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Although high-efficiency targeted delivery is investigated for years, the efficiency of tumor targeting seems still a hard core to smash. To overcome this problem, we design a three-step delivery strategy based on streptavidin-biotin interaction with the help of c(RGDfK), magnetic fields and lasers. The ultrasmall superparamagnetic iron oxide nanoparticles (USIONPs) modified with c(RGDfK) and biotin are delivered at step 1, followed by streptavidin and the doxorubicin (Dox) loaded nanosystems conjugated with biotin at steps 2 and 3, respectively. The delivery systems were proved to be efficient on A549 cells. The co-localization of signal for each step revealed the targeting mechanism. The external magnetic field could further amplify the endocytosis of USPIONs based on c(RGDfK), and magnify the uptake distinctions among different test groups. Based on photoacoustic imaging, laser-heating treatment could enhance the permeability of tumor venous blood vessels and change the insufficient blood flow in cancer. Then, it was noticed that only three-step delivery with laser-heating and magnetic fields realized the highest tumor distribution of nanosystem. Finally, the magnetism/laser-auxiliary cascaded delivery exhibited the best antitumor efficacy. Generally, this study demonstrated the necessity of combining physical, biological and chemical means of targeting.
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Se presenta el caso de una paciente que, durante los estudios por búsqueda de fertilidad y posterior embarazo, mostraba un perfil tiroideo alterado con niveles elevados de T4 libre y TSH normal. Luego de descartar un adenoma tirotropo y ante la ausencia de sintomatología clínica de hipertiroidismo, se investigó la posibilidad de interferencias analíticas en los inmunoensayos utilizados para la medición de las hormonas. Se han descrito interferencias causadas por anticuerpos heterófilos, macro TSH, anticuerpos anti-tiroideos, biotina, y en menor medida anticuerpos anti-estreptavidina y anti-rutenio. Los análisis de la paciente se realizaron en autoanalizador cuya plataforma emplea el sistema estreptavidina-biotina que es muy susceptible a varios interferentes. Un algoritmo propuesto incluye una serie de pruebas simples de realizar e interpretar que permiten detectar o descartar la presencia de interferentes. De acuerdo al mismo, se efectuó la comparación con una plataforma analítica diferente (que no utiliza el sistema estreptavidina-biotina), diluciones seriadas, precipitación con polietilenglicol 6000 y tratamiento con micropartículas recubiertas con estreptavidina. Los resultados obtenidos confirmaron la presencia de anticuerpos anti-estreptavidina en el suero de la paciente. Ante discordancias entre las manifestaciones clínicas y los resultados de laboratorio, se debe investigar la posibilidad de interferencias metodológicas para evitar el riesgo iatrogénico potencial que implica una interpretación bioquímica errónea.
We present the case of a patient who, during studies for fertility and subsequent pregnancy, showed an altered thyroid profile with elevated levels of free T4 and normal TSH. After ruling out a thyrotropic adenoma and in the absence of clinical symptoms of hyperthyroidism, the possibility of analytical interference in the immunoassays used to measure hormones was investigated. Interferences caused by heterophile antibodies, macro TSH, anti-thyroid antibodies, biotin, and to a lesser extent anti-streptavidin and anti-ruthenium antibodies have been described. The analysis of the patient was carried out in a self-analyzer whose platform uses the streptavidin-biotin system that is very susceptible to several interferents. A proposed algorithm includes a series of simple tests to perform and interpret that allow detecting or ruling out the presence of interferents. Accordingly, a comparison was made with a different analytical platform (which does not use the streptavidin-biotin system), serial dilutions, precipitation with polyethylene glycol 6000 and treatment with microparticles coated with streptavidin. Results obtained confirmed the presence of anti-streptavidin antibodies in the patient's serum. In the case of disagreements between clinical manifestations and laboratory results, the possibility of methodological interferences should be investigated in order to avoid the potential iatrogenic risk involved in an erroneous biochemical interpretation.
