22 results on '"Gonzalez-Sapienza G"'
Search Results
2. ITREOH building of regional capacity to monitor recreational water: Development of a non-commercial microcystin ELISA and its impact on public health policy
- Author
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Brena, B M, Diaz, L, Sienra, D, Ferrari, G, Ferraz, N, Hellman, U, Gonzalez-Sapienza, G, and Last, Jerold A
- Subjects
ELISA ,microcystins ,cyanobacteria ,Fogarty International Center ,Uruguay ,Rio de la Plata ,water pollution ,monitoring - Abstract
In 2001, a University of California, Davis-University of the Republic, Montevideo, partnership created a Fogarty ITREOH program to exploit the potential of ELISA to provide a low-cost environmental analysis attractive to economically distressed countries of temperate South America. This paper describes the development and validation of an ELISA method for the determination of Cyanobacteria microcystin toxins in algal blooms, which release hepatotoxic metabolites that can reach toxic levels in rivers, lakes, or coastal estuaries used for recreation or water supplies. The assay made possible the first systematic monitoring of water from the Rio de la Plata at Montevideo over two summers. The project has been integrated into a bi-national effort to monitor the Rio de la Plata.
- Published
- 2006
3. Screening of dioxin-like compounds by complementary evaluation strategy utilising ELISA, micro-EROD, and HRGC-HRMS in soil and sediments from Montevideo, Uruguay
- Author
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Levy, W., Brena, B.M., Henkelmann, B., Bernhöft, S., Pirez, M., González-Sapienza, G., and Schramm, K.-W.
- Published
- 2014
- Full Text
- View/download PDF
4. Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis
- Author
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Segovia-de Los Santos P, Quintero-Campos P, Morais S, Echaides C, Maquieira Á, Lassabe G, and Gonzalez-Sapienza G
- Abstract
Commonly, serological immunoassays and diagnostic kits include reference standard reagents (calibrators) that contain specific antibodies to be measured, which are used for the quantification of unknown antibodies present in the sample. However, in some cases, such as the diagnosis of allergies or autoimmune diseases, it is often difficult to have sufficient quantities of these reference standards, and there are limitations to their lot-to-lot reproducibility and standardization over time. To overcome this difficulty, this study introduces the use of surrogate recombinant calibrators formulated on the basis of two single-domain antibodies (nanobodies) combined through a short peptide linker to produce a recombinant bispecific construct. One of the nanobodies binds to the cognate analyte of the target antibody and the second is specific for the paratope of the secondary detecting antibody. The bispecific nanobody inherits the outstanding properties of stability and low-cost production by bacterial fermentation of the parent nanobodies, and once calibrated against the biological reference standard, it can be reproduced indefinitely from its sequence in a highly standardized manner. As a proof of concept, we present the generation and characterization of two bispecific calibrators with potential application for the diagnosis of allergy against the antibiotics aztreonam and amoxicillin in humans.
- Published
- 2022
5. Polyclonal antibody-based noncompetitive immunoassay for small analytes developed with short peptide loops isolated from phage libraries
- Author
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Gonzalez-Techera, A., Kim, H.J., Gee, S.J., Last, J.A., Hammock, B.D., and Gonzalez-Sapienza, G.
- Subjects
Enzyme-linked immunosorbent assay -- Research ,Monoclonal antibody probes -- Usage ,Chemistry - Abstract
To date, there are a few technologies for the development of noncompetitive immunoassays for small molecules, the most common of which relies on the use of anti-immunocomplex antibodies. This approach is laborious, case specific, and relies upon monoclonal antibody technology for its implementation. We recently demonstrated that, in the case of monoclonal antibody-based immunoassays, short peptide loops isolated from phage display libraries can be used as substitutes of the anti-immunocomplex antibodies for noncompetitive immunodetection of small molecules. The aim of this work was to demonstrate that such phage ligands can be isolated even when the selector antibodies are polyclonal in nature. Using phenoxybenzoic acid (PBA), a major pyrethroid metabolite, as a model system, we isolated the CFNGKDWLYC peptide after panning a cyclic peptide library on the PBA/anti-PBA immunocomplex. The sensitivity of the noncompetitive enzyme-linked immunosorbent assay (ELISA) setup with this peptide was 5-fold (heterologous) or 400-fold (homologous) higher than that of the competitive assay setup with the same antibody. Phage anti-inmunocomplex assay (PHAIA) was also easily adapted into a rapid and highly sensitive dipstick assay. The method not only provides a positive readout but also constitutes a major shortcut in the development of sensitive polyclonal-based assays, avoiding the need of synthesizing heterologous competing haptens.
