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1.
Anal Chim Acta ; 1189: 338907, 2022 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-34815045

RESUMO

The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.


Assuntos
Anticorpos Imobilizados , Técnicas Biossensoriais , Anticorpos , Imunoensaio , Fragmentos Fc das Imunoglobulinas
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 264: 120236, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34358781

RESUMO

Authors performed investigation on "antigen-antibody" interaction of chicken infectious bronchitis coronavirus (IBV) by a method based on the surface plasmon resonance (SPR). Presence of space-size effect related to a difference between antigen and antibody particle sizes has been theoretically grounded and experimentally proven. Herewith, the difference between responses of the SPR-sensor to specific and non-specific interactions is considerably less (up to 6.3 times) than the expected one (8 - 11 times). An impact of functionalization of sensor's sensitive element surface, as well as acidity of buffer solution on the activity of antigen-antibody interaction was studied here. The difference between sensor's responses to specific and non-specific interactions increased two-fold from 200 to 432ang sec due to this treatment. When changing the acidity of analyzed solution from pH7.3 to pH6.8, the corresponding difference between sensor's responses increased by 6.3 times from 194 up to 1235ang.sec. Thus, an impact of space-size effect on interaction between IBV antigen and specific antibody can be considerably (almost in 3 times) decreased by reducing the acidity of used buffer solution. The results of our investigation can be successfully applied to develop new methods for detection of pathogens and specific antibodies using SPR.


Assuntos
Infecções por Coronavirus , Vírus da Bronquite Infecciosa , Animais , Anticorpos , Galinhas , Infecções por Coronavirus/veterinária , Ressonância de Plasmônio de Superfície
3.
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
4.
Food Chem ; 370: 131027, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34537432

RESUMO

The pollution caused by estrogens in the environment and food has received increasing attention. It is still challenging for on-site immunochromatographic assay (ICA) detection of estrogens. The performance of the prepared probes plays a decisive role in the sensitivity and stability of the ICA system. The published probes usually directly couple the detection antibody to the label, ignoring the influence of the label on the activity of the antibody. In this study, 17ß-estradiol (E2) was used as a model analyte for the ICA system. Two universal probes were constructed based on quantum dot nanobeads (QBs), recombinant protein A (SPA, from Staphylococcus aureus), and rabbit anti-mouse immunoglobulin G antibody (anti-IgG). The probes were prepared by coupling QBs with SPA, releasing anti-E2 monoclonal antibody (mAb), and maintaining its activity. The prepared universal probes can orient recognize the Fc region of mAb and fully expose its Fab region, improving the detection sensitivity of the ICA system. The free anti-E2 mAb and the universal probe (QBs@SPA or QBs@SPA@anti-IgG) were used as the detection antibodies and signal donors, respectively. The results show that the proposed ICA based on QBs@SPA and QBs@SPA@anti-IgG probes could detect E2 with IC50 of 8.83 and 0.93 ng/mL, respectively, within 15 min under optimal conditions. The recovery results of ICA based on QBs@SPA and QBs@SPA@anti-IgG probes showed good agreement with the findings of the high-performance liquid chromatography (HPLC) analysis for spiked samples. The developed ICA system based on universal probes was superior in terms of sensitivity, rapidity, and applicability, and held great promise for its implementation in detecting environmental and food small-molecule pollutants.


Assuntos
Leite , Pontos Quânticos , Animais , Anticorpos , Estradiol , Imunoensaio , Camundongos , Coelhos
5.
Methods Mol Biol ; 2414: 37-45, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34784030

RESUMO

Immunoprecipitation is an affinity purification technique that exploits the highly specific interactions formed between antibodies and their cognate antigens to purify molecules of interest from complex biological solutions. The generation of an effective humoral response provides protection against a wide range of gram-positive pathogens, and thus immunoprecipitation using antibodies purified from immune humans or animals provides a simple but effective means of isolating prospective vaccine antigens from fractionated bacterial cells for downstream identification. The commercial availability of antibody preparations from donated human plasma, containing antibodies against many common gram-positive pathogens, allows the protocol to be performed in the absence of bespoke vaccination experiments. Thus, immunoprecipitation has the potential to reduce the number of animals used in vaccine studies by allowing an initial screen for promising antigens to be conducted in vitro.