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Humans , Female , Pregnancy , Adult , Pituitary Neoplasms/diagnosis , Adenoma/diagnosis , Antibodies, Anti-Idiotypic/immunology , Streptavidin/immunology , Hyperthyroidism/diagnosis , Pituitary Neoplasms/immunology , Thyroxine/blood , Triiodothyronine/blood , Thyrotropin/blood , Adenoma/immunology , Diagnostic Errors , Hyperthyroidism/immunologyABSTRACT
Objective@#To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen.@*Methods@#According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method.@*Results@#In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml.In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen.@*Conclusion@#A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.
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Objective To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen.Methods According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method. Results In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml. In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen.Conclusion A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.
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Objective:The polyclonal antibody of aldosterone (ALD) for immunoassay was developed.And a chemiluminescence immunoassay (CLIA) for the determination of ALD in human blood was established.Methods:Aldosterone oxime was prepared by chemical modification and then conjugated with BSA to prepare immunogen.Rabbit anti ALD polyclonal antibody was prepared by immunizing rabbits with the ALD-BSA.The CLIA of ALD was performed using biotin streptavidin amplification system and competition method.Results:After identification,rabbit No.3 received the highest sensitivity to ALD antibody,and the 50％ binding inhibition (IC50) value for ALD concentration was 268 pg/ml.The measuring range of CLIA method using the antibody was 62.5-2 000 pg/ml.The assay sensitivity was 23.7 pg/ml.The intra-and inter-assay coefficients of variation were 6.9％-9.5％ and 8.5％-12.7％,respectively.Analytical recovery rate was in the range of 93.1％-104.1％.The correlation coefficient between measured and expected values were 0.996 after serial dilution.Compared with radioimmunoassay kit,the correlative equation was y =0.932x+4.596,the correlation coefficient was 0.948 (n =95).Conclusion:The result of methodological identification shows that it was in line with the basic requirements of clinical application.
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Objective To study the value of progesterone,β-HCG and serum streptavidin 125 (CA125) in predicting the outcome of pregnancy in patients with early threatened abortion by Logistic regression and ROC curve.Methods A total of 150 pregnant women who were pregnant with infertility from 5 to 7 weeks of gestation were enrolled in this study from August 2014 to October 2016.According to the outcome of pregnancy,the patients were divided into two groups:pregnancy group and spontaneous abortion group.Serum levels of progesterone,β-HCG and CA125 were detected in all patients with threatened abortion before treatment.The levels of progesterone,β-HCG and CA125 were detected in all the pregnant women with normal pregnancy in our hospital for the same period.All patients were followed up for 12 weeks.Logistic regression and ROC curve were used to analyze the value of progesterone,β-HCG and CA125 in the diagnosis of pregnant patients with threatened abortion.Results Seven cases of threatened abortion patients off.The levels of progesterone,β-HCG and CA125 in the normal pregnant group and the pregnant group were significantly higher than those in the abortion group (P ＜0.05).The area under the ROC curve of progesterone,β-HCG and CA125 in the early stage of pregnancy were 0.764,0.688 and 0.870 respectively.The area under the ROC curve of the early threatened abortion was 0.916.The specificity of progesterone,β-HCG,CA125 and the combined detection were 76.54％,61.05％,73.15％,89.09％,the specificity were 78.63％,72.38％,69.54％ and 93.25％ respectively,and the accuracy were 74.43％,70.15％,65.33％,89.77％.Conclusion The combination of progesterone,β-HCG and serum CA125 in the 5 ～ 7 weeks of pregnancy has a high clinical diagnostic value in prediction inevitable abortion.
