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1.
Mol Genet Genomic Med ; 8(2): e1079, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31867841

RESUMO

BACKGROUND: One of the major challenges in managing invasive breast cancer (BC) is the lack of reliable biomarkers to track response. Circulating tumor DNA (ctDNA) from liquid biopsy, as a candidate biomarker, provides a valuable assessment of BC patients. In this retrospective study, we evaluated the utility of ctDNA to reflect the efficacy of treatment and to monitor resistance mechanisms. METHODS: Targeted next-generation sequencing (NGS) of 416 cancer-relevant genes was performed on 41 plasma biopsy samples of 19 HER2+ and 12 HER2- BC patients. Longitudinal ctDNA samples were analyzed in three BC patients over the treatment course for detecting acquired mutations. RESULTS: In HER2+ BC patients, ERBB2 somatic copy numbers in ctDNA samples were significantly higher in patients progressed on HER2-targeted therapy than those who were still responding to the treatment. Recurrent acquired mutations were detected in genes including ERBB2, TP53, EGFR, NF1, and SETD2, which may contribute to trastuzumab resistance. In longitudinal analyses, the observed mutation allele frequencies were tracked closely in concordance with treatment responses. A novel ERBB2 p.(Leu869Arg) mutation was acquired in one patient upon resistant to trastuzumab therapy, which was further validated as an oncogenic mutation in vitro and contributed to resistance. In HER2- BC patients with chemotherapy resistance, genetic alterations on TP53, PIK3CA, and DNA damage repair genes were frequently observed. CONCLUSIONS: In summary, ctDNA monitoring, particularly longitudinal analyses, provides valuable insights into the assessment of targeted therapy efficacy and gene alterations underlying trastuzumab resistance and chemotherapy resistance in HER2+ and HER2- BC patients, respectively.

2.
Oncogene ; 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831835

RESUMO

The original version of this Article omitted the following from the Acknowledgements: Professor Stebbing sits on SABs for Celltrion, Singapore Biotech, Vor Biopharma, TLC Biopharmaceuticals and Benevolent AI, has consulted with Lansdowne partners, Vitruvian and Social Impact Capital and Chairs the Board of Directors for BB Biotech Healthcare Trust and Xerion Healthcare. This has now been corrected in both the PDF and HTML versions of the Article.

3.
Oncogene ; 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754213

RESUMO

EGFR-mutant non-small-cell lung cancer (NSCLC) patients inevitably develop drug resistance when treated with EGFR tyrosine kinase inhibitors (TKIs). Systematic genetic analysis is important to understand drug-resistant mechanisms; however, the clinical significance of co-occurring genetic alterations at baseline, co-acquired mutations at progressive disease (PD), and the clonal evolution remain underinvestigated. We performed targeted sequencing of pre-treatment and PD tumor samples from 54 EGFR-mutant NSCLC patients. Ten additional patients were sequenced using whole-exome sequencing to infer the clonal evolution patterns. We observed a domain-dependent effect of PIK3CA mutation at baseline on patient progression-free survival (PFS). In addition, at baseline, 9q34.3/19p13.3 (NOTCH1/STK11/GNA11) showed a co-deletion pattern, which was associated with a significantly worse PFS (p = 0.00079). T790M-postive patients with other concurrent acquired oncogenic mutations had a significantly shorter PFS (p = 0.005). Besides acquired T790M mutation, chromosomal instability (CIN) related genes, including AURKA and TP53 alterations, were the most frequently acquired events. CIN significantly increased during TKI treatment in T790M-negative patients and is a candidate resistance mechanism to the first-generation TKIs. Clonal evolution analyses suggest that the composition and relationship among resistant subclones, particularly relationship with T790M subclone, affect patients' outcomes. Overall, our findings of novel co-occurring alterations and clonal evolution patterns can be served as predictive biomarkers to stratify patients and help to better understand the drug-resistant mechanism to TKIs.

