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1.
J Chromatogr A ; 1640: 461962, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33582517

RESUMO

In-tube solid-phase microextraction (IT-SPME) with capillary column as extraction device is a well-established green extraction technique with a lot of applications in the fields of biomedicine, food and environment. This article reviews the research contributions of IT-SPME for analysis of proteins. The paper first briefly describes the history of IT-SPME. Then, the development and principle of IT-SPME for analysis of proteins are introduced, in which capillary column configurations of IT-SPME and instruments for quantitative analysis of proteins are summarized. Subsequently, the synthesis strategy and recognition principle of different recognition units, including antibodies, aptamers, molecularly imprinted polymers, and boronate affinity materials, are discussed in detail. This part also introduces several rare recognition units, including lectins, restricted access materials, lysine modified with ß-cyclodextrin and cell membrane. The development trend and possible future direction of IT-SPME for analysis of proteins are mentioned.


Assuntos
Proteínas/análise , Proteínas/isolamento & purificação , Microextração em Fase Sólida/métodos , Anticorpos/isolamento & purificação , Ácidos Borônicos/química , Impressão Molecular , Polímeros/química
2.
Methods Mol Biol ; 2178: 251-284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33128755

RESUMO

Nowadays, monolithic stationary phases, because of their special morphology and enormous permeability, are widely used for the development and realization of fast dynamic and static processes based on the mass transition between liquid and solid phases. These are liquid chromatography, solid-phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including the bioaffinity mode, represents unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In the edited chapter, the examples of the application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by the combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents the original and practically valuable method that can be used in biotechnology. In addition, macroporous monoliths were adapted to the immobilization of such different enzymes as polynucleotide phosphorylase, ribonuclease A, α-chymotrypsin, chitinolytic biocatalysts, ß-xylosidase, and ß-xylanase. The possibility of use of immobilized enzyme reactors based on monoliths for different purposes is demonstrated.


Assuntos
Anticorpos , Ácidos Nucleicos , Peptídeos , Vírus , Anticorpos/química , Anticorpos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Ácidos Nucleicos/química , Ácidos Nucleicos/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Vírus/química , Vírus/isolamento & purificação
3.
J Chromatogr A ; 1635: 461632, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33333349

RESUMO

Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as ß-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.


Assuntos
Produtos Biológicos/isolamento & purificação , Química Farmacêutica/métodos , Cromatografia de Afinidade , Ligantes , Anticorpos/isolamento & purificação , Características da Família , Humanos , Peptídeos/isolamento & purificação , Peptoides/química , Proteínas/isolamento & purificação
4.
Methods Mol Biol ; 2247: 59-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301112

RESUMO

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.


Assuntos
Anticorpos/genética , Formação de Anticorpos/genética , Expressão Gênica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Anticorpos/isolamento & purificação , Baculoviridae/genética , Cromatografia de Afinidade , Ordem dos Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/genética , Imunoglobulina G/isolamento & purificação , Calicreínas/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Transfecção
5.
ACS Appl Mater Interfaces ; 12(52): 58191-58200, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-33319977

RESUMO

Purification of antibodies has become a critical factor in antibody production. A high-purity specific antibody against antigens, especially small molecules, seems to be difficult to obtain, even with the help of a protein A affinity column, which is a conventional and broadly used ligand for the separation of antibody and non-antibody proteins. Therefore, it is urgent to develop a cheap, simple, efficient, and stable method to separate the specific antibody from other antibodies. In this study, to improve the sensitivity and accuracy of immunoassay results, enrofloxacin (ENR) was grafted onto polyethylenimine (PEI) by the abundant amino groups and then the whole ligand (ENR-PEI) was conjugated to CNBr-Sepharose 4B to prepare the affinity column for the purification of the specific antibody against ENR from polyclonal antibodies. Scanning electron microscopy and Fourier transform infrared spectroscopy verification showed that Sepharose 4B was successfully modified by ENR-PEI with excellent uniformity. The capacity of the prepared column could reach to 6.15 mg of specific antibody with high purity per milliliter resin due to the high coupling ratio (49.3:1) of ENR on PEI, and the IC50 value of the antibody after purification was 47.58 ng/mL with a lowest limit of detection (IC10) of 1.099 ng/mL-18 times lower than those of the antibody purified through the protein A column. All the results showed that this new kind of resin could be used as the potential ligand in the purification of the trace-specific antibody against antigens in complex mixtures with high efficiency and specificity.


