32 research outputs found
Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay
The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL−1, which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.Fil: González Techera, Andrés. Universidad de la República; UruguayFil: Zon, MarÃa Alicia. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica. Area ElectroquÃmica; Argentina. Consejo Nacional de Investigaciones CientÃficas y Técnicas; ArgentinaFil: Molina, Patricia Gabriela. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica. Area ElectroquÃmica; Argentina. Consejo Nacional de Investigaciones CientÃficas y Técnicas; ArgentinaFil: Fernandez, Hector. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica. Area ElectroquÃmica; Argentina. Consejo Nacional de Investigaciones CientÃficas y Técnicas; ArgentinaFil: González Sapienza, Gualberto. Universidad de la República; UruguayFil: Arevalo, Fernando Javier. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica. Area ElectroquÃmica; Argentina. Consejo Nacional de Investigaciones CientÃficas y Técnicas; Argentin
Ultra-sensitive electrochemical immunosensor using analyte peptidomimetics selected from phage display peptide libraries
Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC 50 of 0.15ngmL -1 (linear range from 4.4×10 -3 to 10ngmL -1). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9×1000nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors.Fil: Arevalo, Fernando Javier. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; ArgentinaFil: González Techera, Andrés. Universidad de la República; UruguayFil: Zon, MarÃa Alicia. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; ArgentinaFil: González Sapienza, Gualberto. Universidad de la República; UruguayFil: Fernández, Héctor. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; Argentin
Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells
Background Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. Methods Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. Results The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. Conclusions This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. General significance This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.Fil: Rossotti, MartÃn. Universidad de la República. Facultad de QuÃmica; UruguayFil: Tabares, SofÃa. Universidad de la República. Facultad de QuÃmica; UruguayFil: Alfaya, LucÃa. Universidad de la República. Facultad de QuÃmica; UruguayFil: Leizagoyen, Carmen. No especifÃca;Fil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Córdoba. Centro de Investigaciones en BioquÃmica ClÃnica e InmunologÃa; ArgentinaFil: González Sapienza, Gualberto. Universidad de la República. Facultad de QuÃmica; Urugua
Development of an impedimetric immunosensor to determine microcystin-LR: New approaches in the use of the electrochemical impedance spectroscopy was used in determining to determine kinetic parameters of immunoreactions
The development of new electrochemical immunosensors for the detection of environmental contaminants is of great interest due to their simplicity, high sensitivity, and extended analytical range. Because of the antibody is immobilized on the electrode, it is important to determine its loss of reactivity after immobilization. In this work, two aspects were addressed. First, we developed a new methodology based on electrochemical impedance (EI) to determine the kinetic parameters associated with immunoreactions carried out on the electrode. Second, an electrochemical immunosensor based on electrochemical impedance spectroscopy (EIE) was developed to determine microcystin-LR in drinking water samples. Microcystin-LR determination was based on a label-free non-competitive immunoassay. The electrochemical immunosensor shows a limit of detection (LOD) of 33 pg mL−1 (3.32 × 10−11 mol L−1 or 0.033 μg L−1) which is well below the WHO guideline recommendation of 1 μg L−1 and 40 times better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA assay. In addition, an acceptable accuracy, with recovery percentages close to 100% were found. The label-free immunosensor is a valuable tool to monitor microcystin-LR in drinking water samples and the environment.Fil: Boffadossi, Micaela. Universidad Nacional de RÃo Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; ArgentinaFil: Di Tocco, Aylen. Universidad Nacional de RÃo Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; ArgentinaFil: Lassabe, Gabriel. Universidad de la República; UruguayFil: Pirez Schirmer, Macarena. Universidad de la República; UruguayFil: Robledo, Sebastian Noel. Universidad Nacional de RÃo Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de IngenierÃa. Departamento de TecnologÃa QuÃmica; ArgentinaFil: Fernandez, Hector. Universidad Nacional de RÃo Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; ArgentinaFil: Zon, MarÃa Alicia. Universidad Nacional de RÃo Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; ArgentinaFil: González Sapienza, Gualberto. Universidad de la República; UruguayFil: Arevalo, Fernando Javier. Universidad Nacional de RÃo Cuarto. Instituto para el Desarrollo Agroindustrial y de la Salud. - Consejo Nacional de Investigaciones CientÃficas y Técnicas. Centro CientÃfico Tecnológico Conicet - Córdoba. Instituto para el Desarrollo Agroindustrial y de la Salud; Argentina. Universidad Nacional de RÃo Cuarto. Facultad de Ciencias Exactas FisicoquÃmicas y Naturales. Departamento de QuÃmica; Argentin
Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation
Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1β, IL-6, IL-12p40, IFN-β) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid’s effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages
Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity
Immune checkpoint blockade using monoclonal antibodies (mAbs) able to block
programmed death-1 (PD-1)/PD-L1 axis represents a promising treatment for cancer. However,
it requires repetitive systemic administration of high mAbs doses, often leading to adverse effects.
We generated a novel nanobody against PD-1 (Nb11) able to block PD-1/PD-L1 interaction for
both mouse and human molecules. Nb11 was cloned into an adeno-associated virus (AAV) vector
downstream of four different promoters (CMV, CAG, EF1α, and SFFV) and its expression was
analyzed in cells from rodent (BHK) and human origin (Huh-7). Nb11 was expressed at high levels
in vitro reaching 2–20 micrograms/mL with all promoters, except SFFV, which showed lower levels.
Nb11 in vivo expression was evaluated in C57BL/6 mice after intravenous administration of AAV8
vectors. Nb11 serum levels increased steadily along time, reaching 1–3 microgram/mL two months
post-treatment with the vector having the CAG promoter (AAV-CAG-Nb11), without evidence of
toxicity. To test the antitumor potential of this vector, mice that received AAV-CAG-Nb11, or saline
as control, were challenged with colon adenocarcinoma cells (MC38). AAV-CAG-Nb11 treatment
prevented tumor formation in 30% of mice, significantly increasing survival. These data suggest that
continuous expression of immunomodulatory nanobodies from long-term expression vectors could
have antitumor effects with low toxicity
Local delivery of optimized nanobodies targeting the PD-1/PD-L1 axis with a self-amplifying RNA viral vector induces potent antitumor responses
Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond
adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using
small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based
on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and
mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for antiPD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a
potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better
therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in
the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic
nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing
infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid
electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect
A Family of Diverse Kunitz Inhibitors from Echinococcus granulosus Potentially Involved in Host-Parasite Cross-Talk
The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H+-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 – EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (KI*) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection