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1.
Food Chem ; 371: 131071, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34537613

RESUMO

A growing number of ß-agonists are illegally using for reducing animal fat deposition in animals, but the development of analytical methods always lags behind the emergence of new illegal compounds. Therefore, class specificity antibody-based immunoassays that can detect a great many ß-agonists are important for timely supervision. In this study, a competitive inhibition enzyme-linked immunosorbent assay (ciELISA) based on a clenbuterol monoclonal antibody was developed to recognize 23 ß-agonists and analogues. Holographic and three-dimensional quantitative structure-activity relationship (HQSAR and 3D QSAR) revealed that there are two critical binding epitopes on ß-agonist hapten affecting antibody specificity, and these epitopes have been further validated using a ractopamine antibody with narrow specificity. Tert-butyl at C-2' epitope is needed to generate class specific antibodies, and different characteristics of substituents at benzene ring epitope would adjust antibody specificity. This investigation could provide reference for future design of ß-agonist haptens.


Assuntos
Haptenos , Relação Quantitativa Estrutura-Atividade , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Epitopos , Imunoensaio
2.
MAbs ; 14(1): 2005507, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34923915

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Células CHO , COVID-19/prevenção & controle , COVID-19/virologia , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular , Células Clonais , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Ponto Isoelétrico , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
MAbs ; 14(1): 2002236, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34967699

RESUMO

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Técnicas de Visualização da Superfície Celular , Imunoglobulina G/imunologia , Biblioteca de Peptídeos , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Epitopos , Feminino , Interações Hospedeiro-Patógeno , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Mesocricetus , SARS-CoV-2/patogenicidade , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/farmacologia , Células Vero
4.
Bull Cancer ; 108(10S): S4-S17, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34920806

RESUMO

Chimeric antigen receptors (CAR)-T cells are genetically engineered T-lymphocytes redirected with a predefined specificity to any target antigen, in a non-HLA restricted manner, therefore combining antibody-type specificity with effector T-cell function. This strategy was developed some thirty years ago, after extensive work established the key role of the immune system against cancer. The first-engineered T-cell with chimeric molecule was designed in 1993 by Israeli immunologist Zelig Eshhar. Since then, several modifications took place, including the addition of co-stimulatory domain, to further improve CAR-T cell anti-tumor potency. The first clinical application of CAR-T cell was done in Rotterdam in 2005 for metastatic renal cell carcinoma and simultaneously at the National Cancer Institute (NCI) for metastatic ovarian cancer. These pioneered studies failed to demonstrate a therapeutic benefit, but warning emerged concerning their safety of use. The real clinical success came with anti-CD19 CAR-T cells, used since 2009 by Steven Rosenberg at the NCI in a patient with refractory follicular lymphoma and in 2011 by Carl June and David Porter from the University of Pennsylvania in patients with chronic lymphocytic leukemia and B-cell acute lymphoblastic leukemia. From that time, large centers in North America have embarked in several early phase and pivotal trials that have demonstrated unprecedent response rate in heavily pretreated chemo refractory patient with B-cell malignancies. Theses clinical success have led to the approval of three anti-CD19 CAR-T cells products for the management of B-cell malignancies in the United States and in Europe as of December 2020.


Assuntos
Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Especificidade de Anticorpos , Antígenos CD19/imunologia , Carcinoma de Células Renais/secundário , Carcinoma de Células Renais/terapia , Ensaios Clínicos como Assunto , Europa (Continente) , Feminino , História do Século XVIII , História do Século XIX , História do Século XX , Humanos , Imunoterapia Adotiva/história , Israel , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Leucemia Linfocítica Crônica de Células B/terapia , Depleção Linfocítica/métodos , Linfócitos do Interstício Tumoral/transplante , Linfoma Folicular/terapia , Mieloma Múltiplo/terapia , Neoplasias/imunologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T/imunologia , Estados Unidos
5.
Clin Transl Med ; 11(12): e668, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34898055

RESUMO

The level of postvaccine protection depends on two factors: antibodies and T-cell responses. While the first one is relatively easily measured, the measuring of the second one is a difficult problem. The recent studies indicate that the first one may be a good proxy for the protection, at least for SARS-CoV-2. The massive data currently gathered by both researcher and citizen scientists may be pivotal in confirming this observation, and the collective body of evidence is growing daily. This leads to an acceptance of IgG antibody levels as an accessible biomarker of individual's protection. With enormous and immediate need for assessing patient condition at the point of care, quantitative antibody analysis remains the most effective and efficient way to assess the protection against the disease. Let us not discount importance of reference points in the turmoil of current pandemics.