Assuntos
Anticorpos/imunologia , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Haptenos/química , Haptenos/imunologia , Imunoensaio/métodos , Polietilenoimina/química , Enrofloxacina/química , Ligantes
6.
Arq. ciências saúde UNIPAR ; 24(3): 133-138, set-dez. 2020.
Artigo em Português | LILACS | ID: biblio-1129455

RESUMO

Quando um indivíduo é exposto a antígenos eritrocitários não próprios, ocorre uma resposta imunológica, que leva à produção de anticorpos irregulares voltados contra esses antígenos. Esse processo é conhecido como aloimunização eritrocitária e acontece em decorrência de transfusões de sangue ou gestações incompatíveis. Na medicina transfusional a pesquisa de anticorpos irregulares é fundamental, pois a falha na detecção de um aloanticorpo pode provocar reações transfusionais, aloimunizações, anemias hemolíticas autoimunes e doença hemolítica perinatal. Este estudo tem por objetivo analisar a frequência de anticorpos irregulares de pacientes atendidos no Hemocentro Regional de Francisco Beltrão, Paraná, no ano de 2017. Os dados foram coletados a partir da revisão de registros em arquivos do Laboratório de Imunohematologia do Hemonúcleo. Foram avaliados dados de 49 protocolos de pacientes que apresentaram dificuldades transfusionais no ano de 2017. Dentre os pesquisados, 37 pacientes (75,5%) apresentaram anticorpos irregulares. Dentre os anticorpos anti-eritrocitários observados neste estudo, evidenciou-se a presença de doze pacientes com anti-D (27,2%), seis pacientes com anti-K (13,6%), quatro pacientes com anti-C (9,0%) e em seis pacientes (13,6%) foi observada a presença de autoanticorpos. Este estudo indica que, nos pacientes transfundidos, os anticorpos mais frequentes foram os aloanticorpos Anti-D do Sistema Rh, provavelmente devido ao seu alto grau de imunogenicidade. A prevalência desses anticorpos é semelhante a vários estudos encontrados na literatura.


When an individual is exposed to not-self red blood cell antigens, an immune response occurs, which leads to the production of irregular antibodies directed against these antigens. This process is known as erythrocyte alloimmunization and occurs as a result of blood transfusions or incompatible pregnancies. In transfusion medicine, the search for irregular antibodies is essential, since failure to detect an alloantibody can cause transfusion reactions, alloimmunizations, autoimmune hemolytic anemias, and perinatal hemolytic disease. This study aims at analyzing the frequency of irregular antibodies of patients seen at the Regional Blood Center of Francisco Beltrão, Paraná, in 2017. The data were collected from the review of records in files of the Immunohematology Laboratory of Hemonúcleo. Data from 49 protocols of patients who had transfusion difficulties in 2017 were evaluated. Among those surveyed, 37 patients (75.5%) had irregular antibodies. Among the anti-erythrocyte antibodies observed in this study, the presence of twelve patients with anti-D (27.2%), six patients with anti-K (13.6%), four patients with anti-C (9.0 %), and in six patients (13.6%) with the presence of autoantibodies were observed. This study indicates that, in transfused patients, the most frequent antibodies were the Rh System Anti-D alloantibodies, probably due to their high degree of immunogenicity. The prevalence of these antibodies is similar to several studies found in the literature.


Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Adulto Jovem , Autoanticorpos/imunologia , Isoanticorpos/imunologia , Autoanticorpos/isolamento & purificação , Transfusão de Sangue , Estudos Retrospectivos , Distribuição por Sexo , Distribuição por Idade , Eritrócitos/imunologia , Reação Transfusional/imunologia , Isoanticorpos/isolamento & purificação , Anticorpos/isolamento & purificação , Anticorpos/imunologia
7.
PLoS Negl Trop Dis ; 14(9): e0008701, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956365

