addition, Nb@IC-NPs showed a stable selectivity toward NTR. Our results also suggested a high efficient uptake of Nb@IC-NPs in A549 cells, which was more efficient than IC-NPs and IR1048-MZ alone. In vitro assays confirmed the effects of Nb@IC-NPs on catalytic O2 generation even in hypoxic cells. The cell viability was upregulated with the nanocomplex at the absence of the laser, whereas it was dramatically declined with laser treatment that excited at 980 nm. Nb@IC-NPs achieved tumor hypoxia NIR-II/PA imaging through assisting A549 gathering. When NIR was applied, Nb@IC-NPs can significantly relieve A549 cellular/tumor hypoxia by generating more reactive oxygen species (ROS), which in turn helps lower the expression level of HIF-1α. In summary, Nb@IC-NPs based PDT can efficiently decimate A549 primary tumor, inhibit metastatic lung cancer, and prolong the lifespan of the mice under tolerable dosage. At last, in vivo toxicity tests of the nanocomplex showed its biosafety to the main organs and normal blood indices values. Conclusion Nb@IC-NPs improves tumor hypoxia through catalytic reaction and lowers the expression level of HIF-1α. It achieves tumor PA imaging through intensified NIR-II fluorescence signal that caused by response of the complex to the lesion’s nitroreductase (NTR). Nb@IC-NPs based PDT can efficiently kill A549 primary tumor, inhibit a lung metastasis, as well as prolong mice’ survival cycle.

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Oriented Functionalization of Magnetic Beads with in Vivo Biotinylated Nanobodies for Rapid MALDI-TOF MS Ultrasensitive Quantitation of Microcystins in Biological Samples

Cited by 13 publications

References 25 publications

Nuevas metodologías para el análisis de microcistinas en peces. Estudio de Astraloheros Facetus expuestos in vitro.

Nuevas metodologías para el análisis de microcistinas en peces. Estudio de Astraloheros Facetus expuestos in vitro.

Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody

Targeted nanobody complex enhanced photodynamic therapy for lung cancer by overcoming tumor microenvironment

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