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1.
Ann Lab Med ; 42(1): 3-23, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374345

RESUMO

Immunoassays are powerful qualitative and quantitative analytical techniques. Since the first description of an immunoassay method in 1959, advances have been made in assay designs and analytical characteristics, opening the door for their widespread implementation in clinical laboratories. Clinical endocrinology is closely linked to laboratory medicine because hormone quantification is important for the diagnosis, treatment, and prognosis of endocrine disorders. Several interferences in immunoassays have been identified through the years; although some are no longer encountered in daily practice, cross-reaction, heterophile antibodies, biotin, and anti-analyte antibodies still cause problems. Newer interferences are also emerging with the development of new therapies. The interfering substance may be exogenous (e.g., a drug or substance absorbed by the patient) or endogenous (e.g., antibodies produced by the patient), and the bias caused by interference can be positive or negative. The consequences of interference can be deleterious when clinicians consider erroneous results to establish a diagnosis, leading to unnecessary explorations or inappropriate treatments. Clinical laboratories and manufacturers continue to investigate methods for the detection, elimination, and prevention of interferences. However, no system is completely devoid of such incidents. In this review, we focus on the analytical interferences encountered in daily practice and possible solutions for their detection or elimination.


Assuntos
Biotina , Hormônios , Anticorpos , Reações Cruzadas , Humanos , Imunoensaio
2.
Talanta ; 236: 122847, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635237

RESUMO

Nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV2 and is highly conserved, and there are no homologous proteins in the human body, making it an ideal biomarker for the early diagnosis of SARS-CoV2. However, early detection of clinical specimens for SARS-CoV2 remains a challenge due to false-negative results with viral RNA and host antibodies based testing. In this manuscript, a microfluidic chip with femtoliter-sized wells was fabricated for the sensitive digital detection of N protein. Briefly, ß-galactosidase (ß-Gal)-linked antibody/N protein/aptamer immunocomplexes were formed on magnetic beads (MBs). Afterwards, the MBs and ß-Gal substrate fluorescein-di-ß-d-galactopyranoside (FDG) were injected into the chip together. Each well of the chip would only hold one MB as confined by the diameter of the wells. The MBs in the wells were sealed by fluorocarbon oil, which confines the fluorescent (FL) product generated from the reaction between ß-Gal and FDG in the individual femtoliter-sized well and creates a locally high concentration of the FL product. The FL images of the wells were acquired using a conventional inverted FL microscope. The number of FL wells with MBs (FL wells number) and the number of wells with MBs (MBs wells number) were counted, respectively. The percentage of FL wells was calculated by dividing (FL wells number) by (MBs wells number). The higher the percentage of FL wells, the higher the N protein concentration. The detection limit of this digital method for N protein was 33.28 pg/mL, which was 300 times lower than traditional double-antibody sandwich based enzyme-linked immunosorbent assay (ELISA).


Assuntos
Imunoensaio/métodos , Proteínas do Nucleocapsídeo , SARS-CoV-2 , Anticorpos , COVID-19/diagnóstico , Humanos , Proteínas do Nucleocapsídeo/isolamento & purificação , RNA Viral
3.
J Nanobiotechnology ; 19(1): 305, 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34615526

RESUMO

Molecular imprinting (MI) is a technique that creates a template of a molecule for improving complementary binding sites in terms of size and shape to a peptide, protein, bacteria, mammalian cell, or virus on soft materials (such as polymers, hydrogels, or self-assembled materials). MI has been widely investigated for over 90 years in various industries but is now focused on improved tissue engineering, regenerative medicine, drug delivery, sensors, diagnostics, therapeutics and other medical applications. Molecular targets that have been studied so far in MI include those for the major antigenic determinants of microorganisms (like bacteria or viruses) leading to innovations in disease diagnosis via solid-phase extraction separation and biomimetic sensors. As such, although not widely investigated yet, MI demonstrates much promise for improving the detection of and treatment for the current Coronavirus Disease of 2019 (COVID-2019) pandemic as well as future pandemics. In this manner, this review will introduce the numerous applications of MI polymers, particularly using proteins and peptides, and how these MI polymers can be used as improved diagnostic and therapeutic tools for COVID-19.


