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To gain further knowledge of the structure-activity relationship and druggability of novel oxazolidinone-based UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) inhibitors as Gram-negative antibacterial agents, compounds containing the hydrophobic tails with different lengths and terminal substitutions were synthesized and their antibacterial activities against standard and clinically isolated Gram-negative strains were evaluated. We summarized their structure-activity relationships and found that oxazolidinone-based compounds exhibited a narrower antibacterial spectrum compared with threonine-based compounds. Furthermore, we parallelly compared the metabolic stabilities of the compounds with the classic threonine scaffold and the novel oxazolidinone scaffold in liver microsomes. The results indicated that the druggability of the oxazolidinone scaffold may be inferior to the classic threonine scaffold in the design of LpxC inhibitors.
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Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Oxazolidinonas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oxazolidinonas/síntese química , Oxazolidinonas/química , Relação Estrutura-AtividadeRESUMO
Elevated serum levels of high-sensitive C-reactive protein (hs-CRP) and total cholesterol (TC) correlate with atherosclerotic vascular disease and increased frequency of vascular events. In this study, we investigated the effect of atorvastatin treatment on serum hs-CRP and TC levels, and the recurrence rate of atrial fibrillation (AF) in patients. Furthermore, a meta-analysis was performed to confirm the findings in this study. A total of 105 patients with AF were recruited to this study, including 55 patients with AF who were treated with amiodarone and atorvastatin (the treatment group) and 50 patients with AF who were treated with only amiodarone (the control group). Patients were treated for 12 months and followed up regularly for 1 year. Serum hs-CRP and TC levels in patients before and after treatment were recorded, and AF recurrence rate at 3, 6, and 12 months of treatment was obtained. Statistical analyses were performed with R 3.1.0 software and STATA 12.0 software. For patients in both treatment and control groups, serum hs-CRP and TC levels were high before the treatments began (both P < 0.05). However, after 12 months of treatment, serum hs-CRP and TC levels in the treatment group was dramatically reduced compared with the control group (hs-CRP: 3.63 ± 2.14 mg/L vs. 2.75 ± 1.89 mg/L, t = 2.24, P = 0.027; TC: 4.66 ± 1.13 mmol/L vs. 4.20 ± 1.06 mmol/L, t = 2.15, P = 0.034). After 12 months of treatment, the AF recurrence rate in the treatment group was significantly lower than the control group (16.4% vs. 34.0%; χ = 4.37; P = 0.037). In addition, 13 studies were selected for meta-analysis. Pooled results of the meta-analysis showed that serum hs-CRP and TC levels decreased significantly in the treatment group compared with the case group [hs-CRP: SMD = 0.95, 95% confidence interval (CI) = 0.62-1.29, and P < 0.001; TC: SMD = 1.39, 95% CI = 0.65-2.13, and P < 0.001]. Our study presents compelling evidence that atorvastatin is highly effective in reducing serum hs-CRP and TC levels and lowering the recurrence rate of AF.
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Povo Asiático , Atorvastatina/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Proteína C-Reativa/metabolismo , Colesterol/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Idoso , Amiodarona/uso terapêutico , Antiarrítmicos/uso terapêutico , Fibrilação Atrial/metabolismo , China , Quimioterapia Combinada , Feminino , Humanos , Masculino , Metanálise como Assunto , Pessoa de Meia-Idade , Recidiva , Resultado do TratamentoRESUMO
Background: Colorectal signet-ring cell carcinoma (SRCC) is a rare subtype of malignant adenocarcinoma, accounting for approximately 1 % of colorectal cancer (CRC) cases. Its biomarkers and molecular characteristics remain controversial, and there are no specific therapeutic targets or strategies for its clinical treatment. Methods: A retrospective study was conducted between January 2010 and December 2021. 1058 colorectal cancer cases from the Sun Yat-sen University Cancer Center and 489 cases from the Tumor Genome Atlas Project were included in the analysis, of which 64 were SRCC. Data extraction included patient demographics, blood types and risk factors, including clinical variables and genomics (either a 19-gene panel NGS or 1021-gene panel NGS). Univariate analyses were performed to identify factors significantly associated with overall survival. Results: The blood groups of 27 (42.2 %), 18 (28.1 %), 12 (18.8 %), and seven (10.9 %) patients were classified as O, A, B, and AB, respectively. We found that O was a unique blood group characterized by a low frequency of KRAS mutations, a high frequency of heterozygosity at each HLA class I locus, and a high tumor mutational burden (TMB). Patients in blood group A with high-frequency KRAS mutations and those in blood group B with anemia and metabolic abnormalities required targeted treatment. Furthermore, genetic alterations in SRCC differed from those in adenocarcinoma and mucinous adenocarcinoma. Conclusions: Our study revealed genomic changes in SRCC patients across different blood groups, which could advance the understanding and precise treatment of colorectal SRCC.
