RESUMEN
AIM: Novel long-acting drugs for type 2 diabetes mellitus may optimize patient compliance and glycaemic control. Exendin-4-IgG4-Fc (E4F4) is a long-acting glucagon-like peptide-1 receptor agonist. This first-in-human study investigated the safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of a single subcutaneous injection of E4F4 in healthy subjects. METHODS: This single-centre, randomized, double-blind, placebo-controlled phase 1 clinical trial included 96 subjects in 10 sequential cohorts that were provided successively higher doses of E4F4 (0.45, 0.9, 1.8, 3.15, 4.5, 6.3, 8.1, 10.35, 12.6 and 14.85 mg) or placebo (ChinaDrugTrials.org.cn: ChiCTR2100049732). The primary endpoint was safety and tolerability of E4F4. Secondary endpoints were pharmacokinetic, pharmacodynamic and immunogenicity profiles of E4F4. Safety data to day 15 after the final subject in a cohort had been dosed were reviewed before commencing the next dose level. RESULTS: E4F4 was safe and well tolerated among healthy Chinese participants in this study. There was no obvious dose-dependent relationship between frequency, severity or causality of treatment-emergent adverse events. Cmax and area under the curve of E4F4 were dose proportional over the 0.45-14.85 mg dose range. Median Tmax and t1/2 ranged from 146 to 210 h and 199 to 252 h, respectively, across E4F4 doses, with no dose-dependent trends. For the intravenous glucose tolerance test, area under the curve of glucose in plasma from time 0 to 180 min showed a dose-response relationship in the 1.8-10.35 mg dose range, with an increased response at the higher doses. CONCLUSION: E4F4 exhibited an acceptable safety profile and linear pharmacokinetics in healthy subjects. The recommended phase 2 dose is 4.5-10.35 mg once every 2 weeks.
Asunto(s)
Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Exenatida/efectos adversos , Voluntarios Sanos , Área Bajo la Curva , Prueba de Tolerancia a la Glucosa , Método Doble Ciego , Relación Dosis-Respuesta a DrogaRESUMEN
Changes in the intestinal microbiota have been observed in patients with anti-N-methyl-D-aspartate receptor encephalitis (NMDARE). However, whether and how the intestinal microbiota is involved in the pathogenesis of NMDARE susceptibility needs to be demonstrated. Here, we first showed that germ-free (GF) mice that underwent fecal microbiota transplantation (FMT) from NMDARE patients, whose fecal microbiota exhibited low short-chain fatty acid content, decreased abundance of Lachnospiraceae, and increased abundance of Verrucomicrobiota, Akkermansia, Parabacteroides, Oscillospirales, showed significant behavioral deficits. Then, these FMT mice were actively immunized with an amino terminal domain peptide from the GluN1 subunit (GluN1356-385) to mimic the pathogenic process of NMDARE. We found that FMT mice showed an increased susceptibility to an encephalitis-like phenotype characterized by more clinical symptoms, greater pentazole (PTZ)-induced susceptibility to seizures, and higher levels of T2 weighted image (T2WI) hyperintensities following immunization. Furthermore, mice with dysbiotic microbiota had impaired blood-brain barrier integrity and a proinflammatory condition. In NMDARE-microbiota recipient mice, the levels of Evan's blue (EB) dye extravasation increased, ZO-1 and claudin-5 expression decreased, and the levels of proinflammatory cytokines (IL-1, IL-6, IL-17, TNF-α and LPS) increased. Finally, significant brain inflammation, mainly in hippocampal and cortical regions, with modest neuroinflammation, immune cell infiltration, and reduced expression of NMDA receptors were observed in NMDARE microbiota recipient mice following immunization. Overall, our findings demonstrated that intestinal dysbiosis increased NMDARE susceptibility, suggesting a new target for limiting the occurrence of the severe phenotype of NMDARE.