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An enhanced gold immunochromatographic assay ( GICA ) with simplicity, rapidity and high sensitivity was developed to detect imidaclothiz by using the high affinity between biotin and streptavidin. The 13-nm AuNPs were double-labeled with anti-imidaclothiz antibody and biotinylated DNA, and the 41-nm AuNPs were labeled with streptavidin to prepare an enhanced gold immunochromatographic test strip for imidaclothiz. The working conditions of the strip were systematically optimized, and the sensitivity, specificity, precision and accuracy were assessed by testing the cross-reactivity ( CR) , spiked recovery and validation with high performance liquid chromatography ( HPLC) . Under the optimal conditions, the detection could be completed in 10 min with visual result, and the limit of detection ( LOD ) was 25 ng/mL. The analysis showed no cross-reactivity with analogues of imidaclothiz except for imidacloprid. The detection results of GICA agreed with the spiked concentrations of imidaclothiz at spiked levels of 0 . 05 , 0 . 5 and 5 μg/g in river water, rice, cucumber, tomato, pear, cabbage and apple samples. The detection results of GICA for imidaclothiz in unknown concentration river water and pear samples were consistent with that of HPLC.
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Objective:The chemiluminescence immunoassay for AngiotensinⅠ ( AngⅠ ) was developed by the competition method. The renin activity was calculated by the determination of Ang Ⅰ. Methods: AngⅠ antigen was labeled with biotin and fluorescein labeled AngⅠantibody,and anti fluorescein antibody was used to coated with the microporous plate . Results:The standard range of the method was 100-7 800 pg/ml. The assay sensitivity was 24. 3 pg/ml. The intra- and inter-assay coefficients of variance were 5. 6%-8. 7% and 6. 8%-10. 4% respectively. Analytical recovery was 95. 4%-105. 3%. The correlation coefficients between measured and expected values were 0. 999 after serial diluted. Compared with radioimmunoassay kit,the correlative equation was y=0. 95x+235. 70,correlation coefficient was 0. 973. Coated microporous plate and biotin labeled Ang Ⅰ antigen,and fluorescein labeled Ang Ⅰantibody at 37℃ for a week with good stability. Conclusion: The results of the method were in accord with the basic requirements of immunoassay.
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Objective To investigate the expression and clinical significance of Smac and HtrA2 in children with acute leukemia(AL).Methods Bone marrow samples were obtained from 77 children with AL (including 32 newly diagnosed children,33 complete remission children and 12 relapsed children)and the control group of 15 children without malignant blood disease.The expressions of Smac and HtrA2 protein were measured by streptavidin/peroxidase immunoperoxidase technique(SP) in all children.SPSS 13.0 software was applied to analyze the statistical data.Results Protein Smac was detected only in some samples,but HtrA2 was detected in all samples.The levels of Smac and HtrA2 protein in newly diagnosed AL children were both higher than those of the complete remission children (x2 =17.38,F =2.36,all P ＜ 0.05) and normal controls (x2 =12.89,F =5.26,all P ＜ 0.05),there was a statistical significance,but compared with those in the relapsed children,the difference had no statistical significance (x2 =1.18,F =1.57,all P ＞ 0.05).The levels of Smac and HtrA2 protein in complete remission children were both higher than those of the normal controls,and the difference had no statistical sigmficance(x2 =1.20,F =2.23,all P ＞ 0.05).In the newly diagnosed children,the levels of Smac and HtrA2 protein in children with acute lymphocytic leukemia(ALL) were higher than those of the acute myeloid leukemia(AML),but the differences had no statistical significance(x2 =0.113,t =1.024,all P ＞ 0.05).In newly diagnosed AL children,the complete remission(CR) rate of the negative expression of Smac(Smac-,90.9％) and the low expression of HtrA2(HtrA2low,84.6％) in the level of protein were higher than those of the positive expression of Smac(Smac +,47.6％) and the high expression of HtrA2 (HtrA2high,47.4％),and there was statistical significance respectively(x2 =5.772,4.596,all P ＜ 0.05).The CR rate of Smac-HtrA2low group (100％) was higher than that of Smac+ HtrA2high group(30.8％)in the children with AL,and the statistical data were of great significance(x =9.692,P ＜0.01).The protein level of Smac in newly diagnosed AL children was correlatedwith HtrA2 (r =0.979,P ＜ 0.001).Conclusions Pro-apoptotic protein Smac and HtrA2 may be involved in and af-fected each other in the pathogenesis and progression in AL,but levels of Smac and HtrA2 protein may be not correlatedwith the types of AL.In newly diagnosed AL children,the high expression of protein Smac and HtrA2 predicts poorprognosis.