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Ouabain is a bioactive hapten and is very difficult to be accurately quantified because of the lack of useful reagents. Furthermore, where ouabain is produced in the adrenal glands has not been identified. In this study, ouabain-BSA was generated for immunizing the laying hens to generate ouabain-specific IgY antibodies in chicken eggs. The anti-ouabain IgY antibodies were detected in eggs 1 week after the last immunization and their concentrations increased with time. The highest concentrations of anti-ouabain IgY antibodies reached at 1:10,240 for ELISA 5 weeks after immunization and maintained for 4 weeks in chicken eggs. Following PEG precipitation, an average of 8.5 mg of anti-ouabain IgY antibodies with a purity of 87.6% was achieved from a single egg. Further analysis revealed that the anti-ouabain IgY antibodies had little immunoreactivity to hydrocortisone, dexamethasone, cedilanid, and digoxin, indicating their high specificity, and the purified IgY antibodies effectively detected endogenous ouabain in the cytoplasm of cells predominately in the zona reticularis of rat and human adrenal glands, indicating their high immunoreactivity. Given that IgY has an unique structure and bioactive features, the generated anti-ouabain IgY antibodies may be used as a new reagent for accurately quantifying ouabain in biological studies.
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Anticorpos Anti-Idiotípicos/biossíntese , Imunoglobulinas/biossíntese , Ouabaína/imunologia , Glândulas Suprarrenais/metabolismo , Animais , Western Blotting , Galinhas , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulinas/isolamento & purificação , Imuno-Histoquímica , Cinética , Óvulo/metabolismo , RatosRESUMO
AIM: To determine the value of the extensor digitorum reflex in neurologic examination. METHODS: The extensor digitorum, biceps, and brachioradialis reflexes were elicited in 65 patients with hemiplegia and upper-limb paralysis and in a control group of 120 apparently healthy people. Reflexes were elicited by both conventional means and a new method for the extensor digitorum reflex. The sensitivity and specificity of the extensor digitorum reflex were compared with that of the conventional biceps and brachioradialis reflexes to evaluate the value of the extensor digitorum reflex for neurologic examination. RESULTS: The sensitivity of the extensor digitorum, biceps, and brachioradialis reflexes were 93.65%, 90.48%, and 90.48%, respectively. The specificity of the extensor digitorum, biceps, and brachioradialis reflexes were 95.83%, 94.17%, and 93.33%, respectively. The diagnostic efficacies of the extensor digitorum, biceps, and brachioradialis reflexes were 95.08%, 92.90%, and 91.26%, respectively. There were no significant differences (p > 0.05) in sensitivity, specificity, or accuracy among the extensor digitorum, biceps or brachioradialis reflexes in neurologic examination. CONCLUSIONS: The extensor digitorum reflex is a sensitive and useful deep tendon reflex and is suitable for widespread use in neurological examination.