Asunto(s)
Encefalitis Antirreceptor N-Metil-D-Aspartato , Humanos , Ratones , Animales , Barrera Hematoencefálica , Disbiosis , Homeostasis , PermeabilidadRESUMEN
Background: Intrauterine adhesions (IUAs) are a common illness of the uterine cavity. Endometrial fibrosis is the main pathological feature. In addition to a high recurrence rate, patients with severe IUAs have a low pregnancy rate. However, there are few effective treatments for IUAs. This study aims to confirm the influence of apoptotic bodies of bone marrow mesenchymal stem cells (BMSCs) on endometrial stromal cell fibrosis by mediating the Wnt/ß-catenin signaling pathway and to provide new insight for the clinical treatment of IUAs. Methods: Human endometrial stromal cells (HESCs) were used to establish an IUA cell model by treatment with TGF-ß1, and a rat IUA model was established by the double injury method. Apoptosis of BMSCs was detected by TUNEL assays, and cell morphology was observed by the CM-DiI tracer. The morphology of apoptotic vacuoles and apoptotic bodies (ABs) was detected by TEM. We used Western blotting to detect the expression of histone H3.3, histone H2B, C3b, cyclin D1, C1QC, α-SMA, COL1A1, COL5A2, FN, CTGF, Wnt2b, c-MYC, CK-18 and VIM. The expression levels of α-SMA, COL1A1, COL5A2, FN and CTGF were detected by RTâqPCR. The expression levels of α-SMA, COL1A1, FN and CTGF were detected by immunofluorescence. Immunohistochemistry was used to detect the expression of TGF-ß, CK-18 and VIM. Flow cytometry, cell scratch assays, CCK-8 assays, and H & E and Masson staining were used to detect the cell cycle, cell migration, cell proliferation, and endometrial pathology, respectively. Results: We found that ultraviolet light (UV) irradiation induced apoptosis of BMSCs and increased the production of ABs. TGF-ß1 treatment can induce HESCs to form extracellular matrix (ECM), and aggravate cell fibrosis, and adding ABs or FH535, an inhibitor of the Wnt/ß-catenin signaling pathway, can inhibit TGF-ß1-induced HESC fibrosis. However, the inhibitory effect of ABs on TGF-ß1-induced fibrosis of HESCs was attenuated by the addition of LiCl. In the Wnt/ß-catenin signaling pathway, LiCl is an activator after coculture with TGF-ß1. In vivo, IUA-induced narrowing of the uterine cavity was accompanied by intrauterine adhesions, increased deposition of collagen fibers, upregulation of TGF-ß1, VIM, α-SMA, COL1A1 and COL5A2, and downregulation of CK-18. These changes in expression were reversed after treatment with ABs or FH535. When ABs and LiCl were added at the same time, the inhibitory effect of ABs on IUA fibrosis was weakened. Conclusion: BMSC-derived ABs inhibit the fibrosis of HESCs by inhibiting the Wnt/ß-catenin signaling pathway. These results provide a new direction for the clinical treatment of IUAs.
RESUMEN
Sensitive and accurate analysis of exosomes is important for many biological processes including as a biomarker for cerebral venous thrombosis (CVT) diagnosis and therapy. Herein, we established a sensitive and specific exosome detection approach based on target recognition initiated cascade signal amplification. In this method, an allosteric probe was designed with a hairpin structure for specific recognition of the exosome followed by signal amplification. After the cascade signal amplification process, spinach RNA sequences bind to DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one) to produce enhanced fluorescence signal (approximate 2000 fold than that of inactive DFHBI). Compared with former proposed exosome detection techniques, this method exhibited a comparable detection range, but with an easy-to-design toolbox. Therefore, we believe that the proposed approach holds great potential for exosome based early diagnosis and prognosis of disease.
Asunto(s)
Técnicas Biosensibles , Exosomas , Técnicas Biosensibles/métodos , Exosomas/genética , Exosomas/metabolismo , Fluorescencia , Técnicas de Amplificación de Ácido NucleicoRESUMEN
Sulfide and persulfide are chemically different and one might expect persulfide to be more effective in mediating sulfur signaling because persulfide can directly modify protein cysteine residue. However, rapid scrambling, and interconversions occur among sulfur species. Then there is the question of whether the chemical reactivity differences between sulfide and persulfide would translate into pharmacological differences. Utilizing a delivery system to generate pure hydrogen sulfide (H2 S), hydrogen persulfide (H2 S2 ), and N-acetyl-l-cysteine persulfide (N-CysSSH), we examined the activities of sulfide and persulfide in vitro and in vivo. Persulfide prodrugs exhibited increased activities compared to the H2 S prodrug. In particular, the H2 S2 prodrug offers much-elevated analgesic effects compared to the H2 S prodrug in vivo. Persulfide prodrugs also possess a reduced level of toxicity compared to the H2 S prodrug in vivo, indicating persulfide might represent a better therapeutic paradigm than H2 S.