4.
Transl Lung Cancer Res ; 8(4): 392-400, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31555514

RESUMO

Background: Recently, plasma-derived exosomal DNA (exoDNA) has been successfully used in clinical genetic testing. However, the clinical utility of pleural effusion-derived exoDNA (PE-exoDNA) was still unknown. This study aimed to assess the feasibility of using PE-exoDNA for genetic testing in patients with advanced lung adenocarcinoma. Methods: Twenty PE-exoDNA samples and 18 pleural effusion-derived cell-free DNA (PE-cfDNA) samples were obtained from 20 stage IV lung adenocarcinoma patients. Using targeted next-generation sequencing (NGS) of 416 cancer-relevant genes, the genomic alterations between PE-exoDNA and PE-cfDNA were identified and compared. Results: NGS results showed highly similar mutation profiles between exoDNA and cfDNA, with TP53, EGFR, PKD1, and ALK as the top 4 mutated genes in both samples. A total of 304 genetic mutations were identified in 18 cfDNA samples and 276 genetic mutations were identified in 20 exoDNA samples. Forty-seven mutations from 8 genes (EGFR, ALK, KARS, BRAF, MET, PTEN, TP53, and RB1) were identified in 18 patients who had both exoDNA and cfDNA samples. Of the 47 mutations, 43 were shared between the two types of samples, yielding a concordance rate of 89.6%. Collectively, 78% of the mutations were shared between exoDNA and cfDNA samples, and this frequency increased to 94.2% when copy number variations (CNVs) were excluded from the analysis. Conclusions: In patients with advanced lung adenocarcinoma, the genetic profile of PE-exoDNA and PE-cfDNA were comparable, except for CNVs that had lower similarities between these two samples. Our findings support the clinical utility of exoDNA and could motivate further exploration of using exoDNA as an alternative source for genetic testing.

5.
Lung Cancer ; 124: 110-116, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30268447

RESUMO

INTRODUCTION: Increasing evidence leads to a ratiocination that genetic heterogeneity of the lung adenocarcinoma with EGFR mutations may impact clinical responses and outcomes to EGFR tyrosine kinase inhibitor (TKI) treatments. METHODS: We performed genetic profiling of pre-treatment samples of 69 lung adenocarcinoma patients, including tumor FFPE and cell-free DNA (cfDNA), targeting 416 cancer-related genes using next generation sequencing. We analyzed mutation concordance across sample types and investigated potential mechanisms that confer primary resistance to EGFR-TKIs in patients with short progression-free survival (PFS) versus those with long PFS. RESULTS: We detected a total of 200 actionable genetic alterations (mean: 2.9 variants/patient, range: 1-7 variants) in tumor FFPE and 140 actionable genetic alterations (mean: 2.0 variants/patient, range: 0-5 variants) in matched cfDNA, respectively. All patients had EGFR TKI-sensitizing mutations, including EGFR Ex19del, L858R, G719S/C, and L861Q. Concurrent TP53 mutations were most commonly observed in 72.5% of patients, followed by EGFR amplification (20.3%), RB1 (10.1%), PIK3CA (7.2%), and MYC (5.8%). For EGFR activating mutations, the concordance rate was 88.2% between cfDNA and FFPE samples. Furthermore, we identified genes that potentially confer primary resistance to EGFR-TKIs including CDC73, SMAD4, RB1 and PIK3CA. We also report signaling pathways enriched in patients with TKI primary resistance. CONCLUSIONS: We note the genetic complexity and heterogeneity of EGFR-mutated lung adenocarcinoma and underscore that mutation status is highly concordant between tumor FFPE and cfDNA samples. This study also highlights the alterations that potentially confer primary resistance to EGFR TKI treatments in patients who demonstrated short PFS.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Ácidos Nucleicos Livres/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Perfilação da Expressão Gênica , Humanos , Biópsia Líquida , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Proteínas de Ligação a Retinoblastoma/genética , Estudos Retrospectivos , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética
6.
Transl Oncol ; 11(6): 1364-1369, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30196239

RESUMO

Non-small cell lung cancer (NSCLC) with activating EGFR mutations in exon 19 and 21 typically responds to EGFR tyrosine kinase inhibitors (TKI); however, for some patients, responses last only a few months. The underlying mechanisms of such short responses have not been fully elucidated. Here, we sequenced the genomes of 16 short-term responders (SR) that had progression-free survival (PFS) of less than 6 months on the first-generation EGFR TKI and compared them to 12 long-term responders (LR) that had more than 24 months of PFS. All patients were diagnosed with advanced lung adenocarcinoma and harbored EGFR 19del or L858R mutations before treatment. Paired tumor samples collected before treatment and after relapse (or at the last follow-up) were subjected to targeted next-generation sequencing of 416 cancer-related genes. SR patients were significantly younger than LR patients (P < .001). Collectively, 88% of SR patients had TP53 variations compared to 13% of LR patients (P < .001). Additionally, 37.5% of SR patients carried EGFR amplifications compared to 8% of LR patients. Other potential primary resistance factors were also identified in the pretreatment samples of 12 SR patients (75%), including PTEN loss; BIM deletion polymorphism; and amplifications of EGFR, ERBB2, MET, HRAS, and AKT2. Comparatively, only three LR patients (25%) were detected with EGFR or AKT1 amplifications that could possibly exert resistance. The diverse preexisting resistance mechanisms in SR patients revealed the complexity of defining treatment strategies even for EGFR-sensitive mutations.