RESUMO

Russell's vipers (RVs) envenoming is an important public health issue in South-East Asia. Disseminated intravascular coagulopathy, systemic bleeding, hemolysis, and acute renal injury are obvious problems that develop in most cases, and neuromuscular junction blocks are an additional problem caused by western RV snakebite. The complex presentations usually are an obstacle to early diagnosis and antivenom administration. Here, we tried to produce highly specific antibodies in goose yolks for use in a paper-based microfluidic diagnostic kit, immunochromatographic test of viper (ICT-Viper), to distinguish RVs from other vipers and even cobra snakebite in Asia. We used indirect ELISA to monitor specific goose IgY production and western blotting to illustrate the interaction of avian or mammal antibody with venom proteins. The ICT-Viper was tested not only in prepared samples but also in stored patient serum to demonstrate its preliminary efficacy. The results revealed that specific anti-Daboia russelii IgY could be raised in goose eggs effectively without inducing adverse effects. When it was collocated with horse anti-Daboia siamensis antibody, which broadly reacted with most of the venom proteins of both types of Russell's viper, the false cross-reactivity was reduced, and the test showed good performance. The limit of detection was reduced to 10 ng/ml in vitro, and the test showed good detection ability in clinical snake envenoming case samples. The ICT-Viper performed well and could be combined with a cobra venom detection kit (ICT-Cobra) to create a multiple detection strip (ICT-VC), which broadens its applications while maintaining its detection ability for snake envenomation identification. Nonetheless, the use of the ICT-Viper in the South-East Asia region is pending additional laboratory and field investigations and regional collaboration. We believe that the development of this practical diagnostic tool marks the beginning of positive efforts to face the global snakebite issue.


Assuntos
Antivenenos/imunologia , Aves/imunologia , Mamíferos/imunologia , Mordeduras de Serpentes/diagnóstico , Mordeduras de Serpentes/imunologia , Peçonhas/imunologia , Lesão Renal Aguda , Animais , Anticorpos/isolamento & purificação , Ásia , Ásia Sudeste , Testes Diagnósticos de Rotina , Venenos Elapídicos , Ensaio de Imunoadsorção Enzimática , Gansos/imunologia , Hemorragia , Cavalos/imunologia , Humanos , Imunoglobulinas , Víbora de Russell
8.
Gene ; 756: 144911, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32574756

RESUMO

Enolase, a multifunctional glycolytic enzyme, is known to act as a plasminogen receptor in many species, involved in the pivotal processes such as motility, adhesion, invasion, growth, and differentiation of the parasites. Knowledge on the function of enolase from Dermanyssus gallinae is very limited. Here we report on the molecular cloning, enzymatic activity, tissue distribution and plasminogen binding activity of enolase from D. gallinae (DgENO). The full-length of cDNA was 1305 bp, specifying a peptide of 434 amino acids. Bioinformatics analysis showed that DgENO was highly conserved compared with a range of organisms, indicating the potentially similar functions in D. gallinae. A recombinant DgENO (rDgENO) protein was produced and characterized, it catalyzed the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, the optimal pH was 7.5. Polyclonal antibodies were generated in mice and western blotting indicated that antiserum specifically recognized the native enolase in the somatic extracts from D. gallinae. Immunohistochemical staining of mite sections revealed that the distribution of DgENO was ubiquitous with high level in salivary gland, mite digestive tissues and fat bodies in D. gallinae. Expression level of DgENO was observed mostly in engorged adult mites. Moreover, ELISA binding assay showed that rDgENO could bind plasminogen, and lysine analog ε-aminocaproic acid significantly inhibited this binding activity, indicating that D. gallinae enolase is a receptor of plasminogen. The present study provided foundation for understanding of the biological functions of DgENO and its application in development of vaccines against D. gallinae.


Assuntos
Antígenos/imunologia , Ácaros/imunologia , Fosfopiruvato Hidratase/química , Vacinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Antígenos/química , Antígenos/genética , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Ácaros/enzimologia , Ácaros/genética , Ácaros/crescimento & desenvolvimento , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/genética , Plasminogênio/metabolismo , Alinhamento de Sequência
9.
Proc Natl Acad Sci U S A ; 117(18): 9851-9856, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32327606

RESUMO

Toward the goal of increasing the throughput of high-resolution mass characterization of intact antibodies, we developed a RapidFire-mass spectrometry (MS) assay using electrospray ionization. We achieved unprecedented screening throughput as fast as 15 s/sample, which is an order of magnitude improvement over conventional liquid chromatography (LC)-MS approaches. The screening enabled intact mass determination as accurate as 7 ppm with baseline resolution at the glycoform level for intact antibodies. We utilized this assay to characterize and perform relative quantitation of antibody species from 248 samples of 62 different cell line clones at four time points in 2 h using RapidFire-time-of-flight MS screening. The screening enabled selection of clones with the highest purity of bispecific antibody production and the results significantly correlated with conventional LC-MS results. In addition, analyzing antibodies from a complex plasma sample using affinity-RapidFire-MS was also demonstrated and qualified. In summary, the platform affords high-throughput analyses of antibodies, including bispecific antibodies and potential mispaired side products, in cell culture media, or other complex matrices.