- Published
- 2007
6. Phage anti-immune complex assay: general strategy for noncompetitive immunodetection of small molecules
- Author
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Gonzalez-Techera, A., Vanrell, L., Last, J.A., Hammock, B.D., and Gonzalez-Sapienza, G.
- Subjects
Microbiological assay -- Properties ,Molecular dynamics -- Research ,Chemistry - Abstract
Due to their size, small molecules cannot be simultaneously bound by two antibodies, precluding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. This has prompted the development of anti-immune complex antibodies, but these are difficult to produce, and often exhibit high cross-reactivity with the unliganded primary antibody. This work demonstrates that anti-immune complex antibodies can be substituted by phage particles isolated from phage display peptide libraries. Phages bearing specific small peptide loops allowed to focus the recognition to changes in the binding area of the immune complex. The concept was tested using environmental and drug analytes; with improved sensitivity and ready adaptation into on-site formats. Peptides specific for different immune complexes can be isolated from different peptide libraries in a simple and systematic fashion allowing the rapid development of noncompetitive assays for small molecules
- Published
- 2007
7. The apo form of the triclocarban-binding single domain camelid nanobody VHH T10
- Author
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Tabares-da Rosa, S., primary, Gonzalez-Sapienza, G., additional, and Wilson, D.K., additional
- Published
- 2018
- Full Text
- View/download PDF
8. The hapten triclocarban bound to the single domain camelid nanobody VHH T9
- Author
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Wogulis, L.A., primary, Wogulis, M.D., additional, Tabares-da Rosa, S., additional, Gonzalez-Sapienza, G., additional, and Wilson, D.K., additional
- Published
- 2018
- Full Text
- View/download PDF
9. The hapten triclocarban bound to the single domain camelid nanobody VHH T10
- Author
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Tabares-da Rosa, S., primary, Gonzalez-Sapienza, G., additional, and Wilson, D.K., additional
- Published
- 2018
- Full Text
- View/download PDF
10. A highly sensitive nanobody-based immunoassay detecting SARS-CoV-2 nucleocapsid protein using all-recombinant reagents.
- Author
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Segovia-de Los Santos P, Padula-Roca C, Simon X, Echaides C, Lassabe G, and Gonzalez-Sapienza G
- Subjects
- Humans, SARS-CoV-2, Indicators and Reagents, Pandemics, Antibodies, Viral, Immunoassay methods, Nucleocapsid Proteins, COVID-19 diagnosis
- Abstract
Antigen tests have been crucial for managing the COVID-19 pandemic by identifying individuals infected with SARS-CoV-2. This remains true even after immunity has been widely attained through natural infection and vaccination, since it only provides moderate protection against transmission and is highly permeable to the emergence of new virus variants. For this reason, the widespread availability of diagnostic methods is essential for health systems to manage outbreaks effectively. In this work, we generated nanobodies to the virus nucleocapsid protein (NP) and after an affinity-guided selection identified a nanobody pair that allowed the detection of NP at sub-ng/mL levels in a colorimetric two-site ELISA, demonstrating high diagnostic value with clinical samples. We further modified the assay by using a nanobody-NanoLuc luciferase chimeric tracer, resulting in increased sensitivity (detection limit = 61 pg/mL) and remarkable improvement in diagnostic performance. The luminescent assay was finally evaluated using 115 nasopharyngeal swab samples. Receiver Operating Characteristic (ROC) curve analysis revealed a sensitivity of 78.7% (95% confidence interval: 64.3%-89.3%) and specificity of 100.0% (95% confidence interval: 94.7%-100.0%). The test allows the parallel analysis of a large number of untreated samples, and fulfills our goal of producing a recombinant reagent-based test that can be reproduced at low cost by other laboratories with recombinant expression capabilities, aiding to build diagnostic capacity., Competing Interests: XS is employed by ATGen SRL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Segovia-de los Santos, Padula-Roca, Simon, Echaides, Lassabe and Gonzalez-Sapienza.)
