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@#Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein). One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen. Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and antimouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established. The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined. Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL. No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80% — 120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than15%. For the residual protein in GLP preparation,about 62% of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than0. 01% — 0. 1% in biological preparations.
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@#Objective To establish and verify a universal and stable potency test method in vitro for SARS-CoV-2 mRNA vaccine,so as to use it for the quality control of SARS-CoV-2 mRNA vaccine.Methods ELISA kits that could bind well to S protein of SARS-CoV-2 variants,as well as transfected cells,cell plating concentrations and doses for transfection were screened,and then a potency test method for SARS-CoV-2 mRNA vaccine in vitro was established and verified.Results An ELISA kit was found with good binding ability to S protein of each variant,and HEK293T cells were determined as the transfection cells,with the plating concentration of 2.5 × 10~5 cells/mL and the transfection dose of 4 μg/well in the 6-well plate.An universal and stable potency test method for SARS-CoV-2 mRNA vaccine in vitro was established.The verification results showed that the method met the quality control needs.Conclusion The established potency test method in vitro for SARS-CoV-2 mRNA vaccine has good relative accuracy,linearity,intermediate precision and range,and can be applied to the quality control of SARS-CoV-2 mRNA vaccines
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Objective To investigate the changes and clinical significance of serum tumor necrosis fac-tor-α(TNF-α),adiponectin(ADP)and their ratio in patients with depression.Methods A total of 40 patients with depression admitted to Shandong Daizhuang Hospital from January to December 2020 were selected as the observation group,and 26 healthy volunteers were selected as the control group.The observation group was treated with oral antidepressants for four weeks,and the severity of depressive symptoms was assessed by Hamilton depression scale(HAMD).The levels of serum TNF-α and ADP in the observation group before and after treatment and the control group were detected by ELISA,and the change of TNF-α/ADP was ana-lyzed.Results HAMD scores of the observation group were 25.5(21.0,30.0)before treatment and 2.0(1.0,4.0)after treatment,the difference was statistically significant(P<0.05).The levels of TNF-α and TNF-α/ADP in the observation group before treatment were higher than those in the control group,which af-ter treatment were also higher than those before treatment(P<0.05).The level of ADP in the observation group after treatment was lower than that before treatment and in the control group,the differences were sta-tistically significant(P<0.05).Conclusion The level of TNF-α/ADP in patients with depression is more ob-vious than TNF-α.
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OBJECTIVE To investigate the effect of SAMC on the killing function of CD8+T cells against nasopharyngeal carcinoma cells and its mechanism.METHODS HK-1 and C666-1 are divided into 0,25,50,and 100 mol/L SAMC group,Western blot was used to detcet PD-L1 expression in tumor cells.CD8+T cells were co-cultured with HK-1 and C666-1 cells in a ratio of 10∶1,and 0,25,50,and 100 μmol/L SAMC were added and detect the killing ability of CD8+T on HK-1 and C666-1.ELISA was used to detect INF-γ and TNF-α concentration.Construct a subcutaneous transplant tumor model of nasopharyngeal HK-1 cells,and divide it into a control group,CD8+T cell group,SAMC group,and SAMC+CD8+T cell group.The tumor volume was measured during treatment in each group of mice.Western blot was used to detect the expression of PD-L1 in the tumor tissue,ELISA was used to detect INF-γ and TNF-α concentration of mouse serum.RESULTS Compared to 0 μmol/L SAMC group,the expression of PD-L1 in 25,50,100μmol/L SAMC group were significantly downregulated(P<0.05).Compared to 0 μmol/L SAMC+CD8+T group,the INF-γ and TNF-α concentration were significantly increased in 25,50,100 μmol/L SAMC+CD8+T group,the lysis rates of HK-1 and C666-1 cells were significantly increased.Compared with the control group,the tumor volume and weight in the CD8+T cell group and SAMC+CD8+T cell group were significantly reduced(P<0.05),the concentration of INF-γ and TNF-α were significantly increased.Compared with the CD8+T cell group,the tumor volume and weight in the SAMC+CD8+T cell group mice significantly decreased(P<0.05),while the serum INF-γ and TNF-α concentration significantly increased(P<0.05),and the expression of PDL-1 in tumor tissue was significantly downregulated(P<0.01).CONCLUSION SAMC can promote the killing function of CD8+T cells by inhibiting the expression of PD-L1 in human nasopharyngeal carcinoma cells.
