Atrazine（AT,2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine）has been detected in ground water in several areas of the United States for many years,as well as in China,wherein the growth rate of its gross
A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an...A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.展开更多
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
GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succini...GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.展开更多
Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain an...Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.展开更多
After primary analyses on the serum glycocon-jugates of lung cancer and normal individul using the enzyme-linked lectin assay (ELLA) with 12 kinds of lectins, PHA and LCA were selected to further study in the sera of ...After primary analyses on the serum glycocon-jugates of lung cancer and normal individul using the enzyme-linked lectin assay (ELLA) with 12 kinds of lectins, PHA and LCA were selected to further study in the sera of 8 kinds of cancers, 4 kinds of non-malignant disease and 1 kind of postoperative cancer. It was found that the test values of 7 kinds of cancers with PHA or LCA were significantly higher than that of the normal (P<0.01); the values of 4 kinds of non-malignant diseases with PHA were not higher (P>0.05); the values of the postoperative cancer with PHA were obviously lower than that of the preoperative (P<0.05). The results showed that the serum glycoconjugates which can bind to PHA seemed related to the cancerous existence in human bodies. The significance of the findings was 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
Objective: to compare and analyze the application effect of chemiluminescence and enzyme-linked immunosorbent assay in hepatitis B virus serological test. Methods: fifty patients with hepatitis B virus carriers admitt...Objective: to compare and analyze the application effect of chemiluminescence and enzyme-linked immunosorbent assay in hepatitis B virus serological test. Methods: fifty patients with hepatitis B virus carriers admitted to our hospital from August 2019 to September 2021 were enrolled. Peripheral blood was collected in fasting state in the morning after admission and sent to the laboratory for serological examination. Two detection techniques, namely chemiluminescence and enzymic immunosorbent assay, were used to evaluate and compare the specific detection results. Results: the positive rate of HBeAg, HBsAg, anti-HBE and other serological markers detected by chemiluminescence was higher than that by ELISA, and the difference was statistically significant (P < 0.05). Conclusion: chemiluminescence is better than elisa in the diagnosis of hepatitis B virus serology.展开更多
A rapid enzyme-linked immunosorbent assay(ELiSa)system for the detection of fipronil was studied.artificial antigens were synthesized through the glutaraldehyde method,and monoclonal antibodies were obtained via immun...A rapid enzyme-linked immunosorbent assay(ELiSa)system for the detection of fipronil was studied.artificial antigens were synthesized through the glutaraldehyde method,and monoclonal antibodies were obtained via immunization and fusion techniques.the concentration of the coated antigen and antibody and the reaction time were determined,and the recovery rate was determined by an ELiSa detection system.the cross-reaction rate of the antibody against fipronil metabolites was lower than 21.46%.the response time of the selected ELiSa system was 30 min shorter than that of the competitive ELiSa system.the iC20 to iC80 of the standard curve ranged from 0.139 to 1.433μg/L.the recovery rate of fipronil spiked in eggs was 76.7%-93.7%.this study provides support for the rapid ELiSa of fipronil.展开更多
Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in ...Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.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
LIF is a cytokine with leiotropic activities. In order to understand better the physiological and patho-physiological role of LIF. we have developed a simple and specific enzyme-linked immunosorbent assay (ELISAI for ...LIF is a cytokine with leiotropic activities. In order to understand better the physiological and patho-physiological role of LIF. we have developed a simple and specific enzyme-linked immunosorbent assay (ELISAI for detecting LIF in human plasma and serum and in tissue culture media. A monoclonal ami-LIF antibody 8B11 (IgGl) produced in our laboratory was coated onto microtiter plates. After block-展开更多