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Músculo Esquelético/fisiologia , Exame Neurológico , Reflexo , Tendões/fisiologia , Adulto , Idoso , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To construct prokaryotic expression system for expressing, purifying and identifying truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology. METHODS: According to the conservative sequence of M1-M2 and M3-M4 extra-cellular gene fragments of sodium pump a3 subunit, which published in GenBank, a serial of primers and gene fragments was designed, and directly synthesized to fuse the above two gene fragments. The fusion gene was fused with gene-specific primers by PCR, and then fusion gene fragment was fused into the single stranded homology regions of vector pGEX-6P-1 by in-fusion cloning to construct recombinant vector pGEX-Trf-alpha3 (Truncated fragment of extracellular segment of sodium pump alpha3 subunit, Trf-alpha3). After DH10bac was transferred with it, the pGEX-Trf-alpha3 plasmid was purified and identified by PCR and sequenced. Then the recombinant plasmid pGEX-Trf-alpha3 was expressed in E. coli BL21 cells, inducted by IPTG. GST-Trf-alpha3 fusion protein was purified with Glutathione Sepharose 4B purifying system and analyzed by SDS-PAGE. RESULTS: The results of PCR and sequencing demonstrated that the M1-M2 and M3-M4 extra-cellular gene was inserted in plasmid pGEX-6P-1 vector successfully. And the sequence was correct. Protein sequence analysis showed that the GST-Trf-alpha3 fusion protein was consisted of 262 amino-acid residues. Relative molecular mass in theory was 33.22 X 10(3). The amount of recombinant protein was 10% of the total bacteria protein. The soluble fusion protein was about 80.8%. After affinity purification, the purity of GST-Trf-alpha3 fusion protein was over 95%. There was some extent binding activity between GST-Trf-alpha3 fusion protein and ouabain, but the activity was very low. CONCLUSION: Prokaryotic expression system for expressing truncated fragment of extra-cellular segment of sodium pump alpha3 subunit with pGEX-6P-1 GST gene fusion system in Escherichia coli by in-fusion technology had been constructed. The purified method had also established. High purified GST-Trf-alpha3 fusion protein was obtained. These have found the foundation of further study on its biological function and potential pharmacology function.
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Escherichia coli/metabolismo , Espaço Extracelular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Escherichia coli/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
In order to explore the activity of a peptide containing rat sodium pump α2 subunit M1-M2 extramembrane fragment (RES2 derivative) in vitro, the peptide (Leu-Ala-Ala-Met-Glu-Asp-Glu-Pro-Ser-Asn-Asp-Asn-Gly-Gly-Gly-Ser) was synthesized by peptide synthesizer with Fmoc method and purified by high performance liquid chromatography (HPLC). Its binding activity was identified by radioligand-receptor binding assay (RRA) and its bioactivity was measured by erythrocyte (86)Rb uptake. The results of saturation binding experiment and competitive binding experiment showed that the synthesized RES2 derivative had the capability to bind to (3)H-ouabain. The dissociation constant (K(d)) was 38.46 nmol/L and IC(50) was 6.353 nmol/L. Erythrocyte (86)Rb uptake experiment showed that the RES2 derivative blocked the inhibitory effect of ouabain on the sodium pump on erythrocyte membrane in a dose-dependent manner. The results showed that the RES2 derivative is capable of binding to ouabain and improving the activity of sodium pump on erythrocyte membrane, suggesting that the RES2 derivative might become an effective antihypertensive drug in the future.
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Membrana Eritrocítica/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Ouabaína/farmacologia , RatosRESUMO
Natural marine products are useful candidates for the treatment of oxidative and inflammatory diseases, including myocardial ischemia. 3-bromo-4,5 - dihydroxybenzaldehyde (BDB), a natural bromophenol isolated from marine red algae, has been shown to display anti-microbial, anti-oxidative, anti-cancer, anti-inflammatory, and free radical scavenging activities. In this study, the potential protective effects of BDB against myocardial ischemia and reperfusion (IR) injury was investigated in an in vitro model mimicked by oxygen and glucose deprivation (OGD) in cardiomyocytes and in an in vivo model induced by coronary artery ligation in rats. The results showed that BDB attenuated the OGD-induced cytotoxicity in a dose-dependent manner, with no toxic effect when treated alone. BDB significantly decreased apoptosis and the cleavage of caspase-3 after OGD. We found that OGD-induced oxidative stress, as evidenced by increases of reactive oxygen species (ROS) and lipid peroxidation, as well as mitochondrial dysfunction, as measured by mitochondrial reporter gene, cytochrome c release and ATP synthesis, were markedly attenuated by BDB treatment. In addition, BDB increased the enzymatic activities of mitochondrial antioxidant enzymes, including IDH2, GSH-Px and SOD2. Western blot analysis showed that BDB increased Akt phosphorylation and upregulated the expression of Sirt3 and PGC1α after OGD. Furthermore, BDB-induced protection in cardiomyocytes was partially reversed by the Akt inhibitor and downregulation of PGC1α. BDB also attenuated myocardial contractile dysfunction and activated the Akt-PGC1α-Sirt3 pathway in vivo. All these data suggest that BDB protects against myocardial IR injury through activating the Akt-PGC1α-Sirt3 pathway.