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Objective To investigate the effects and mechanisms of peimine(PME)on chronic obstructive pulmonary disease(COPD)in mice.Methods The mice were randomly divided into 4 groups(20 mice in each group),control group,PME group,chronic obstructive pulmonary disease group and treatment group.Animal models of COPD were induced in mice by lipopolysaccharide combined with smoke.The effects of PME on COPD model mice was analyzed by HE staining,transmission electron microscopy and the ratio of wet/dry weight of mouse lung tissue.The effects of PME on COPD model mice were analyzed by HE staining,transmission electron microscopy and the ratio of wet/dry weight of mouse lung tissue.The effects of PME on inflammatory factor tumor necrosis factor(TNF)-α,interleukin(IL)-6 and IL-1β in lung tissue were analyzed by ELISA and Western blotting.The effects of PME on oxidative stress in lung tissue were analyzed by dihydroethidium(DHE)staining and Western blotting.The effects of PME on nuclear factor kappa-B(NF-κB)pathway and nuclear factor erythroid 2-related factor 2(Nrf2)pathway were analyzed by protein immunoblotting.Results Compared with the COPD group,PME treatment could significantly improve the lung tissue injury and the number of inflammatory cells in mice,and the wet/dry weight ratio of lung tissue was significantly reduced.Compared with the control group,the levels of TNF-α,IL-6 and IL-1β in the alveolar lavage fluid of COPD mice significantly increased,and the level of TNF-α,IL-6 and IL-1β in the alveolar lavage fluid of mice after PME treatment was significantly reduced.In addition,compared with the control group,the protein expression of TNF-α,IL-6 and IL-1β in the lung tissue of COPD mice significantly increased,and the level of TNF-α,IL-6 and IL-1β in the lung tissue of COPD mice after PME treatment were significantly reduced.Immunohistochemistry and Western blotting showed that the level of superoxide dismutase 2(SOD2)protein in COPD group was significantly lower than that in control group,while PME treatment could improve the level of superoxide dismutase protein.The analysis of MDA content in lung tissue showed that compared with the COPD group,the production of MDA in lung tissue of COPD mice was significantly inhibited after PME treatment.Protein Western blotting showed that PME treatment could prevent the phosphorylation of inhibitor of NF-κB(IκBα)and the transfer of NF-κB p65 to the cell nucleus,and the expression of Nrf2 and its main downstream target heme oxygenase-1(HO-1)in the lung tissue of mice treated with PME significantly increased.Conclusion PME is able to inhibit inflammation and oxidative stress and improve lung tissues damage in the COPD model in vivo and this protection effect might be both the Nrf2 pathway activation and NF-κB pathway inhibition.
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In the quality control of Chinese medicine, the detection of active components and toxic and harmful components are two important links. Although conventional methods such as high performance liquid chromatography and liquid chromatography-mass spectrometry can accurately quantify the above substances, they have shortcomings such as complicated operation, high costs, inability of detection at any time, difficult detection of insoluble and macromolecular substances. Enzyme-linked immunosorbent assay (ELISA) can adsorb antigens or antibodies on the surface of solid carriers and realize qualitative or quantitative analysis of targets by using the specific reactions of antigens and antibodies. This method is praised for the simple operation, high sensitivity, strong specificity, simple requirements for experimental equipment, a wide application range, and low costs. In recent years, ELISA has been widely used in the quality control of Chinese medicine, especially in the content determination of mycotoxins represented by aflatoxin and the qualitative and quantitative analysis of active components. ELISA plays an increasingly important role with its unique advantages, providing new methods and ideas for the rapid quality examination of large quantities of Chinese medicines. This paper reviews the research progress in ELISA for the quality control of Chinese medicine in recent years and prospects its technical development and application prospects, aiming to provide reference and research ideas for further using this method to ensure the quality, safety, and controllability of Chinese medicine.
