A selective and sensitive competitive enzyme-linked immunosorbent assay(ELISA) method was developed and validated for the quantification of erlotinib in 50 mL of samples of human serum. Anti-erlotinib serum was obtain...A selective and sensitive competitive enzyme-linked immunosorbent assay(ELISA) method was developed and validated for the quantification of erlotinib in 50 mL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N-succinimidyl ester method. Enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity(6.7%) with a major metabolite, O-desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.展开更多
Afatinib is an oral tyrosine kinase inhibitor(TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug ...Afatinib is an oral tyrosine kinase inhibitor(TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring(TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay(ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using(S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine(CTQD), which has the same substructure as afatinib, as a hapten. Enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods(Y=0.976 X – 0.207, r=0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.展开更多
Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra f...Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.展开更多
Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneousl...Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.展开更多
A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELI...A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).展开更多
The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia.Thirty healthy wom...The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia.Thirty healthy women of childbearing age,22 second trimester pregnant women,30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study.ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay.The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women,between third and second trimester pregnant women (P<0.05).The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P<0.05).Nevertheless,no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P>0.05).In conclusion,plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels.Prothrombotic state is involved in the pathogenesis of severe preeclampsia,as a result of endothelial injury.展开更多
Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxaci...Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxacin(OFL).The linear range of the CI-ELISAwas from 0.5 to 128 ng/mL with a limit of detection(LOD)of 0.35 ng/mL.Good recoveries were obtained in analyzing simulated swine urine samples.The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min,and test results were read visually without any instrument.展开更多
Plasma quinolone concentrations are not routinely measured in clinical practice.However,in order to optimize quinolone treatment,monitoring of plasma concentrations could sometimes be useful particularly in critically...Plasma quinolone concentrations are not routinely measured in clinical practice.However,in order to optimize quinolone treatment,monitoring of plasma concentrations could sometimes be useful particularly in critically ill patients.In this study,anti-sparfloxacin antibody was obtained by immunizing rabbits with sparfloxacin conjugated with bovine serum albumin using isobutyl chloroformate method.After the assay procedure was optimized,the standard curve of sparfloxacin was established.The practical measuring range of the competitive ELISA extended from 5 ng/mL to 2 mg/mL.The recovery rates and coefficients of variation for rat plasma,urine and tissues were 87.7-106.2% and 4.8-15.3%,respectively.To demonstrate the potential of the ELISA,a preliminary pharmacokinetics and tissue distribution study of sparfloxacin in rats and quantitative analysis of sparfloxacin in several pharmaceuticals were performed and compared with high-performance liquid chromatography(HPLC).The experimental data indicated that the proposed method would be a valuable tool in therapeutic drug monitoring(TDM) for sparfloxacin.展开更多
Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient c...Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies.Therefore,a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101.In this study,a sensitive bioanalytical method was developed and validated,using a Quanterix single molecular array(Simoa)assay.Moreover,to thoroughly assess the platform,enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed,and their performance was compared with that of this novel technology platform.The assay was validated in compliance with the current guidelines.Measurements with the Simoa assay were precise and accurate,presenting a valid assay range from 6.55 to 4000 pg/mL.The intra-and inter-run accuracy and precision were within-19.3%to 15.3%and 5.5%to 17.0%,respectively.S3101 was stable in human serum for 280 days at-20℃and-70℃,for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature(22℃-28℃),respectively,and after five and two freeze-thaw cycles at-70℃and-20oC,respectively.The Simoa assay also demonstrated sufficient dilution linearity,assay sensitivity,and parallelism for quantifying S3101 in human serum.The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.展开更多
Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective...Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A(PVA)is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies(MAbs)(2D4,8E11,14A6 and 16H10)using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680(w/v,g m L^(-1))by ACP-ELISA or up to1:10240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.展开更多
BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by th...BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.展开更多
Microcystins(MCs)are a group of closely related toxic cyclic heptapeptides produced by common cyanobacte-ria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked...Microcystins(MCs)are a group of closely related toxic cyclic heptapeptides produced by common cyanobacte-ria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immu-nosorbent assay(ic-ELISA)was established and used to detect microcystin-LR(MC-LR)in drinking and surface waters.The concentration of coating antigen was 5 mg/mL,the dilution of monoclonal antibody MC10E7 was 1:3000,the dilution of enzyme tracer(goat anti-mouse IgG-peroxidase)was 1:3000,the standard concentration of MC-LR ranged from 0.001 mg/L to 30 mg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC)with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 mg/L and up to 5.1 mg/L.The quantitative detection range was from 0.03 mg/L to 3 mg/L,and the antibody had high specificity for[4-arginine]microcystins.It performed well in spite of the influence of the real samples.展开更多
Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory int...Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory interference dial,and laboratory precision dial were constructed.The linear range,sensitivity,specificity,precision and other performances of ELISA for detecting syphilis dry blood spot samples were comprehensively evaluated,and the stability of dry blood spot samples at 37℃was detected.In addition,112 suspected syphilis antibody-positive plasma samples were selected as the control,and dry blood spot samples were prepared accordingly.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples and plasma samples in ELISA for syphilis detection were compared,and the consistency and correlation between the two samples were analyzed by the Kappa consistency test and Spearman rank correlation analysis.Results:The results of the linear analysis showed that the serial dilution of dry blood spot samples in ELISA for syphilis antibody ranged from 23to 27,and there was a good linear range[R2=0.9862(P<0.05)].Sensitivity and specificity analysis showed that the positive coincidence rate and negative coincidence rate of the two detection methods were 100%(15/15).The results of the interference dial test showed that ELISA based on dry blood spot samples could accurately detect 6 syphilis antibody samples from 18samples,and the detection accuracy rate was 100.00%(6/6).The results of the precision test showed that the RSD of syphilis antibody detection in dry blood spot samples with different dilution times(23,25and 27)was 0.24%to 3.87%between spots,0.06%to4.07%between batches and 0.49%to 3.88%between days.Within 7 days,the inter-day RSD of dry blood spots with different dilution times(23,25,27)were 0.27%,0.65%and 0.95%,respectively.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples in ELISA detection of syphilis antibody were 96.00%(72/75),100.00%(37/37),100.00%(72/72)and 92.50%,respectively.The results of the Kappa consistency test and Spearman rank correlation analysis showed that the Kappa value of the two methods was 0.941(P<0.01),and the correlation coefficientρbetween the S/CO ratios of the two methods was 0.211(P<0.01).The comparison of S/CO ratio results showed that the distribution characteristics of the S/CO ratio between the two methods were similar,and the ratio distribution was relatively concentrated.Conclusion:using dry blood spot samples to perform ELISA for syphilis detection has good precision,strong anti-interference ability and excellent stability.Although false-positive results appear in weak positive samples,it still has a high application value in ELISA for syphilis antibody detection,which can provide an important reference for disease diagnosis.展开更多
The use of tetra-substituted amino aluminum phthalocyanine (TAAlPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAlPc displayed...The use of tetra-substituted amino aluminum phthalocyanine (TAAlPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAlPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H 2O 2 could rapidly and significantly react with TAAlPc, thus quenching the fluorescence of TAAlPc. The Michaelis-Menten parameters K m and V max were measured to be 2.82×10 -6 mol/L -1 and 6.0×10 -9 mol·L -1·s -1, respectively. In this paper, TAAlPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5-200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.展开更多
Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent...Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-展开更多
文摘A selective and sensitive competitive enzyme-linked immunosorbent assay(ELISA) method was developed and validated for the quantification of erlotinib in 50 mL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N-succinimidyl ester method. Enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity(6.7%) with a major metabolite, O-desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.
文摘Afatinib is an oral tyrosine kinase inhibitor(TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring(TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay(ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using(S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine(CTQD), which has the same substructure as afatinib, as a hapten. Enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods(Y=0.976 X – 0.207, r=0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.
