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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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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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@#Objective To compare three methods for detection of antibody level in serum immunized with SARS-CoV-2mRNA vaccine. Methods Enzyme-linked immunosorbent assay(ELISA),pseudo virus-based neutralization assay(PBNA)and micro-cytopathic effect neutralization test(MCPENT)were used to detect the antibody levels of a total of 120 serum samples(40 before immunization and 80 after immunization)before and after 2 doses of mRNA vaccine immunization,and the consistency and correlation of the three methods were analyzed. Results The consistency rates of the three methods detecting 120 serum samples were all over 90%,the Kappa coefficients were all more than 0. 7,and each P was less than0. 01. The correlation coefficient(r)between the antibody potency results of positive serum samples detected by the three methods was 0. 825～0. 902,and each P was less than 0. 01. Conclusion The three methods have good consistency and correlation in detecting antibody level of serum immunized with SARS-CoV-2 mRNA vaccine.
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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.
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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 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 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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Objetivo: Evaluar la validez diagnóstica del ensayo de inmunoabsorción ligado a enzima (Enzyme-Linked Immunosorbent Assay, ELISA) para el virus de inmunodeficiencia humana (VIH) en bancos de sangre, con base en estudios publicados entre 2000 y 2020. Metodología: Se realizó una revisión sistemática y metaanálisis de pruebas diagnósticas, mediante un modelo de efectos aleatorios para la sensibilidad, la especificidad, el cociente de probabilidad positivo y negativo, la razón de odds (OR) diagnóstica y la curva ROC, con sus intervalos de confianza del 95 %. La heterogeneidad se evaluó con el estadístico Q(χ2) DerSimonian-Laird y el I2 de inconsistencia, y la incertidumbre, con el porcentaje de peso de cada estudio. Resultados: Se incluyeron 15 investigaciones; la ELISA de tercera generación (detección de anticuerpos) se aplicó en 2992 infectados y 4076 sanos; las de cuarta generación (determinación simultánea de antígeno-anticuerpo), en 967 infectados y 154 264 sanos; ambas presentaron sensibilidad cercana al 100 %, pero la especificidad fue mejor en los ensayos de cuarta generación (98 vs. 100 %). Para ambas tecnologías, los cocientes de probabilidad, OR diagnóstica y curva ROC evidenciaron excelente discriminación de sanos e infectados. Conclusión: Se confirmó que las ELISA de tercera y cuarta generación presentan excelente validez y utilidad diagnóstica en donantes de sangre, lo que es importante para las políticas de sangre segura y control del VIH.
Objective: To evaluate the diagnostic validity of the enzyme-linked immunosorbent assay (ELISA) for human immunodeficiency virus (HIV) in blood banks, based on studies published between 2000 and 2020. Methodology: We performed a systematic review and meta-analysis of diagnostic tests, using a random-effects model for the sensitivity, specificity, positive and negative likelihood ratio, diagnostic odds ratio (DOR), and ROC curve, with 95% confidence intervals. Heterogeneity was assessed with the DerSimonianLaird Q(χ2) statistic and the I2 inconsistency statistic. Uncertainty was assessed using percentage study weights. Results: 15 studies were included. The third-generation ELISA (antibody detection) was applied for 2,992 infected and 4,076 healthy individuals, and the fourth-generation ELISA (simultaneous antigen-antibody detection) was used for 967 infected and 154,264 healthy individuals. Both showed close to 100% sensitivity, but there was an improved specificity in the fourth-generation assays (98% vs. 100%). Both technologies' likelihood ratios, DOR, and ROC curve aptly distinguished between healthy and infected individuals. Conclusion: The third and fourth-generation ELISA were confirmed to have excellent validity and diagnostic utility in blood donors, which is important for HIV control and blood safety policies.