Asunto(s)
Sulfuro de Hidrógeno , Profármacos , Cisteína/química , Sulfuro de Hidrógeno/química , Profármacos/química , Sulfuros/química , Azufre/metabolismoRESUMEN
BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a hereditary monogenic peripheral nerve disease. Variants in the gene encoding myelin protein zero (MPZ) lead to CMT, and different variants have different clinical phenotypes. A variant site, namely, c.389A > G (p.Lys130Arg), in the MPZ gene has been found in Chinese people. The pathogenicity of this variant has been clarified through pedigrees, and peripheral blood-related functional studies have been conducted. METHOD: Whole-exome sequencing and Sanger sequencing were used to detect the c.389A > G (p.Lys130Arg) variant in the MPZ gene in family members of the proband. Physical examination was performed in the case group to assess the clinical characteristics of MPZ site variants. The expression of MPZ and phosphorylated MPZ in the blood of 12 cases and 12 randomly selected controls was compared by RT-qPCR, Western blotting, and ELISA. RESULTS: The proband and 12 of her family members presented the AG genotype with different clinical manifestations. The expression of MPZ mRNA in the case group was increased compared with that in the control group, and the levels of MPZ and phosphorylated MPZ in peripheral blood were higher than those in normal controls. CONCLUSION: The heterozygous genotype of the c.389A > G (p.Lys130Arg) variant in the MPZ gene mediated the increase in MPZ and phosphorylated MPZ levels in peripheral blood and was found to be involved with CMT.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Proteína P0 de la Mielina , Enfermedad de Charcot-Marie-Tooth/genética , China , Femenino , Humanos , Mutación , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/metabolismo , FenotipoRESUMEN
BACKGROUND: Post-stroke insomnia (PSI) is a common and severe illness among the complications of stroke. Although there are plenty of drugs currently used for PSI treatment, they generate several side effects and other problems. Bright light therapy (BLT) is thought to be relatively safe and effective in treating PSI patients. Despite this, there is still a lack of systematic review on BLT in the treatment of PSI. Allowing for this, the aim of this study is to assess the efficacy and safety of BLT for PSI. METHODS: The meta-analysis and systematic review will perform a comprehensive electronic search for items fulfilling the required criteria in Web of Science, Google Scholar, Wan Fang database, MEDLINE, Baidu Scholar, PubMed, SinoMed, Embase, Chinese Biomedical Literature Database (CBM), China national knowledge infrastructure database (CNKI), Cochrane Library Central Register of Controlled Trials (CENTRAL), and Wei Pu database from establishment to January 1, 2022. We will select articles, collect data, and assess the methodology quality. And we will set the primary outcome and secondary outcomes in this research. RevMan 5.3 software will be used to analyze the data for this investigation. RESULTS: The work of this research will be published in peer-reviewed scientific journals. CONCLUSION: The aim of this study is to assess the efficacy and safety of BLT for PSI and present robust scientific evidence concerning BLT for PSI. REGISTRATION: INPLASY2021100065.