- Published
- 2023
- Full Text
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11. Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody.
- Author
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Badagian N, Pírez Schirmer M, Pérez Parada A, Gonzalez-Sapienza G, and Brena BM
- Subjects
- Animals, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Enzyme-Linked Immunosorbent Assay, Microcystins analysis, Single-Domain Antibodies
- Abstract
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.
- Published
- 2023
- Full Text
- View/download PDF
12. Induction of antigen-specific tolerance by nanobody-antigen adducts that target class-II major histocompatibility complexes.
- Author
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Pishesha N, Harmand T, Smeding LY, Ma W, Ludwig LS, Janssen R, Islam A, Xie YJ, Fang T, McCaul N, Pinney W 3rd, Sugito HR, Rossotti MA, Gonzalez-Sapienza G, and Ploegh HL
- Subjects
- Animals, Autoantigens, Histocompatibility, Major Histocompatibility Complex, Mice, Encephalomyelitis, Autoimmune, Experimental, Immune Tolerance
- Abstract
The association of autoimmune diseases with particular allellic products of the class-II major histocompatibility complex (MHCII) region implicates the presentation of the offending self-antigens to T cells. Because antigen-presenting cells are tolerogenic when they encounter an antigen under non-inflammatory conditions, the manipulation of antigen presentation may induce antigen-specific tolerance. Here, we show that, in mouse models of experimental autoimmune encephalomyelitis, type 1 diabetes and rheumatoid arthritis, the systemic administration of a single dose of nanobodies that recognize MHCII molecules and conjugated to the relevant self-antigen under non-inflammatory conditions confers long-lasting protection against these diseases. Moreover, co-administration of a nanobody-antigen adduct and the glucocorticoid dexamethasone, conjugated to the nanobody via a cleavable linker, halted the progression of established experimental autoimmune encephalomyelitis in symptomatic mice and alleviated their symptoms. This approach may represent a means of treating autoimmune conditions., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2021
- Full Text
- View/download PDF
13. Sortase-Mediated Phage Decoration for Analytical Applications.
- Author
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Ding Y, Chen H, Li J, Huang L, Song G, Li Z, Hua X, Gonzalez-Sapienza G, Hammock BD, and Wang M
- Subjects
- Enzyme-Linked Immunosorbent Assay, Immunoassay, Immunoglobulin Fragments, Peptide Library, Bacteriophages
- Abstract
Phage-borne peptides and antibody fragments isolated from phage display libraries have proven to be versatile and valuable reagents for immunoassay development. Due to the lack of convenient and mild-condition methods for the labeling of the phage particles, isolated peptide/protein affinity ligands are commonly removed from the viral particles and conjugated to protein tracers or nanoparticles for analytical use. This abolishes the advantage of isolating ready-to-use affinity binders and creates the risk of affecting the polypeptide activity. To circumvent this problem, we optimized the phage display system to produce phage particles that express the affinity binder on pIII and a polyglycine short peptide fused to pVIII that allows the covalent attachment of tracer molecules employing sortase A. Using a llama heavy chain only variable domain (VHH) against the herbicide 2,4-D on pIII as the model, we showed that the phage can be extensively decorated with a rhodamine-LPETGG peptide conjugate or the protein nanoluciferase (Nluc) equipped with a C-terminal LPETGG peptide. The maximum labeling amounts of rhodamine-LPETGG and Nluc-LPETGG were 1238 ± 63 and 102 ± 16 per phage, respectively. The Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of 2,4-D. The limit of detection and 50% inhibition concentration of P-BLEIA were 0.491 and 2.15 ng mL
-1 , respectively, which represent 16-fold and 8-fold improvement compared to the phage enzyme-linked immunosorbent assay. In addition, the P-BLEIA showed good accuracy for the detection of 2,4-D in spiked samples.- Published