Assuntos
Anticorpos Antivirais/química , Anticorpos/química , Biomarcadores/metabolismo , COVID-19/sangue , COVID-19/imunologia , Especificidade de Anticorpos , Humanos , Sistema Imunitário , Imunidade , Imunoglobulina G/metabolismo , Unidades de Terapia Intensiva , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , SARS-CoV-2 , Testes Sorológicos/métodos , Testes Sorológicos/normas , Vacinas
6.
PLoS One ; 16(12): e0261082, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34910746

RESUMO

INTRODUCTION: Multiple previous studies have shown the monoclonal antibody Das-1 (formerly called 7E12H12) is specifically reactive towards metaplastic and carcinomatous lesions in multiple organs of the gastrointestinal system (e.g. Barrett's esophagus, intestinal-type metaplasia of the stomach, gastric adenocarcinoma, high-grade pancreatic intraepithelial neoplasm, and pancreatic ductal adenocarcinoma) as well as in other organs (bladder and lung carcinomas). Beyond being a useful biomarker in tissue, mAb Das-1 has recently proven to be more accurate than current paradigms for identifying cysts harboring advanced neoplasia. Though this antibody has been used extensively for clinical, basic science, and translational applications for decades, its epitope has remained elusive. METHODS: In this study, we chemically deglycosylated a standard source of antigen, which resulted in near complete loss of the signal as measured by western blot analysis. The epitope recognized by mAb Das-1 was determined by affinity to a comprehensive glycan array and validated by inhibition of a direct ELISA. RESULTS: The epitope recognized by mAb Das-1 is 3'-Sulfo-Lewis A/C (3'-Sulfo-LeA/C). 3'-Sulfo-LeA/C is broadly reexpressed across numerous GI epithelia and elsewhere during metaplastic and carcinomatous transformation. DISCUSSION: 3'-Sulfo-LeA/C is a clinically important antigen that can be detected both intracellularly in tissue using immunohistochemistry and extracellularly in cyst fluid and serum by ELISA. The results open new avenues for tumorigenic risk stratification of various gastrointestinal lesions.


Assuntos
Anticorpos Monoclonais/imunologia , Transformação Celular Neoplásica/imunologia , Epitopos de Linfócito B/imunologia , Neoplasias Gastrointestinais/imunologia , Mucosa Intestinal/imunologia , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Oligossacarídeos/imunologia , Especificidade de Anticorpos , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica
7.
PLoS Comput Biol ; 17(12): e1009675, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34898603

RESUMO

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.


Assuntos
Antígenos Virais/química , COVID-19/imunologia , COVID-19/virologia , Epitopos de Linfócito B/química , SARS-CoV-2/química , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Reações Antígeno-Anticorpo/genética , Reações Antígeno-Anticorpo/imunologia , Biologia Computacional , Coronavirus/química , Coronavirus/genética , Coronavirus/imunologia , Bases de Dados de Compostos Químicos , Mapeamento de Epitopos , Epitopos de Linfócito B/genética , Humanos , Camundongos , Modelos Moleculares , Pandemias , SARS-CoV-2/genética , Anticorpos de Domínio Único/imunologia
8.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(11): 1026-1031, 2021 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-34809742

