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Recombinant human interferon α2b(rhIFNα2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNα2b is complex.In this study,an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b.RhIFNα2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNα2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other com-mercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.
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T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies.
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Objective@#To explore the effect of multimodal guidance based on the concept of five senses and six senses on the sleep phase of postoperative patients with intestinal tumor.@*Methods@#A total of 110 patients with intestinal tumor after surgery from January 2018 to July 2019 in Qilu Hospital of Shandong University (Qingdao) were randomly divided into two groups, 55 patients in each group. The control group received routine daily care. Instructed, the experimental group conducted multimodal guided training based on the five senses and six senses on the basis of daily care, and continued intervention for 3 weeks. Polysomnography (PSG) was used to compare the sleep phase differences before and after intervention.@*Results@#The ratio of NREM to total sleep time, the ratio of stage 1 sleep time to total sleep time, the ratio of stage 3 sleep time to total sleep time, the ratio of stage 4 sleep time to total sleep time, and the ratio of REM to total sleep time after intervention. The number of NREM micro-wakes (Z value was -5.178--2.157, all P<0.05), the difference was statistically significant. The proportion of NREM in total sleep time, the proportion of sleep time in total sleep time, the number of NREM micro-wakes, and the number of REM micro-wakes decreased compared with the pre-intervention period; the proportion of sleep time in total sleep time in stage 3, sleep in stage 4 The proportion of time to total sleep time and the proportion of REM to total sleep time were increased compared with those before intervention (Z value was -6.029--4.064, all P<0.05), and the difference was statistically significant. In the control group, the proportion of sleep time in total sleep time was increased compared with that before intervention. The number of NREM micro-wake and the number of REM micro-wakes were lower than those before intervention (Z value was -2.948, -5.632, -2.475, all P<0.05). The difference was statistically significant.@*Conclusion@#Multimodal guided training based on the concept of five senses and six senses can effectively improve the sleep structure of patients with intestinal tumors and improve their sleep quality.
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Objective:To explore the effect of multimodal guidance based on the concept of five senses and six senses on the sleep phase of postoperative patients with intestinal tumor.Methods:A total of 110 patients with intestinal tumor after surgery from January 2018 to July 2019 in Qilu Hospital of Shandong University (Qingdao) were randomly divided into two groups, 55 patients in each group. The control group received routine daily care. Instructed, the experimental group conducted multimodal guided training based on the five senses and six senses on the basis of daily care, and continued intervention for 3 weeks. Polysomnography (PSG) was used to compare the sleep phase differences before and after intervention.Results:The ratio of NREM to total sleep time, the ratio of stage 1 sleep time to total sleep time, the ratio of stage 3 sleep time to total sleep time, the ratio of stage 4 sleep time to total sleep time, and the ratio of REM to total sleep time after intervention. The number of NREM micro-wakes ( Z value was -5.178--2.157, all P<0.05), the difference was statistically significant. The proportion of NREM in total sleep time, the proportion of sleep time in total sleep time, the number of NREM micro-wakes, and the number of REM micro-wakes decreased compared with the pre-intervention period; the proportion of sleep time in total sleep time in stage 3, sleep in stage 4 The proportion of time to total sleep time and the proportion of REM to total sleep time were increased compared with those before intervention ( Z value was -6.029--4.064, all P<0.05), and the difference was statistically significant. In the control group, the proportion of sleep time in total sleep time was increased compared with that before intervention. The number of NREM micro-wake and the number of REM micro-wakes were lower than those before intervention ( Z value was -2.948, -5.632, -2.475, all P<0.05). The difference was statistically significant. Conclusion:Multimodal guided training based on the concept of five senses and six senses can effectively improve the sleep structure of patients with intestinal tumors and improve their sleep quality.
