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Objective: Fetomaternal hemorrhage (FMH) is an alloimmunization resulting caused by the incompatibility between fetal and maternal blood. For the prevention of newborn haemolytic disease (HDN), it is crucial to quantify the amount of fetomaternal hemorrhage. However, the classical Kleihauer-Betke test (K-B test) for detecting fetomaternal hemorrhage is limited by experimental tools and conditions and is not suitable for routine clinical use. Consequently, the method of prenatal diagnosis of fetomaternal hemorrhage applicable to the clinic is a topic worthy of further study. Therefore, it is worthwhile to further investigation on the clinically applicable prenatal diagnosis method for fetomaternal hemorrhage. Methods: This experiment demonstrates hydrogel's ability to separate sensitized red blood cells from soluble antibodies. Using flow cytometry the fluorescence values of sensitized red blood cells and fluorophore-labeled antibodies were measured, and the testing steps for the detection products of a novel technology were determined. The properties of a hydrogel fluoroimmunoassay were evaluated by distinguishing between the amounts of fetal and adult haemoglobin. The precision of this technology is evaluated using the Kleihauer-Betke test as a comparison. Results: This experiment compared the detection of haemoglobin fluorescence in adults (n = 2) and fetuses (n = 6). At the same time, the fluorescence intensity of different fetal haemoglobin (HbF) in adult haemoglobin (HbA) was calculated. The fluorescence value is 1.6% when the fetal hemoglobin concentration is 0.1%. Conclusion: The novel hydrogel fluoroimmunoassay can accurately determine the fluorescence intensity by flow cytometry to differentiate fetal haemoglobin from adult haemoglobin, quantitatively prenatally diagnose fetal haemoglobin, address the incompatibility between fetal and maternal blood types, and prevent alloimmunization.
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Urinary tract infections caused by urinary catheter implantations are becoming more serious. Therefore, the construction of a responsive antibacterial biomaterial that can not only provide biocompatible conditions, but also effectively prevent the growth and metabolism of bacteria, is urgently needed. In this work, a benzophenone-derived phosphatase light-triggered antibacterial agent is designed and synthesized, which is tethered to the biological materials using a one-step method for in vivo antibacterial therapy. This surface could kill gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). More importantly, because this material exhibited a zwitterion structure, it does not damage blood cells and tissue cells. When the bacteria interact with this surface, the initial fouling of the bacteria is reduced by zwitterion hydration. When the bacteria actively accumulate and metabolize to produce a certain amount of alkaline phosphatase, the surface immediately started the sterilization performance, and the bactericidal effect is achieved by destroying the bacterial cell membrane. In summary, an antibacterial biomaterial that shows biocompatibility with mammalian cells is successfully constructed, providing new ideas for the development of intelligent urinary catheters.
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Materiales Biocompatibles , Infecciones Urinarias , Animales , Materiales Biocompatibles/farmacología , Fosfatasa Alcalina , Antibacterianos/farmacología , Antibacterianos/química , Staphylococcus aureus , Infecciones Urinarias/tratamiento farmacológico , Infecciones Urinarias/prevención & control , Escherichia coli , MamíferosRESUMEN
BACKGROUND: AKI is related to severe adverse outcomes and mortality with Coronavirus Disease 2019 (COVID-19) patients, that early diagnosed and intervened is imperative. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the most promising biomarkers for detection of acute kidney injury (AKI), but current detection methods are inadequacy, so more rapid, convenient and accuracy methods are needed to detect NGAL for early diagnosis of AKI. Herein, we established a rapid, reliable and accuracy lateral flow immunoassay (LFIA) based on europium nanoparticles (EU-NPS) for the detection of NGAL in human urine specimens. METHODS: A double-antibody sandwich immunofluorescent assay using europium doped nanoparticles was employed and the NGAL monoclonal antibodies (MAbs) conjugate as labels were generated by optimizing electric fusion parameters. Eighty-three urine samples were used to evaluate the clinical application efficiency of this method. RESULTS: The quantitative detection range of NGAL in AKI was 1-3000 ng/mL, and the detection sensitization was 0.36 ng/mL. The coefficient of variation (CV) of intra-assay and inter-assay were 2.57-4.98 % and 4.11-7.83 %, respectively. Meanwhile, the correlation coefficient between europium nanoparticles-based lateral fluorescence immunoassays (EU-NPS-LFIA) and ARCHITECT analyzer was significant (R2 = 0.9829, n = 83, p < 0.01). CONCLUSIONS: Thus, a faster and easier operation quantitative assay of NGAL for AKI has been established, which is very important and meaningful to diagnose the early AKI, suggesting that the assay can provide an early warning of final outcome of disease.
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Lesión Renal Aguda/diagnóstico , Europio , Fluoroinmunoensayo/métodos , Lipocalina 2/orina , Nanopartículas del Metal , Lesión Renal Aguda/virología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , COVID-19/complicaciones , Ensayo de Inmunoadsorción Enzimática , Humanos , Lipocalina 2/inmunología , Ratones , Proteínas Recombinantes/aislamiento & purificación , Reproducibilidad de los Resultados , SARS-CoV-2RESUMEN
Objective To prepare and identify mouse monoclonal antibodies against human vasorin (VASN) protein using electrofusion method. Methods The mice were immunized with human recombinant protein VASN-His, and then the cells were fused by electrofusion apparatus. Indirect ELISA was used to screen the positive hybridoma cells which could bind natural protein VASN. The titer and affinities of the antibodies were detected by ELISA, and Western blotting was used to determine whether the antibody could recognize VASN protein in HepG2 cells. Results The fusion rate reached 0.31% when the ratio of spleen cells and Sp2/0 myeloma cells was 2:1, the alternating electric field intensity was 50 V, 2 MHz for 20 seconds, and the direct current pulse intensity was 500 V for 0.5 second. Two mouse anti-human VASN monoclonal antibodies (4H1and 8B9) were obtained, with the highest titer of 1:256 000 and the highest affinity constant (Ka) of 4.9×106 L/mol. Western blotting showed that both monoclonal antibodies could specifically recognize VASN in HepG2 cells. Conclusion Two mouse anti-human VASN monoclonal antibodies have been successfully prepared by the cell electrofusion method.