7.
Lung Cancer ; 121: 1-4, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29858019

RESUMO

Missense mutations in EGFR exon 20 are rare in non-small-cell lung cancer (NSCLC), and mostly insensitive to the first generation tyrosine kinase inhibitors (TKIs) of EGFR. However, their responses to the third generation TKI are unclear. Here, we reported a patient with advanced NSCLC harboring a rare EGFR H773L/V774M mutation complex. Although he was irresponsive to the first generation TKI gefitinib, he demonstrated sustained disease control to osimertinib, suggesting that this complex is an activating mutation of EGFR and can be suppressed by osimertinib. The follow-up genetic profiling revealed multiple acquired new mutations that might be related to his resistance to osimertinib. This finding would provide valuable experience for future treatment of the same mutations.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Mutação de Sentido Incorreto/genética , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Acrilamidas , Compostos de Anilina , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Feminino , Seguimentos , Gefitinibe/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias
8.
ACS Sens ; 2(9): 1267-1271, 2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-28884572

RESUMO

Alzheimer's disease (AD) biomarkers can reflect the neurochemical indicators used to estimate the risk in clinical nephrology. Apolipoprotein E (ApoE) is an early biomarker for AD in clinical diagnosis. In this research, through bactrian camel immunization, lymphocyte isolation, RNA extraction, and library construction, ApoE-specific Nbs with high affinity were successfully separated from an immune phage display nanobody library. Herein, a colorimetric immunosensor was developed for the point-of-care testing of ApoE by layer-by-layer nanoassembly techniques and novel nanobodies (Nbs). Using highly oriented Nbs as the capture and detection antibodies, an on-site immunosensor was developed by detecting the mean gray value of fade color due to the glutaraldehyde@3-aminopropyltrimethoxysilane oxidation by H2O2. The detection limit of AopE is 0.42 pg/mL, and the clinical analysis achieves a good performance. The novel easily operated immunosensor may have potential application in the clinical diagnosis and real-time monitoring for AD.

9.
ACS Appl Mater Interfaces ; 8(22): 13830-9, 2016 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-27196036

RESUMO

A phage display library of variable domain of the heavy chain only antibody or nanobody (Nb) was constructed after immunizing a bactrian camel with testosterone. With the smaller molecular size (15 kDa), improved solubility, good stability, high affinity, specificity, and lower immunogenicity, Nbs are a promising tool in the next generation of diagnosis and medical applications. Testosterone is a reproductive hormone, playing an important role in normal cardiac function and being the highly predictive marker for many diseases. Herein, a simple and sensitive immunosensor based on electrochemical impedance spectroscopy (EIS) and Nbs was successfully developed for the determination of testosterone. We successfully isolated the antitestosterone Nbs from an immune phage display library. Moreover, one of the Nbs was biotinylated according to in vivo BirA system, which showed the highest production yield and the most stable case. Further, the EIS immunosensor was set up for testosterone detection by applying the biotinylated antitestosterone Nb. As a result, the biosensor exhibited a linear working range from 0.05 to 5 ng mL(-1) with a detection limit of 0.045 ng mL(-1). In addition, the proposed immunosensor was successfully applied in determining testosterone in serum samples. In conclusion, the proposed immunosensor revealed high specificity of testosterone detection and showed as a potential approach for sensitive and accurate diagnosis of testosterone.


Assuntos
Técnicas Biossensoriais/métodos , Espectroscopia Dielétrica , Testosterona/análise , Animais , Camelus , Limite de Detecção
10.
Anal Chim Acta ; 902: 107-114, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26703259

RESUMO

Cystatin C (CysC) is a sensitive marker for the estimation of the glomerular filtration rate and the clinical diagnosis of different diseases. In this paper, CysC-specific nanobodies (Nbs) were isolated from a phage display nanobody library. A simple and sensitive photoelectrochemical immunosensor based on TiO2 nanotube arrays (TNAs) was proposed for the sensitive detection of CysC. The TiO2 nanotube arrays deposited by electrochemical anodization displayed a high and stable photocurrent response under irradiation. After coupling CysC-specific nanobody to TNA (Nb/TNA), the proposed immunosensor for CysC can be utilized for tracking the photocurrent change of Nb/TNA caused by immunoreactions between CysC and the immobilized CysC-specific Nb. This allowed for the determination of CysC with a calibration range from 0.72 pM to 7.19 nM. The variation of the photocurrent was in a linear relationship with the logarithm of the CysC concentration in the range of 0.72 pM-3.6 nM. The immunosensor had a correlation coefficient of 0.97 and a detection limit of 0.14 pM at a signal-to-noise ratio of 3. The proposed immunosensor showed satisfactory intra- and inter-assay accuracy, high selectivity and good stability. As a result, this proposed strategy would offer a novel and simple approach for the detection of immunoreactions, provide new insights in popularizing the diagnosis of CysC, and extend the application of TiO2 nanotubes.