Assuntos
Anticorpos Biespecíficos/sangue , Anticorpos/sangue , Ensaios de Triagem em Larga Escala/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Anticorpos/isolamento & purificação , Anticorpos Biespecíficos/isolamento & purificação , Linhagem Celular , Cromatografia Líquida/métodos , Humanos
10.
Sci Rep ; 10(1): 4611, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165720

RESUMO

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.


Assuntos
Anticorpos/imunologia , Dissacarídeos/imunologia , Imunoglobulina G/imunologia , Anticorpos/isolamento & purificação , Anticorpos/farmacologia , Dissacarídeos/antagonistas & inibidores , Ensaio de Imunoadsorção Enzimática , Escherichia coli/imunologia , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/farmacologia , Sepse/sangue , Sepse/diagnóstico , Sepse/etiologia
11.
Sensors (Basel) ; 20(3)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-32028629

RESUMO

Since the norovirus is the main cause of acute gastroenteritis all over the world, its fast detection is crucial in medical diagnostics. In this work, a rapid, sensitive, and selective optical fiber biosensor for the detection of norovirus virus-like particles (VLPs) is reported. The sensor is based on highly sensitive long-period fiber gratings (LPFGs) coated with antibodies against the main coat protein of the norovirus. Several modification methods were verified to obtain reliable immobilization of protein receptors on the LPFG surface. We were able to detect 1 ng/mL norovirus VLPs in a 40-min assay in a label-free manner. Thanks to the application of an optical fiber as the sensor, there is a possibility to increase the user's safety by separating the measurement point from the signal processing setup. Moreover, our sensor is small and light, and the proposed assay is straightforward. The designed LPFG-based biosensor could be applied in both fast norovirus detection and in vaccine testing.


Assuntos
Anticorpos/isolamento & purificação , Técnicas Biossensoriais , Gastroenterite/genética , Norovirus/isolamento & purificação , Gastroenterite/diagnóstico , Gastroenterite/imunologia , Gastroenterite/virologia , Humanos , Norovirus/patogenicidade , Proteínas Virais/imunologia , Proteínas Virais/isolamento & purificação
12.
Mol Biotechnol ; 62(4): 228-239, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31981039

RESUMO

In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. We show that single chain variable fragment (scFv) libraries with adequate qualities can readily be cloned in a 'scar-less' manner and that the isolation of antigen-specific antibodies from immunized chickens is feasible within three selection rounds. Moreover, we demonstrate the general applicability of this method by rapidly constructing and panning VHH single domain antibody phage display libraries from immunized Llama repertoires.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Anticorpos de Cadeia Única/genética , Anticorpos de Domínio Único/genética , Animais , Anticorpos/imunologia , Anticorpos/isolamento & purificação , Bacteriófagos/genética , Camelídeos Americanos , Galinhas , Desoxirribonucleases de Sítio Específico do Tipo II , Receptores ErbB/imunologia , Escherichia coli , Anticorpos de Cadeia Única/imunologia , Anticorpos de Domínio Único/imunologia
13.
J Immunol Methods ; 477: 112688, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31676342

RESUMO

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.


Assuntos
Anticorpos/isolamento & purificação , Toxinas Bacterianas/imunologia , Monitoramento de Medicamentos/métodos , Exotoxinas/imunologia , Leucemia de Células Pilosas/tratamento farmacológico , Domínios Proteicos/imunologia , ADP Ribose Transferases/imunologia , Anticorpos/sangue , Anticorpos/imunologia , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/efeitos adversos , Ensaios Clínicos Fase III como Assunto , Exotoxinas/administração & dosagem , Exotoxinas/efeitos adversos , Reações Falso-Negativas , Estudos de Viabilidade , Humanos , Imunoensaio/métodos , Leucemia de Células Pilosas/sangue , Leucemia de Células Pilosas/imunologia , Sensibilidade e Especificidade , Resultado do Tratamento , Fatores de Virulência/imunologia
14.
Sensors (Basel) ; 19(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842479

RESUMO

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.