Assuntos
COVID-19/diagnóstico , Polímeros Molecularmente Impressos/uso terapêutico , SARS-CoV-2/isolamento & purificação , Anticorpos , Portadores de Fármacos , Humanos , Impressão Molecular , Polímeros Molecularmente Impressos/química , Peptídeos , Proteínas , Receptores de Superfície Celular
4.
Anal Chim Acta ; 1184: 339054, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34625272

RESUMO

Immobilized antibodies with site-specific, oriented, and covalent pattern are of great significance to improve the sensitivity of solid-phase immunoassay. Here, we developed a novel antibody conjugation strategy that can immobilize antibodies in a directional and covalent manner. In this study, an IgG-Fc binding protein (Z domain) carrying a site-specific photo-crosslinker, p-benzoyl-L-phenylalanine, and a single C-terminal cysteine (Cys) handle was genetically engineered. Upon UV irradiation, the chimeric protein enables the Cys handle to couple with the native antibody in Fc-specific and covalent conjugation pattern, resulting in a novel thiolated antibody. Thus, an approach for the covalent, directional immobilization of antibodies to maleimide-modified magnetic nanoparticles (MNPs) was developed on the basis of the crosslinking between sulfhydryl and maleimide groups. The antibody-conjugated MNPs were applied in MNP-based enzyme-linked immunosorbent assay (ELISA) for the detection of carcinoembryonic antigen. The MNP-based ELISA presented a quantification linear range of 0.1-100 ng mL-1 and detection limit of 0.02 ng mL-1, which was approximately 100 times more sensitive than the traditional microplate ELISA (2.0 ng mL-1). Thus, the proposed antibody immobilization approach can be used in surface functionalization for the sensitive detection of various biomarkers.


Assuntos
Proteínas de Transporte , Nanopartículas de Magnetita , Anticorpos , Antígenos , Magnetismo
5.
Anal Chim Acta ; 1182: 338714, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602193

RESUMO

Antibody-based affinity capture has become the gold standard in sample preparation for determination of low-abundance protein biomarkers in biological matrices prior to liquid chromatography-mass spectrometry (LC-MS) determination. This comprises both capture of intact proteins prior to the digestion step and capture of proteolytic peptides after digestion of the sample. The latter can be performed both using antibodies specifically developed to capture target proteolytic peptides, as well as by the less explored use of anti-protein antibodies to capture the proteolytic epitope peptide. Molecularly imprinted polymers (MIPs), also called plastic antibodies are another affinity-based approach emerging as sample preparation technique in LC-MS based protein biomarker analysis. The current review gives a critical and comprehensive overview of proteolytic peptide capture using antibodies and MIPs in LC-MS based protein biomarker determination during the last five years. The main emphasis is on capture of non-modified peptides, while a brief overview of affinity capture of peptides containing post-translational modifications (PTMs) is provided.


Assuntos
Polímeros Molecularmente Impressos , Peptídeo Hidrolases , Anticorpos , Peptídeos , Proteólise
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(10): 936-941, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34670671

RESUMO

Objective To obtain the prokaryotic expression of Helicoverpa armigera sulfatase 1 (HaSulf1) protein and prepare its polyclonal antibodies. Methods A prokaryotic expression vector was firstly constructed. After inducing the protein expression, the crude recombinant protein was collected and purified with a nickel column, which was injected in immunized mice. Mouse blood was collected for detecting the titers of polyclonal antibodies by ELISA and the immune specificity was detected by Western blotting. Results The recombinant plasmid was obtained and the fusion protein was positively expressed and purified. ELISA data showed that the polyclonal antibody titer was 1:819 200. Western blotting showed that the prepared antibodies could bind to both the recombinant protein expressed in vitro and natural sulfatase from cotton bollworms. Conclusion Prokaryotic expression of recombinant HaSulf1 protein was obtained and specific mouse polyclonal antibodies were prepared.


Assuntos
Anticorpos , Mariposas , Animais , Especificidade de Anticorpos , Western Blotting , Camundongos , Sulfatases
7.
Nat Commun ; 12(1): 5596, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552086

RESUMO

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.


Assuntos
Coagulação Sanguínea , Fator XII/química , Fator XII/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Fator XII/genética , Fator XII/imunologia , Fator XIIa/metabolismo , Camundongos , Mutação , Tempo de Tromboplastina Parcial/normas , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo , Trombose/diagnóstico , Trombose/genética , Trombose/metabolismo
8.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576131

RESUMO

The cyclical proliferation of the wild fossorial rodent Arvicola terrestris scherman (ATS) is critical in mid-mountain ecosystems of several European countries. Our goal is to develop an immunocontraceptive vaccine to control their fertility, as a sustainable alternative to chemical poisons currently used. Indeed, these chemicals cause the death of ATS predators and animals sharing their ecosystem, and current laws progressively limit their use, making the development of a targeted vaccination strategy an interesting and efficient alternative. In order to identify species-specific sperm antigens, male and female ATS received subcutaneous injections of whole ATS spermatozoa to elicit an immune response. The analysis of the immune sera led to the identification of 120 immunogenic proteins of sperm cells. Of these, 15 were strictly sperm-specific and located in different regions of the male gamete. Some of these antigens are proteins involved in molecular events essential to the reproductive process, such as sperm-egg interaction, acrosomal reaction, or sperm motility. This approach not only identified a panel of immunogenic proteins from ATS sperm cells, but also demonstrated that some of these proteins trigger an immune response in both male and female ATS. These spermatic antigens are good candidates for the development of a contraceptive vaccine.


