Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Anal Chem ; 94(51): 17843-17852, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36519948

RESUMO

The pursuit of the limit between dimensionalities is a scientific goal with high applicability. Sandwich immunoassay, usually based on two antibodies binding two epitopes, is one of the most popular mainstay tools in both academic and industrial fields. Herein, we determined and evaluated the minimum distance of two epitopes in sandwich immunoassays for small molecules. Briefly, nine model analytes comprising two hapten epitopes, that is, melamine (MEL) and p-nitroaniline (NIA), were designed by increasing the linear chain linkers brick by brick. Two groups of monoclonal antibodies (mAbs) were produced with different recognition properties toward MEL and NIA using 12 new haptens with different spacer arms. The results indicated that two epitopes of the analyte with a distance of only 2.4 Å could be simultaneously bound by two mAbs, which is the known limit of epitope distance in sandwich immunoassays thus far. We further found that an epitope distance of below 8.8 Å for the analyte generally induces noticeable steric hindrance of antibodies, preventing a sandwich immunoassay with high probability. These observations were investigated and evaluated by molecular docking, molecular dynamics, and surface plasmon resonance and using model and real analytes. Altogether, we determined the minimum distance of two epitopes and explored the molecular mechanism of the antibody-analyte-antibody ternary complex in sandwich immunoassays, providing a theoretical basis for hapten design, antibody discovery and development, and sandwich immunoassay establishment for small molecules.


Assuntos
Anticorpos Monoclonais , Haptenos , Epitopos , Simulação de Acoplamento Molecular , Imunoensaio/métodos , Anticorpos Monoclonais/química
2.
Foods ; 11(11)2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35681298

RESUMO

To monitor the contamination of a type A trichothecene, diacetoxyscirpenol (DAS), one monoclonal antibody (mAb) 8A9 with high affinity and specificity was prepared in the present study. The mAb 8A9 showed a 50% inhibition concentration (IC50) of 0.31 µg/L, which is of the highest affinity reported to date. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral flow immunoassay (LFIA) based on mAb 8A9 were developed and exhibited limits of detection as low as 0.65 µg/kg and 100 µg/kg in rice samples, respectively. The molecular recognition mechanism of mAb 8A9 to DAS was explored by molecular docking. The results showed that the hydrophobic amino acids of mAb 8A9 interacted with DAS by forming hydrogen bonds and a pi-sigma bond, which lead to a highly specific recognition of DAS. In summary, we produced one mAb, developed ELISA and LFIA for DAS detection in rice with significantly sensitivity, specificity, accuracy, and precision.

3.
Biosens Bioelectron ; 204: 114093, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35180688

RESUMO

Sandwich lateral flow immunoassays (LFIAs) based on paired antibodies are the most frequently used platform for food-borne pathogen detection. Although label-free strategies are used in LFIAs to avoid the utilization of paired antibodies, challenges of probe design and detection reliability still remain. Here, we report a new label-free and dual-readout LFIA (LD-LFIA) mediated by a 'Three-To-One' multi-functional nanocomposite with a unique combination of magnetic-adhesion-color-nanozyme properties. The strengths of the new designed nanocomposite are: (i) the Fe3O4 magnetic core simplifies the separation processes; (ii) surface adherent polydopamine (PDA) films exhibit a strong adhesion to pathogenic bacteria and provide colorimetric detection signal; and (iii) the deposited platinum nanoparticles (Pt NPs) can function as nanozymes to generate an extra catalytic signal for constructing a dual-readout mode to improve the detection accuracy. The resulting Fe3O4@PDA@Pt nanocomposite-based LD-LFIA can detect highly pathogenic Escherichia coli O157:H7 with limits of detection of 102 and 10 CFU mL-1 for colorimetric and catalytic quantitative analyses, respectively. Systematic results also reveal that the proposed method exhibited high specificity and applicability for drinking water and chicken samples, serving as a promising tool for real bacterial sample testing. The multi-functional Fe3O4@PDA@Pt nanocomposite-based LD-LFIA can provide new ideas for designing new multi-functional probes for improving detection performance of conventional label-free LFIA and constructing more accurate and sensitive detection systems.


Assuntos
Técnicas Biossensoriais , Escherichia coli O157 , Nanopartículas Metálicas , Nanocompostos , Imunoensaio , Limite de Detecção , Platina , Reprodutibilidade dos Testes
4.
J Agric Food Chem ; 70(4): 976-991, 2022 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-34990134

RESUMO

As antibodies are the main biological binder for hazards in food samples, their performance directly determines the sensitivity, specificity, and reproducibility of the developed immunoassay. The overwhelmingly used mammalian-derived antibodies usually suffer from complicated preparation, high cost, frequent bleeding of animals, and sometimes low titer and affinity. Chicken yolk antibody (IgY) has recently attracted considerable attention in the bioanalytical field owing to its advantages in productivity, animal welfare, comparable affinity, and high specificity. However, a broad understanding of the application of IgY-based immunoassay for the detection of chemical and biological hazards in food samples remains limited. Here, we briefly summarized the diversity, structure, and production of IgY including polyclonal and monoclonal formats. Then, a comprehensive overview of the principles, designs, and applications of IgY-based immunoassays for these hazards was reviewed and discussed, including food-borne pathogens, food allergens, veterinary drugs, pesticides, toxins, endocrine disrupting chemicals, etc. Thus, the trend of IgY-based immunoassays is expected, and more IgY types, higher sensitivity, and diversification of recognition-to-signal manners are necessary in the future.