Assuntos
Anticorpos/isolamento & purificação , Técnicas Biossensoriais , Técnicas de Diagnóstico Cardiovascular , Imunoensaio/métodos , Anticorpos/genética , Proteína C-Reativa/genética , Proteína C-Reativa/isolamento & purificação , Cromatografia de Afinidade , Produtos de Degradação da Fibrina e do Fibrinogênio/genética , Produtos de Degradação da Fibrina e do Fibrinogênio/isolamento & purificação , Ouro/química , Humanos , Nanopartículas Metálicas/química , Mioglobina/sangue , Mioglobina/isolamento & purificação , Fitas Reagentes/química
15.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1134-1135: 121832, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31790917

RESUMO

Separations of complex peptide mixtures have been a common target application for two-dimensional liquid chromatography over the last few decades. These separations have most frequently been carried out at the capillary scale, with columns on the order of 75 µm i.d. and flow rates on the order of 500 nL/min. Recently, however, several groups have worked to optimize comprehensive 2D-LC (LC × LC) separations of peptides at the analytical scale (i.e., 2 mm i.d. columns, and ca. 1 mL/min flow rates) and demonstrated peak capacities on the order of 5000 in analysis times of a few hours, using reversed-phase separations in both dimensions. In this paper we aim to advance the performance of such separations in two primary ways. First, we demonstrate that active solvent modulation (ASM) can be used to improve the 2D peak capacity by both enabling use of long and highly efficient first dimension (1D) columns, and by mitigating the deleterious effects of injecting large fractions of 1D effluent into the small columns that are required for fast and highly sensitive second dimension (2D) separations. Taken together these two benefits enable the realization of a peak capacity of 10,000 in an analysis time of four hours. This comes at the cost of increased instrument complexity compared to 1D-LC separations, but the 2D-LC approach is unquestionably the most efficient way to improve upon the resolving power of existing 1D-LC. Second, we have systematically studied the compromise between the peak capacity of each 2D separation and the operating pressure required to achieve that peak capacity. Understanding this compromise will be important to the development of LC × LC methods that both produce high peak capacities, and are sufficiently robust to operate for days at a time without significant losses in separation performance. Based on the results of this study we chose conditions for subsequent separations that required less than 400 bar operating pressure in the second dimension, but yielded a 2D peak capacity of about 3500 in 2 h. After 160 h of continuous operation of the LC × LC separation under these conditions (and about 20,000 injections into the 2D column) the 2D column had only lost about 18% of its initial isocratic efficiency. These results should motivate further development and implementation of such high performing and robust separations for the identification and quantification of peptides in a variety of application areas, including digests of therapeutic proteins such as monoclonal antibodies.


Assuntos
Anticorpos/análise , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Peptídeos/análise , Anticorpos/química , Anticorpos/isolamento & purificação , Humanos , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Reprodutibilidade dos Testes , Solventes/química
16.
Sensors (Basel) ; 19(24)2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31835479

RESUMO

Aptamers are synthetic bio-receptors of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) origin selected by the systematic evolution of ligands (SELEX) process that bind a broad range of target analytes with high affinity and specificity. So far, electrochemical biosensors have come up as a simple and sensitive method to utilize aptamers as a bio-recognition element. Numerous aptamer based sensors have been developed for clinical diagnostics, food, and environmental monitoring and several other applications are under development. Aptasensors are capable of extending the limits of current analytical techniques in clinical diagnostics, food, and environmental sample analysis. However, the potential applications of aptamer based electrochemical biosensors are unlimited; current applications are observed in the areas of food toxins, clinical biomarkers, and pesticide detection. This review attempts to enumerate the most representative examples of research progress in aptamer based electrochemical biosensing principles that have been developed in recent years. Additionally, this account will discuss various current developments on aptamer-based sensors toward heavy metal detection, for various cardiac biomarkers, antibiotics detection, and also on how the aptamers can be deployed to couple with antibody-based assays as a hybrid sensing platform. Aptamers can be used in various applications, however, this account will focus on the recent advancements made toward food, environmental, and clinical diagnostic application. This review paper compares various electrochemical aptamer based sensor detection strategies that have been applied so far and used as a state of the art. As illustrated in the literature, aptamers have been utilized extensively for environmental, cancer biomarker, biomedical application, and antibiotic detection and thus have been extensively discussed in this article.