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Objective:To study on the levels of CXCL8 and its receptors (CXCR1 and CXCR2) in peripheral blood neutrophils of the patients with chronic hepatitis B.Methods:The neutrophils were isolated and purified by neutrophil isolation medium,and the loads of HBV-DNA in neutrophils were detected by PCR,and the levels of HBeAg in serum were measured by ELISA.The patients were divided into different groups according to the detective results so that the expressions of CXCL8 and its receptors ( CXCR1,CXCR2) in neutrophils were detected by the methods of streptavidin-biotin complex ( SABC ) immunocytochemistry stain.Results:The data of SABC immunocytochemical stain showed that the positive color of CXCL8 was mainly located in the cytoplasm of PMNs.However,the most positive color of CXCR1and CXCR2 was mainly expressed in the cytoplasm and cell membrane.Interestingly,the deeper immune coloring of CXCL8 and CXCR1, and relatively shallow immune coloring of CXCR2 were explored in the group with positive of HBeAg.The similar detective results also had been found in the cases with positive of HBV DNA in neutrophils.Compared with the normal control group,the levels of CXCL8 and CXCR1 in the patients were significantly increased ( P0.05).Conclusion:After neutrophils occult infected by HBV,not only the secretion of CXCL8 can be promoted, but also the expression of CXCR1 will be further increased.The data of immunohistochemical staining have been shown that the color degree of CXCL8 and its receptors ( CXCR1, CXCR2 ) are positive correlation to the level of HBeAg and the loads of HBV DNA.More PMNs can be chemotactic attraction to lesion so as to participate in the local inflammatory injury and tissue repair via the interactive pathway of the high expression of CXCR1 on surface of neutrophils with CXCL8.
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Objective:To develop a biotin-streptavidin system (BAS)-mediated folate receptor (FR)-targeted quantum dot (QD) fluorescent probe and preliminarily validate the targeting ability and signal amplification effect of the probe. Methods: Streptavidin (SA) was covalently coupled with QD through the active ester method;the physical characteristics of the prepared QD-SA were veri-fied. Biotinylated folate was synthesized through the carrier bovine serum albumin using the same method and then reacted with QD-SA to form the special probe. The probe was used to identify SKOV3 cells and FR-negative A549 cells to verify its targeting speci-ficity. QD-SA was used as the contrast. SKOV3 cells were imaged using the BAS-mediated FR-targeted QD probe with a biotinylated folate incubation time of 1 or 4 h. Various reaction times were also tested between the probe and the QD-FA that was formed without BAS mediation. Results:The BAS-mediated FR-targeted QD probe specifically recognized FR-positive SKOV3 cells. The probe ob-tained higher fluorescent intensity after 4 h than after 1 h of biotinylated folate incubation. The BAS-mediated FR-targeted QD probe al-so had a stronger fluorescent signal than the QD-FA probe. Conclusion:The proposed probe presents a great potential in the early diag-nosis of ovarian cancer because of its high specificity and sensitivity.