Assuntos
Imunoprecipitação , Animais , Anticorpos , Antígenos , Antígenos de Bactérias , Humanos , Vacinação
6.
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
7.
Orv Hetil ; 162(47): 1897-1901, 2021 11 21.
Artigo em Húngaro | MEDLINE | ID: mdl-34801984

RESUMO

Összefoglaló. Bevezetés: A gyermekkori májtranszplantációk hosszú távú kimenetelének javítása érdekében az immunológiai mechanizmusok kerültek a kutatások középpontjába. A donorspecifikus antitesteknek (DSA-k) fontos szerepük van a graft túlélésében a szervtranszplantációk után, a májtranszplantáció esetén azonban ez még vitatott. Célkituzés: Tanulmányunk célja májtranszplantált gyermekeknél a DSA-k meghatározása, valamint a DSA-k jelenléte és a graft állapota közötti összefüggés vizsgálata volt. Módszer: A Semmelweis Egyetem I. Sz. Gyermekgyógyászati Klinikáján gondozott 54 májtranszplantált gyermek vérmintájából történt a humán leukocytaantigén (HLA) elleni antitestek meghatározása. Vizsgáltuk, hogy a laboratóriumi vérvizsgálat eredményei - szérumbilirubin (összes, direkt), alkalikus foszfatáz (ALP), transzaminázok, gamma-glutamil-transzferáz (GGT), immunglobulin-G (IgG) -, az aszpartát-aminotranszferáz/thrombocyta hányadosindex (APRI) és a 4 tényezon alapuló fibrosisindex (FIB4) tekintetében van-e eltérés a DSA-pozitív, illetve a HLA-immunizált betegek esetén a nem immunizált csoporthoz képest. Eredmények: A vizsgált paraméterekben nem találtunk szignifikáns különbségeket a DSA-pozitív, a HLA-immunizált és a nem immunizált betegek csoportjai között. Következtetés: Bár a jelen vizsgálatban nem volt szignifikáns különbség a vizsgált paraméterek esetén, de ez a kis esetszámból is adódhat. A DSA-knak a graftfibrosis kialakulásában való szerepe tisztázására több páciens vizsgálata szükséges, ezért megkezdtük az összes páciensnél a DSA- és HLA- (donor, recipiens) meghatározást, valamint ennek a klinikai gyakorlatunkba való beépítését. Orv Hetil. 2021; 162(47): 1897-1901. INTRODUCTION: To improve the long-term survival of liver-transplanted children, immunological mechanisms became the main interest of researchers. Donor-specific antibodies (DSAs) play a significant role in graft survival after solid organ transplantation, although their role in liver transplantation is controversial. OBJECTIVE: The aim of our study was to determine the presence of DSAs in liver-transplanted children and to examine their effect on graft's condition. METHOD: The determination of anti-human leukocyte antigen (HLA) antibodies was performed using the blood samples of 54 liver-transplanted children. We analysed the difference between the results of the laboratory blood examination - serum bilirubin (all, direct), alkaline-phosphatase (ALP), transaminases, gamma-glutamyl transferase (GGT), immunoglobulin-G (IgG) -, aspartate aminotransferase to platelet ratio index (APRI) and fibrosis-4 index (FIB4) according to DSA and HLA immunization. RESULTS: We did not find any significant difference in the examined parameters regarding DSA and HLA immunization. CONCLUSION: Although this study was not able to provide significant difference in the examined parameters, this can be explained with the low number of cases. To clarify the significance of DSA in graft fibrosis, we need a larger dataset. We started regular DSA and HLA (donor and recipient) determination during follow-up in liver-transplanted children. Orv Hetil. 2021; 162(47): 1897-1901.