RESUMO

Objective To prepare mouse-derived monoclonal antibody against human suppression of tumorigenicity 2 (ST2) molecule and make initial identification. Methods BALB/c mice were immunized with the recombinant human ST2 molecule, and the conventional B-cell hybridoma technology was used to prepare the anti-ST2 monoclonal antibodies (mAbs). Their application in western blotting, immunohistochemistry, and flow cytometry were evaluated. The sandwich ELISA detecting soluble ST2 was established to test the serum levels of ST2 in patients with heat stroke. And the ST2 luciferase reporter gene detection system was established to detect their neutralization activity. Results Thirty-eight hybridoma cell lines secreting mouse anti-human ST2 mAb were obtained and named from XA325.1 to XA325.38. Preliminary screening and identification of them showed that they can be used to identify the purified recombinant ST2 proteins and cellular expressed ST2 using western blotting and immunohistochemistry. Two of them can be used for flow cytometry to identify the exogenously transfected ST2 molecule on the cell surface. Using XA325.16 mAb coating, combined with XA325.5-labeled biotin, an ELISA kit detecting soluble ST2 in serum was established. It was found that the serum levels of ST2 in patients with heat stroke increased significantly. Moreover, XA325.5 was found with neutralizing activity which can block the biological effect of IL-33. Conclusion A set of mouse anti-human ST2 mAbs was prepared, which can be used in a variety of immunological detection techniques. Besides, XA325.5 neutralizing antibody has a potential value in clinical application.


Assuntos
Anticorpos Monoclonais , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C
9.
J Korean Med Sci ; 36(43): e294, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34751011

RESUMO

BACKGROUND: In Korea, the first community outbreak of coronavirus disease 2019 (COVID-19) occurred in Daegu on February 18, 2020. This study was performed to investigate the prevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies in healthcare workers (HCWs) at 6 major hospitals in Daegu. METHODS: Blood specimens of 2,935 HCWs at 6 major hospitals in Daegu from January 2021 to February 2021 were collected. Every specimen was tested for antibody against SARS-CoV-2 using both Elecsys Anti-SARS-CoV-2 electrochemiluminescence immunoassay (Roche Diagnostics, Rotkreuz, Switzerland) and R-FIND COVID-19 IgG/M/A enzyme-linked immunosorbent assay kit (SG medical Inc., Seoul, Korea) as screening tests. If 1 or more of these screening test results was positive, 2 additional antibody tests were performed using Abbott Anti-SARS-CoV-2 IgG assay (Abbott, Abbott Park, IL, USA) and cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript USA Inc., Piscataway, NJ, USA). If 2 or more of the total 4 test results were positive, it was determined as positive for the antibody against SARS-CoV-2. RESULTS: According to the criteria of SARS-CoV-2 antibody positivity determination, 12 subjects were determined as positive. The overall positive rate of the SARS-CoV-2 antibody was 0.41% (12/2,935). Of the 12 subjects determined as positive, 7 were diagnosed with COVID-19, and the remaining 5 were nondiagnosed cases of COVID-19. CONCLUSION: In early 2021, the overall seroprevalence of SARS-CoV-2 antibody among HCW located in Daegu was 0.41%, and 0.17% excluding COVID-19 confirmed subjects. These results were not particularly high compared with the general public and were much lower than HCWs in other countries.


Assuntos
Anticorpos Antivirais/sangue , COVID-19/diagnóstico , COVID-19/imunologia , Pessoal de Saúde/estatística & dados numéricos , Imunoglobulina G/sangue , Adulto , Idoso , Anticorpos Neutralizantes , Especificidade de Anticorpos , COVID-19/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hospitais , Humanos , Imunoglobulina A/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Prevalência , República da Coreia/epidemiologia , SARS-CoV-2
10.
Front Immunol ; 12: 752003, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34646276

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have become a major concern in the containment of current pandemic. The variants, including B.1.1.7 (Alpha), B.1.351 (Beta), P1 (Gamma) and B.1.617.2 (Delta) have shown reduced sensitivity to monoclonal antibodies, plasma and/or sera obtained from convalescent patients and vaccinated individuals. Development of potent therapeutic monoclonal antibodies (mAbs) with broad neutralizing breadth have become a priority for alleviating the devastating effects of this pandemic. Here, we review some of the most promising broadly neutralizing antibodies obtained from plasma of patients that recovered from early variants of SARS-CoV-2 that may be effective against emerging new variants of the virus. This review summarizes several mAbs, that have been discovered to cross-neutralize across Sarbecoviruses and SARS-CoV-2 escape mutants. Understanding the characteristics that confer this broad and cross-neutralization functions of these mAbs would inform on the development of therapeutic antibodies and guide the discovery of second-generation vaccines.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/sangue , COVID-19/sangue , COVID-19/virologia , Reações Cruzadas , Interações Hospedeiro-Patógeno , Humanos , Mutação , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade
11.
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
12.
Cells ; 10(10)2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34685704