Assuntos
Técnicas Biossensoriais , Cistatina C/sangue , Nanotubos , Anticorpos de Domínio Único , Titânio/química , Eletroquímica , Humanos , Limite de Detecção , Microscopia Eletrônica de Varredura , Fotoquímica , Ressonância de Plasmônio de Superfície , Temperatura Ambiente
11.
J Nanobiotechnology ; 13: 33, 2015 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-25944262

RESUMO

BACKGROUND: Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). Their unique advantageous properties make Nbs highly attractive in various applications. The general approach to obtain Nbs is to isolate them from immune libraries by phage display technology. However, it is unfeasible when the antigens are toxic, lethal, transmissible or of low immunogenicity. Naïve libraries could be an alternative way to solve the above problems. RESULTS: We constructed a large camel naïve phage display Nanobody (Nb) library with great diversity. The generated library contains to 6.86 × 10(11) clones and to our best of knowledge, this is the biggest naïve phage display Nb library. Then Nbs against human procalcitonin (PCT) were isolated from this library. These Nbs showed comparable affinity and antigen-binding thermostability at 37°C and 60°C compared to the PCT Nbs from an immune phage-displayed library. Furthermore, two PCT Nbs that recognize unique epitopes on PCT have been successfully applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect PCT, which showed a linear working range from 10-1000 ng/mL of PCT. CONCLUSION: We have constructed a large and diverse naïve phage display Nb library, which potentially functioning as a good resource for selecting antigen-binders with high quality. Moreover, functional Nbs against PCT were successfully characterized and applied, providing great values on medical application.


Assuntos
Calcitonina/imunologia , Biblioteca de Peptídeos , Precursores de Proteínas/imunologia , Anticorpos de Domínio Único/farmacologia , Sequência de Aminoácidos , Animais , Biotinilação , Peptídeo Relacionado com Gene de Calcitonina , Camelus/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Escherichia coli/genética , Humanos , Linfócitos/imunologia , Dados de Sequência Molecular , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos de Domínio Único/metabolismo
12.
Anal Chem ; 87(3): 2007-15, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25557870

RESUMO

Acute renal failure (ARF) represents a very important and potentially devastating disorder in clinical nephrology. Neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for ARF in a wide range of different disease processes, which is frequently detected in clinical diagnosis. Herein, we present a label-free and sensitive photoelectrochemical (PEC) immunosensor for NGAL by utilizing a biotinylated anti-NGAL Nanobody (Nb) orientedly immobilized to streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2 electrode. The Nb was biotinylated at the C-terminus, which is situated at the opposite site of the antigen binding region. Using highly oriented Nb as receptor molecules, a label-free PEC immunosensor for NGAL was developed by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Immobilization of Nb to streptavidin-coated CoPc-sensitized TiO2 electrode surface provides high binding capacity to NGAL; thus, it can lead to a high sensitivity. The limit of detection (LOD) of the proposed immunosensor has been significantly lowered to 0.6 pg mL(-1). This proposed immunosensor reveals high specificity to detect NGAL, with acceptable intra-assay precision and excellent stability. In addition, the present work provides a new approach to design Nb-based PEC immunosensor and increases versatility of Nbs.


Assuntos
Lesão Renal Aguda/sangue , Lesão Renal Aguda/diagnóstico , Lipocalinas/sangue , Proteínas Proto-Oncogênicas/sangue , Anticorpos de Domínio Único/química , Proteínas da Fase Aguda/imunologia , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Camelus , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Lipocalina-2 , Lipocalinas/imunologia , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/imunologia , Anticorpos de Domínio Único/imunologia
13.
Biosens Bioelectron ; 64: 111-8, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25203942

RESUMO

Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15 kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL(-1) with a detection limit of 0.03 ng mL(-1). This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis.


Assuntos
Apolipoproteína B-100/sangue , Espectroscopia Dielétrica/métodos , Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Animais , Anticorpos , Apolipoproteína B-100/análise , Técnicas Biossensoriais/métodos , Biotinilação , Camelus , Humanos , Imunoensaio/métodos , Limite de Detecção , Dados de Sequência Molecular , Reprodutibilidade dos Testes
14.
J Transl Med ; 12: 343, 2014 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-25496223

RESUMO

BACKGROUND: Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones. METHODS: We constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study. RESULTS: A large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 10(9) CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL. CONCLUSION: This proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.


Assuntos
Bacteriófagos/genética , Regiões Determinantes de Complementaridade , Anticorpos de Domínio Único , Repetições de Trinucleotídeos , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia
15.
Toxicon ; 92: 186-92, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25448390

RESUMO

The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.


Assuntos
Proteínas de Bactérias/imunologia , Camelus/imunologia , Endotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Imunoensaio/métodos , Anticorpos de Domínio Único/isolamento & purificação , Animais , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Escherichia coli , Adjuvante de Freund , Biblioteca de Peptídeos
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