Development of polyclonal antibodies for the detection of recombinant human erythropoietin

Recombinant human erythropoietin (rHuEPO) is detected by using direct pharmacological assays and indirect haematological assays. However, both methods have several limitations including technical challenges and  cost-related issues. The aim of this study was to develop polyclonal antibodies against rHuEPO (anti-rHuEPO pAb) that can be used in immunoassays. In this study, we purified anti-rHuEPO pAb that could be used in immunoblotting assays to efficiently detect rHuEPO. Furthermore, these antirHuEPO pAb which could also detect rHuEPO that was expressed in a eukaryotic expression system (CHO cells). Thus, the anti-rHuEPO pAb developed in this study may be useful for rHuEPO detection.Keywords: Antibodies, rHuEPO, immunoassays, pAb.African Journal of Biotechnology Vol. 12(37), pp. 5595-559

Large-Scale Evidence for the Effect of the COLIA1 Sp1 Polymorphism on Osteoporosis Outcomes: The GENOMOS Study

BACKGROUND: Osteoporosis and fracture risk are considered to be under genetic control. Extensive work is being performed to identify the exact genetic variants that determine this risk. Previous work has suggested that a G/T polymorphism affecting an Sp1 binding site in the COLIA1 gene is a genetic marker for low bone mineral density (BMD) and osteoporotic fracture, but there have been no very-large-scale studies of COLIA1 alleles in relation to these phenotypes. METHODS AND FINDINGS: Here we evaluated the role of COLIA1 Sp1 alleles as a predictor of BMD and fracture in a multicenter study involving 20,786 individuals from several European countries. At the femoral neck, the average (95% confidence interval [CI]) BMD values were 25 mg/cm (2) (CI, 16 to 34 mg/cm (2)) lower in TT homozygotes than the other genotype groups ( p < 0.001), and a similar difference was observed at the lumbar spine; 21 mg/cm (2) (CI, 1 to 42 mg/cm (2)), ( p = 0.039). These associations were unaltered after adjustment for potential confounding factors. There was no association with fracture overall (odds ratio [OR] = 1.01 [CI, 0.95 to 1.08]) in either unadjusted or adjusted analyses, but there was a non-significant trend for association with vertebral fracture and a nominally significant association with incident vertebral fractures in females (OR = 1.33 [CI, 1.00 to 1.77]) that was independent of BMD, and unaltered in adjusted analyses. CONCLUSIONS: Allowing for the inevitable heterogeneity between participating teams, this study—which to our knowledge is the largest ever performed in the field of osteoporosis genetics for a single gene—demonstrates that the COLIA1 Sp1 polymorphism is associated with reduced BMD and could predispose to incident vertebral fractures in women, independent of BMD. The associations we observed were modest however, demonstrating the importance of conducting studies that are adequately powered to detect and quantify the effects of common genetic variants on complex diseases

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Production and characterization of monoclonal antibodies against the identical region of LigA and LigB proteins from Leptospira interrogans