Objetivo: Avaliar a validade diagnóstica do ensaio de imunoabsorção ligado à enzima (Enzyme-Linked Immunosorbent Assay, ELISA) para o vírus de imunodeficiência humana (VIH) em bancos de sangue, com base em estudos publicados entre 2000 e 2020. Medotologia: Realizou-se uma revisão sistemática e meta-análise de provas diagnósticas, por meio de um modelo de efeitos aleatórios para a sensibilidade, a especificidade, o cociente de probabilidade positivo e negativo, a razão de odds (OR) diagnóstica e a curva de ROC, com seus intervalos de confiança do 95%. A heterogeneidade foi avaliada com o estatístico Q(χ2) DerSimonian-Laird e o I2 de inconsistência, e a incerteza, com a porcentagem de peso de cada estudo. Resultados: Foram incluídas 15 pesquisas; a ELISA de terceira geração (detecção de anticorpos) aplicouse em 2992 infetados e 4076 sadios; as de quarta geração (determinação simultânea de antígeno-anticorpos), em 967 infetados e sadios; ambas as duas apresentaram sensibilidade próxima ao 100%, mas a especificidade foi melhor nos ensaios de quarta geração (98 vs. 100%). Para ambas as tecnologias, os cocientes de probabilidade, OR diagnóstica e curva ROC evidenciaram excelente discriminação de sadios e infetados. Conclusão: Confirmou-se que as ELISA de terceira e quarta geração apresentam excelente validade e utilidade diagnóstica em doadores de sangue, o que é importante para as políticas de sangue seguro e controle do VIH.
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ObjectiveTo compare the effect of different solvent extracts of spore powder and fruiting body of Lasiosphaera Calvatia on fibroblasts and wound healing of full-thickness skin defect， in order to screen the optimal medication part of Lasiosphaera Calvatia. MethodThe effect of water extract and alcohol extract of spore powder and fruiting body on cell proliferation and cell migration of mouse skin fibroblasts （MSF） were examined in vitro. Cell proliferation and activity test （CCK-8） method was used for cell proliferation， scratch assay was used for cell migration， flow cytometry was conducted to explore cell cycle， enzyme-linked immunosorbent assay （ELISA） was used to determine the production of collagen Ⅰ and Ⅲ. At the same time， a full-thickness skin defect wound model was established to investigate the therapeutic effect of different solvent extracts of spore powder. Ultraviolet-visible spectrophotometry was used to measure the contents of index components in different solvent extracts. ResultThe water extract of spore powder and fruiting body had certain cytotoxicity， while the alcohol extract could promote proliferation， migration and production of collagen Ⅰ and Ⅲ of MSF， and the effect of spore powder was significantly higher than that of fruiting body. When the concentration was 10 mg·L-1， the cell proliferation rate of alcohol extract of spore powder was as high as （159.22±15.95）%， and could promote MSF from the G0/G1 phase to S phase and G2/M phase with an increased proliferation index. The alcohol extract also promoted the migration of fibroblasts， secreted collagen Ⅰ and Ⅲ. On in vivo model， the alcohol extract of spore powder significantly accelerated wound healing on mice， effectively promoted the complete epithelialization of wound tissue， and generated new collagen fiber. The results of determination showed that the contents of polyphenols and flavonoids in the alcohol extract were higher than the alcohol extract of fruiting body. ConclusionThe alcohol extract of spore powder in Lasiosphaera Calvatia has active components in the treatment of wounds with good development prospect， and the medicinal components may be polyphenols and flavonoids.
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【Objective】 To explore the viability of classification management of HIV reactive blood donors based on test results in blood screening laboratory. 【Methods】 According to the HIV test results of blood donors (including twice ELISA and once NAT), the HIV reactive blood donors were divided into three groups. Group 1 was all-test reactive (both ELISA and NAT were reactive), group 2 serological reactive (only ELISA was reactive), and group 3 NAT reactive (only NAT was reactive). The HIV test results of 191 628 blood donors from May to December 2017 were analyzed. Samples with positive RIBA results and / or the repeated reactive NAT results were determined as HIV true positive. The yielding rates of HIV true positivity in each group were analyzed. Receiver operating characteristic curve (ROC curve) was used to elevate the S/CO limit under 99% specificity as the blood donor deferral limit for ELISA. 【Results】 A total of 180 HIV reactive samples were detected out of 191 628 blood donors, including 77 positive cases in group 1, 100 in group 2 and 3 in group 3. 1) The HIV reactive results were diverse. Among the 82 true positive blood donors, 4 were early HIV infection (3 HIV antibody+ antigen window period yield, 1 HIV antibody window period yield), 2 were suspected elite controllers, and 76 cases were both serology and NAT reactive. 2) The overall yielding rate of HIV was 47.67%, with group 1 (100%) = group 3 (100%) > group 2 (2.17%), showing statistically significant (P0.05). All true positive blood donors in group 1 and group 2 could be accurately screened by using the blood donor deferral limit for ELISA1 and ELISA2 simultaneously. 【Conclusion】 The composition of HIV results among blood donors is diverse and complex. It is necessary to continuously improve the awareness of HIV prevention and control. The classification of HIV reactive blood donors is conducive to conduct fine and scientific management. The blood donors in group 1 and group 3 should be permanently deferral, and the suspected HIV elite controllers in group 2 should be paid attention to and permanently deferral.