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@#Objective To establish and verify a universal and stable potency test method in vitro for SARS-CoV-2 mRNA vaccine,so as to use it for the quality control of SARS-CoV-2 mRNA vaccine.Methods ELISA kits that could bind well to S protein of SARS-CoV-2 variants,as well as transfected cells,cell plating concentrations and doses for transfection were screened,and then a potency test method for SARS-CoV-2 mRNA vaccine in vitro was established and verified.Results An ELISA kit was found with good binding ability to S protein of each variant,and HEK293T cells were determined as the transfection cells,with the plating concentration of 2.5 × 10~5 cells/mL and the transfection dose of 4 μg/well in the 6-well plate.An universal and stable potency test method for SARS-CoV-2 mRNA vaccine in vitro was established.The verification results showed that the method met the quality control needs.Conclusion The established potency test method in vitro for SARS-CoV-2 mRNA vaccine has good relative accuracy,linearity,intermediate precision and range,and can be applied to the quality control of SARS-CoV-2 mRNA vaccines.
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Background & objectives: The diagnosis of scrub typhus (ST) is usually done using enzyme-linked immunosorbent assay (ELISA) due to its ease of performance and reading objectivity. The cut-off value for ELISA needs to be calculated for each geographical location as it depends on zonal endemicity of the disease. This study was, therefore, undertaken to calculate the pan-India cut-off for anti-Orientia tsutsugamushi (OT) immunoglobulin M (IgM) by ELISA. Methods: Samples from cases (cases of ST) and controls (voluntary, consenting, healthy adults) were collected by a network of 29 laboratories across India and tested for anti-OT IgM by immunofluorescence assay (IFA), the considered gold standard test. These samples were retested by ELISA for anti-OT IgM and their optical densities (ODs) were used for cut-off estimation by receiver operating characteristic (ROC) curve. Results: Anti-OT IgM ELISA ODs from 273 controls and 136 cases were used for the cut-off estimation. The ODs of the anti-OT IgM ELISA on healthy individuals and those of confirmed ST cases ranged from 0.1 to 0.75 and 0.5 to 4.718, respectively. ROC curve-based cut-off for ELISA was calculated as 0.554 at a sensitivity of 95.2 per cent and specificity of 95.1 per cent. A value of >1 was noted to have a specificity of 100 per cent in diagnosing ST. Interpretation & conclusions: The cut-off calculated for India was similar to the previous cut-off that was used until now.
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ABSTRACT Serodiagnosis of strongyloidiasis is usually performed by ELISA for the detection of IgG antibodies due to its high sensitivity and practicality, but its main limitation is a constant source of S. stercoralis antigens. The use of S. venezuelensis as a heterologous source of antigens has facilitated several published studies on the serodiagnosis and epidemiology of human strongyloidiasis. The main objective of this study was to evaluate the diagnostic accuracy of surface cuticle antigens of infective larvae of S. venezuelensis extracted with CTAB detergent (L3-CTAB) in comparison with soluble somatic extracts (L3-SSE) using a panel of sera from immunocompetent and immunocompromised individuals, at three different cut-offs. ROC curve analysis showed that L3-CTAB had an AUC of 0.9926. At the first cut-off value (OD 450 nm = 0.214), sensitivity and specificity were 100% and 90.11%, respectively, with a diagnostic accuracy of 0.93. At a second cut-off value (OD 450 nm = 0.286), sensitivity and specificity were 70% and 100%, respectively, with a diagnostic accuracy of 0.91. However, at an alternative third cut-off value (OD 450 nm = 0.589), sensitivity and specificity were 95% and 97.8%, respectively, with a diagnostic accuracy of 0.97. Using L3-CTAB as an antigenic source, the seropositivity rate in immunocompromised patients was 28.13% (9/32) whereas a seropositivity rate of 34.38% (11/32) was found when L3-SSE was used in ELISA. Therefore, the L3-CTAB is simple and practical to obtain and was found to be highly sensitive and specific.