Assuntos
Antígenos/metabolismo , Arvicolinae/imunologia , Anticoncepcionais , Espermatozoides/imunologia , Animais , Anticorpos/sangue , Feminino , Ontologia Genética , Imunidade , Imunização , Masculino , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Proteômica , Especificidade da Espécie
9.
Nat Commun ; 12(1): 5456, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526511

RESUMO

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.


Assuntos
Anticorpos/imunologia , Complemento C3/antagonistas & inibidores , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Rim/métodos , Macaca mulatta/imunologia , Piridonas/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Complemento C3/imunologia , Complemento C3/metabolismo , Citocinas/sangue , Citocinas/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transplante Homólogo
10.
Clin Appl Thromb Hemost ; 27: 10760296211040110, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34541935

RESUMO

Since the outbreak of Covid-19 in December, 2019, scientists worldwide have been committed to developing COVID-19 vaccines. Only when most people have immunity to SARS-CoV-2, COVID-19 can reduce even wholly overcome. So far, nine kinds of COVID-19 vaccines have passed the phase III clinical trials and have approved for use. At the same time, adverse reactions after COVID-19 vaccination have also reported. This paper focuses on the adverse effects of thrombosis and thrombocytopenia caused by the COVID-19 vaccine, especially the adenovirus-vector vaccine from AstraZeneca and Pfizer, and discusses its mechanism and possible countermeasures.


Assuntos
Adenoviridae/genética , Vacinas contra COVID-19/efeitos adversos , Vetores Genéticos , Trombocitopenia/induzido quimicamente , Trombose/induzido quimicamente , Vacinação/efeitos adversos , Anticorpos/sangue , Vacinas contra COVID-19/genética , Vacinas contra COVID-19/imunologia , Humanos , Fator Plaquetário 4/imunologia , Medição de Risco , Fatores de Risco , Trombocitopenia/sangue , Trombocitopenia/imunologia , Trombose/sangue , Trombose/imunologia
11.
BMC Res Notes ; 14(1): 359, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526111

RESUMO

OBJECTIVE: Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). RESULTS: The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.


Assuntos
Colágeno Tipo XI , Peixe-Zebra , Animais , Anticorpos , Proteínas da Matriz Extracelular , Peptídeos , Coelhos
12.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575943

RESUMO

Worldwide, cancer is a serious health concern due to the increasing rates of incidence and mortality. Conventional cancer imaging, diagnosis and treatment practices continue to substantially contribute to the fight against cancer. However, these practices do have some risks, adverse effects and limitations, which can affect patient outcomes. Although antibodies have been developed, successfully used and proven beneficial in various oncology practices, the use of antibodies also comes with certain challenges and limitations (large in size, poor tumor penetration, high immunogenicity and a long half-life). Therefore, it is vital to develop new ways to visualize, diagnose and treat cancer. Nanobodies are novel antigen-binding fragments that possess many advantageous properties (small in size, low immunogenicity and a short half-life). Thus, the use of nanobodies in cancer practices may overcome the challenges experienced with using traditional antibodies. In this review, we discuss (1) the challenges with antibody usage and the superior qualities of nanobodies; (2) the use of antibodies and nanobodies in cancer imaging, diagnosis, drug delivery and therapy (surgery, radiotherapy, chemotherapy and immunotherapy); and (3) the potential improvements in oncology practices due to the use of nanobodies as compared to antibodies.


Assuntos
Anticorpos/uso terapêutico , Imunoterapia , Neoplasias/terapia , Anticorpos de Domínio Único/uso terapêutico , Anticorpos/imunologia , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias/diagnóstico , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Anticorpos de Domínio Único/imunologia
13.
PLoS One ; 16(9): e0249254, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34570776

RESUMO

Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among the spike protein mutants, we confirmed a low mutation rate exhibiting less than 10 non-synonymous mutations in 99.6% of the analyzed sequences, but the mean and median number of spike protein mutations per sample increased over time. 5,472 distinct variants were found in total. The majority of the observed variants were recurrent, but only 21 and 14 recurrent variants were found in at least 1% of the mutant genome assemblies and NGS samples, respectively. Further, we found high-confidence subclonal variants in about 2.6% of the NGS data sets with mutant spike protein, which might indicate co-infection with various SARS-CoV-2 strains and/or intra-host evolution. Lastly, some variants might have an effect on antibody binding or T-cell recognition. These findings demonstrate the continuous importance of monitoring SARS-CoV-2 sequences for an early detection of variants that require adaptations in preventive and therapeutic strategies.