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BACKGROUND: Recently, numerous investigations have shown that endogenous ouabain plays an important role in primary and secondary hypertension. The purpose of this study was to find ouabain-conjugated peptides (OCP) to block or to antagonize actions between endogenous ouabain (EO) and sodium pump, to serve as theoretical and experimental bases of EO in hypertension. METHODS: The study involved screening the phage displayed 12-peptide library by biopanning for OCP. The DNA sequence of each selected peptide was determined and the amino acid sequences were deduced. The highest consistent of the polypeptide was synthesized by peptide synthesizer. Synthetic OCP was identified, its binding activity determined by radioligand binding assay, and bioactivity of ouabain conjugated peptide was measured by erythrocyte 86Rb uptake. RESULTS: Three kinds of peptides were identified. Peptide A (12 peptide, Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg-Pro-Trp-Thr) was the highest consistence of peptide sequences, occupied in 66.7% (8/12). Peptide A was synthesized. The results verified that there was binding activity between synthetically OCP and 3H-ouabain, and that OCP was capable of suppressing the inhibition action between ouabain and sodium pump on the surface of erythrocyte. The results showed that efficacy was in a dose-dependent manner. CONCLUSION: It is important that the results not only obtain distinctive OCP, but also supply valuable experimental data in detection of ouabain and therapy in hypertension.
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Inibidores Enzimáticos/administração & dosagem , Ouabaína/administração & dosagem , Ouabaína/metabolismo , Biblioteca de Peptídeos , Peptídeos/efeitos dos fármacos , Peptídeos/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Humanos , Ouabaína/antagonistas & inibidores , Ovalbumina/administração & dosagem , Fragmentos de Peptídeos/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
OBJECTIVE: Paclitaxel was used in a phase II trial in combination with cisplatin for esophageal cancer. The anti-tumor response, toxicity and survival of the treated patients were evaluated. METHODS: Thirty patients with advanced, unresectable, or complicated with metastasis were allotted, twenty-seven patients had no prior chemotherapy while 3 patients had received adjuvant chemotherapy. Patients were given paclitaxel 175 mg/m(2) by 3-hour infusion on D1, and cisplatin 40 mg/m(2) daily on D2 and D3. Granulocyte colony-stimulating factor (G-CSF) was not routinely administered unless the patient had neutropenia. Treatment was recycled every 21 days. RESULTS: Thirty patients (male/female, 28/2; median age 58) completed a median of 3 cycles and 27 patients were evaluable for response. Major objective responses were observed in 16 patients (59.3%; 95% confidence interval, 38.9% to 75.5%), including 5 complete responses (18.5%) and 11 partial responses (40.7%). The median time to tumor progression was 5.0 months (range, 1 to 23 months). The median actuarial survival was 9.7 months (range, 1 to 23 months). Twenty-eight patients were assessable for toxicity. The most common nonhematologic toxicity was alopecia. Grade 3 to 4 neutropenia was observed in 17.9% of the patients. Toxicity was manageable with dose attenuation and G-CSF support. CONCLUSION: The combination of paclitaxel and cisplatin can be considered as a main regimen in the treatment of advanced esophageal cancer.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Esofágicas/tratamento farmacológico , Adulto , Idoso , Alopecia/induzido quimicamente , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Carcinoma de Células Escamosas/secundário , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Esquema de Medicação , Neoplasias Esofágicas/patologia , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neutropenia/induzido quimicamente , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Indução de Remissão , Taxa de SobrevidaRESUMO
A percutaneous visceral tumor puncture director is introduced in this paper, which can be used for needle biopsy and brush biopsy in kinds of visceral tumor. The clinical informations show that the application of the director can increase the positive ratio of puncture, and it has important clinical value. The director is safe, practical and easy to operate.