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Purpose: Remote ischemic preconditioning (RIPC) confers cardioprotection against ischemia reperfusion (IR) injury. However, the precise mechanisms involved in RIPC-induced cardioprotection are not fully explored. The present study was aimed to identify the role of melatonin in RIPC-induced late cardioprotective effects in rats and to explore the role of H2 S, TNF-α and mitoKATP in melatoninmediated effects in RIPC. Methods: Wistar rats were subjected to RIPC in which hind limb was subjected to four alternate cycles of ischemia and reperfusion of 5 min duration by using a neonatal blood pressure cuff. After 24 h of RIPC or ramelteon-induced pharmacological preconditioning, hearts were isolated and subjected to IR injury on the Langendorff apparatus. Results: RIPC and ramelteon preconditioning protected the hearts from IR injury and it was assessed by a decrease in LDH-1, cTnT and increase in left ventricular developed pressure (LVDP). RIPC increased the melatonin levels (in plasma), H2 S (in heart) and decreased TNF-α levels. The effects of RIPC were abolished in the presence of melatonin receptor blocker (luzindole), ganglionic blocker (hexamethonium) and mitochondrial KATP blocker (5-hydroxydecanoic acid). Conclusion: RIPC produce delayed cardioprotection against IR injury through the activation of neuronal pathway, which may increase the plasma melatonin levels to activate the cardioprotective signaling pathway involving the opening of mitochondrial KATP channels, decrease in TNF-α production and increase in H2 S levels. Ramelteon-induced pharmacological preconditioning may also activate the cardioprotective signaling pathway involving the opening of mitochondrial KATP channels, decrease in TNF-α production and increase in H2 S levels.
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Animals , Rats , Troponin/physiology , Cardiotonic Agents , Ischemic Preconditioning , Melatonin/analysis , Myocardial Infarction/veterinary , Enzyme-Linked Immunosorbent Assay/veterinary , Rats, Wistar/physiology , MitochondriaABSTRACT
Objetivo: la sensibilidad subóptima de las pruebas coproparasitológicas dificulta el diagnóstico de la estrongiloidiasis. Los métodos serológicos son más sensibles, pero los estudios en pacientes inmunodeprimidos son escasos. El objetivo del estudio fue de evaluar la sensibilidad de una prueba ELISA comercial en pacientes inmunodeprimidos. Métodos: se realizó en Bolivia un estudio multicéntrico en pacientes con cáncer, VIH, enfermedades reumatológicas y hematológicas. 88 pacientes con larvas de S.stercoralis en heces identificadas mediante técnicas coproparasitológicas tuvieron una prueba serológica ELISA (Bordier Affinity Products). Resultados: la sensibilidad de la técnica ELISA fue de 77,3% (61/88) (CI95%: 67,7-85,1). La sensibilidad de este test serológico fue identificada más baja en pacientes HIV+ con CD4300 o una serología VIH desconocida (84,2%) (p=0,035). Conclusiones: la sensibilidad del ELISA es inversamente proporcional al grado de inmunosupresión. Este resultado refuerza la recomendación de diagnosticar la estrongiloidiasis mediante una combinación de técnicas serológicas y coproparasitológicas.
