SARS-CoV-2 Rapid Antigen Test Based on a New Anti-Nucleocapsid Protein Monoclonal Antibody: Development and Real-Time Validation.

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants-Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients' follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Crohn's Disease in a Patient with Tuberculosis: A Diagnostic and Therapeutic Challenge

Introduction: Crohn's disease (CD) is unusual in Colombia. Tuberculosis (TB) occurs more frequently, but intestinal involvement is rare. Differentiating these two entities and treating the cases in which they coexist is a challenge. Clinical case: A 28-year-old man with three months of constitutional, respiratory, and GI symptoms was initially diagnosed with pulmonary tuberculosis, and all the clinical manifestations were attributed to this entity. Given the absence of improvement with treatment and the sum of radiological, endoscopic, and pathological findings, CD was confirmed. Treatment was complex due to the coexistence of the two entities, although he finally went into remission with the use of biologicals. Discussion: Diagnosing CD requires the sum of clinical and paraclinical findings. A therapeutic test may be necessary to differentiate it from intestinal TB. The treatment of CD in a patient with TB has some limitations; steroids are not contraindicated, and biologicals must be initiated cautiously. Conclusions: Differentiating CD from intestinal TB is a diagnostic challenge. Therapeutic management when these two entities coexist requires an interdisciplinary approach.

Introducción: la enfermedad de Crohn (EC) es inusual en Colombia. La tuberculosis (TB) se presenta con mayor frecuencia, pero el compromiso intestinal es raro. Diferenciar estas dos entidades y el tratamiento de los casos en los que coexisten es un reto. Caso clínico: hombre de 28 años con 3 meses de síntomas constitucionales, respiratorios y gastrointestinales al que en un inicio se le confirmó el diagnóstico de tuberculosis pulmonar y se le atribuyó a esta entidad todas las manifestaciones clínicas. Ante la ausencia de mejoría con el tratamiento y la suma de hallazgos radiológicos, endoscópicos y patológicos, se confirmó EC. El tratamiento fue difícil debido a la coexistencia de las dos entidades, aunque finalmente presentó remisión con el uso de biológicos. Discusión: diagnosticar la EC requiere de la sumatoria de hallazgos clínicos y paraclínicos. Para diferenciarla de TB intestinal puede llegar a ser necesaria una prueba terapéutica. El tratamiento de EC en un paciente con TB tiene algunas limitaciones, los esteroides no se contraindican de manera absoluta y el inicio de biológicos debe hacerse con precaución. Conclusiones: diferenciar la EC de la TB intestinal es un reto diagnóstico. El enfoque terapéutico cuando coexisten estas dos entidades requiere un abordaje interdisciplinario.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

SARS-CoV-2 rapid antigen test based on a new anti-nucleocapsid protein monoclonal antibody: development and real-time validation

SARS-CoV-2 diagnostic tests have become an important tool for pandemic control. Among the alternatives for COVID-19 diagnosis, antigen rapid diagnostic tests (Ag-RDT) are very convenient and widely used. However, as SARS-CoV-2 variants may continuously emerge, the replacement of tests and reagents may be required to maintain the sensitivity of Ag-RDTs. Here, we describe the development and validation of an Ag-RDT during an outbreak of the Omicron variant, including the characterization of a new monoclonal antibody (anti-DTC-N 1B3 mAb) that recognizes the Nucleocapsid protein (N). The anti-DTC-N 1B3 mAb recognized the sequence TFPPTEPKKDKKK located at the C-terminus of the N protein of main SARS-CoV-2 variants of concern. Accordingly, the Ag-RDT prototypes using the anti-DTC-N 1B3 mAB detected all the SARS-CoV-2 variants—Wuhan, Alpha, Gamma, Delta, P2 and Omicron. The performance of the best prototype (sensitivity of 95.2% for samples with Ct ≤ 25; specificity of 98.3% and overall accuracy of 85.0%) met the WHO recommendations. Moreover, results from a patients’ follow-up study indicated that, if performed within the first three days after onset of symptoms, the Ag-RDT displayed 100% sensitivity. Thus, the new mAb and the Ag-RDT developed herein may constitute alternative tools for COVID-19 point-of-care diagnosis and epidemiological surveillance.