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To develop fusion protein of a GnRH Fc fragment and the integrin targeting AP25 antitumor peptide for GnRH receptor-expressing cancer therapy. The LMRAP fusion protein was constructed. A transwell invasion assay was performed. The gene mRNA and protein levels of GnRHR-I, 51, and v3 in different cancer cell lines were assessed. Cell proliferation was measured using a cell counting kit-8. An antagonist assay was performed on GnRH receptors. Anti-tumor activity was evaluated with a mouse xenograft tumor model. Immunohistochemistry (IHC) was applied to detect CD31 and CD34 expressions. Pharmacokinetic characteristics were determined with an indirect competition ELISA. The developed bifunctional fusion protein LMRAP not only inhibited HUVEC invasion, but also inhibited proliferation of GnRHR-I, 51, and v3 high expression cancer cells. The IC for LMRAP in the GnRH receptor was 6.235 × 10 mol/L. LMRAP significantly inhibited human prostate cancer cell line 22RV1 proliferation and . LMRAP significantly inhibited CD31 and CD34 expressions. The elimination half-life of the fusion protein LMRAP was 33 h in rats. The fusion protein made of a GnRH Fc fragment and the integrin targeting AP25 peptide retained the bifunctional biological activity of GnRHR blocking, angiogenesis inhibition, prolonged half-life and good tolerance.
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Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor?40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p＝0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.
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Objective: To analyze the chemical constituents of Morchella esculenta, and to determine the main activeingredient of it, so as to better control the quality of Morchella esculenta and promote the development and utilization of Morchella. Methods: The compounds were extracted and percolated by ethanol. The samples were separated using silicagel and identified by13C-NMR data. HPLC was used to assay the contents of 4-Hydroxybenzyl alcohol. Results: A totalof 5 compounds were isolated and their structures were identified as 11, 15, 19-trimethyl-5, 9-eicosadienoic acid, cis-13-Docosenoic acid&Erucic acid, 4-Hydroxybenzyl alcohol, Ergosta-5, 7, 22-triene-3β-ol and Mannitol. Conclusion: 4-Hydroxybenzyl alcohol was first isolated from the Morchella esculenta fruiting body, and HPLC method for thedetermination of 4-Hydroxybenzyl alcohol was established. The content of 4-Hydroxybenzyl alcohol were no less than0.40％. The determination method can be used for quality control of Morchella esculenta.
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Objective:To study the correlation among lipoprotein (a) [Lp (a)] ,its gene polymorphism and degenera‐tive calcific aortic valve disease (DCAVD) .Methods :From Feb 2010 to Jan 2014 ,a total of 164 DCAVD cases trea‐ted in our hospital were enrolled as DCAVD group ,another 164 healthy subjects undergoing physical examination in the same period were selected as normal control group .Relationship among Lp (a) level ,its gene polymorphism and aortic valvular calcification was compared and analyzed ,and Logistic regression analysis was used to analyze in‐dependent risk factors of DCAVD . Results :Lp (a ) gene possesses four point mutations in population , namely rs10455872 ,rs6415084 ,rs3798221 and rs7770628. In DCAVD group ,Lp (a) level of AG genotype was significantly higher than that of AA genotype [ (325.5 ± 108.2) mg/L vs .(211.7 ± 135.4) mg/L] in rs10455872 gene pheno‐type ,Lp (a) level of CC+TT genotype was significantly higher than that of CT genotype [ (287.9 ± 144.1) mg/L vs .(240.7 ± 127.2) mg/L] in rs6415084 gene phenotype ,and Lp (a) level of TT+ CC genotype was significantly higher than that of CT genotype [(304.1 ± 124.1) mg/L vs .(226.8 ± 101.6) mg/L] in rs7770628 gene phenotype , P<0.05 or <0.01 ;patient′s percentage of valvular calcification of AG genotype was significantly higher than that of AA genotype (90.0% vs .61.7% ) in rs10455872 , patients percentage of valvular calcification of CC +TT geno‐type was significantly higher than that of CT genotype (83.8% vs .66.7% ) in rs6415084 ,and patient′s percentage of valvular calcification of TT+CC genotype was significantly higher than that of CT genotype (87.3% vs .63.4% ) in rs7770628 , P<0.05 or <0.01. Logistic regression analysis indicated that rs10455872 ,rs6415084 ,rs7770628 and Lp (a) level were independent risk factors for valvular calcification of DCAVD (OR=1.67~2.31 , P<0.01 all) . Conclusion :Lp (a) gene polymorphism (rs10455872 ,rs6415084 and rs7770628) and plasma Lp (a) level are signifi‐cantly correlated to valvular calcification of DCAVD ,which may be susceptible genes for DCAVD occurrence .