文摘Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.
文摘Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.
基金supported by the National Basic Research Program of China (973 Program,no. 2007CB714507)National Nature Science Foundation of China (no. 90813015)
文摘A chemiluminescence enzyme immunoassay based on magnetic microparticles (MmPs-CLEIA) was developed to evaluate serum a-fetoprotein (AFP) in parallel with traditional colorimetric enzyme-linked immunosorbent assay (ELISA).A systematic comparison between the MmPs-CLEIA and colorimetric ELISA concluded that the MPs-CLEIA exhibited fewer dosages of immunoreagents,less total assay time,and better linearity,recovery,precision,sensitivity and validity.AFP was detected in forty human serum samples by the proposed MPs-CLEIA and ELISA,and the results were compared with commercial electrochemiluminescence immunoassay (ECLIA) kit.The correlation coefficient between MPs-CLEIA and ELISA was obtained with R 2 0.6703;however,the correlation between MPs-CLEIA and ECLIA (R 2 0.9582) was obviously better than that between colorimetric ELISA and ECLIA (R 2 0.6866).
基金supported by grants from National Natural Sciences Foundation of China(No.30672243,No.30671093)Natural Sciences Foundation of Hubei Province(No.ZR-Y1271)
文摘The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia.Thirty healthy women of childbearing age,22 second trimester pregnant women,30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study.ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay.The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women,between third and second trimester pregnant women (P<0.05).The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P<0.05).Nevertheless,no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P>0.05).In conclusion,plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels.Prothrombotic state is involved in the pathogenesis of severe preeclampsia,as a result of endothelial injury.
文摘Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxacin(OFL).The linear range of the CI-ELISAwas from 0.5 to 128 ng/mL with a limit of detection(LOD)of 0.35 ng/mL.Good recoveries were obtained in analyzing simulated swine urine samples.The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min,and test results were read visually without any instrument.
文摘Plasma quinolone concentrations are not routinely measured in clinical practice.However,in order to optimize quinolone treatment,monitoring of plasma concentrations could sometimes be useful particularly in critically ill patients.In this study,anti-sparfloxacin antibody was obtained by immunizing rabbits with sparfloxacin conjugated with bovine serum albumin using isobutyl chloroformate method.After the assay procedure was optimized,the standard curve of sparfloxacin was established.The practical measuring range of the competitive ELISA extended from 5 ng/mL to 2 mg/mL.The recovery rates and coefficients of variation for rat plasma,urine and tissues were 87.7-106.2% and 4.8-15.3%,respectively.To demonstrate the potential of the ELISA,a preliminary pharmacokinetics and tissue distribution study of sparfloxacin in rats and quantitative analysis of sparfloxacin in several pharmaceuticals were performed and compared with high-performance liquid chromatography(HPLC).The experimental data indicated that the proposed method would be a valuable tool in therapeutic drug monitoring(TDM) for sparfloxacin.
文摘Recombinant batroxobin(S3101)is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system.A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies.Therefore,a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101.In this study,a sensitive bioanalytical method was developed and validated,using a Quanterix single molecular array(Simoa)assay.Moreover,to thoroughly assess the platform,enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed,and their performance was compared with that of this novel technology platform.The assay was validated in compliance with the current guidelines.Measurements with the Simoa assay were precise and accurate,presenting a valid assay range from 6.55 to 4000 pg/mL.The intra-and inter-run accuracy and precision were within-19.3%to 15.3%and 5.5%to 17.0%,respectively.S3101 was stable in human serum for 280 days at-20℃and-70℃,for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature(22℃-28℃),respectively,and after five and two freeze-thaw cycles at-70℃and-20oC,respectively.The Simoa assay also demonstrated sufficient dilution linearity,assay sensitivity,and parallelism for quantifying S3101 in human serum.The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.
基金supported by the National Key Research and Development Program of China(2017YFD0201604)the National Natural Science Foundation of China(31571976)。
文摘Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A(PVA)is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies(MAbs)(2D4,8E11,14A6 and 16H10)using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680(w/v,g m L^(-1))by ACP-ELISA or up to1:10240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.