Assuntos
Transplante de Fígado , Transplante de Órgãos , Anticorpos , Criança , Humanos , Hungria , Fígado , Doadores de Tecidos
8.
Anal Chem ; 93(45): 15049-15057, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34726904

RESUMO

We report a low-cost and convenient microchannel resistance (MCR) biosensing platform that uses current signal to report biorecognition. The biorecognition behavior between targets and biometric molecules (antigens, antibodies, or oligonucleotides) immobilized on magnetic beads and polystyrene (PS) microspheres induces a quantitative change in the unreacted PS microspheres. After magnetic separation, the unreacted PS microsphere solution is passed through the microchannel, leading to an obvious blocking effect, resulting in an increase in resistance, which can in turn be measured by monitoring the electric current. Thus, the biorecognition is directly converted into a detectable current signal without any bulky instruments or additional chemical reactions. The MCR biosensing platform is cost-effective and user-friendly with high accuracy. It can be an appropriate analysis technique for point-of-care testing in resource-poor settings.


Assuntos
Técnicas Biossensoriais , Anticorpos , Separação Imunomagnética , Microesferas , Poliestirenos
9.
J Agric Food Chem ; 69(46): 13691-13699, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34783242

RESUMO

Currently, the infection with Helicobacter pylori affects about half of the world's population, and the most common therapy to treat H. pylori is the first line clarithromycin-based triple therapy or the quadruple therapy. However, drug resistance, eradication in a low level, high rate of reinfection, and gastrointestinal side effects among the causative organisms for H. pylori infection pose a critical challenge to the global health care community. Therefore, new approaches to treat H. pylori infections are urgently needed. Chicken egg yolk constituting a source of immunoglobulin Y (IgY) has attracted noticeable attention for its advantages of cost-effective extraction, minimization of animal harm and suffering, and induction of no specific resistance and is, therefore, being regarded as an alternative therapy for H. pylori infection. This review is intended to summarize various H. pylori antigens for IgY preparation in terms of their application, mechanism, and limitations.


Assuntos
Helicobacter pylori , Animais , Anticorpos , Gema de Ovo , Imunoglobulinas , Urease
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(11): 1032-1037, 2021 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-34809743

RESUMO

Objective To investigate the immunological functions of SARS-CoV-2 main protease (Mpro) in coronavirus disease 2019 (COVID-19), polyclonal antibody against Mpro was developed. Methods A codon-optimized SARS-CoV-2 Mpro gene was synthesized and ligated into a pET-28a vector for construction of a recombinant plasmid named by pET-28a-Mpro. Subsequently, this plasmid was transformed into E.coli Rosetta (DE3) competent cells for Mpro expression in an optimized condition, and then Mpro was purified using a HisTrap chelating column. The purified Mpro was used as immunogen to inoculate rats and the serum was collected after third immunization cycle. The titer, selectivity and sensitivity of polyclonal antibody against Mpro were analyzed using the ELISA and Western blot analysis. Results An optimized expression condition in E.coli cells for Mpro was determined, and the recombinant Mpro was purified by a HisTrap chelating column. The ELISA and Western blot analysis demonstrated that the highly sensitive polyclonal antibody against Mpro specially recognized the recombinant Mpro, and the titer reached 1:256 000. Conclusion The highly specific polyclonal antibody against SARS-CoV-2 Mpro is successfully prepared, which lays an experimental foundation for investigating the immunological function of Mpro in COVID-19.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Anticorpos , Western Blotting , Humanos , Peptídeo Hidrolases , Ratos
11.
Sensors (Basel) ; 21(21)2021 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-34770405