RESUMO

Patients with prostate cancer (PCa) receiving docetaxel chemotherapy invariably develop chemoresistance. The transcription co-activator lens epithelium-derived growth factor p75 (LEDGF/p75), also known as DFS70 and PSIP1, is upregulated in several human cancers, including PCa and promotes resistance to docetaxel and other drugs. The C-terminal region of LEDGF/p75 contains an integrase binding domain (IBD) that tethers nuclear proteins, including the HIV-1 integrase and transcription factors, to active chromatin to promote viral integration and transcription of cellular survival genes. Here, we investigated the contribution of the LEDGF/p75 IBD interactome to PCa chemoresistance. Quantitative immunoblotting revealed that LEDGF/p75 and its IBD-interacting partners are endogenously upregulated in docetaxel-resistant PCa cell lines compared to docetaxel-sensitive parental cells. Using specific human autoantibodies, we co-immunoprecipitated LEDGF/p75 with its endogenous IBD-interacting partners JPO2, menin, MLL, IWS1, ASK1, and PogZ, as well as transcription factors c-MYC and HRP2, in docetaxel-resistant cells, and confirmed their nuclear co-localization by confocal microscopy. Depletion of LEDGF/p75 and selected interacting partners robustly decreased the survival, clonogenicity, and tumorsphere formation capacity of docetaxel-resistant cells. These results implicate the LEDGF/p75 IBD interactome in PCa chemoresistance and could lead to novel therapeutic strategies targeting this protein complex for the treatment of docetaxel-resistant tumors.


Assuntos
Docetaxel/farmacologia , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Esferoides Celulares/patologia , Especificidade de Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Autoanticorpos/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Esferoides Celulares/efeitos dos fármacos
13.
J Clin Invest ; 131(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34499051

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Little is known about the interplay between preexisting immunity to endemic seasonal coronaviruses and the development of a SARS-CoV-2-specific IgG response. We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed antibody reactivity to nucleocapsid and spike antigens and correlated this IgG response to SARS-CoV-2 neutralization. Patients with COVID-19 mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against a seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings indicate a boost of poorly protective CoV-specific antibodies in patients with COVID-19 that correlated with disease severity, revealing "original antigenic sin."


Assuntos
Linfócitos B/imunologia , Linfócitos B/virologia , COVID-19/imunologia , COVID-19/virologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Estudos de Casos e Controles , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Reações Cruzadas , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunoglobulina G/sangue , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pandemias , Fosfoproteínas/imunologia , Estações do Ano , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia
14.
MAbs ; 13(1): 1978130, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34586015

RESUMO

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Microfluídica/métodos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos , Antígenos/imunologia , Antígenos de Neoplasias/imunologia , Preservação de Sangue , COVID-19/imunologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Hibridomas/imunologia , Separação Imunomagnética , Dispositivos Lab-On-A-Chip , Camundongos , Microfluídica/instrumentação , Muromonab-CD3/imunologia , Plasmócitos , Proteínas Recombinantes/imunologia , SARS-CoV-2/imunologia , Toxoide Tetânico/imunologia , Vacinação
15.
Blood Cancer J ; 11(9): 151, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521813

RESUMO

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.


Assuntos
COVID-19/imunologia , Neoplasias Hematológicas/imunologia , Imunidade Humoral/efeitos dos fármacos , Rituximab/farmacologia , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/efeitos dos fármacos , Anticorpos Antivirais/metabolismo , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/fisiologia , Especificidade de Anticorpos/efeitos dos fármacos , COVID-19/complicações , COVID-19/epidemiologia , COVID-19/terapia , Estudos de Casos e Controles , Feminino , Seguimentos , Neoplasias Hematológicas/complicações , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/epidemiologia , Hospitalização , Humanos , Itália/epidemiologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Rituximab/uso terapêutico
17.
J Immunol Methods ; 498: 113144, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34481824

RESUMO

Bivalent VHHs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both VHHs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity.