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Anticuerpos Monoclonales , Animales , Western Blotting , Humanos , Hibridomas , Ratones , Ratones Endogámicos BALB C , Proteínas RecombinantesRESUMEN
A large human natural singlechain fragment variable (scFv) phage library was constructed based on CreLoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcriptionquantitative PCR. Twostep overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through CreLoxP enzymemediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56â ¹1010 M towards PCSK9. In vitro, the PCSK9/lowdensity lipoprotein receptor (LDLR) pathway of HepG2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human CreLoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.
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Técnicas de Visualización de Superficie Celular , Integrasas , Inhibidores de PCSK9 , Recombinación Genética , Anticuerpos de Cadena Única/inmunología , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/inmunología , Anticuerpos de Cadena Única/genéticaRESUMEN
T2 toxin is a type A trichothecene mycotoxin. In order to reduce the side effects of T2 toxin and increase the tumor targeting ability, a pHsensitive liposome of T2 toxin (LPpHST2) was prepared and characterized in the present study. The cytotoxicity of LPpHST2 on A549, HepG2, MKN45, K562 and L929 cell lines was tested by 3(4,5dimethylthiazolyl2)2,5diphenyltetrazolium bromide assay, with T2 toxin as the control. The apoptotic and migratory effects of LPpHST2 on HepG2 cells were investigated. The preparation process of LPpHST2 involved the following parameters: Dipalmitoyl phosphatidylcholine: dioleoylphosphatidylethanolamine, 1:2; total phospholipid concentration, 20 mg/ml; phospholipid:cholesterol, 3:1; 4(2hydroxyethyl)1piperazineethanesulfonic acid buffer (pH 7.4), 10 ml; drug:lipid ratio, 2:1; followed by ultrasound for 10 min and extrusion. The encapsulation efficiency reached 95±2.43%. The average particle size of LPpHST2 after extrusion was 100 nm; transmission electron microscopy showed that the shape of LPpHST2 was round or oval and of uniform size. The release profile demonstrated a twophase downward trend, with fast leakage of T2 toxin in the first 6 h (~20% released), followed by sustained release up to 48 h (~46% released). From 4872 h, the leakage rate increased (~76% released), until reaching a minimum at 72 h. When LPpHST2 was immersed in 0.2 mol/l disodium phosphatesodium dihydrogen phosphate buffers (pH 6.5), the release speed was significantly increased and the release rate reached 91.2%, demonstrating strong pH sensitivity. Overall, antitumor tests showed that LPpHST2 could promote the apoptosis and inhibit the migration of HepG2 cells. The present study provided a new approach for the development of T2 toxinbased anticancer drugs.
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Antineoplásicos/farmacología , Toxina T-2/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Estabilidad de Medicamentos , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Células K562 , Liposomas , Ratones , Tamaño de la Partícula , Toxina T-2/químicaRESUMEN
D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.
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Anticuerpos Antiidiotipos/inmunología , Productos de Degradación de Fibrina-Fibrinógeno/inmunología , Ensayos Analíticos de Alto Rendimiento , Mediciones Luminiscentes , Animales , Anticuerpos Antiidiotipos/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Antígenos/sangre , Productos de Degradación de Fibrina-Fibrinógeno/aislamiento & purificación , Fibrinógeno/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Luz , RatonesRESUMEN
BACKGROUND: This study aimed to further investigate the role of circulating TGF-beta1 during radiation therapy (RT) in predicting radiation-induced lung toxicity (RILT). METHODS AND MATERIALS: Patients with stages I-III non-small cell lung cancer treated with RT based therapy were included in this study. Platelet poor plasma was obtained pre-RT, at 2 and 4 weeks during-RT, and at the end of RT. TGF-beta1 was measured using an enzyme-linked immunosorbent assay. The primary endpoint for RILT was >or=grade 2 radiation pneumonitis or fibrosis. RESULTS: Twenty-six patients with a minimum follow-up of 12 months were included. Six patients (23.1%) experienced >or=grade 2 RILT. There was no significant difference in absolute TGF-beta1 levels pre-RT, at 2 and 4 weeks during-RT, or at the end of RT between patients with and without RILT. The TGF-beta1 ratios (over the pre-RT levels) for patients with and without RILT at 2, 4 weeks during-, and the end of RT were 2.8+/-2.2 and 1.0+/-0.6 (P=0.123), 2.3+/-1.3 and 0.8+/-0.5 (P=0.001), 1.5+/-0.9 and 0.8+/-0.5 (P=0.098), respectively. Using 2.0 as a cut-off, the TGF-beta1 ratio at 4 weeks during-RT predicted RILT with a sensitivity and specificity of 66.7% and 95.0%, respectively. CONCLUSION: Elevation of plasma TGF-beta1 level 4 weeks during-RT is significantly predictive of RILT. The role of plasma TGF-beta1 in predicting RILT deserves further study.