Assuntos
Galinhas , Imunoglobulinas , Animais , Anticorpos , Gema de Ovo , Imunoensaio , Reprodutibilidade dos Testes
5.
Foods ; 10(12)2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34945657

RESUMO

Albendazole (ABZ) is one of the benzimidazole anthelmintics, and the overuse of ABZ in breeding industry can lead to drug resistance and a variety of toxic effects in humans. Since the residue markers of ABZ are the sum of ABZ and three metabolites (collectively referred to as ABZs), albendazole-sulfone (ABZSO2), albendazole-sulfoxide (ABZSO), and albendazole-2-amino-sulfone (ABZNH2SO2), an antibody able to simultaneously recognize ABZs with high affinity is in urgent need to develop immunoassay for screening purpose. In this work, an unreported hapten, 5-(propylthio)-1H-benzo[d]imidazol-2-amine, was designed and synthesized, which maximally exposed the characteristic sulfanyl group of ABZ to the animal immune system to induce expected antibody. One monoclonal antibody (Mab) that can simultaneously detect ABZs was obtained with IC50 values of 0.20, 0.26, 0.77, and 10.5 µg/L for ABZ, ABZSO2, ABZSO, and ABZNH2SO2 in ic-ELISA under optimized conditions respectively, which has been never achieved in previous reports. For insight into the recognition profiles of the Mab, we used computational chemistry method to parameterize cross-reactive molecules in aspects of conformation, electrostatic fields, and hydrophobicity, revealing that the hydrophobicity and conformation of characteristic group of molecules might be the key factors that together influence antibody recognition with analytes. Furthermore, the practicability of the developed ic-ELISA was verified by detecting ABZs in spiked milk, beef, and liver samples with recoveries of 60% to 108.8% and coefficient of variation (CV) of 1.0% to 15.9%.

6.
Anal Chem ; 93(31): 10940-10946, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34319068

RESUMO

With a nearly 100% mortality rate, African swine fever (ASF) has devastated the pork industry in many countries. Without a vaccine in sight, mitigation rests on rapid diagnosis and immediately depopulating infected or exposed animals. Unfortunately, current tests require centralized laboratories with well-trained personnel, take days to report the results, and thus do not meet the need for such rapid diagnosis. In response, we developed a portable, sample-to-answer device that allows for ASF detection at the point of need in <30 min. The device employs droplet magnetofluidics to automate DNA purification from blood, tissue, or swab samples and utilizes fast thermal cycling to perform real-time quantitative polymerase chain reaction (qPCR), all within an inexpensive disposable cartridge. We evaluated its diagnostic performance at six farms and slaughter facilities. The device exhibits high diagnostic accuracy with a positive percent agreement of 92.2% and a negative percent agreement of 93.6% compared with a lab-based reference qPCR test.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Febre Suína Africana/diagnóstico , Vírus da Febre Suína Africana/genética , Animais , Reação em Cadeia da Polimerase em Tempo Real , Suínos
7.
Food Chem ; 351: 129270, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33640770

RESUMO

Small molecules are immunochemically classified as hapten that lacking of at least two epitopes, usually using competitive format for establishing immunoassays. However, theoretically, noncompetitive immunoassay format is more sensitive and has a wider analytical range. In the present study, a novel hapten of halofuginone was synthesized and used to produce a monoclonal antibody (mAb). By analyzing the binding kinetics, we found that the affinity of analyte-enzyme to mAb was much greater than that of analyte, which could result in a low sensitivity of competitive assay format. Based on this, we established a novel noncompetitive immunoassay by using a replacement approach. The noncompetitive format has obvious advantages in sensitivity and analytical range, which promoted approximately 3.5- and 5-fold, respectively, compared to the competitive immunoassay. Ultimately, the newly designed noncompetitive immunoassay in this work will provide insights as well as alternative method to traditional small molecule competitive assays.


Assuntos
Imunoensaio/métodos , Limite de Detecção , Piperidinas/análise , Quinazolinonas/análise , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Haptenos/imunologia , Piperidinas/imunologia , Quinazolinonas/imunologia
8.
Front Chem ; 9: 813219, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35004629

RESUMO

Mushrooms containing Amanita peptide toxins are the major cause of mushroom poisoning, and lead to approximately 90% of deaths. Phallotoxins are the fastest toxin causing poisoning among Amanita peptide toxins. Thus, it is imperative to construct a highly sensitive quantification method for the rapid diagnosis of mushroom poisoning. In this study, we established a highly sensitive and automated magnetic bead (MB)-based chemiluminescence immunoassay (CLIA) for the early, rapid diagnosis of mushroom poisoning. The limits of detection (LODs) for phallotoxins were 0.010 ng/ml in human serum and 0.009 ng/ml in human urine. Recoveries ranged from 81.6 to 95.6% with a coefficient of variation <12.9%. Analysis of Amanita phalloides samples by the automated MB-based CLIA was in accordance with that of HPLC-MS/MS. The advantages the MB-based CLIA, high sensitivity, repeatability, and stability, were due to the use of MBs as immune carriers, chemiluminescence as a detection signal, and an integrated device to automate the whole process. Therefore, the proposed automated MB-based CLIA is a promising option for the early and rapid clinical diagnosis of mushroom poisoning.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...