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Objective:To explore effects of different extracts and monomers of <italic>Lepidium meyenii </italic>（Maca） on the proliferation of mouse splenic lymphocytes and induction of interleukin-2 （IL-2） and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） by observing their immunomodulatory effects. Method:An octadecylsilyl （ODS） column was used to enrich the methanol extract of <italic>L. meyenii</italic> in stages to obtain six fractions and three monomers. Different groups of extracts and monomers of <italic>L. meyenii </italic>at different doses were set up. Cell counting Kit-8 （CCK-8） was used to detect the effect on the proliferation of mitogen-free， concanavalin A （Con A）-induced， and lipopolysaccharides （LPS）-induced mouse splenic lymphocytes. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of IL-2 and TNF-<italic>α</italic>. Result:<italic>L. meyenii </italic>extracts Fr<sub>3</sub> and Fr<sub>6</sub>， and monomers <italic>N</italic>-benzyl hexadecanamide and 1，2-dihydro-4-carboxaldehyde-3-benzyl-<italic>N</italic>-hydroxypyridine slightly promoted the proliferation of Con A-induced T lymphocytes and LPS-induced B lymphocytes （<italic>P</italic><0.01） as compared with the conditions in the model group. <italic>L. meyenii</italic> extracts and monomers significantly induced the secretion of IL-2 and TNF-<italic>α</italic> by splenic lymphocytes （<italic>P</italic><0.01）. Conclusion:<italic>L. meyenii</italic> extracts and monomers can achieve immunological enhancement by promoting the secretion of IL-2 and TNF-<italic>α</italic>， and facilitate the proliferation of splenic lymphocytes. The active components are presumedly macamides and pyridine alkaloids， and the specific mechanism still needs to be further explored.
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Introducción: En la supervivencia del corazón trasplantado son de importancia el empleo de los anticuerpos contra el sistema principal de histocompatibilidad (anticuerpos anti-HLA). Hace seis años se introdujo en Cuba el porcentaje de anticuerpos anti-HLA frente a panel (PRA) por método de ensayo de inmunoabsorción ligado a enzima (ELISA) como parte de las pruebas de compatibilidad pretrasplante de los receptores de trasplante cardiaco. Objetivo: Caracterizar los anticuerpos anti-HLA en pacientes receptores cubanos de trasplante cardiaco. Métodos: Entre septiembre de 2013 y abril de 2017 se les realizó el PRA por ELISA a 38 muestras de pacientes recibidas en el laboratorio de histocompatibilidad del Instituto de Hematología e Inmunología. Se utilizó la comparación de proporciones para el análisis estadístico. Resultados: El 47,4 por ciento de los pacientes estudiados presentó anticuerpos anti-HLA, fueron los más frecuentes los de clase I. La proporción de pacientes con PRA del 0 por ciento fue mayor en PRA clase II que en I (p: 0,0027). Mientras que fue mayor la proporción de pacientes con PRA clase I entre el 20 y el 75 por ciento (p: 0,0046). El 77,8 por ciento de los pacientes tuvo un PRA clase I mayor al 10 por ciento y en el PRA clase II alcanzó el 80 por ciento. Conclusiones: El porcentaje de anticuerpos anti-HLA frente a panel por método de ensayo de inmunoabsorción ligado a enzima permitió una mejor caracterización de los anticuerpos anti-HLA, lo que contribuyó a mejorar la compatibilidad en este tipo de paciente(AU)
Introduction: In survival after heart transplantation, the use of antibodies against the main histocompatibility system (anti-HLA antibodies) is important. Six years ago, the percentage of anti-HLA antibodies against panel (PRA) by enzyme-linked immunosorbent assay (ELISA) method was introduced in Cuba as part of the pre-transplant compatibility tests of heart transplant recipients. Objective: To characterize anti-HLA antibodies in Cuban heart transplant recipients. Methods: Between September 2013 and April 2017, PRA by ELISA was performed on 38 patient samples received in the histocompatibility laboratory of the Institute of Hematology and Immunology. Comparison of proportions was used for statistical analysis. Results: 47.4 percent of the study patients presented anti-HLA antibodies; those in class were the most frequent. The proportion of patients with PRA of 0 percent was higher in PRA class II than in class I (p=0.0027). The proportion of patients with PRA class I was greater, accounting for 20-75 percent (p=0.0046). 77.8 percent of the patients had a class I PRA greater than 10 percent, while in class II PRA it reached 80 percent. Conclusions: The percentage of anti-HLA antibodies versus a panel of enzyme linked immunosorbent assay method allowed better characterization of anti-HLA antibodies, which contributed to improving compatibility in this type of patient(AU)
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Humans , Male , Female , Heart Transplantation/methods , Transplant Recipients , Antibodies/therapeutic use , Enzyme-Linked Immunosorbent Assay/methods , Survival Analysis , CubaABSTRACT