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Objective To establish a novel TRFIA for the measurement of heparanase (HPA) in serum samples,and investigate its clinical application.Methods The micro-pore plate wells were first coated with partially recombinant murine anti-human HPA monoclonal antibody.Biotin-labeled recombinant HPA protein was then used to compete with HPA in serum samples,and the prepared europium (III)-labeled streptavidin (Eu3+-SA) was used as signal readout for establishing the BSA-TRFIA assay.Using this assay,the serum HPA levels in healthy subjects (n=32) and tumor patients (n=54) were measured.The results of BSA-TRFIA were compared with those of ELISA.Two-sample t test (or t' test),and linear correlation analysis were used to analyze the data.Results The sensitivity of BSA-TRFIA for measuring HPA was 0.33 ug/L.The CV values for intra-batch and inter-batch were 5.29% and 7.54%,respectively.The average recovery rate was 105.5%.The standard curve range was 0-1 000 ug/L.The serum HPA level measured by the BSA-TRFIA method in healthy subjects was (2.03_+ 1.47) Iug/L.In tumor patients,the HPA level was significantly higher:(22.13_+7.38) ug/L (t'=19.388,P
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Objective To explore a novel quantitative detection method for the concentration of specific IgG (sIgG) in food intolerance, taking egg sIgG detection for the example. Methods A total of 173 patients underwent food allergen sIgG detection were included in this study, and 78 healthy subjects were used as negative controls. The microtiter plates were coated with biotinylated bovine serum albumin (BSA) and linked with streptavidin. Then, the biotinylated egg antigen and specimen were successively added into the wells of plate.After washing, enzyme labeled anti-human IgG was added to establish an antigen indirectly coated liquid-phase reaction patterns of ELISA method. The concentration of the biotinylated allergens and enzyme labeled antibody were optimized and the reaction conditions were determined. This method was used to detect the sIgG in serum samples. Results The biotinylated egg white was selected as antigen, the optimal dilution rate was 1∶2 000,and the most suitable enzyme labeled antibody dilution ratio was 1∶12 000 in this method. The within-run and the between-run coefficients of variation were 4.83%-8.55%and 4.88%-7.93%respectively. The specificity is preferable, the species-crossed reaction rates with crab, cow milk and goat milk were＜10%. There was a good correlation between the assay developed in this study and the food intolerance detection kit provided by United States BIOMERICA Inc ( =0.977X+8.45, r=0.961, P＜0.05). Conclusion The detection method can be used to detect serum sIgG for egg intolerance patients with easy operation, highly accuracy and specificity. Furthermore, it showed the capacity of excellent repeatability and flexibility potential, which provided a good foundation for developing a kind of“personalized”random combination of food varieties ELISA kit.
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EGF family growth factors consists of growth factors, such as transforming growth factor (TGF)-a, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin (AR) and epiregulin, autocrine growth factors in normal human keratinocytes. HB-EGF is mitogen for epithelial cells and like other members of the EGF family, HB-EGF exerts its biological effects via interaction with the EGF receptor (EGFR). HB-EGF is an autocrine growth factor for human keratinocytes, and has a possible role as a paracrine growth factor for fibroblast. Our report concerning immunohistochemical localization of HB-EGF in normal skin by using the streptavidin-peroxidase (HRP) conjugate method, confirms previous data, revealing specific patterns of HB-EGF localization. Identification of HB-EGF in cells of epithelial origin suggests its autocrine and/or paracrine roles in epithelial cell maintenance. Our report especially wants to give a technical contribution, easy to manage and with evident results. A simple technique that does not require use of sophisticated equipment.
La familia factores de crecimiento EGF se compone de representantes como el factor de crecimiento transformante (TGF)-a, factor epidérmico vinculante a la heparina (HB-EGF), anfiregulina (AR) y epirregulina, factores autocrinos de crecimiento en queratinocitos humanos normales. HB-EGF es mitógeno para células epiteliales y al igual que otros miembros de la familia EGF, HB-EGF ejerce sus efectos biológicos a través de la interacción con el receptor de EGF (EGFR). HB-EGF es un factor de crecimiento autocrino de queratinocitos humanos, y tiene un posible papel como factor de crecimiento paracrino de los fibroblastos. Nuestro reporte sobre la localización inmunohistoquímica de HB-EGF en la piel normal mediante el método de conjugado estreptavidina-peroxidasa (HRP), confirma datos anteriores, revelando patrones de localización específicos para HB-EGF. La identificación de HB-EGF en las células de origen epitelial sugiere su papel autocrino y/o paracrinos en el mantenimiento de las células epiteliales. Nuestro informe quiere dar una contribución técnica, fácil de manejar y con resultados evidentes. Una técnica simple que no requiera el uso de equipo sofisticado.