A cyanidin-based horseradish peroxidase(HRP)-catalyzed reaction system was established in this work.In B-R buffer solutions(pH 6.8),a UV-visible absorbance peak of cyanidin(CAG) at 540 nm(Ap1) appeared.After the oxida...A cyanidin-based horseradish peroxidase(HRP)-catalyzed reaction system was established in this work.In B-R buffer solutions(pH 6.8),a UV-visible absorbance peak of cyanidin(CAG) at 540 nm(Ap1) appeared.After the oxidation reaction of CAG catalyzed by HRP in the presence of H2O2,a significant absorbance peak at 482 nm(Ap2) occurred.The ratio R(AP2/AP1)was proportional to the HRP concentration.The application of CAG in the enzyme-linked immunosensing assays was investigated using food and mouth disease virus antigen(FMDVAg) as a model analyte.In sandwich immunoreaction,the analyte FMDVAg and food and mouth disease virus antibody(FMDVAb)-modified magnetic nanoparticles bound the supported conconvalina(Con A) bound with HRP-FMDVAb.After de-absorbing and separating,the HRP-FMDVAb-FMDVAg-FMDVAb-magnetic nanoparticles complexes were subject to enzymatic reaction and UV-visible absorbance measurements.The HRP moiety of the immunoreaction complexes can catalyze the oxidation reaction of CAG by H2O2,and the substrate CAG is converted to products.Based on this principle,a sandwich assay model has been employed to determine FMDVAg in rabbit serum samples with the aid of FMDVAb-Fe3O4 magnetic nanoparticles.The linear range of the FMDVAg determination is 1.5×10-8-2.7×10-6 g/mL with the relatively standard deviation of 3.7%(n = 11).The detection limit is 3.1×10-9 g/mL.Additional advantages of the typical substrate such as OPD,OAP and TMB are good water-solubility and stability.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 ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc displayed ...The use of tetra-substituted ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc 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 H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis-Menten parameters Km and Vmax were measured to be 2.82 × 10?6 mol/L?1 and 6.0 × 10?9 mol·L?1, respectively. In this paper, TAAIPc 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
Objective:To explore the mechanism of action of Zangjiangzhi capsule(ZJZC)in treating hyperlipidemia(HLP).Methods:The components of ZJZC were analyzed and identified using ultra-high performance liquid chromatography ...Objective:To explore the mechanism of action of Zangjiangzhi capsule(ZJZC)in treating hyperlipidemia(HLP).Methods:The components of ZJZC were analyzed and identified using ultra-high performance liquid chromatography with Q-Exactive Orbitrap tandem mass spectrometry(UHPLC-Q-Exactive-Orbitrap-MS/MS).Network pharmacology analysis was used to explore the mechanism of action of ZJZC in HLP treatment.The SwissTargetPrediction database was used to predict compound targets,and GeneCards,DisGeNet,OMIM,and DRUGBANK databases were used to identify HLP-related targets.Proteineprotein interaction diagrams were constructed using the STRING database.The targets were subjected to gene ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The“herbingredient-target”network was visualized using Cytoscape.Preliminary validation was performed using molecular docking and enzyme-linked immunosorbent assay.Results:Ninety compounds were identified in ZJZC,including 34 flavonoids,12 phenols,10 terpenoids,10 alkaloids,8 organic acids,8 anthraquinones,and 9 other compounds.In total,904 targets were identified for these compounds.Among them,158 targets intersected with the HLP target network.Network pharmacology analysis showed that MAPK1,PPAR-a,RXRA,HSP90AA1,PIK3R1,AKT1,PIK3CA,IL6,TNF,and ESR1 are the key targets of action.KEGG enrichment analysis identified 164 pathways.Among these,the AGE-RAGE signaling pathway in diabetic complications,lipid and atherosclerosis pathways,regulation of lipids in adipocytes,and insulin resistance are related to HLP.Molecular docking showed good affinity between the key targets and ingredients.Further,ZJZC treatment in mice resulted in lower expression of MAPK1 protein and increased expression of PPAR-a protein,which have been shown to be strongly associated with HLP.Conclusions:This study showed that ZJZC contains various active ingredients and can modulate multiple targets and pathways associated with HLP,providing evidence at the molecular level for its clinical application in the treatment of HLP.展开更多
The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability ...The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability to boost host immunity.In this study,the heterologous expression,purification,and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated.Initially,the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid.SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions,which was further demonstrated by Western blot(WB)and LC-MS/MS.ELISA,WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors(TNF-α,IL-6,NO)and ROS via MAPK and NF-κB signaling pathways.The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.展开更多
基金supported by the National Natural Science Foundation of China(No.81030052,20907074)National Science & Technology Supporting Program(2012BAJ25B03-02)Tianjin Science & Technology Program(11ZCKFSF01100)
文摘Atrazine（AT,2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine）has been detected in ground water in several areas of the United States for many years,as well as in China,wherein the growth rate of its gross
基金the grants from the Ministry of Sciences and Technology of China, No. 2006AA02A408, 2008ZX09312-014
文摘A total of 66 samples （from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, aa 13 cases with optic neuritis） were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay. The sensitivities and specificities of the two assays were similar. We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay. A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the se of neuromyelitis optica patients. No significant correlations were identified with onset age or disea duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica. The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.