- 2021
- Full Text
- View/download PDF
14. Recombinant, Fluorescent, Peptidomimetic Tracer for Immunodetection of Imidaclothiz.
- Author
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Ding Y, Hua X, Du M, Yang Q, Hou L, Wang L, Liu F, Gonzalez-Sapienza G, and Wang M
- Subjects
- Chromatography, High Pressure Liquid, Humans, Peptide Library, Fluorescence, Immunoassay, Peptides chemistry, Peptidomimetics, Thiazoles analysis
- Abstract
Peptidomimetic and anti-immunocomplex peptides, which can be readily isolated from a phage-display library, have shown great potential for small-molecule immunoassay development because they typically improve the sensitivity and avoid the use of chemical haptens as coatings or tracer antigens. However, phage-borne peptides are unconventional immunoassay reagents, which greatly limits their use in commercial applications, and require secondary reagents for detection. In order to overcome these limitations, we used C2-15, a peptidomimetic of imidaclothiz, as a model peptide fused to emerald-green fluorescent protein (EmGFP) at the N-terminus (C2-15-EmGFP) and C-terminus (EmGFP-C2-15) to generate novel fluorescent-peptide tracers. Both recombinant fluorophores reacted with similar affinity to the anti-imidaclothiz monoclonal antibody 1E
7 , but because of its higher expression, C2-15-EmGFP was chosen to develop a competitive magnetic-separation fluorescence immunoassay (MSFIA). After a competitive step with the analyte, the C2-15-EmGFP-antibody complex bound to the magnetic beads was separated with a magnet, and because of the fast dissociation of the peptide-antibody interaction, the fluorescence signal was detected following the spontaneous dissociation of the complex in fresh buffer. The concentration of imidaclothiz causing the 50% inhibitory concentration (IC50 ) was 11.00 ng mL-1 , and the MSFIA performed with excellent recovery and had a good correlation with high-performance liquid chromatography in different matrices.- Published
- 2018
- Full Text
- View/download PDF
15. Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications.
- Author
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Gonzalez-Sapienza G, Rossotti MA, and Tabares-da Rosa S
- Abstract
With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs) has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb)] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.
- Published
- 2017
- Full Text
- View/download PDF
16. Rapid quantitative analysis of microcystins in raw surface waters with MALDI MS utilizing easily synthesized internal standards.
- Author
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Roegner AF, Schirmer MP, Puschner B, Brena B, and Gonzalez-Sapienza G
- Subjects
- Microcystins chemistry, Molecular Structure, Reference Standards, Sensitivity and Specificity, Uruguay, Fresh Water chemistry, Harmful Algal Bloom, Microcystins analysis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Water Pollutants, Chemical analysis
- Abstract
The freshwater cyanotoxins, microcystins (MCs), pose a global public health threat as potent hepatotoxins in cyanobacterial blooms; their persistence in drinking and recreational water has been associated with potential chronic effects in addition to acute intoxications. Rapid and accurate detection of the over 80 structural congeners is challenged by the rigorous and time consuming clean up required to overcome interference found in raw water samples. MALDI-MS has shown promise for rapid quantification of individual congeners in raw water samples, with very low operative cost, but so far limited sensitivity and lack of available and versatile internal standards (ISs) has limited its use. Two easily synthesized S-hydroxyethyl-Cys(7)-MC-LR and -RR ISs were used to generate linear standard curves in a reflectron MALDI instrument, reproducible across several orders of magnitude for MC-LR, -RR and -YR. Minimum quantification limits in direct water samples with no clean up or concentration step involved were consistently below 7 μg/L, with recoveries from spiked samples between 80 and 119%. This method improves sensitivity by 30 fold over previous reports of quantitative MALDI-TOF applications to MCs and provides a salient option for rapid throughput analysis for multiple MC congeners in untreated raw surface water blooms as a means to identify source public health threats and target intervention strategies within a watershed. As demonstrated by analysis of a set of samples from Uruguay, utilizing the reaction of different MC congeners with alternate sulfhydryl compounds, the m/z of the IS can be customized to avoid overlap with interfering compounds in local surface water samples., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
17. Limited analytical capacity for cyanotoxins in developing countries may hide serious environmental health problems: simple and affordable methods may be the answer.