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Humans , Epidermal Growth Factor/metabolism , Heparin/metabolism , Skin/metabolism , Immunohistochemistry , Peroxidase , Keratinocytes/metabolism , StreptavidinABSTRACT
Purpose To prepare streptavidin-tagged mouse interleukin-4(mIL-4-SA)bifunctional fusion protein and to study on its bioactivity.Methods The mIL-4 gene was cloned by RT-PCR and cloned into pET21 vector to get mIL-4-SA-pET21 expression plasmid.The mIL-4-SA fusion protein was expressed in BL21 (DE3)host bacteria and purified through the Ni-NTA affinity chromatography and refolded by dilution and dialysis.The effect of mIL-4-SA fusion protein on mouse thymocytes proliferation was evaluated by MTY.Flow cytometric analysis was performed to detect the mIL-4-SA fusion protein on the biotinylated B16F10 tumor cells.Results The mIL-4-SA-pET21 vector was successful by constructed and the mIL-4-SA fusion protein was expressed in BL21(DE3)at about 35%of total bacterial proteins.The purity of mIL-4-SA Was about 95% through Ni-NTA.The mIL-4-SA fusion protein exhibited bifunctional activities,i.e.,stimulative effect for mouse thymocyte proliferation and SA-mediated high-affinity binding to biotinylated cell surfaces(anchoring modified rate Was about 96.69%).Conclusion The mIL-4-SA fusion protein was expected to be developed for the treatment of tumors.
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Background & objectives: An inability or decreased ability of spermatozoa to bind to the zona pellucida (ZP), an extracellular glycoproteinaceous matrix surrounding egg, is one of the plausible causes of idiopathic infertility. It will be clinically useful to distinguish this condition from other causes of infertility. An assay system, investigating binding of human sperm with ZP glycoprotein may prove useful in this regard. We attempted to develop a simple assay system to analyse the binding of capacitated human spermatozoa to human zona pellucida glycoprotein-3 (ZP3) using baculovirus-expressed recombinant human ZP3 coated beads. Methods: Recombinant baculovirus-expressed ZP3 was purified, labelled with biotin and coated on streptavidin sepharose beads. An in vitro assay system was optimized to study binding of capacitated human sperm to ZP3 coated beads. Results: A higher percentage of baculovirus-expressed recombinant human ZP3 coated beads showed significant (P<0.05) binding of capacitated human sperm as compared to beads coated with fetuin. An inhibition in the binding of sperm to ZP3 coated beads was observed in presence of cold recombinant human ZP3. Further, prior incubation of ZP3 coated beads with monoclonal antibodies (MAbs) against ZP3 but not against ZP2 resulted in the decrease in number of sperm bound to bead. Interpretation & conclusion: An in vitro assay system to study the binding of human sperm to ZP3- primary sperm receptor was established, which may be useful to determine the functional competence of spermatozoa.
Subject(s)
Egg Proteins/genetics , Egg Proteins/metabolism , Humans , Male , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Protein Binding , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sperm Capacitation/physiology , Spermatozoa/cytology , Spermatozoa/metabolism , Zona Pellucida/metabolism , alpha-Fetoproteins/metabolismABSTRACT
Objective To prepare a novel fusion protein of hu3D3 and core-streptavidin(hu3D3/cSA) as a universal carrier targeting to lung cancer,and analyze its activities. MethodscSA gene was acquired by PCR,and inserted into plasmid hu3D3/pET-22b(+)to construct a recombinant plasmid hu3D3/cSA/pET-22b(+)．The fusion gene was expressed in E. Coli BL21(DE3).The fusion protein was purified through nickel-affinity chromatography column and renatured using TEA buffer. The tetrameric hu3D3/cSA complex was analyzed by SDS-PAGE and Western blot.The hu3D3/cSA protein was labeled with FITC,then its antigen-binding activity was analyzed using fluorescence microscopy,and the functional affinity of hu3D3/cSA and hu3D3 were analyzed and compared by flow cytometry. The biotin-binding activity of hu3D3/cSA was tested bv EUSA and Western blot. Results The recombinant plasmid hu3D3/cSA/pET-22b(+)with correct sequence was obtained. The fusion protein was found after expression in E. Coli BL21 (DE3)mainly in the form of inclusion body. After being purified and refolded,tetrameric complex was formed. The purified tetrameric hu3D3/cSA complex retained both antigen-binding activity of hu3D3 moiety and biotin-binding activity of cSA moiety:furthermore,the avidity of the hu3D3/cSA to its target antigen increased by about 14 times as compared with that of monomeric hu3D3. Conclusion The fusion protein hu3D3/cSA with both antigen- and biotin-binding activity is SHCCessfully prepared,and the avidity of hu3D3 moiety to 3D3 antigen is enhanced. Consequently,hu3D3/cSA could be a universal carrier targeting to lung cancer, and could be an alternative,convenient method to realize target therapy to lung cancer by the combination of multiple biotinylated anti-tumor drugs or agents.