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Biópsia por Agulha/instrumentação , Neoplasias/patologia , Biópsia por Agulha/métodos , Desenho de Equipamento , Humanos , Sensibilidade e EspecificidadeRESUMO
PURPOSE: To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB. METHODS: HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence. RESULTS: 0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment. CONCLUSION: Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.
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An investigation was conducted to study the relationships of soil organic carbon (SOC) content with root biomass and soil moisture content as well as the accumulation mechanisms of SOC under the Elacagnus angustifolia-Achnatherum splenden community in Ningxia Hui Autonomous Region of Northwest China. The results showed that the SOC content decreased gradually with increasing soil depth, and changed gently in both horizontal and vertical directions. The correlations of the SOC content and its affecting factors varied with soil depth. In 0-30 cm layer, the SOC content was significantly negatively correlated with soil moisture content; in 60-150 cm layer, the SOC content was significantly positively correlated with soil moisture content and root biomass. Partial regression analysis indicated that the root biomass density in 0-30 cm soil layer contributed significantly to the variance of SOC content. In 60-150 cm layer, the SOC content was mainly affected by root system and soil moisture content; in 30-60 cm layer, no significant correlations were observed between the SOC content and the root biomass and soil moisture content. There was an obvious difference in the accumulation mechanism of SOC in different soil layers and at different locations of E. angustifolia--A. splendens community.
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Carbono/análise , Elaeagnaceae/crescimento & desenvolvimento , Poaceae/crescimento & desenvolvimento , Solo/química , China , Clima Desértico , Ecossistema , Compostos Orgânicos/análise , Raízes de Plantas/crescimento & desenvolvimento , Análise EspacialRESUMO
OBJECTIVE: To assess the binding activity of polypeptide containing human Na+, K+-ATPase alpha1 subunit M1-M2 extracellular segment (HES1 derivative). METHODS: HES1 derivative was synthesized by Fmoc method and purified by high-performance liquid chromatography-mass spectrometry, and its binding activity was identified by radioligand binding assay. RESULTS: 3H-ouabain and synthetic HES1 derivative showed some binding activity with the equilibrium dissociation constant (KD) of 24.58 nmol/L, with the the receptor density of 492.43 fmol x mg(-1) pro. and IC50 of 3.078 x 10(-7) mol/L. CONCLUSION: HES1 derivative can bind to ouabain and has the potential of becoming an effective therapeutic agent.
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Ouabaína/química , Peptídeos/química , ATPase Trocadora de Sódio-Potássio/química , Sítios de Ligação/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos , Ouabaína/farmacologia , Ligação Proteica , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
OBJECTIVE: To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1. METHODS: Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1. RESULTS: After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells. CONCLUSION: High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.
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Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Imunoglobulinas/imunologia , Proteínas Oncogênicas Virais/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/isolamento & purificação , Especificidade de Anticorpos/imunologia , Células CHO , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Galinhas , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunização/métodos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
OBJECTIVE: To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues. METHODS: Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay (ELISA) for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry. RESULTS: The IgY titer increased rapidly after the second immunization, with the highest titer of 1:10240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation (CV) of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex. CONCLUSION: Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.
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Imunização/métodos , Imunoglobulinas/imunologia , Ouabaína/imunologia , Animais , Calibragem , Bovinos , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Ouabaína/análise , RatosRESUMO
AIM: To improve specificity and accuracy of endogenous ouabain measurement assay. METHODS: Anti-ouabain polyclonal antibody egg yolk (IgY) and anti-ouabain rabbit antibody (IgG) were prepared respectively. In the presence of two kinds of antibody, then the specificity and accuracy of enzyme-linked immunosorbent assay (ELISA) were compared. RESULTS: The ELISA, in the presence of IgY, provided a sensitivity of the average intraassay coefficient of variation(CV) was 2.03%, and the inter-assay CV was 2.34% respectively. In contrast, IgG were 2.83% and 3.29%. No significant interferences were observed with hydrocortisone and dexamethasone. There was 3.45% vs. 5.95%, 3.20% vs. 5.20% of crossreaction with cedilanid and digoxin. CONCLUSION: The specificity and accuracy of ELISA, in which IgY was used, were more better than IgG.