RESUMO

Many studies have found that gold nanoparticles with branched surfaces (nanoflowers) are markers for immunosensors that provide higher sensitivity gains than the commonly used spherical gold nanoparticles. Although the analytical characteristics of nanoparticle-using systems vary significantly depending on their size and shape, the question of choosing the best gold nanoflowers remains open. This work presents a comparative study of a panel of 33 preparations of gold nanoflowers formed by varying several parameters: the size of spherical nanoparticles-nuclei, the concentrations of nuclei, and tetrachloroauric acid during growth. The sizes of the resulting particles, their sorption capacity under antibody immobilization, mobility along membranes for lateral flow assays, and the effects of these parameters on the limits of detection of lateral flow immunoassay are characterized. The optimality of preparations obtained by growing a 0.2% v/v solution of nuclei with a diameter of 10 or 20 nm with tetrachloroauric acid at a concentration of 0.12 mM was shown. With their use, lateral flow immune tests were developed to determine markers of acute myocardial infarction-fatty acids binding protein and troponins I and T. The use of gold nanoflowers obtained under the proposed protocols led to significant gains in the limits of detection-3 to 10 times under visual detection and over 100 times under instrumental detection-compared to spherical gold nanoparticles. The significant increase under instrumental detection is due to the label's low nonspecific binding.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Anticorpos , Ouro , Imunoensaio
12.
Int J Nanomedicine ; 16: 7449-7461, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34785893

RESUMO

Purpose: There has been a substantial global market for antibodies, which are based on extracellular targets. Binding intracellular targets by antibodies will bring new chances in antibody therapeutics and a huge market increase. We aim to evaluate the efficiency of a novel delivery system of His6-metal assembly (HmA) in delivering intracellular antibodies and biofunctions of delivered antibodies. Methods: In this study, the physicochemical properties of HmA@Antibodies generated through co-assembling with antibodies and HmA were well characterized by dynamic light scatter. The cytotoxicity of HmA@Antibodies was investigated by Cell Counting Kit-8 (CCK-8). The endocytic kinetics and lysosome escape process of HmA@Antibodies were studied by flow cytometry and fluorescent staining imaging, respectively. Compared to the commercialized positive control, the intracellular delivery efficiency by HmA@Antibodies and biofunctions of delivered antibodies were evaluated by fluorescent imaging and CCK-8. Results: Various antibodies (IgG, anti-ß-tubulin and anti-NPC) could co-assemble with HmA under a gentle condition, producing nano-sized (~150 nm) and positively charged (~+30 eV) HmA@Antibodies particles with narrow size distribution (PDI ~ 0.15). HmA displayed very low cytotoxicity to divers cells (DCs, HeLa, HCECs, and HRPE) even after 96 h for the feeding concentration ≤100 µg mL-1, and fast escape from endosomes. In the case of delivery IgG, the delivery efficiency into alive cells of HmA was better than a commercial protein delivery reagent (PULSin). For cases of the anti-ß-tubulin and anti-NPC, HmA showed comparable delivery efficiency to their positive controls, but HmA with ability to deliver these antibodies into alive cells was still superior to positive controls delivering antibodies into dead cells through punching holes. Conclusion: Our results indicate that this strategy is a feasible way to deliver various antibodies intracellularly while preserving their functions, which has great potential in various applications and treating many refractory diseases by intracellular antibody delivery.


Assuntos
Histidina , Oligopeptídeos , Anticorpos , Humanos , Proteínas
13.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638989

RESUMO

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination's effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C-C, Im-C, C-Im, and Im-Im. The pre-implantation losses were significantly lower in the Im-C group than in the C-Im group. At the same time, the resorption rates reduced markedly in the C-Im compared to the Im-C group. Embryo and placenta weights were significantly higher in the Im-Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im-Im group, they were strongly correlated with embryo mass. The number-size trade-off was only significant in the Im-Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males' antigenic challenge on the IVF success rate.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Transferência Embrionária/métodos , Desenvolvimento Embrionário/imunologia , Fertilização In Vitro/métodos , Hemocianinas/administração & dosagem , Sêmen/imunologia , Espermatozoides/imunologia , Vacinação/métodos , Animais , Anticorpos/sangue , Blastocisto/imunologia , Blastocisto/metabolismo , Divisão Celular/imunologia , Implantação do Embrião/imunologia , Feminino , Hemocianinas/imunologia , Imunoglobulina G/sangue , Masculino , Camundongos , Gravidez , Vasectomia/métodos
14.
J Agric Food Chem ; 69(45): 13331-13338, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34714660