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The GFP(green fluorescence protein)-streptavidin(SA) bi-functional fusion protein was generated and characterized in order to demonstrate novel platform for efficiently and durably modifying the cell surface with SA-tagged bi-functional proteins.The GFP-SA/pET24d construct was generated and expressed in BL21(DE3) host bacteria at the high level.The recombinant protein GFP-SA was purified through the Ni-NTA affinity chromatography,and then refolded.After biotinylation B16 tumor cells were modified with GFP-SA bi-functional fusion protein and then subjected to fluorescent microscopy and FACS analysis.The effect of surface modification on the viability and growth of B16.F10 tumor cells was evaluated by MTT staining.The GFP-SA recombinant fusion protein was expressed in BL21(DE3) at about 20 % of total bacterial proteins.The GFP-SA bi-functional fusion protein exhibited the bi-functionality,i.e.,SA-mediated high-affinity binding to biotinylated cell surfaces and GFP-emitted green fluorescence.The cell surface modification with GFP-SA bi-functional fusion protein did not affect the viability and growth of the modified B16.F10 tumor cells significantly.The GFP-SA bi-functional fusion protein was obtained and could be displayed efficiently on the surface of the biotinylated B16.F10 tumor cells through the specific and tight interaction between streptavidin and biotin,thus can be used as good trace protein and experimental control in the development of surface-modified tumor vaccine.
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In the present study, we inserted the core-streptavidin cDNA into downstream of multi-cloning site of plasmid pOPE101-8E5 by DNA gene recombination technology. And then, the variable fragments of heavy and light chain of the scFv-8E5 were replaced by the scFv-C4 variable fragments to construct the expression vector pOPE101-C4∷core-streptavidin. After transformed the vector pOPE101-C4∷core streptavidin into E.coil, the fusion protein C4∷core streptavidin-His-tag can be expressed by inducing with IPTG, and the expression level and activity of the expressed fusion protein analyzed by SDS-PAGE and Western blot. The results show that a scFv-C4∷core-streptavidin fusion protein of 45kDa was obtained, which can bind proteins of 60kDa & 45kDa from the KG1a cells lysate simultaneously. The binding function can be detected by the binding of core-streptavidin and biotin directly.
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BACKGROUND: The aim of our study was to optimize and establish erythropoietin (EPO) enzyme linked immunosorbent assay (ELISA) system. METHODS: We prepared several monoclonal and polyclonal antibodies specific to human-EPO. The best combinations of antibodies for coating and detecting antibodies were selected for the establishment of ELISA. We tested several methods such as a competitive EIA and a sandwich ELISA. RESULTS: The best sandwich ELISA was optimized compared to competitive EIA when purified polyclonal antibody (PoAb) was used as a coating antibody and biotinylated PoAb as a detecting antibody. This sandwich ELISA easily detected EPO when PoAb pairs were used compared to the ELISA using monoclonal antibody and PoAb. There were no significant differences between the effects of various blocking solutions on the performance of sandwich ELISA using biotinylated antibody. The ELISA system using PBST containing 3% BSA as a blocking solution can sensitively detect EPO (10 mU/mL) in a broad range of EPO concentrations (10-2,000 mU/mL) and there were cross-reactions with other cytokines). CONCLUSIONS: EPO can be easily determined by using biotinylated PoAb as a detecting antibody and another PoAb as a coating antibody.