Objectives: the sensitivity of coproparasitological tests for the diagnosis of strongyloidiasis are suboptimal. Serological methods are more sensitive, but studies among immunocompromised patients are scarce. The aim of this study was to evaluate the sensitivity of a commercial ELISA test among immunocompromised patients. Methods: a multicenter study was conducted in Bolivia among patients with cancer, HIV, rheumatologic or hematologic diseases. 88 patients with S. stercoralis larvae in stool identified by coproparasitological techniques had an ELISA serological test (Bordier Affinity Products). Results: the sensitivity of the ELISA technique was 77,3% (61/88) (CI95%: 67,7-85,1), and was identified lower among HIV+ patients with CD4300 or unknown HIV serology (84,2%) (p=0,035). Conclusions: the sensitivity of ELISA is inversely proportional to the degree of immunosuppression. This result reinforces the recommendation to diagnose strongyloidiasis by a combination of serological and coproparasitological techniques.
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ObjectiveTo investigate the mechanism of Magnolia officinalis cortex for constipation-type irritable bowel syndrome(IBS-C) rats before and after sweating. MethodIBS-C rat model was established by gavage of ice water, and rats were randomly divided into the blank group, model group, mosapride group(1 mg·kg-1), M. officinalis cortex group(10 g·kg-1) and sweated M. officinalis cortex group(10 g·kg-1). The changes of body weight, fecal number and fecal water content of rats were observed, 16S rRNA sequencing was used to detect the structural changes of fecal intestinal flora in rats, the levels of 5-hydroxytryptamine(5-HT) and substance P(SP) in colonic tissues of rats were determined by enzyme-linked immunosorbent assay(ELISA). ResultCompared with the model group, the fecal water content and fecal number of mosapride group, M. officinalis cortex group and sweated M. officinalis cortex group were significantly increased(P<0.05). At the phylum level, the top four species of flora abundance were Firmicutes, Bacteroidetes, Spirochaetes and Proteobacteria. Compared with the blank group, the proportion of Firmicutes in the model group was significantly reduced(P<0.05), while the proportion of Spirochaetes was significantly increased(P<0.05). Compared with the model group, the proportion of Firmicutes and Spirochaetes in M. officinalis cortex group and sweated M. officinalis cortex group tended to be similar to that in the blank group, and the proportion of Spirochaetes in sweated M. officinalis cortex group was lower than that of M. officinalis cortex group. At the family level, the top four species of flora abundance were Lactobacillaceae, S24_7, Ruminococcaceae, Bacteroidaceae, compared with the blank group, the proportion of Lactobacillaceae in the model group decreased significantly(P<0.05), and its proportion in the M. officinalis cortex group and sweated M. officinalis cortex group increased significantly after administration(P<0.05), and the flora structure of the two groups tended to be similar to that of the blank group. At the genus level, the top four species of flora abundance were Lactobacillus, Unspecified_S24_7, Bacteroides and Treponema. Compared with the blank group, the proportion of Lactobacillus in the model group decreased significantly(P<0.05), while the proportion of Treponema increased significantly(P<0.05). Compared with the model group, ratio of bacterial structure of Lactobacillus and Treponema in the M. officinalis cortex group and sweated M. officinalis cortex group tended to be similar to those in the blank group, indicating that M. officinalis cortex could restore the intestinal microbial structure of IBS-C rats before and after sweating. Compared with the model group, the 5-HT content in mosapride group was significantly reduced(P<0.05), the contents of 5-HT and SP in the M. officinalis cortex group and sweated M. officinalis cortex group were significantly increased(P<0.01), and the sweated M. officinalis cortex group was higher than the M. officinalis cortex group. ConclusionM. officinalis cortex can play a therapeutic role on IBS-C rats by regulating 5-HT pathway and intestinal flora structure before and after sweating.
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@#ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap2=0.995 6,P=0.000 1)。结论 建立的双抗体夹心ELISA法具有良好的准确性、精密性和特异性,可用于VZV疫苗中g E抗原含量的快速检测。 ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap(TM)Mabselect(TM)Mabselect(TM)Su Re and Hi Trap(TM)Su Re and Hi Trap(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1~14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of10(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1~14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of106~106~107with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95~1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)7with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95~1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)(1.49)]+3.99,and R(1.49)]+3.99,and R2value was 0.999.The recoveries of accuracy verification were 94.9%~114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2value was 0.999.The recoveries of accuracy verification were 94.9%~114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2=0.995 6,P=0.000 1).ConclusionThe developed double antibody sandwich ELISA had good accuracy,precision and specificity,which might be used for rapid detection of gE antigen content in VZV vaccine.