Assuntos
Técnicas Biossensoriais/métodos , Monitoramento Ambiental , Análise de Alimentos , Patologia Molecular , Anticorpos/isolamento & purificação , Aptâmeros de Nucleotídeos/química , Biomarcadores/análise , Humanos , Ligantes , Técnica de Seleção de Aptâmeros/métodos
17.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1134-1135: 121850, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31783251

RESUMO

Antibodies are widely used as therapeutic drugs in the treatment of various diseases. Currently, Protein A affinity chromatography is still the most popular technique in antibody purification. However, it has some limitations such as high cost, leakage of ligands and requirement of harsh elution conditions. Hydrophobic charge-induction chromatography (HCIC) provides an alternative to Protein A affinity chromatography. The binding between HCIC resins and target proteins can be achieved via hydrophobic interactions at neutral pH, and proteins can be eluted via electrostatic repulsion between proteins and charged ligands under acidic conditions. HCIC is applied to the purification of antibodies and some specific proteins successfully, which is a promising technique with economic benefits and high efficiency. In this review, theoretical analysis and factors affecting HCIC adsorption are presented that provides functional mechanism of HCIC. HCIC ligands with different structures available on the market and reported in the literature are discussed and their recent applications in antibody purification are reviewed. Moreover, affecting factors such as ligand density, preparation approaches and additives of HCIC resins on their adsorption performance are summarized and discussed. In addition, future development of HCIC such as polymer-grafted HCIC resins, membrane chromatography with HCIC and continuous chromatography are proposed.


Assuntos
Cromatografia Líquida/métodos , Animais , Anticorpos/isolamento & purificação , Células CHO , Fenômenos Químicos , Cricetinae , Cricetulus , Humanos , Eletricidade Estática
18.
J Chromatogr A ; 1604: 460474, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31493850

RESUMO

Biomimetic affinity chromatography with short peptide ligands is a developing technology, which has great potential for antibody separation and purification. In this study, a tetrapeptide library with critical residues of natural ligands to hIgG was constructed and a novel tetrapeptide ligand (Ac-FYHE) with high LibDock scores was selected by molecular docking. Then, Ac-FYHE ligand was linked to agarose bead to prepare a new chromatography resin. The properties of antibody adsorption were measured and evaluated by static/dynamic adsorption. It was found that the resin with ligand Ac-FYHE has high binding capacity and selectivity for hIgG. The results showed the Qm-hIgG of Ac-FYHE-4FF resin was 87.9 mg/g resin while the Qm-BSA of this resin was only 16.5 mg/g resin at pH 7.0. Moreover, at pH 7.0, Q10% of Ac-FYHE-4FF resin was 24.1 mg/mL for hIgG but just 2.1 mg/mL for BSA, which presented high selectivity of the screened resin at pH 7.0. Subsequently, the adsorption and separation properties of the Ac-FYHE-4FF resin were further investigated. As a result, with the addition of 0.5 M NaCl, Qm decreased by less than 20% but Qm decreased by 70% with the addition of 50% (v/v) ethylene glycol, which indicated that hydrophobic interaction would be the driving force for the binding between resin and hIgG. Besides, pH 7.5 and pH 4.5 could be the optimal loading and elution condition for hIgG, respectively. Finally, the Ac-FYHE-4FF resin was applied to separate mAb or/and hIgG from BSA containing feedstock, CHO cell culture supernatant and human serum, and the purity and recovery were both more than 90% with only one-step separation. The results indicate that the Ac-FYHE-4FF resin developed in this work would be promising for antibody separation and purification.


Assuntos
Anticorpos/isolamento & purificação , Biomimética , Técnicas de Química Analítica/métodos , Cromatografia de Afinidade , Adsorção , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento Molecular , Peptídeos/química , Sefarose/química
19.
J Vis Exp ; (150)2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31498318

RESUMO

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.


Assuntos
Anticorpos/isolamento & purificação , Linfócitos B/fisiologia , Citometria de Fluxo/métodos , Leucócitos Mononucleares/fisiologia , Especificidade de Anticorpos , Antígenos/imunologia , Linfócitos B/imunologia , Biomarcadores , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Proteínas Luminescentes/química
20.
Biochem Biophys Res Commun ; 517(3): 421-426, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31378371

RESUMO

Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5' splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance.


Assuntos
Processamento Alternativo , Antitrombina III/química , Heparina/química , Neuropeptídeos/química , Serpinas/química , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Antitrombina III/genética , Antitrombina III/metabolismo , Sequência de Bases , Sítios de Ligação , Encéfalo/metabolismo , Expressão Gênica , Heparina/metabolismo , Humanos , Fígado/metabolismo , Simulação de Dinâmica Molecular , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Coelhos , Serpinas/genética , Serpinas/metabolismo
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