Assuntos
COVID-19/virologia , Genoma Viral , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos/imunologia , COVID-19/prevenção & controle , COVID-19/transmissão , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Taxa de Mutação , Pandemias , Domínios Proteicos , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Linfócitos T/imunologia
14.
Front Immunol ; 12: 728513, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484238

RESUMO

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.


Assuntos
Anticorpos/efeitos adversos , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Púrpura Trombocitopênica Idiopática/induzido quimicamente , Púrpura Trombocitopênica Idiopática/fisiopatologia , Adenoviridae/genética , Animais , Plaquetas/imunologia , Plaquetas/patologia , Vacinas contra COVID-19/imunologia , Modelos Animais de Doenças , Vetores Genéticos , Humanos , Camundongos , Ativação Plaquetária/imunologia , Fator Plaquetário 4 , Coelhos
15.
Infectio ; 25(3): 169-175, jul.-set. 2021. tab
Artigo em Espanhol | LILACS, COLNAL | ID: biblio-1250088

RESUMO

Resumen Objetivo: Verificación del desempeño de las pruebas serológicas rápidas utilizadas en el departamento de Risaralda, Colombia. Métodos: Estudio analitico, de corte transversal. Incluyó muestras de sueros de trabajadores de la salud de la ciudad de Pereira, quienes tuvieron sospecha clínica y epidemiológica por SARS-CoV-2. El procesamiento y validación de las pruebas fue realizado en las instalaciones de la Universidad Tecnológica de Pereira. Se calculó sensibilidad y especificidad de las pruebas rápidas serológicas IgM/IgG usando como prueba de oro la RT-PCR. Resultados: Se incluyeron las muestras de 144 profesionales de la salud. Las pruebas serológicas rápidas evidenciaron ser útiles para identificar o descartar la presencia de anticuerpos IgM e IgG, especialmente en pacientes sintomáticos, en quienes el inicio de los síntomas es superior a 11 días. Discusión: El uso de pruebas rápidas se encuentra en aumento, no solo por la rapidez de sus resultados, sino también por los bajos costos asociados y la necesidad de identificar pacientes no susceptibles, quienes deben priorizar su retorno a actividades laborales en comunidad como parte de la reactivación económica de Colombia. Es necesario confirmar el desempeño de la prueba para aumentar la probabilidad de una adecuada clasificación antes de proceder a su uso rutinario.


Abstract Objective: We aimed to realize a verification of the performance of the rapid serological tests used in Risaralda department. Methods: Analytical, cross-sectional study. Serum samples from health workers in Pereira city, who had a clinical and epidemiological suspicion for SARS-CoV-2 were included. The processing and validation of the tests was carried out at Universidad Tecnológica de Pereira. Sensitivity and specificity of rapid IgM / IgG sero logical tests were calculated using RT-PCR as the gold standard test. Results: 144 samples of health professionals were included. Rapid serological tests useful to identify or rule out the presence of IgM and IgG antibodies, especially in symptomatic patients, in whom the onset of symptoms is longer than 11 days. Discussion: The use of rapid tests is increasing, not only due to the speed of their results, but also due to the low associated costs and the need to identify non-susceptible patients, who must prioritize their return to work activities in the community as part of the economic reactivation of Colombia. It is necessary to confirm the adequate performance of the test to increase the probability of an adequate classification before proceeding with the routine use of this test.


Assuntos
Humanos , Masculino , Feminino , Adulto , Testes Sorológicos , Pessoal de Saúde , SARS-CoV-2 , Estudos Transversais , COVID-19/diagnóstico , Anticorpos , Categorias de Trabalhadores , Antígenos
16.
Recurso na Internet em Português | LIS - Localizador de Informação em Saúde | ID: lis-48399

RESUMO

A equipe do projeto PROTECTCoV-19 recruta grávidas, a partir da 28º semana de gestação, e puérperas, com idade maior ou igual a 18 anos, para participar do estudo, que pretende saber o papel dos anticorpos do leite materno na proteção contra Covid-19


Assuntos
Leite Humano/imunologia , COVID-19 , Anticorpos/imunologia , Gestantes
17.
Front Immunol ; 12: 639008, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394070