Toxoplasmosis is a neglected tropical disease with a global distribution that is estimated to infect one third of the world’s human population. This study was a comparison of ELISA and rapid Immunochromatographic tests (ICT) in diagnosis of toxoplasmosis in Port Harcourt Nigeria. Eight hundred patients grouped in four categories from three Health Care Centres were randomly sampled after due ethical approval was obtained. Samples were analysed using Toxo IgG-IgM rapid test (ICT) and Enzyme linked Immunosorbent Assay (ELISA) technique. Socio Demo graphic Data were obtained using well-structured questionnaires. The seroprevalence of toxoplasmosis based on ICT was 28.1% while that of ELISA was 34.5% both significant (P < 0.05) with a relative risk of 0.815. The diagnostic parameters of ICT versus ELISA IgG were sensitively 46.7% specificity 81.7% positive predictive value (PPV) 57.3%, Negative predictive value (NPV) 74.4with a diagnostic efficiencyof 69.6%Cohen Kappas indicate good to moderate agreement between the two tests for detecting IgG. Although ELISA is the gold standard for diagnosing toxoplasmosis,ICT being less expensive, faster with high specificity and good diagnostic efficiency indetecting IgG is recommended as a preliminary screening tool for diagnosing toxoplasmosis in remote areas and facilities because ELISAislaborious, expensive and not readily available
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Objective To investigate the antibody levels against diphtheria among healthy population in Jiading District of Shanghai, and to make evaluation of the effect of diphtheria toxoid vaccination strategy. Methods Serum samples were collected from healthy population in Jiading District of Shanghai in 2010-2013.Antibody against diphtheria was tested by the enzyme-linked immunosorbent assay (ELISA).Geometric mean titer (GMT) of diphtheria antibody was compared between different population characteristics. Results A total of 1 029 serum samples were collected from the healthy population.With 1 003 of 1 029 diphtheria antibody titer of IgG (over 0.01 IU/mL), the positive rate was 97.47%, reaching the level of security protection (over 0.1 IU/mL) that accounted for 70.36%, and achieving the long-term protection level (over 1 IU/mL) that accounted for 35.57%;IgG GMT 0.325 IU/mL.There was significant difference in the positive rates and antibody levels between city residents and floating population in the field of diphtheria antibodies (χ2=4.708, P < 0.05;t=2.787, P < 0.05).There were statistically significant differences between different age groups in diphtheria antibody positive rates (χ2=30.262, P < 0.001) and statistically significant differences in GMT diphtheria antibody levels between different age groups (F=99.500, P < 0.001), GMT ranged in 0.029 IU/mL (30 years age group)~1.526 IU/mL (5 years age group). Conclusion The investigation of diphtheria antibody levels in Shanghai Jiading District truly reflects the effect of routine diphtheria immunization in Jiading District.DT (diphtheria & tetanus) vaccination for third grade junior is effective.We suggest a booster dose of diphtheria-containing vaccine for adolescents or adults so as to maintain immune levels.
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Objective : To investigate the expression and clinical significance of α1-antitrypsin (α1-AT) in tears of thyroid-associated ophthal-mopathy (TAO) patients. Methods ¡¤ Patients diagnosed with TAO at Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine from January to December 2019 were included and divided into active TAO group (n=33) and inactive TAO group (n=22). Thirty eyes of 30 healthy volunteers were included (n=30). Unstimulated tear samples were collected from all subjects and the concentration of α1-AT was analyzed by enzyme-linked immunosorbent assay (ELISA). Difference of α1-AT concentration among 3 groups was compared by using Kruskal-Wallis H test. Mann-Whitney U test was performed for further comparison between each 2 groups. Correlationship between α1-AT con-centration and clinical activity score (CAS) was analyzed by Spearman relation. Statistical significance was accepted at a value of P<0.05. Re-sults ¡¤ α1-AT was significantly higher in active TAO group than that in inactive TAO group and control group (both P<0.05). There was no significant difference in α1-AT between inactive TAO group and control group. Tear α1-AT level was significantly correlated with CAS (r=0.846, P=0.000). ROC curve showed that the optimal diagnostic cut off value of α1-AT was 939.48 ng/mL, and the sensitivity and specificity were 81.8% and 100% (AUC=0.959, P<0.05). Conclusion ¡¤ α1-AT is significantly elevated in the tears of active TAO patients. Tear α1-AT level has important value in the diagnosis of clinical activity of TAO.