基金Supported by the German Federal Ministry of Education and Research(BMBF-Wachstumskern-PRAEMED.BIO),03WKDB2Csupported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences,BO/00232/17/5+1 种基金Research Grants of National Research Development and Innovation Office,K115818/2015/1New National Excellence Program of the Ministry of Human Capacities,ÚNKP-18-4 Bolyai Plus.
文摘BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.
基金This work was supported by the National High-Tech Research and Development(863)Program of China(2002AA649160).
文摘Microcystins(MCs)are a group of closely related toxic cyclic heptapeptides produced by common cyanobacte-ria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immu-nosorbent assay(ic-ELISA)was established and used to detect microcystin-LR(MC-LR)in drinking and surface waters.The concentration of coating antigen was 5 mg/mL,the dilution of monoclonal antibody MC10E7 was 1:3000,the dilution of enzyme tracer(goat anti-mouse IgG-peroxidase)was 1:3000,the standard concentration of MC-LR ranged from 0.001 mg/L to 30 mg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC)with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 mg/L and up to 5.1 mg/L.The quantitative detection range was from 0.03 mg/L to 3 mg/L,and the antibody had high specificity for[4-arginine]microcystins.It performed well in spite of the influence of the real samples.
文摘Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory interference dial,and laboratory precision dial were constructed.The linear range,sensitivity,specificity,precision and other performances of ELISA for detecting syphilis dry blood spot samples were comprehensively evaluated,and the stability of dry blood spot samples at 37℃was detected.In addition,112 suspected syphilis antibody-positive plasma samples were selected as the control,and dry blood spot samples were prepared accordingly.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples and plasma samples in ELISA for syphilis detection were compared,and the consistency and correlation between the two samples were analyzed by the Kappa consistency test and Spearman rank correlation analysis.Results:The results of the linear analysis showed that the serial dilution of dry blood spot samples in ELISA for syphilis antibody ranged from 23to 27,and there was a good linear range[R2=0.9862(P<0.05)].Sensitivity and specificity analysis showed that the positive coincidence rate and negative coincidence rate of the two detection methods were 100%(15/15).The results of the interference dial test showed that ELISA based on dry blood spot samples could accurately detect 6 syphilis antibody samples from 18samples,and the detection accuracy rate was 100.00%(6/6).The results of the precision test showed that the RSD of syphilis antibody detection in dry blood spot samples with different dilution times(23,25and 27)was 0.24%to 3.87%between spots,0.06%to4.07%between batches and 0.49%to 3.88%between days.Within 7 days,the inter-day RSD of dry blood spots with different dilution times(23,25,27)were 0.27%,0.65%and 0.95%,respectively.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples in ELISA detection of syphilis antibody were 96.00%(72/75),100.00%(37/37),100.00%(72/72)and 92.50%,respectively.The results of the Kappa consistency test and Spearman rank correlation analysis showed that the Kappa value of the two methods was 0.941(P<0.01),and the correlation coefficientρbetween the S/CO ratios of the two methods was 0.211(P<0.01).The comparison of S/CO ratio results showed that the distribution characteristics of the S/CO ratio between the two methods were similar,and the ratio distribution was relatively concentrated.Conclusion:using dry blood spot samples to perform ELISA for syphilis detection has good precision,strong anti-interference ability and excellent stability.Although false-positive results appear in weak positive samples,it still has a high application value in ELISA for syphilis antibody detection,which can provide an important reference for disease diagnosis.
文摘The use of tetra-substituted amino aluminum phthalocyanine (TAAlPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAlPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H 2O 2 could rapidly and significantly react with TAAlPc, thus quenching the fluorescence of TAAlPc. The Michaelis-Menten parameters K m and V max were measured to be 2.82×10 -6 mol/L -1 and 6.0×10 -9 mol·L -1·s -1, respectively. In this paper, TAAlPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5-200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.
文摘Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-