Leptospirosis is a zoonotic disease caused by pathogenic bacteria belonging to the Leptospira genus. Several mammals may carry the agent, and rats are the most important source of human infection in urban settings. The wide spectrum of clinical manifestations varies from mild cases, with fever and headaches, to severe presentations, with liver and kidney failure, which may lead to death. As a result of the various degrees of severity, leptospirosis is frequently mistaken, in its acute stage, with other tropical diseases such as influenza and dengue. The microscopic agglutination test (MAT) is considered the gold standard when diagnosing leptospirosis; however, the test presents limitations regarding sensitivity in the acute phase of the disease. Recently, surface proteins LigA and LigB have been identified to be related with leptospiral virulence. These proteins have been characterized as adhesins, with a proteic structure similar to Escherichia coli intimin and Yersinia pseudotuberculosis invasin, which are important virulence factors in these organisms. The goal of this study was to produce and characterize monoclonal antibodies (MAbs) against a truncated fragment of approximately 54 kDa, named rLigBrep, that comprise a identical portion of LigA and LigB (domains 2-7). The 5 MAbs obtained were of the IgG1 (2) and IgG2b (3) isotypes and their affinity constants for rLigBrep varied from 7 x 107 M-1 to 4 x 108 M-1. The MAbs were able to react with the native antigen in L. interrogans serovar Copenhageni strain Fiocruz L1-130 by indirect immunofluorescence, immunoblotting, whole-cell ELISA and immunoelectron microscopy. These results allow concluding that these MAbs are important tools for studies aiming understanding the role of Lig proteins in Leptospira pathogenesis and in the development of tests for diagnosis of leptospirosis.A leptospirose é uma zoonose causada por bactérias patogênicas pertencentes ao gênero Leptospira. Diversos mamíferos podem albergar o agente, sendo o rato a principal fonte de infecção para humanos no ambiente. As manifestações clínicas da doença variam desde os sintomas leves, como febre e dores de cabeça, até os mais graves com insuficiência renal e hepática que podem levar o indivíduo à morte. Esse amplo espectro de sintomas faz com que a leptospirose seja freqüentemente confundida em sua fase aguda com outras doenças febris, como gripe e dengue. O teste de aglutinação microscópica (MAT) é considerado o padrão ouro para o diagnóstico laboratorial da leptospirose, entretanto, o teste apresenta limitações relacionadas com a sensibilidade na fase aguda da doença. Recentemente, as proteínas de superfície LigA e LigB foram identificadas e relacionadas com a virulência de leptospiras patogênicas. Estas proteínas foram caracterizadas como adesinas, possuindo estrutura protéica semelhante a intimina de Escherichia coli e invasina de Yersinia pseudotuberculosis, que são importantes fatores de virulência nestes microrganismos. O objetivo desse estudo foi produzir e caracterizar anticorpos monoclonais (MAbs) contra um fragmento truncado de aproximadamente 54 kDa, que codifica a região idêntica das proteínas LigA e LigB (domínios 2-7), denominado rLigBrep. Foram obtidos 5 MAbs dos isotipos IgG1 (2) e IgG2b (3), com afinidades por rLigBrep que variaram entre 7 x 107 M-1 e 4 x 108 M-1. Foi comprovado através de imunofluorescência indireta, immunoblotting, ELISA whole-cell e microscopia imunoeletrônica que os MAbs foram capazes de detectar o antígeno nativo presente em L. interrogans sorovar Copenhageni cepa Fiocruz L1-130. Estes resultados permitem concluir que os MAbs produzidos são importantes ferramentas para serem utilizados em estudos que visam entender o papel das proteínas Lig na patogênese das leptospiras e em testes diagnósticos para leptospirose