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Objective@#To establish an indirect enzyme-linked immunosorbent assay (ELISA)and to compare the efficiency of receptor binding domain (RBD) proteins in different forms for Middle East respiratory syndrome coronavirus (MERS-CoV) antibody detection.@*Methods@#The monomeric and trimeric forms of MERS-CoV RBD were expressed in Bac-insect cells, 293T cells and ExpiCHO-S™ expression system and then purified. The purified RBD proteins were identified with native gel electrophoresis and Western blot. Then, an equal amount of each RBD protein was used as coating antigen to establish an ELISA for detecting MERS-CoV IgG titer. For comparison, the newly developed ELISA and the commercial MERS-CoV IgG antibody detection kit (Euroimmune with S1 as the coating antigen) were used to measure the MERS-CoV antibody reference panel supplied by World Health Organization (WHO).@*Results@#The purified monomeric and trimeric MERS-CoV RBD were successfully prepared using 293T cells and ExpiCHO-S™ system. RBD antigens of different forms and from different systems could recognize MERS-CoV specific antibody without having any cross reaction with the sera from healthy adults. The in-house RBD-based ELISA had good detection consistency with the Euroimmune commercial kit. The positive samples showed higher and more concentrated values based on the RBD trimer than the monomer.@*Conclusions@#Novel indirect ELISA methods based on the monomeric and trimeric forms of RBD protein were established. The trimetric form-based ELISA achieved higher detection efficiency than the one using the monomer antigen, suggesting that it could be uses as a competent alternative to the commercial kit.
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Con el objetivo de incrementar la precisión diagnóstica, la Organización Mundial de la Salud recomienda la realización de dos o más pruebas inmunoserológicas para el diagnóstico de la enfermedad de Chagas en etapa crónica. El objetivo de este trabajo fue realizar una revisión sistemática rápida acerca del desempeño de las técnicas inmunoserológicas y métodos moleculares en la población general. Se identificaron 178 estudios de los cuales fueron incluidos nueve. Las técnicas de ELISA mostraron la mayor sensibilidad (82-98%) y especificidad (96-100%). Los métodos rápidos mostraron valores de sensibilidad entre 88-93% y especificidad 97- 100%, mientras que los métodos moleculares (PCR) presentaron niveles muy variables de sensibilidad (22-92%) y especificidad (70-100%). Estos resultados muestran que las técnicas de ELISA cuentan con una sensibilidad y especificidad adecuadas. La PCR, al igual que los métodos rápidos, mostró una gran variabilidad en los resultados, debido principalmente a la heterogeneidad de la técnicas y profusión de métodos elaborados de manera in house.
In order to increase diagnostic accuracy, the World Health Organization recommends performing two or more immunoserological tests for the diagnosis of Chagas disease in chronic stage. The aim of this work was to make a rapid systematic review of the performance of immunoserological techniques and molecular methods in the general population. A total of 178 studies were identified, nine ofwhich were included. ELISA techniques showed the highest sensitivity (82-98%) and specificity (96-100%). Rapid methods presented values of sensitivity between 88-93% and 97-100% of specificity, while the molecular methods (PCR) showed highly variable levels of sensitivity (22-92%) and specificity (70-100%). These results indicate that ELISA techniques have adequate sensitivity and specificity. PCR, as well as rapid methods, showed great variability in the results, mainly due to the heterogeneity of the techniques and abundance of "in-house" methods.