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Objective To explore the correlation between childhood trauma and plasma adiponectin levels in patients with depression.Methods A total of 121 patients with depression and 39 healthy controls(control group)were enrolled.Childhood trauma questionnaire(CTQ-SF)was used to assess the experience of childhood abuse and neglect,and the patients with depression were divided into trauma group(n=53)and non-trauma group(n=68)according to the CTQ-SF score.The 17-item Hamilton depression scale-17(HAMD17)and the Hamilton anxiety scale(HAMA)were used to evaluate the severity of depression and anxiety symptoms,respectively.Plasma adiponectin levels of subjects were measured by enzyme-linked immunosorbent assay.Results The plasma adiponectin level of trauma group[3.82(2.44,4.92)μg/mL]was significantly lower than that of non-trauma group[4.64(2.98,6.43)μg/mL,P=0.01]and the control group[6.29(4.54,7.51)μg/mL,P<0.01].The plasma adiponectin level of non-trauma group was lower than that of the control group(P<0.01).Correlation analysis showed that plasma adiponectin level in patients with depression was negatively correlated with childhood trauma(r=-0.34,P<0.01).Multivariate linear regression analysis showed that plasma adiponectin level was negatively correlated with childhood trauma scores in patients with depression(β=-0.05,P<0.01).Conclusions Patients with depression who have experienced childhood trauma have lower plasma levels of adiponectin,and childhood trauma may be associated with decreased plasma adiponectin levels in patients with depression.
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Zearalenone(ZEN) is a toxic metabolite produced by Fusarium culmorum, F. graminearum, F. tricinctum, and other fungi, with estrogenic characteristics. Exposure to or ingestion of ZEN during pregnancy can cause reproductive dysfunction, miscarriage, stillbirth, and malformation, and seriously endanger human life and health. The detection methods for ZEN in the Chinese Pharmacopoeia(2020 edition) are liquid chromatography(LC) and liquid chromatography-mass spectrometry(LC-MS), and it is stipulated that ZEN should not exceed 500 μg in 1 000 g of Coicis Semen. Although these detection methods by instruments can achieve the qualitative and quantitative analysis of ZEN in Coicis Semen, their high detection cost and long periods hinder the rapid screening of a large number of samples in the field. In this study, the synthesized ZEN hapten was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) to obtain the complete ZEN antigen. By virtue of antibody preparation techniques, ZEN monoclonal antibody 4F6 was prepared, which showed 177.5%, 137.1%, and 109.7% cross-reactivity with ZEN structural analogs zearalanol, zearalenone, and α-zearalenol, respectively, and no cross-reactivity with other fungal toxins such as aflatoxin. Direct competitive enzyme-linked immunosorbent assay(dcELISA) based on ZEN monoclonal antibody 4F6 was developed for the determination of ZEN in Coicis Semen with an IC_(50) of 1.3 μg·L~(-1) and a detection range of 0.22-21.92 μg·L~(-1). The recoveries were 83.91%-105.3% and the RSD was 4.4%-8.0%. The established dcELISA method was used to determine the ZEN residuals in nine batches of Coicis Semen samples, and the results were validated by LC-MS. The correlation between the two detection methods was found to be 0.993 9, indicating that the established dcELISA could be used for the rapid qualitative and quantitative detection of ZEN residuals in Coicis Semen.