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The study aims to characterize and compare interferon reference standards from 5 manufacturers. By testing molecular mass and trypsin-digested peptide mass mapping, the amino acid sequence was verified and post-translational modifications such as disulfide bond were identified. Results show that the molecular mass and amino acid sequence were consistent with theory; the disulfide bonds of 4 lots of interferon were Cys1-Cys98/Cys29-Cys138, 1 lot was Cys29-Cys139/Cys86-Cys99; N-terminal "+Met", acetyl N-terminal and Met oxidation were identified in part of the sample. UPLC-MS can be used to characterize and compare interferon reference standards from different manufacturers.
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Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.
Subject(s)
Animals , Humans , Acids , Chemistry , Amino Acid Sequence , Capsid Proteins , Chemistry , Genetics , Metabolism , Enterovirus A, Human , Genetics , Metabolism , Physiology , HEK293 Cells , Host-Pathogen Interactions , Hydrogen-Ion Concentration , Lysosomal Membrane Proteins , Chemistry , Genetics , Metabolism , Molecular Docking Simulation , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , RNA, Viral , Genetics , Metabolism , Receptors, Scavenger , Chemistry , Genetics , Metabolism , Sequence Homology, Amino Acid , Sf9 Cells , Static Electricity , Virion , Genetics , Metabolism , Virus AttachmentABSTRACT
This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.
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Objective To establish a method for rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin,and to study the possibility of using it in single radial immunodiffusion (SRID) assay for quantitative detection of antigen in H7N9 influenza vaccine.Methods Hemagglutinin proteins expressed in eukaryotic cells were used to immunize sheep.Serum samples were collected to detect antibody titers by ELISA and double immunodiffusion assay.Different concentrations of antiserum were used in SRID assay to get the optimized concentration.Results After 4 times of immunization,the antiserum titers achieved 1 ∶ 1 000 000 and 1 ∶ 32 as indicated by ELISA and double immunodiffusion assay,respectively.The antiserum could form a clear precipitation line in SRID assay.The detection of antigen in the range of 10 to 40 μg/ml showed good linearity in the standard curve.The antigen titers in six batches of H7N9 vaccine detected by this SRID assay were identical with those by SDS-PAGE assay.Conclusion The antiserum against H7N9 hemagglutinin for SRID assay was developed successfully,and could be used as a reagent for the quantitative detection of antigen in H7N9 influenza vaccine.
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The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS). Firstly, the molecular mass of deglycosylated protein was measured. Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications. Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with -80% occupancy; N103, N448 and N184 (-15% occupancy) were glycosylated with complex-type oligosaccharides. LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.
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Objective To study the possibility of using heterogeneous antiserum in single radial immunodiffusion (SRID) for quantitative detection of HA in H7N9 influenza vaccine product when H7N9-specific antiserum is not available in order to establish a testing method for the detection of H 7N9 antigen in any urgent situation.Methods Antisera specific for H7N1, H7N2, H7N3 and H7N7 were obtained from NIBSC and used for SRID assay .Amino acid sequences of hemagglutinins were comparatively analyzed be-tween H7N9 virus and other viruses used to prepare heterogeneous antiserum .The titers of antisera against H7N9 and their homogenous antigens were detected by double immunodiffusion method .Based on the results of homology analysis and cross-reaction, a suitable antiserum was selected and its applicability was further validated by the SRID assay using H7N9 antigen.Results Influenza A virus subtype H7N3 that used for preparation of 07/278 antiserum showed the highest HA homology with H7N9 (97.14%).The titer of 07/278 antiserum against H7N9 antigen was 1 ∶8 as detected by double immunodiffusion assay .The H7N9 anti-gen and the 07/278 antiserum could form a clear precipitation line in SRID assay .The detection of H7N9 antigen in the range of 10 to 40μg/ml showed a good linearity in the standard curve .Conclusion The 07/278 antiserum from NIBSC can be used as an alternative reagent for the quantitative detection of hemaggluti -nin in H7N9 influenza virus vaccine .