Recombinant human TIM-3 ectodomain expressed in bacteria and recovered from inclusion bodies as a stable and active molecule

Introduction: Microbial systems, such as Escherichia coli, as host recombinant expression is the most versatile and the cheapest system for protein production, however, several obstacles still remain, such as recovery of soluble and functional proteins from inclusion bodies, elimination of lipopolysaccharides (LPS) contamination, incomplete synthesis, degradation by proteases, and the lack of post-translational modifications, which becomes even more complex when comes to membrane proteins, because they are difficult not only to produce but also to keep in solution in its active state. T-cell Immunoglobulin and Mucin domain 3 (TIM-3) is a type I transmembrane protein that is predominantly expressed on the surface of T lymphocytes, natural killer (NK) cells, dendritic cells, and macrophages, playing a role as a negative immune checkpoint receptor. TIM-3 comprises a single ectodomain for interaction with immune system soluble and cellular components, a transmembrane domain, and a cytoplasmic tail, responsible for the binding of signaling and scaffolding molecules. TIM-3 pathway holds potential as a therapeutic target for immunotherapy against tumors, autoimmunity, chronic virus infections, and various malignancies, however, many aspects of the biology of this receptor are still incompletely understood, especially regarding its ligands. Methods: Here we overcome, for the first time, the challenge of the production of active immune checkpoint protein recovered from bacterial cytoplasmic inclusion bodies, being able to obtain an active, and non-glycosylated TIM-3 ectodomain (TIM-3-ECD), which can be used as a tool to better understand the interactions and roles of this immune checkpoint. The TIM-3 refolding was obtained by the association of high pressure and alkaline pH. Results: The purified TIM-3-ECD showed the correct secondary structure and was recognized from anti-TIM-3 structural-dependent antibodies likewise commercial TIM-3-ECD was produced by a mammal cells system. Furthermore, immunofluorescence showed the ability of TIM-3-ECD to bind to the surface of lung cancer A549 cells and to provide an additional boost for the expression of the lymphocyte activation marker CD69 in anti-CD3/CD28 activated human PBMC. Discussion: Taken together these results validated a methodology able to obtain active checkpoint proteins from bacterial inclusion bodies, which will be helpful to further investigate the interactions of this and others not yet explored immune checkpoints.

Identification of zika virus ns1-derived peptides with potential applications in serological tests

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Therapeutic Antibodies Against Shiga Toxins: Trends and Perspectives.

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

Therapeutic antibodies against shiga toxins: trends and perspectives

Shiga toxins (Stx) are AB5-type toxins, composed of five B subunits which bind to Gb3 host cell receptors and an active A subunit, whose action on the ribosome leads to protein synthesis suppression. The two Stx types (Stx1 and Stx2) and their subtypes can be produced by Shiga toxin-producing Escherichia coli strains and some Shigella spp. These bacteria colonize the colon and induce diarrhea that may progress to hemorrhagic colitis and in the most severe cases, to hemolytic uremic syndrome, which could lead to death. Since the use of antibiotics in these infections is a topic of great controversy, the treatment remains supportive and there are no specific therapies to ameliorate the course. Therefore, there is an open window for Stx neutralization employing antibodies, which are versatile molecules. Indeed, polyclonal, monoclonal, and recombinant antibodies have been raised and tested in vitro and in vivo assays, showing differences in their neutralizing ability against deleterious effects of Stx. These molecules are in different phases of development for which we decide to present herein an updated report of these antibody molecules, their source, advantages, and disadvantages of the promising ones, as well as the challenges faced until reaching their applicability.

The Deleterious Effects of Shiga Toxin Type 2 Are Neutralized In Vitro by FabF8:Stx2 Recombinant Monoclonal Antibody.

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

Human-modified landscapes narrow the isotopic niche of neotropical birds.