RESUMO

The xMAP food allergen detection assay (xMAP FADA) is an advanced multiplex immunoassay with multiple antibodies for each of 15 target food allergens and gluten, allowing for signal confirmation and antigenic profiling to occur in a single analysis. Botanicals used as spices are complex matrices for allergen analysis because they can exhibit inherent cross-reactivity with antibodies employed by the assays. Preliminary examinations of botanicals revealed chili peppers to have notably high levels of cross-reactivity with Brazil nut and hazelnut antibody bead sets in the xMAP FADA. This in-depth investigation of 29 pre-ground and whole chili peppers indicated Brazil nut and hazelnut cross-reactivity to be consistent among most members of genusCapsicum, although cross-reactive signals generated by chili peppers were distinguishable from signals indicative of target allergen detection. Using the requirements that complementary antibodies used in the assay generated positive responses and that the various secondary end points were characteristic of the target analytes, xMAP FADA reactivity to chilis of the genus Capsicum was categorized as cross-reactivity instead of confirmed detection of target allergenic foods.


Assuntos
Capsicum , Hipersensibilidade Alimentar , Alérgenos , Anticorpos , Reações Cruzadas
15.
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
16.
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
17.
FASEB J ; 35(11): e21855, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34644430

RESUMO

Glutamate is the most pivotal excitatory neurotransmitter in the central nervous system. Metabotropic glutamate receptors (mGluRs) dimerize and can couple to inhibitory intracellular signal cascades, thereby protecting glutamatergic neurons from excessive excitation and cell death. MGluR7 is correlated with age-related hearing deficits and noise-induced hearing loss; however its exact localization in the cochlea is unknown. Here, we analyzed the expression and localization of mGluR7a and mGluR7b in mouse cochlear wholemounts in detail, using confocal microscopy and 3D reconstructions. We observed a presynaptic localization of mGluR7a at inner hair cells (IHCs), close to the synaptic ribbon. To detect mGluR7b, newly generated antibodies were characterized and showed co-localization with mGluR7a at IHC ribbon synapses. Compared to the number of synaptic ribbons, the numbers of mGluR7a and mGluR7b puncta were reduced at higher frequencies (48 to 64 kHz) and in older animals (6 and 12 months). Previously, we reported a presynaptic localization of mGluR4 and mGluR8b at this synapse type. This enables the possibility for the formation of homo- and/or heterodimeric receptors composed of mGluR4, mGluR7a, mGluR7b and mGluR8b at IHC ribbon synapses. These receptor complexes might represent new molecular targets suited for pharmacological concepts to protect the cochlea against noxious stimuli and excitotoxicity.


Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Pré-Sinápticos/metabolismo , Sinapses/metabolismo , Animais , Anticorpos/imunologia , Ácido Glutâmico/metabolismo , Células HEK293 , Perda Auditiva Provocada por Ruído/metabolismo , Humanos , Imageamento Tridimensional/métodos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal/métodos , Receptores de Glutamato Metabotrópico/imunologia , Transfecção
18.
BMC Neurol ; 21(1): 414, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34706651

RESUMO

BACKGROUND: While Covid-19 predominantly affects the respiratory system, neurological manifestations including encephalitis occur in some patients, possibly affecting the course and outcome of the disease. Here, we describe a unique case of a young man with Covid-19 and transient MOG-positive encephalitis, with a benign course. CASE PRESENTATION: A 22-year-old male, with PCR confirmed Covid-19 infection was admitted because of persistent headache. The clinical examination was normal. Neuropsychological testing revealed distinct executive deficits. Brain MRI and cerebrospinal fluid (CSF) analysis were suggestive for encephalitis. Further laboratory examination revealed a serum MOG antibody titre. The headache improved with analgetic treatment and i.v. methylprednisolone. Consequently, the MOG antibody titer decreased and MRI lesions were resolving. The patient made a full recovery, with no signs of deterioration over the following months. CONCLUSIONS: Covid-19 manifestations in the CNS include encephalitis with variable course and prognosis. This case highlights a possible association between inflammation due to COVID-19 and transient secondary autoimmunity with transient MOG antibodies and atypical clinical presentation.