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The amino acid sequence of the fusion protein FP3 was measured by two types of LC-MS/MS and its primary structure was confirmed. After reduction and alkylation, the protein was digested with trypsin and glycosyl groups in glycopeptide were removed by PNGase F. The mixed peptides were separated by LC, then Q-TOF and Ion trap tandem mass spectrometry were used to measure b, y fragment ions of each peptide to analyze the amino acid sequence of fusion protein FP3. Seventy-six percent of full amino acid sequence of the fusion protein FP3 was measured by LC-ESI-Q-TOF with the remaining 24% completed by LC-ESI-Trap. As LC-MS and tandem mass spectrometry are rapid, sensitive, accurate to measure the protein amino acid sequence, they are important approach to structure analysis and identification of recombinant protein.
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Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.
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Objective To establish the universal antibody against all subtypes of the influenza A viral hemagglutinins(HA). Methods All HA sequences from influenza viruses in common domains were analyzed to search for the most conservative re,on of the HA gene. The peptides conjugated to carrier proteins were used to immunize rabbits in order to obtain high titer antibody. Results The most conservative region among all influenza viruses HA genes were 14 amino acids located at the N-terminal of HA2. The antibody titer reached to 1 : 80 000 after 4 injections of the coupled peptides. The achieved antibody was demonstrated by Western blot to bind HA proteins of influenza virus subtypes H1-H13 specifically. Conclusion The universal antibody has been successfully established by immunizing the rabbits with most conservative peptides of HA protein coupled to carrier protein.
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With two recipes,i.e.nodule disease treated with revised Maimengdong Decoction and Xiaoluo Pill,sub-acute thyroid inflammation of granuloma treated with Haizao Yuhu Decoction and Xiaoluo Pill,it makes clinical research to discuss the causa morbi,mechanism,differentiation thought and the experiences of removing toxin,stasis,swelling,sputum,and softening mass in treating such refractory diseases.
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Objective To evaluate the clinical efficacy of circumferential ablation at orifice of pulmonary veins with ultrasound energy in the cure of paroxysmal atrial fibrillation(PAF).Methods The study group included 5 patients with PAF who had significant symptom and were drug resistance,and ultrasound ablation was applied at the orifice of their pulmonary veins to get electrical isolation.Results Nineteen pulmonary veins got electrical isolation and among them 3 were combined with radiofrequency ablation.PAF recurred in one patients and atrial premature beats occurred in other two patients but all disappeared after being treated with amiodarone.During the ablation atrial perforation occurred in one patients and severity vague response in another patient.All patients reached a minimum of six months of follow up,and remained in sinus rhythm.Conclusions The circumferential ablation at orifice of pulmonary veins with ultrasound energy is effective in restoration and maintenance of sinus rhythm in patients with PAF.
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Objective To observe and compare the influences of radiofrequency catheter ablation (RFCA) and through-the-balloon ultrasound ablation (TTB-USA) on coagulable states of blood and evaluate their safety. Methods Eleven mongrel canines of either sex were divided into TTB-USA group (n=5) and RFCA group (n=6). We measured the expression of alphagranule membrane protein (GMP-140) on the surface of activated platelets by flow cytomety before and after cannulation, immediately after ablation, 30 minutes and 48 hours later, respectively. And at the same time,we also examined the change of tissue type plasminogen activatior (T-PA)? plasmingen activator inhibitor-1 (PAI-1). Results The expression of GMP-140 elevated before ablation, reached its peak value at hour 48, but the degree of uprising was lower in TTB-USA group than in RFCA group. Both t-PA and PAI-1 uprising just after ablation, restored to normal 48 hours later, t-PA reached its peak just after ablation, but PAI-1 was 30 minutes later and there was no difference in uprising degree between the two groups. Conclusion Both TTB-USA and RFCA could activate platelets and result in the change of blood coagulable states, but TTB-USA was slighter than RFCA, so TTB-USA seems to be more safely in ablating pulmonary vein orifice than RFCA in treatment of focal atrial fibrillation.