- Author
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Pírez M, Gonzalez-Sapienza G, Sienra D, Ferrari G, Last M, Last JA, and Brena BM
- Subjects
- Chlorophyll analysis, Chlorophyll A, Cyanobacteria Toxins, Developing Countries, Environmental Monitoring economics, Enzyme-Linked Immunosorbent Assay, Risk Management, Uruguay, Bacterial Toxins analysis, Cyanobacteria, Environmental Monitoring methods, Harmful Algal Bloom, Marine Toxins analysis, Microcystins analysis, Rivers microbiology, Water Microbiology
- Abstract
In recent years, the international demand for commodities has prompted enormous growth in agriculture in most South American countries. Due to intensive use of fertilizers, cyanobacterial blooms have become a recurrent phenomenon throughout the continent, but their potential health risk remains largely unknown due to the lack of analytical capacity. In this paper we report the main results and conclusions of more than five years of systematic monitoring of cyanobacterial blooms in 20 beaches of Montevideo, Uruguay, on the Rio de la Plata, the fifth largest basin in the world. A locally developed microcystin ELISA was used to establish a sustainable monitoring program that revealed seasonal peaks of extremely high toxicity, more than one-thousand-fold greater than the WHO limit for recreational water. Comparison with cyanobacterial cell counts and chlorophyll-a determination, two commonly used parameters for indirect estimation of toxicity, showed that such indicators can be highly misleading. On the other hand, the accumulated experience led to the definition of a simple criterion for visual classification of blooms, that can be used by trained lifeguards and technicians to take rapid on-site decisions on beach management. The simple and low cost approach is broadly applicable to risk assessment and risk management in developing countries., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
18. Nanopeptamers for the development of small-analyte lateral flow tests with a positive readout.
- Author
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Vanrell L, Gonzalez-Techera A, Hammock BD, and Gonzalez-Sapienza G
- Subjects
- Molecular Structure, Azepines analysis, Isoxazoles analysis, Nanotechnology, Oxazolidinones analysis, Thiocarbamates analysis
- Abstract
There is a great demand for rapid tests that can be used on-site for the detection of small analytes, such as pesticides, persistent organic pollutants, explosives, toxins, medicinal and abused drugs, hormones, etc. Dipsticks and lateral flow devices, which are simple and provide a visual readout, may be the answer, but the available technology for these compounds requires a competitive format that loses sensitivity and produces readings inversely proportional to the analyte concentration, which is counterintuitive and may lead to potential misinterpretation of the result. In this work, protein-multipeptide constructs composed of anti-immunocomplex peptides selected from phage libraries and streptavidin/avidin as core protein were used for direct detection of small compounds in a noncompetitive two-site immunoassay format that performs with increased sensitivity and positive readout. These constructs that we termed "nanopeptamers" allow the development of rapid point-of-use tests with a positive visual end point of easy interpretation. As proof of concept, lateral flow assays for the herbicides molinate and clomazone were developed and their performance was characterized with field samples.
- Published
- 2013
- Full Text
- View/download PDF
19. A monoclonal antibody-based copro-ELISA kit for canine echinococcosis to support the PAHO effort for hydatid disease control in South America.
- Author
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Morel N, Lassabe G, Elola S, Bondad M, Herrera S, Marí C, Last JA, Jensen O, and Gonzalez-Sapienza G
- Subjects
- Animals, Dog Diseases parasitology, Dogs, Echinococcosis diagnosis, Echinococcosis parasitology, Enzyme-Linked Immunosorbent Assay methods, Feces chemistry, Female, Male, Pan American Health Organization, Peru, Reagent Kits, Diagnostic, Reproducibility of Results, Sensitivity and Specificity, Antibodies, Monoclonal, Antigens, Helminth analysis, Dog Diseases diagnosis, Echinococcosis veterinary, Feces parasitology, Parasitology methods, Veterinary Medicine methods
- Abstract
Cystic echinococcosis is still a major concern in South America. While some regions show advances in the control of the disease, others have among the highest incidence in the world. To reverse this situation the Pan American Health Organization (PAHO) has launched a regional project on cystic echinococcosis control and surveillance. An early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. Under this premise, we have developed a new copro-ELISA test after extensive screening of a large panel of monoclonal antibodies (MAbs) and polyclonal sera, which performs with high standards of sensitivity (92.6%) and specificity (86.4%) as established by necropsy diagnosis of dogs. The key component of the test, MAbEg9 has a convenient IgG isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. Time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day 20 post infection. The test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of 3 months at room temperature. This characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in Peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion.
- Published
- 2013
- Full Text
- View/download PDF
20. Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR.