Visando a aumentar a precisão do diagnóstico, a Organização Mundial da Saúde recomenda executar dois ou mais testes imunoserológicos para o diagnóstico na fase crônica da doença de Chagas. O objetivo deste trabalho foi fazer uma revisão sistemática rápida sobre o desempenho de técnicas imunoserológicas e métodos moleculares na população em geral. Foram identificados 178 estudos dos quais se incluíram nove. Técnicas de ELISA mostraram a maior sensibilidade (82-98%) e especificidade (96-100%). Métodos rápidos apresentaram valores de sensibilidade e especificidade de 88-93% e 97-100%, respectivamente enquanto que os métodos moleculares (PCR) tinham níveis extremamente variáveis de sensibilidade (22-92%) e especificidade (70-100%). Estes resultados mostram que as técnicas de ELISA possuem sensibilidade e especificidade adequadas. A PCR, bem como os métodos rápidos, mostrou grande variabilidade nos resultados, principalmente devido à heterogeneidade das técnicas e profusão de métodos desenvolvidos de modo in house.
Subject(s)
Humans , Chagas Disease/diagnosis , Clinical Laboratory Techniques/methods , Immunoglobulins , Serologic TestsABSTRACT
Objective Comparison of ELISA for detection of Mycoplasmapneumoniae IgM(MP-IgM)and cold agglutination test for early diagnosis of Mycoplasma pneumoniae infection in clinical value. Methods ELISA and cold agglutination test were used to detect 500 cases of serum samples infected with Mycoplasma pneumoniae. Results The positive rate of ELISA(54.8%)was significantly higher than that of cold agglutination(28.4%), and there was significant difference(χ2=71.72,P<0.01).The positive rates of ELISA in Fever 3 days(44.2%), 4~7days(46.4%),8~14 days(71.3%),and 15 days(78.6%)were significantly higher than that of cold aggluti-nation 22.1%,25.4%,35.7%,35.7%.The positive rates were increased with fever days extended.These is no sig-nificant difference(P > 0.05)in 1 ~ 3 days and 4 ~ 7 days,8~14 days and more than 15 days(P > 0.05) by ELISA,while there was a significant difference(P < 0.05)in 8~14 days with 1 ~ 3 days and 4 ~ 7 days. Conclution ELISA of detection MP-IgM should be selected prior in clinical detection,especially in the fever 8~14 days.It can obviously improve the early detection rate of mycoplasma infection and give the most useful value for clinical diagnosis.
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Objective To examine the interleukin-27 (IL-27) levels in the serum of the patients with neuromyelitis optica (NMO) and healthy controls (HCs) and explore the correlation of the serum IL-27 level with disease severity.Methods Serum concentrations of IL-27 from 39 patients with NMO and 39 HCs were measured by using enzyme-linked immunosorbent assay (ELISA).Mann-Whitney U test was performed to analyze the difference in the IL-27 levels between the NMO group and the HCs.Spearman's rank correlation analysis was used to analyze the correlations of serum IL-27 levels with clinical parameters (EDSS,spinal cord lesion length,annual relapse rate and antibody titers of AQP-4) of NMO.Results The serum IL-27 levels were significantly lower in NMO group than in HCs (P＜0.001).Serum IL-27 levels were negatively correlated with EDSS,total length of spinal cord lesion identified by MRI at the sampling,and the average relapse rate during two-year follow-up (r=-0.439,P=0.010;r=-0.434,P=0.006;r=-0.451,P=0.031).There was no significant correlation between IL-27 levels and antibody titers of AQP-4 (r=-0.027,P=0.871).Multivariate regression analysis showed that serum IL-27 levels were negatively correlated with EDSS (B=-0.025,P=0.023).Conclusion IL-27 may participate in the pathogenic process of NMO and might be a prognosis marker of the disease.
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Objective To establish an animal model for evaluating immunogenicity of pneumococcal conjugate vaccine.Methods New Zealand rabbits were intramuscularly administrated with three doses of 13-valent pneumococcal conjugate vaccine (PCV13) with two weeks interval between each injection.Serum samples were collected at different time points before and after vaccination.Quantitative enzyme-linked immunosorbent assay (ELISA) and opsonophagocytosis assay (OPA) that were in conformity with the World Health Organization (WHO) standards were used to detect the concentrations of serotype-specific antibodies and their bactericidal activities.Results The concentrations (Geometric mean concentration, GMC) of serotype-specific antibodies in rabbit serum samples were well correlated with their bactericidal activities (Geometric mean titer, GMT) following vaccination.Moreover, the dynamic changes of GMC and GMT of the same serotype-specific antibody remained consistent as time went by.Conclusion Rabbit model can be used to analyze the immunogenicity of PCV13 vaccine with quantitative ELISA and OPA, which indicates that it is a suitable animal model for evaluating immunogenicity of pneumococcal conjugate vaccine.