Asunto(s)
Trastornos del Inicio y del Mantenimiento del Sueño/terapia , Accidente Cerebrovascular/complicaciones , Humanos , Metaanálisis como Asunto , Fototerapia , Proyectos de Investigación , Trastornos del Inicio y del Mantenimiento del Sueño/etiología , Rehabilitación de Accidente Cerebrovascular , Revisiones Sistemáticas como Asunto , Resultado del TratamientoRESUMEN
The capacity of targeted anticancer agents to exert immunomodulatory effects provides a strong rationale to develop novel agents suitable for combinatorial regimens with immunotherapy to improve clinical outcomes. In this study, we developed a dual-targeting PI3K and HDAC inhibitor BEBT-908 that potently inhibits tumor cell growth and potentiates anti-PD1 therapy in mice by inducing immunogenic ferroptosis in cancer cells. Treatment with BEBT-908 promoted ferroptotic cell death of cancer cells by hyperacetylating p53 and facilitating the expression of ferroptotic signaling. Furthermore, BEBT-908 promoted a proinflammatory tumor microenvironment that activated host antitumor immune responses and potentiated immune checkpoint blockade therapy. Mechanistically, BEBT-908-induced ferroptosis led to upregulation of MHC class I and activation of endogenous IFNγ signaling in cancer cells via the STAT1 signaling pathway. The dual PI3K/HDAC inhibitor BEBT-908 is a promising targeted therapeutic agent against multiple cancer types that promotes immunogenic ferroptosis and enhances the efficacy of immunotherapy. SIGNIFICANCE: The dual PI3K/HDAC inhibitor BEBT-908 elicits potent antitumor responses, effectively inducing immunogenic ferroptosis of tumor cells and potentiating cancer immunotherapy.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Ferroptosis , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Pirimidinas/farmacología , Animales , Apoptosis , Proliferación Celular , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To analyze changes in volume and position of target regions and organs at risk (OARs) during radiotherapy for esophageal cancer patients. METHODS: Overall, 16 esophageal cancer patients who underwent radiotherapy, including 10 cases of intensity-modulated radiation therapy (IMRT) and six of three-dimensional conformal radiotherapy (3D-CRT), were enrolled. The prescription doses for the planning target volumes (PTVs) were as follows: PTV1, 64 Gy/32 fractions; and PTV2, 46 Gy/23 fractions. Repeat computed tomography (CT) was performed for patients after the 5th, 10th, 15th, 20th, and 25th fractions. Delineation of the gross tumor volume (GTV) and OAR volume was determined using five repeat CTs performed by the same physician. The target and OAR volumes and centroid positions were recorded and used to analyze volume change ratio (VCR), center displacement (ΔD), and changes in the distance from the OAR centroid positions to the planned radiotherapy isocenter (distance to isocenter, DTI) during treatment. RESULTS: No patient showed significant changes in target volume (TV) after the first week of radiotherapy (five fractions). However, TV gradually decreased over the following weeks, with the rate slowing after the fourth week (40 Gy). The comparison of TV from baseline to 40 Gy (20 fractions) showed that average GTVs decreased from 130.7 ± 63.1 cc to 92.1 ± 47.2 cc, with a VCR of -29.21 ± 13.96% (p<0.01), while the clinical target volume (CTV1) decreased from 276.7 ± 98.2 cc to 246.7 ± 87.2 cc, with a VCR of -10.34 ± 7.58% (p<0.01). As TVs decreased, ΔD increased and DTI decreased. After the fourth week of radiotherapy (40 Gy), centroids of GTV, CTV1, and prophylactic CTV (CTV2) showed average deviations in ΔD of 7.6 ± 4.0, 6.9 ± 3.4, and 6.0 ± 3.0 mm, respectively. The average DTI of the heart decreased by 4.53 mm (from 15.61 ± 2.96 cm to 15.16 ± 2.27 cm). CONCLUSION: During radiotherapy for esophageal cancer, Targets and OARs change significantly in volume and position during the 2nd-4th weeks. Image-guidance and evaluation of dosimetric changes are recommended for these fractions of treatment to appropriate adjust treatment plans.
RESUMEN
EGFR mutation-positive NSCLC tumors are highly heterogeneous, therefore, exploring an agent simultaneously targeting multiple EGFR mutations may be valuable for clinical practice. Compared with osimertinib, BEBT-109 shows more sensitive and extensive antitumor activity in EGFR mutant NSCLC, while sparing wild-type EGFR cell lines. Meanwhile, unlike the metabolite of osimertinib AZ5104, the main metabolites of BEBT-109 are found lacking in activity against wild-type EGFR cell lines. Preclinical and clinical studies demonstrate a unique pharmacokinetic profiles of BEBT-109 with rapid absorption and quick in vivo clearance without accumulation, which are conducive to minimizing the off-target toxicity of the covalent irreversible EGFR inhibitor. Oral administration of BEBT-109 induces tumor regression in EGFR exon 20 insertion xenografts, and even tumor disappearance in PC-9, HCC827 and H1975 xenograft models. Furthermore, in clinical trials, the objective responses were observed in NSCLC patients with EGFR T790M mutation in the first and second dosing cohorts. These findings demonstrate that BEBT-109, a potent pan-mutant-selective EGFR inhibitor with improved pharmacokinetic properties, might offer a promising new option for the treatment of multiple mutant-EGFR-driven NSCLC.