Deforestation and habitat loss resulting from land use changes are some of the utmost anthropogenic impacts that threaten tropical birds in human-modified landscapes (HMLs). The degree of these impacts on birds' diet, habitat use, and ecological niche can be measured by isotopic analysis. We investigated whether the isotopic niche width, food resources, and habitat use of bird trophic guilds differed between HMLs and natural landscapes (NLs) using stable carbon (δ13C) and nitrogen isotopes (δ15N). We analyzed feathers of 851 bird individuals from 28 landscapes in the Brazilian Atlantic Forest. We classified landscapes into two groups according to the percentage of forest cover (HMLs ≤ 30%; NLs ≥ 47%), and compared the isotopic niche width and mean values of δ13C and δ15N for each guild between landscape types. The niches of frugivores, insectivores, nectarivores, and omnivores were narrower in HMLs, whereas granivores showed the opposite pattern. In HMLs, nectarivores showed a reduction of 44% in niche width, while granivores presented an expansion of 26%. Individuals in HMLs consumed more resources from agricultural areas (C4 plants), but almost all guilds showed a preference for forest resources (C3 plants) in both landscape types, except granivores. Degraded and fragmented landscapes typically present a lower availability of habitat and food resources for many species, which was reflected by the reduction in niche width of birds in HMLs. Therefore, to protect the diversity of guilds in HMLs, landscape management strategies that offer birds more diverse habitats must be implemented in tropical regions.

The deleterious effects of Shiga toxin type 2 are neutralized in vitro by fabF8:Stx2 recombinant monoclonal antibody

Hemolytic Uremic Syndrome (HUS) associated with Shiga-toxigenic Escherichia coli (STEC) infections is the principal cause of acute renal injury in pediatric age groups. Shiga toxin type 2 (Stx2) has in vitro cytotoxic effects on kidney cells, including human glomerular endothelial (HGEC) and Vero cells. Neither a licensed vaccine nor effective therapy for HUS is available for humans. Recombinant antibodies against Stx2, produced in bacteria, appeared as the utmost tool to prevent HUS. Therefore, in this work, a recombinant FabF8:Stx2 was selected from a human Fab antibody library by phage display, characterized, and analyzed for its ability to neutralize the Stx activity from different STEC-Stx2 and Stx1/Stx2 producing strains in a gold standard Vero cell assay, and the Stx2 cytotoxic effects on primary cultures of HGEC. This recombinant Fab showed a dissociation constant of 13.8 nM and a half maximum effective concentration (EC50) of 160 ng/mL to Stx2. Additionally, FabF8:Stx2 neutralized, in different percentages, the cytotoxic effects of Stx2 and Stx1/2 from different STEC strains on Vero cells. Moreover, it significantly prevented the deleterious effects of Stx2 in a dose-dependent manner (up to 83%) in HGEC and protected this cell up to 90% from apoptosis and necrosis. Therefore, this novel and simple anti-Stx2 biomolecule will allow further investigation as a new therapeutic option that could improve STEC and HUS patient outcomes.

Zebrafish embryo sensitivity test as in vivo platform to anti-Shiga toxin compound screening.

Shiga toxin-producing Escherichia coli (STEC) pathotype secretes two types of AB5 cytotoxins (Stx1 and Stx2), responsible for complications such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in infected patients, which could lead to sequels and death. Currently, there is no effective treatment against the cytotoxic effect of these toxins. However, in order to approve any therapy molecule, an animal experiment is required in order to evaluate the efficacy and safety of therapeutic approaches. The use of alternative small host models is growing among human infectious disease studies, particularly the vertebrate zebrafish model, since relevant results have been described for pathogen-host interaction. In this sense, the present work aimed to analyze the toxic effect of Shiga toxins in zebrafish embryo model in order to standardize this method in the future to be used as a fast, simple, and efficient methodology for the screening of therapeutic molecules. Herein, we demonstrated that the embryos were sensitive in a dose-dependent manner to both Stx toxins, with LD50 of 22 µg/mL for Stx1 and 33 µg/mL for Stx2, and the use of anti-Stx polyclonal antibody abolished the toxic effect. Therefore, this methodology can be a rapid alternative method for selecting promising compounds against Stx toxins, such as recombinant antibodies.

Macrophage inflammatory response mediated by intimin and bundle-Forming pilus from Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are important agents of acute diarrhea in children living in developing countries. A severe dysfunction of the intestinal epithelial barrier occurs during EPEC infection, leading to diarrhea and inflammation as consequences. EPEC main virulence factors include the adhesins intimin and bundle-forming pilus (BFP), as well as several effector proteins translocated to the enterocyte by the type-three secretion system. The initial interaction of EPEC with the host cell and the role of effector proteins in this process are well known. However, the role of the EPEC virulence factors in macrophage activation is not fully understood. Hence, we analyzed the ability of intimin and bundle-forming pilus (BfpA) to activate the innate response mediated by macrophages, where the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, as well as the anti-inflammatory cytokine IL-10 and chemokine MCP-1, were evaluated. Our results showed that recombinant intimin and BfpA activate macrophages in a dose-dependent manner, and the stimulated cells produced TNF-α, IL-12, IL-6, IL-10 and MCP-1, but not IL-1β. No synergistic effect was observed in the production of pro-inflammatory cytokines by combining BfpA and intimin, although production of IL-10, an anti-inflammatory mediator, was potentiated at a higher dose. The effect observed was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. Thus, herein we showed that intimin and BfpA can activate the innate immune response, inducing the production of pro- and anti-inflammatory cytokines, as well as chemokines, playing additional role as inflammatory molecules in the early steps of EPEC infection.