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Humans , Female , Pregnancy , Zearalenone , Coix , Enzyme-Linked Immunosorbent Assay , Mycotoxins , Antibodies, MonoclonalABSTRACT
@#Objective To prepare the second generation internal control reference(B2)for Ig G antibody against severe acute respiratory symptom coronavirus 2(SARS-CoV-2)and evaluate its applicability in ELISA detection method. Methods Among the volunteers vaccinated with SARS-CoV-2 inactivated vaccine(BBIBP-Cor V)produced by Beijing Institute of Biological Products Co.,Ltd.,19 Ig G antibody positive plasma samples with ELISA-Ig G dilution ratio of 20 ~ 60 were screened,and the Ig G antibody,IgM antibody and neutralizing antibody were detected by ELISA,B2 was prepared from nonlipid plasma with ELISA-Ig G dilution ratio of 32 ~ 45,IgM negative and similar neutralizing antibody inhibition rate. The neutralizing antibody potency of the first generation internal control reference(B1)and B2 detected by ELISA was calibrated with the first generation WHO international standard of anti-SARS-CoV-2 immunoglobulin(NIBSC 20/136),and the accelerated stability(storage at 2 ~ 8 ℃ for 5,8,14,20,and 30 d respectively),the service stability(storage at 18 ~25 ℃ for 1,2,and 3 h respectively),the freeze-thaw stability(1,2 and 3 times)and the long-term stability(storage at-25 ℃ for10 months)of B2 were tested. B2 was used as standard to detect plasma after single vaccine immunization and mixed plasma was prepared according to different ELISA-Ig G dilution ratio. The correlation and linear regression analysis between ELISA-Ig G dilution ratio and neutralizing antibody potency of pseudovirus in mixed plasma were carried out. Results Among 19 plasma samples,5 samples were non-lipid plasma with ELISA-Ig G dilution ratio of 32 ~ 45,IgM negative and similar neutralizing antibody inhibition rate. B2 was prepared by mixing every plasma in equal volume fraction,and the dilution ratio of ELISA-Ig G was assigned to 32. The neutralizing antibody potency of B1 calibrated with NIBSC 20/136 was 133. 38 EIU/m L and that of B2 was 122. 14 EIU/m L. The recovery rates of accelerated stability,service stability,freeze-thaw stability and long-term stability of B2 were all in the range of(100 ± 15)%. The ELISA-Ig G dilution ratio of the mixed plasma from the same source was significantly correlated with the neutralizing antibody potency of pseudovirus.(each R~2> 0. 99,each P < 0. 000 1).Conclusion B2 prepared from plasma immunized with SARS-CoV-2 inactivated vaccine can replace B1 prepared from plasma of COVID-19 convalescent patients.
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@#Objective To develop and verify a sandwich ELISA method with bovine polyclonal antibody against rabbit polyclonal antibody for the determination of D antigen content of Sabin strain inactivated poliovirus vaccine(sIPV).Methods The rabbit polyclonal antibodies were prepared with sIPV vaccine bulks of type Ⅰ,Ⅱ and Ⅲ as antigens and detected for the titer and specificity by indirect ELISA.A double antibody sandwich ELISA with bovine polyclonal antibody as coating antibody and rabbit polyclonal antibody as detection antibody was developed to determine D antigen content,and the accuracy,precision and specificity to D antigen of the method were verified.sIPV vaccine samples from five domestic enterprises were detected by the developed method.Results The rabbit polyclonal antibodies for type Ⅰ,Ⅱ and Ⅲ sIPV with good specificity and high titer were prepared,and the double antibody sandwich ELISA method was successfully developed.Using four-parameter fitting,all three standard curves showed good linear relationship,and R~2 values were more than 0.99.The spike recoveries of type Ⅰ,Ⅱ and Ⅲ D antigens were all within 80%~120%,with average values of98.11%,97.41% and 98.66%,respectively.The CVs of repeatability and intermediate precision were all below 10%.The method also distinguished D antigens from C antigens.The developed method determined the D antigen contents of sIPV vaccines from five enterprises.Conclusion A sandwich ELISA method for determination of D antigen content in sIPV vaccine was successfully developed with satisfying accuracy,precision and certain D antigen specificity,which can be used to detect vaccines produced by different manufacturers.