RESUMO

Background: Previous reports identified proteins associated with 'apoptosis' following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated 'apoptosis', however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response. Methods: Initially, we predicted the allergenicity of the epitope sequences associated with 'neurotoxic' anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of mouse primary neurons (MPN), neuroblastoma cells (N2a) and microglia (N11) cell lines lead to a neuronal allergenic response. Results: In-Silico studies showed that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for previously reported 'neurotoxic' antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, we identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, we identified 8 neuronal allergenic-related proteins following treatment of N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells when compared with untreated cells. Antibody treatment of MPN or MPN co-cultured with antibody-treated N11 led to identifying 10 and 7 allergenic-related proteins when compared with untreated cells. However, comparison with 3F4 antibody treatment revealed 5 and 4 allergenic-related proteins respectively. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Finally, co-culture of N2a or MPN with N11-treated with anti-PrP antibodies resulted in significant accumulation of NO and IL6 but not TNF-α in the cell culture media supernatant. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal hypersensitivity response and highlights the important role of microglia in triggering an IgG-mediated neuronal hypersensitivity response. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative disorders to derive safe and targeted biotherapeutics.


Assuntos
Anticorpos/imunologia , Hipersensibilidade/imunologia , Neurônios/imunologia , Proteínas PrPC/imunologia , Proteínas PrPC/metabolismo , Animais , Epitopos de Linfócito B/imunologia , Humanos , Camundongos , Neuroglia/imunologia
18.
Chem Commun (Camb) ; 57(68): 8508-8511, 2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34351331

RESUMO

In this work, we have designed a template-free multiple signal amplification method for the highly sensitive detection of cancer cell-derived exosomes. In this design, DNase I serves as a bridge to link the DNA-based amplification approach and terminal deoxynucleotidyl transferase (TdT)-mediated polymerization reaction. Consequently, a detection limit of 10 particles per µL can be achieved, while a complex nucleic acid sequence design can be avoided. This method also exhibits good performance in a complicated matrix and enables the differentiation of healthy individuals from colorectal cancer (CRC) patients.


Assuntos
Técnicas Biossensoriais , Exossomos/química , Neoplasias/metabolismo , Anticorpos , Aptâmeros de Nucleotídeos , DNA/química , DNA/metabolismo , DNA Nucleotidilexotransferase/metabolismo , Desoxirribonuclease I/metabolismo , Exossomos/metabolismo , Células HeLa , Humanos , Técnicas de Amplificação de Ácido Nucleico
19.
Biomolecules ; 11(8)2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34439834

RESUMO

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.


Assuntos
Glutaratos/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Succinatos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Encéfalo/metabolismo , Cromatografia de Afinidade , Soros Imunes/química , Immunoblotting , Fígado/metabolismo , Masculino , Coelhos , Ratos
20.
Transpl Int ; 34(9): 1689-1702, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34448270

RESUMO

Circulating donor-specific antibodies (DSA) do not necessarily indicate antibody-mediated rejection (ABMR). Here, we evaluated the diagnostic value of donor-derived cell-free DNA (dd-cfDNA) as an add-on to DSA detection. The study included two independent cohorts of DSA+ kidney allograft recipients, 45 subclinical cases identified by cross-sectional antibody screening (cohort 1), and 30 recipients subjected to indication biopsies (cohort 2). About 50% of the DSA+ recipients had ABMR and displayed higher dd-cfDNA levels than DSA+ ABMR- recipients (cohort 1: 1.90% [median; IQR: 0.78-3.90%] vs. 0.52% [0.35-0.72%]; P < 0.001); (cohort 2: 1.20% [0.82-2.50%] vs. 0.59% [0.28-2.05%]; P = 0.086). Receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.89 and 0.69 for dd-cfDNA, and 0.88 and 0.77 for DSA mean fluorescence intensity (MFI), respectively. In combined models, adding dd-cfDNA to DSA-MFI or vice versa significantly improved the diagnostic accuracy. Limited diagnostic performance of dd-cfDNA in cohort 2 was related to the frequent finding of other types of graft injury among ABMR- recipients, like T cell-mediated rejection or glomerulonephritis. For dd-cfDNA in relation to injury of any cause an AUC of 0.97 was calculated. Monitoring of dd-cfDNA in DSA+ patients may be a useful tool to detect ABMR and other types of injury.


Assuntos
Ácidos Nucleicos Livres , Transplante de Rim , Aloenxertos , Anticorpos , Estudos Transversais , Rejeição de Enxerto/diagnóstico , Humanos , Isoanticorpos , Rim , Transplante de Rim/efeitos adversos
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