Comparative analysis of rapid agglutination latex test using single-chain antibody fragments (scFv) versus the gold standard vero cell assay for Shiga toxin (Stx) detection

The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30ng/mL for Stx1 and 10ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.

Comparative analysis of rapid agglutination latex test using single-chain antibody fragments (scFv) versus the gold standard vero cell assay for Shiga toxin (Stx) detection

The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30ng/mL for Stx1 and 10ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.

Zebrafish embryo sensitivity test as in vivo platform to anti-Shiga toxin compound screening

Shiga toxin-producing Escherichia coli (STEC) pathotype secretes two types of AB5 cytotoxins (Stx1 and Stx2), responsible for complications such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in infected patients, which could lead to sequels and death. Currently, there is no effective treatment against the cytotoxic effect of these toxins. However, in order to approve any therapy molecule, an animal experiment is required in order to evaluate the efficacy and safety of therapeutic approaches. The use of alternative small host models is growing among human infectious disease studies, particularly the vertebrate zebrafish model, since relevant results have been described for pathogen-host interaction. In this sense, the present work aimed to analyze the toxic effect of Shiga toxins in zebrafish embryo model in order to standardize this method in the future to be used as a fast, simple, and efficient methodology for the screening of therapeutic molecules. Herein, we demonstrated that the embryos were sensitive in a dose-dependent manner to both Stx toxins, with LD50 of 22 µg/mL for Stx1 and 33 µg/mL for Stx2, and the use of anti-Stx polyclonal antibody abolished the toxic effect. Therefore, this methodology can be a rapid alternative method for selecting promising compounds against Stx toxins, such as recombinant antibodies.

Immunological tests for diarrhoea caused by diarrhoeagenic Escherichia coli targeting their main virulence factors

Gene detection does not assure the expression of the corresponding virulence factor. Therefore, the antibody-based assays comprise the largest group of rapid methods that can be employed for the detection of virulence factor production/secretion. For these antibody-based assays, polyclonal, monoclonal, or recombinant antibodies can be employed. Thus, this chapter presents protocols for antibodies that are generated as well as the strategies of standardized and developed immunoassays for diarrhoeagenic Escherichia coli diagnosis targeting their main virulence factors.

Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo.

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test.

Stx1 toxin is one of the AB5 toxins of Shiga toxin-producing Escherichia coli (STEC) responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv) is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (KD) of 2.26 × 10-7 M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay), and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis.

ATLANTIC BIRDS: a data set of bird species from the Brazilian Atlantic Forest.

South America holds 30% of the world's avifauna, with the Atlantic Forest representing one of the richest regions of the Neotropics. Here we have compiled a data set on Brazilian Atlantic Forest bird occurrence (150,423) and abundance samples (N = 832 bird species; 33,119 bird individuals) using multiple methods, including qualitative surveys, mist nets, point counts, and line transects). We used four main sources of data: museum collections, on-line databases, literature sources, and unpublished reports. The data set comprises 4,122 localities and data from 1815 to 2017. Most studies were conducted in the Florestas de Interior (1,510 localities) and Serra do Mar (1,280 localities) biogeographic sub-regions. Considering the three main quantitative methods (mist net, point count, and line transect), we compiled abundance data for 745 species in 576 communities. In the data set, the most frequent species were Basileuterus culicivorus, Cyclaris gujanensis, and Conophaga lineata. There were 71 singletons, such as Lipaugus conditus and Calyptura cristata. We suggest that this small number of records reinforces the critical situation of these taxa in the Atlantic Forest. The information provided in this data set can be used for macroecological studies and to foster conservation strategies in this biodiversity hotspot. No copyright restrictions are associated with the data set. Please cite this Data Paper if data are used in publications and teaching events.