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Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.
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【Objective】 To establish an enzyme-linked immunosorbent assay (ELISA) method for the determination of residual human coagulation factor Ⅺ in human prothrombin complex and validate the method. 【Methods】 Human factor Ⅺ was reacted with the capture antibody coated on the microtiter plate. After appropriate washing steps, biotinylated primary antibody was bound to the captured protein. Excess primary antibody was washed away and bound antibody was reacted with horseradish peroxidase conjugated streptavidin. TMB substrate was used for color development at 450 nm. The dilution reliability, accuracy, specificity, repeatability, intermediate precision, linearity, range and durability were verified. 【Results】 The verification results showed that the accuracy and specificity of this method met the experimental requirements, with an average recovery rate of 109.2% and RSD of 6.93%. The repeatability RSD was 6.78%, and the intermediate precision RSD was 6.75%, indicating good precision. The linear regression correlation coefficient of standard curve was 0.999 9, showing good accuracy and precision within the linear range. The durability was verified by the incubation time and the validity period of reagent kit opening. The results showed that the RSD of the incubation time change was 6.62%, indicating that the incubation time of this detection method was controlled between 28 to 32 minutes, and there was no significant impact on the results. The RSD of the detection results before and after the reagent kit was opened and stored under conditions for 7 days was 3.84%, indicating that the preservation of the reagent kit according to the conditions for 7 days after opening has no effect on the FⅪ detection results. Both indicated that the method had good durability. The dilution reliability results showed that there was a "hook" effect in the detection of FⅪ residue in human prothrombin complex, which could be solved by diluting 100 to 200 times. 【Conclusion】 This method can be used for the determination of FⅪ residues of human prothrombin complex in laboratory.
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【Objective】 To evaluate the effectiveness of random quality control sampling in blood sample detetion by ELISA. 【Methods】 Blood samples of 5 mL specification of blood donors from our blood station from May to July 2022 were selected for routine operation on a fully automated sampler. J standard substances(3 mL specification) as daily samples were added to A1 well, H12 well and random wells of HBsAg, anti-HCV, anti-HIV, and -TP, and then placed in a fully automated enzyme immunoassay analyzer for testing. With random well quality control as the internal quality control judgment standard, 20 consecutive tests were conducted and were divided into A1 (well) group, H12 (well) group and random (well) group according to different well positions. Quality control maps were drawn using Levey-Jennings quality control chart with random group as the framework, and were compared with the quality control map of A1 well and H12 well results in the same day. 【Results】 The mean quality control levels of infectious indicators of blood transfusion in blood donors by ELISA were: HBsAg 3.87±0.28, anti-HCV 3.79±0.38, anti-HIV 3.64±0.30 and anti-TP 4.53±0.51. 【Comparison】 of HBsAg, anti-HCV, anti-HIV and anti-TP, between random group, A1 group and H12 group were HBsAg 3.87± 0.28 vs 4.09±0.30 vs 3.64±0.26, anti-HCV 3.78±0.37 vs 3.96±0.38 vs 3.63±0.38, anti-HIV 3.63±0.31 vs 3.82±0.32 vs 3.48±0.28 and anti-TP 4.51±0.51 vs 4.71±0.52 vs 4.36±0.51, The S/CO value of each indicator were H12 group<random group<Al group (P<0.05), and the mean quality control levels of random group were similar to each detection indicator (P>0.05) . Using random group as the quality control framework standard, 5 points in group A1 fell outside of +2s, and 1 point in group H12 fell outside of -2s, resulting in a total of 6 alarms. With the quality control substance placed in A1 well of the ELISA plate, the judgment of detection results of the entire ELISA plate could be inevitably affected, especially the last row of low concentration virus marker samples on the ELISA plate. 【Conclusion】 The application of random quality control sampling method in donor blood by ELISA is scientific and reasonable, which can reduce the systematic error caused by artificial setting of ELISA plate fixed well positions and can also discover edge effects that affect the detection results.