Assuntos
COVID-19 , Encefalite , Adulto , Anticorpos , Encefalite/complicações , Humanos , Masculino , Glicoproteína Mielina-Oligodendrócito , SARS-CoV-2 , Adulto Jovem
19.
ACS Nano ; 15(10): 15841-15849, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34596391

RESUMO

Bacterial infectious diseases seriously threaten public health and life. The specific interaction between an antibody and its multivalent antigen is an attractive way to defeat infectious disease. However, due to the high expense and strict storage and applied conditions for antibodies, it is highly desirable but remains an urgent challenge for disease diagnosis and treatment to construct artificial antibodies with strong stability and binding ability and excellent selectivity. Herein, we designed and synthesized antibody-like bio-orthogonal catalysts with the ability to recognize specific bacteria and accomplish in situ drug synthesis in captured bacteria by using improved bacterial imprinting technology. On one hand, the artificial antibody possesses a matching morphology for binding pathogens, and on the other hand, it acts as a bio-orthogonal catalyst for in situ synthesis of antibacterial drugs in live bacteria. Both in vitro and in vivo experiments have demonstrated that our designed antibody can distinguish and selectively bind to specific pathogens and eliminate them on site with the activated drugs. Therefore, our work provides a strategy for designing artificial antibodies with bio-orthogonal catalytic activity and may broaden the application of bio-orthogonal chemistry.


Assuntos
Antibacterianos , Anticorpos , Antibacterianos/uso terapêutico , Bactérias , Catálise
20.
Nat Commun ; 12(1): 5908, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625564

RESUMO

Oncolytic herpes simplex virus-1 is capable of lysing tumor cells while alerting the immune system. CD47, in collaboration with SIRPα, represents an important immune checkpoint to inhibit phagocytosis by innate immune cells. Here we show locoregional control of glioblastoma by an oncolytic herpes virus expressing a full-length anti(α)-human CD47 IgG1 or IgG4 antibody. The antibodies secreted by the virus-infected glioblastoma cells block the CD47 'don't eat me' signal irrespective of the subclass; however, αCD47-IgG1 has a stronger tumor killing effect than αCD47-IgG4 due to additional antibody-dependent cellular phagocytosis by macrophages and antibody-dependent cellular cytotoxicity by NK cells. Intracranially injected αCD47-IgG1-producing virus continuously releases the respective antibody in the tumor microenvironment but not into systemic circulation; additionally, αCD47-IgG1-producing virus also improves the survival of tumor-bearing mice better than control oncolytic herpes virus combined with topical αCD47-IgG1. Results from immunocompetent mouse tumor models further confirm that macrophages, and to a lesser extent NK cells, mediate the anti-tumor cytotoxicity of antibody-producing oncolytic herpesviruses. Collectively, oncolytic herpes simplex virus-1 encoding full-length antibodies could improve immune-virotherapy for glioblastoma.


Assuntos
Anticorpos/farmacologia , Glioblastoma/imunologia , Glioblastoma/terapia , Imunidade Inata , Vírus Oncolíticos/imunologia , Animais , Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígeno CD47 , Modelos Animais de Doenças , Feminino , Herpesvirus Humano 1/imunologia , Humanos , Imunoglobulina G , Imunoterapia , Células Matadoras Naturais , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Terapia Viral Oncolítica/métodos , Fagocitose , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
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