RESUMEN
The exploration of cost-effective and highly efficient electrocatalysts for the hydrogen evolution reaction (HER) is of great significance for realizing sustainable H2 production. As previously proposed, anion incorporation in promising earth-abundant transition metal-based electrocatalysts could be a reasonable and competitive approach to regulate the electronic structure with optimized atomic hydrogen adsorption and desorption for enhanced intrinsic electrocatalytic performance during the HER. Herein, we present the rational design and fabrication of O-incorporated CoP (expressed as O-CoP) nanorods with a controllable component and electronic structure. As demonstrated, when the lattice-incorporated O is at an appropriate concentration, the engineered O-CoP nanocatalysts have more active sites exposed with an increased number of electrochemically active areas and better electron/ion conductivity, leading to boosted HER activity and running stability. Typically, the obtained O-CoP nanorods with an optimal oxygen content exhibit excellent HER activity with an overpotential of 116 mV at a current density of 10 mA cm-2 and a small Tafel slope of 59 mV dec-1 in alkaline media. This anion doping strategy may make a widespread contribution to the efficient engineering of electrocatalysts for energy conversion devices.
RESUMEN
The development of functional as well as nutritional surfactants for the food industry remains a matter of great interest. In the present study, a series of 6â³-O-acylmaltotriose monoesters bearing alkyl side chains of 10-18 carbons was prepared by enzymatic means. The emulsions derived from those monoesters incorporating palmitoyl, stearoyl, and oleoyl side chains generally displayed advantageous shelf-lives, superior resistance to environmental variations, and more favorable droplet size distributions as well as stronger cytotoxic effects against various cancer cell lines. Ester 6 was shown to significantly inhibit the proliferation of MCF-7 breast cancer cells by inducing G1 phase arrest. Specifically, the levels of the G1 phase-related markers cyclin D1 and cyclin E as well as the cycle-dependent kinase 4 were suppressed by this particular ester. This study thus reveals that maltotriose esters can not only serve as novel functional food emulsifiers but also act, in vitro, as notable cytotoxic agents through a well-defined mechanism-of-action.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Emulsiones/química , Emulsiones/farmacología , Ésteres/química , Ésteres/farmacología , Trisacáridos/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Trisacáridos/químicaRESUMEN
Cysteine (Cys) is a semi-essential amino acid that exerts a vital role in numerous biological functions. A noninvasive method for in vivo imaging of cysteine could represent a valuable tool for research cysteine and its complex contributions in living organisms. Thus, we developed a turn-on bioluminescence probe (CBP) not only for detecting exogenous and endogenous cysteine in vitro and in vivo, but also for visualizing these cysteines in whole animal. The current applications may help shed light on the complex mechanisms of cysteine in miscellaneous physiological and pathological processes.
Asunto(s)
Cisteína/química , Colorantes Fluorescentes/química , Animales , Permeabilidad de la Membrana Celular , Humanos , Límite de Detección , Mediciones Luminiscentes , Maleatos/química , Ratones , Modelos Animales , Imagen ÓpticaRESUMEN
BACKGROUND: Three-dimensional ultrasound (3DUS) is an attractive option in image-guided radiotherapy (IGRT) for prostate cancer (PCa) patients. However, the potential factors influencing the accuracy of 3DUS in comparison with cone-beam CT (CBCT) in IGRT for PCa patients haven't been clearly identified. METHODS: The differences between US/US and CBCT/CT registrations were analyzed over 586 and 580 sessions for 24 and 25 PCa patients treated with or without pelvic lymph node irradiation, respectively. The clinical factors that may influence registration differences were also evaluated. RESULTS: The average discrepancies between US/US and CBCT/CT registrations were - 0.28 ± 5.28 mm, - 0.16 ± 3.48 mm, and - 0.47 ± 4.31 mm in the superior-inferior (SI), left-right (LR), and anterior-posterior (AP) directions, respectively. The discrepancies were respectively less than 5 mm longitudinally, laterally, and vertically in 64.4 and 70.1%, 84.9 and 89.2%, and 75.9 and 79.1% of the patients treated with or without pelvic lymph node irradiation, respectively. The registration differences were significantly smaller at least in one direction in patients younger than 70 years, without pelvic lymph node irradiation, guided by transperineal ultrasonography and had a bladder volume smaller than 300 mL. CONCLUSIONS: Age, irradiated regions, 3DUS modality, and bladder volume are important factors that may influence the differences between US/US and CBCT/CT registrations. 3DUS guidance is more feasible for younger PCa patients with a better control of bladder volume during the treatment and those who did not undergo pelvic lymph node irradiation.