Assuntos
Epitopos , Proteínas de Fluorescência Verde/imunologia , Fragmentos de Peptídeos/imunologia , Anticorpos de Domínio Único/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Mutação , Fragmentos de Peptídeos/genética , Multimerização Proteica , Proteína Supressora de Tumor p53/genética
18.
Lab Chip ; 21(19): 3627-3654, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34505611

RESUMO

Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery.


Assuntos
Anticorpos Monoclonais , Antineoplásicos Imunológicos , Especificidade de Anticorpos , Humanos
19.
Acta Cytol ; 65(6): 510-521, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34535585

RESUMO

INTRODUCTION/OBJECTIVE: Liquid-based cytology (LBC) is advantageous as multiple stained specimens can be prepared and used for additional assays such as immunocytochemical and molecular-pathological investigations. Two types of preservative-fixative solutions (fixatives) are used for nongynecologic specimens used in the BD SurePath-LBC (SP-LBC) method, and their components vary. However, few studies have evaluated the differences in antigen-retaining ability between these fixatives. Therefore, we investigated and compared the antigen-retaining ability of the fixatives in immunocytochemical staining (ICC) under long-term storage conditions. MATERIALS AND METHODS: Sediments of cultured RAJI cells (derived from Burkitt's lymphoma) were added to each fixative (red and blue) and stored at room temperature for a specified period (1 h; 1 week; and 1, 3, and 6 months). The specimens were then prepared using the SP-LBC method and subjected to ICC. Positivity rate was calculated using the specimens fixed at room temperature for 1 h as a control. Antibodies against Ki67 expressed in the nucleus and against CD20 and leukocyte common antigen (LCA) expressed on the cell membrane were used. RESULTS: For CD20 and LCA, the positivity rate increased with time in the red fixative compared with that in the control. In the blue fixative, the positivity rate was highest at 1 h and was maintained at a high level throughout the storage period. In contrast, the Ki67 positivity rate was highest at 1 h in both red and blue fixatives and markedly decreased with time. Therefore, although refrigerated (8°C) storage was used, no improvement was noted. CONCLUSIONS: Long-term storage is possible for cell membrane antigens at room temperature; however, it is unsuitable for intranuclear antigens. Therefore, we conclude that suitable fixative type and storage temperature differ based on antigen location. Further investigation is warranted.


Assuntos
Antígenos CD20/análise , Antígenos/análise , Linfoma de Burkitt/imunologia , Fixadores/química , Imuno-Histoquímica , Antígeno Ki-67/análise , Antígenos Comuns de Leucócito/análise , Fixação de Tecidos , Especificidade de Anticorpos , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Humanos , Biópsia Líquida , Valor Preditivo dos Testes , Estabilidade Proteica , Fatores de Tempo
20.
J Immunol Methods ; 498: 113124, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34425081

RESUMO

Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use (Keefe et al., 2009; Leite et al., 2008; Lozier et al., 2015). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes (Garg and Loring, 2017). We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) (Urriola et al., 2021) that utilises the monoclonal antibody mab35(Urriola et al., 2021) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits (Vernino et al., 1998). Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use. We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.


Assuntos
Anticorpos Monoclonais/imunologia , Doenças Autoimunes do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso Autônomo/diagnóstico , Conotoxinas/química , Citometria de Fluxo , Corantes Fluorescentes/química , Gânglios Autônomos/imunologia , Neuroblastoma/imunologia , Receptores Colinérgicos/análise , Especificidade de Anticorpos , Autoanticorpos/sangue , Doenças Autoimunes do Sistema Nervoso/imunologia , Doenças do Sistema Nervoso Autônomo/imunologia , Linhagem Celular Tumoral , Epitopos , Humanos , Valor Preditivo dos Testes , Receptores Colinérgicos/imunologia
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