Magnetic particles: synthesis and applications of 3 immunoseparation assays

The development of reagents for infectious disease diagnosis and prevention is strategic for the scientific and technological advance of the country. Promising results have been obtained with the use of magnetic nanocomposites (MNC) in health for several purposes as separation, purification and detection of cells or biomolecules. In this work, an immunomagnetic separation (IMS) method using carbon and cobalt-based (MNCc), synthesized, and other polystyrene and iron-based (MNCp) commercially available was applied, for the isolation and detection of pathogenic leptospires. The MNCc were synthesized through polymeric precursor method and characterized by scanning electron microscopy/transmission (SEM/TEM), X-ray diffractometry (XRD), infrared spectrometry (FTIR), and immunofluorescence (IF). The MNCc surface was functionalized with carboxylic groups through the incorporation of acrylic acid. MNCc, adsorbed with monoclonal antibodies (MAbs) against pathogenic leptospires, were used in the IMS assays followed by PCR and cell culture was able to detect and isolate the organism. MNCp were used for detecting Leptospira spp. in biological fluids from dogs artificially contaminated with leptospires, and clinical samples from dogs positive for leptospirosis. The introduction of IMS, as a step prior to PCR using MNCp, detected a concentration of leptospires ten times smaller when compared to PCR performed directly on artificially contaminated urine and blood samples. Moreover, the IMS performed with MNCp increased PCR sensitivity in clinical samples from dogs positive for leptospirosis. Data from both studies suggest that MNCc and MNCp are important tools for the development of new diagnosis tests.O desenvolvimento de insumos para o diagnóstico e prevenção de doenças infecciosas é estratégico para o avanço científico e tecnológico do país. Resultados promissores vêm sendo obtidos com o uso de nanocompósitos magnéticos (NCM) aplicados à área da saúde para as mais diversas finalidades, como na separação, purificação e detecção de células ou biomoléculas. Nesse trabalho, um método de imunoseparação magnética (IMS) utilizando NCM a base de carbono e cobalto (NCMc) sintetizados, e outro de poliestireno e ferro (NCMp) disponível comercialmente, foi utilizado para capturar e detectar leptospiras patogênicas. Os NCMc foram sintetizados através do método dos precursores poliméricos e caracterizados por microscopia eletrônica de varredura/transmissão (MEV/MET), difratometria de raio-X (DRX), espectrometria no infravermelho (FTIR) e imunofluorescência (IF). A superfície dos NCMc foi funcionalizada com grupos carboxílicos mediante incorporação de ácido acrílico. Os NCMc adsorvidos com anticorpos monoclonais (MAbs) contra leptospiras patogênicas, quando utilizados em ensaios de IMS seguidos por PCR e cultivo celular, foram capazes de detectar e isolar o organismo. Os NCMp foram utilizados para a detecção de Leptospira spp. em fluidos biológicos de cães artificialmente contaminados com leptospiras e em amostras clínicas de cães positivos para a leptospirose. A introdução da IMS como passo anterior a PCR utilizando os NCMp foi capaz de detectar uma concentração 10 vezes menor de leptospiras quando comparada a PCR realizada diretamente a partir de amostras de urina e sangue artificialmente contaminados. Além disso, a IMS realizada com os NCMp aumentou a sensibilidade da PCR em amostras clínicas de cães positivos para leptospirose. Os dados obtidos em ambos os estudos sugerem que os NCMc e NCMp constituem importantes ferramentas para o desenvolvimento de novos testes de diagnóstico

Differential adhesion between moving particles as a mechanism for the evolution of social groups

The evolutionary stability of cooperative traits, that are beneficial to other individuals but costly to their carrier, is considered possible only through the establishment of a sufficient degree of assortment between cooperators. Chimeric microbial populations, characterized by simple interactions between unrelated individuals, restrain the applicability of standard mechanisms generating such assortment, in particular when cells disperse between successive reproductive events such as happens in Dicyostelids and Myxobacteria. In this paper, we address the evolutionary dynamics of a costly trait that enhances attachment to others as well as group cohesion. By modeling cells as self-propelled particles moving on a plane according to local interaction forces and undergoing cycles of aggregation, reproduction and dispersal, we show that blind differential adhesion provides a basis for assortment in the process of group formation. When reproductive performance depends on the social context of players, evolution by natural selection can lead to the success of the social trait, and to the concomitant emergence of sizeable groups. We point out the conditions on the microscopic properties of motion and interaction that make such evolutionary outcome possible, stressing that the advent of sociality by differential adhesion is restricted to specific ecological contexts. Moreover, we show that the aggregation process naturally implies the existence of non-aggregated particles, and highlight their crucial evolutionary role despite being largely neglected in theoretical models for the evolution of sociality

Differential adhesion between moving particles as a mechanism for the evolution of social groups

The evolutionary stability of cooperative traits, that are beneficial to other individuals but costly to their carrier, is considered possible only through the establishment of a sufficient degree of assortment between cooperators. Chimeric microbial populations, characterized by simple interactions between unrelated individuals, restrain the applicability of standard mechanisms generating such assortment, in particular when cells disperse between successive reproductive events such as happens in Dicyostelids and Myxobacteria. In this paper, we address the evolutionary dynamics of a costly trait that enhances attachment to others as well as group cohesion. By modeling cells as self-propelled particles moving on a plane according to local interaction forces and undergoing cycles of aggregation, reproduction and dispersal, we show that blind differential adhesion provides a basis for assortment in the process of group formation. When reproductive performance depends on the social context of players, evolution by natural selection can lead to the success of the social trait, and to the concomitant emergence of sizeable groups. We point out the conditions on the microscopic properties of motion and interaction that make such evolutionary outcome possible, stressing that the advent of sociality by differential adhesion is restricted to specific ecological contexts. Moreover, we show that the aggregation process naturally implies the existence of non-aggregated particles, and highlight their crucial evolutionary role despite being largely neglected in theoretical models for the evolution of sociality