Asunto(s)
Tomografía Computarizada de Haz Cónico/métodos , Imagenología Tridimensional/métodos , Ganglios Linfáticos/patología , Pelvis/patología , Neoplasias de la Próstata/patología , Radioterapia Guiada por Imagen/métodos , Ultrasonografía/métodos , Anciano , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Ganglios Linfáticos/diagnóstico por imagen , Ganglios Linfáticos/efectos de la radiación , Irradiación Linfática , Masculino , Pelvis/diagnóstico por imagen , Pelvis/efectos de la radiación , Pronóstico , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/radioterapia , Dosificación Radioterapéutica , Planificación de la Radioterapia Asistida por Computador/métodos , Radioterapia de Intensidad Modulada/métodos , Estudios RetrospectivosRESUMEN
Emulsifiers derived from renewable resources such as sucrose and fatty acids are high volume commodity chemicals and currently produced by traditional chemical synthesis techniques that lack the capacity to form the most desirable monoesters (of sucrose) in a selective and efficient fashion. The development of new emulsifiers (surfactants) from alternate, structurally simpler but nevertheless abundant disaccharides such as maltose represents a possible solution to this problem. Herein, we report the facile enzymatic preparation of a homologous series of 6'- O-acylmaltose esters and an in-depth evaluation of them revealing that their surfactant properties and thermal stabilities are largely determined by the length of the fatty acid chain. In the first such comparison, we show that the foaming and emulsifying effects of certain of these maltose monoesters are superior to those of their sucrose-derived and commercially exploited counterparts. As such, maltose esters have considerable potential as emulsifiers for use in, for example, the food industry.
Asunto(s)
Emulsionantes/química , Ésteres/química , Maltosa/química , Ácidos Grasos/química , CalorRESUMEN
AIM: To prepare the monoclonal antibody (mAb)against Pfs25 protein of Plasmodium falciparum, and establish the method of sandwich ELISA for detecting the Pfs25 protein. METHODS: Pfs25 protein the recombinant expressed by Pichia pastoris was purified. The purified Pfs25 protein as the antigen was used to immune the BALB/c mice, The secreting specific mAb positive cell strains, which were prepared by hybridizing the Sp2/0 myeloma cell and the spleen cell from immunized mice, were detected by indirect ELISA method. The ascites of mAb were collected from immunization F1 mice, and their biological properties were identified by indirect ELISA. The anti-Pfs25 antibody was labeled by Horseradish Peroxidase (HRP), the sandwich ELISA method to detect Pfs25 protein was established based on the anti-Pfs25 mAb 4B7 and 1B4 as coating and enzyme antibody, respectively. RESULTS: Three hybridoma cell lines secreting mAb against Pfs25 protein have been selected from the antibody positive hybridizing cells. The two of them have a better stability and specificity. The sandwich ELISA method detecting Pfs25 protein was established. Its detecte range was 0.07-1 mg/mL , and its sensitivity was 41.6 ng/mL. CONCLUSION: The anti-Pfs25 mAb are successfully prepared and the double antibody sandwich ELISA method detecting Pfs25 protein is established. Our study lay a foundation of developing transmission-blocking malaria vaccine with Pfs25 protein as antigen.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Protozoarias/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/análisis , Sensibilidad y EspecificidadRESUMEN
AIM: To purify recombinated GNLY which is expressed by prokaryotic expression system, and to prepare the monoclonal antibody (mAb) against it. METHODS: The solvable protein was purified by affinity chromatography. And employing the fusion protein GNLY immuned BALB/c mice, using conventional hybridoma technology prepared the mAb against human GNLY. Then purified and determined for titer by indirect ELISA method, for antigenic epitopes by additive ELISA, for relative affinity index by sulfocyanate elution method, and analyzed for subclass, specificity and stability. RESULTS: We got the soluble reactive fusion GNLY, and its purity and content were 95%, 0.8 g/L, respectively. Four cell strains secreting mAb against human GNLY were screened, 6C8, 9C6, 5G7and5E5. Their neutralizing titers were 1:100-1:3 200 and (0.1 - 8) x 10(-4) in supernatant and ascites. CONCLUSIONS: We successfully purified the fusion protein of GNLY, and prepared the mAb against human GNLY, lying a certain foundation for its laboratory and clinical research.