- Author
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Ferraz N, Carnevia D, Nande G, Rossotti M, Miguez MN, Last JA, and Gonzalez-Sapienza G
- Subjects
- Animals, Antigens genetics, Biomarkers analysis, Cichlids, Cloning, Molecular, DNA Primers genetics, Endocrine System drug effects, Environmental Monitoring methods, Female, Polymerase Chain Reaction, Recombinant Proteins genetics, Time Factors, Egg Proteins analysis, Endocrine Disruptors analysis, Endocrine System metabolism, Hormone Antagonists analysis, Immunoassay methods, Protein Precursors analysis, Vitellogenins analysis
- Abstract
Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L(-1) of 17-beta-estradiol, 100 ng L(-1) of ethynylestradiol, and 6.6 microg L(-1) of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.
- Published
- 2007
- Full Text
- View/download PDF
21. ELISA as an affordable methodology for monitoring groundwater contamination by pesticides in low-income countries.
- Author
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Brena BM, Arellano L, Rufo C, Last MS, Montaño J, Egaña Cerni E, Gonzalez-Sapienza G, and Last JA
- Subjects
- Agriculture, Atrazine analysis, Carbaryl analysis, Cities, Environmental Monitoring methods, Enzyme-Linked Immunosorbent Assay methods, Herbicides analysis, Naphthols analysis, Seasons, Uruguay, Water Pollutants, Chemical toxicity, Pesticides analysis, Vegetables chemistry, Water Pollutants, Chemical analysis, Water Supply
- Abstract
The traditional instrumental technology for pesticide residue analysis is too expensive and labor-intense to meet the regional needs concerning environmental monitoring. ELISA methodology was used for a pilot scale study of groundwater quality in an agricultural region a few kilometers southwest of Montevideo, the capital city of Uruguay. The study spanned 2 years and examined concentrations (detection limits are given in [ppb]) of two triazine herbicides (simazine [0.3] and atrazine [0.4]) and the carbamate insecticide carbaryl [10] and its major metabolite 1-naphthol [17]. In general, pesticide concentrations were below detection limits in the samples tested and in all cases were well below the maximum contaminant levels set by the U.S. EPA. 1-Naphthol was detected frequently by ELISA, but the assay may have tended to systematically overestimate this analyte. To our knowledge, this is the first study of its type in Uruguay and perhaps the first systematic approach to monitoring for organic pesticides in groundwater water sources in the temperate region of South America.
- Published
- 2005
- Full Text
- View/download PDF
22. Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies selected from an immune b9 allotype rabbit antibody library.
- Author
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Popkov M, Jendreyko N, Gonzalez-Sapienza G, Mage RG, Rader C, and Barbas CF 3rd
- Subjects
- Amino Acid Sequence, Animals, Antibodies metabolism, Humans, Kinetics, Mice, Molecular Sequence Data, Protein Binding, Rabbits, Sequence Alignment, Vascular Endothelial Growth Factor Receptor-2 metabolism, Antibodies immunology, Immunoglobulin Allotypes immunology, Peptide Library, Vascular Endothelial Growth Factor Receptor-2 immunology
- Abstract
Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Models of murine tumor angiogenesis and receptor-specific antibodies are required to evaluate roles of VEGF receptors in mouse models of human cancer. Human VEGFR2 (also known as KDR) and murine VEGFR2 (or Flk-1) share 85% amino acid sequence identity in their extracellular domain. We describe here the development of antibodies that cross-react with mouse and human VEGFR2. High-affinity, species cross-reactive, Fabs specific for KDR/Flk-1 were selected from an antibody phage display library generated from an immunized rabbit of b9 allotype. The selected chimeric rabbit/human Fabs were found to bind to purified KDR and Flk-1 with nanomolar affinity. Three of the selected Fabs detected KDR expression on human endothelial cells as well as Flk-1 on murine endothelial cells. The availability of anti-VEGFR2 Fab with species cross-reactivity will help to decipher the functional role of KDR/Flk-1 in tumor biology as well as facilitate the preclinical evaluation of the suitability of KDR/Flk-1 for drug targeting. This report underscores our earlier finding that b9 rabbits are excellent sources for high-affinity cross-reactive antibodies with therapeutic potential., (Copyright 2004 Elsevier B.V.)
- Published
- 2004
- Full Text
- View/download PDF
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