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GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.
文摘Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.
文摘After primary analyses on the serum glycocon-jugates of lung cancer and normal individul using the enzyme-linked lectin assay (ELLA) with 12 kinds of lectins, PHA and LCA were selected to further study in the sera of 8 kinds of cancers, 4 kinds of non-malignant disease and 1 kind of postoperative cancer. It was found that the test values of 7 kinds of cancers with PHA or LCA were significantly higher than that of the normal (P<0.01); the values of 4 kinds of non-malignant diseases with PHA were not higher (P>0.05); the values of the postoperative cancer with PHA were obviously lower than that of the preoperative (P<0.05). The results showed that the serum glycoconjugates which can bind to PHA seemed related to the cancerous existence in human bodies. The significance of the findings was 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.
文摘Objective: to compare and analyze the application effect of chemiluminescence and enzyme-linked immunosorbent assay in hepatitis B virus serological test. Methods: fifty patients with hepatitis B virus carriers admitted to our hospital from August 2019 to September 2021 were enrolled. Peripheral blood was collected in fasting state in the morning after admission and sent to the laboratory for serological examination. Two detection techniques, namely chemiluminescence and enzymic immunosorbent assay, were used to evaluate and compare the specific detection results. Results: the positive rate of HBeAg, HBsAg, anti-HBE and other serological markers detected by chemiluminescence was higher than that by ELISA, and the difference was statistically significant (P < 0.05). Conclusion: chemiluminescence is better than elisa in the diagnosis of hepatitis B virus serology.
文摘A rapid enzyme-linked immunosorbent assay(ELiSa)system for the detection of fipronil was studied.artificial antigens were synthesized through the glutaraldehyde method,and monoclonal antibodies were obtained via immunization and fusion techniques.the concentration of the coated antigen and antibody and the reaction time were determined,and the recovery rate was determined by an ELiSa detection system.the cross-reaction rate of the antibody against fipronil metabolites was lower than 21.46%.the response time of the selected ELiSa system was 30 min shorter than that of the competitive ELiSa system.the iC20 to iC80 of the standard curve ranged from 0.139 to 1.433μg/L.the recovery rate of fipronil spiked in eggs was 76.7%-93.7%.this study provides support for the rapid ELiSa of fipronil.
文摘Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.
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LIF is a cytokine with leiotropic activities. In order to understand better the physiological and patho-physiological role of LIF. we have developed a simple and specific enzyme-linked immunosorbent assay (ELISAI for detecting LIF in human plasma and serum and in tissue culture media. A monoclonal ami-LIF antibody 8B11 (IgGl) produced in our laboratory was coated onto microtiter plates. After block-
基金Supported by the Scientific Research Foundation of Hunan Province (Grant No. 2008SK3052)the Scientific Research Foundation of Hunan Provincial Education Department (Grant No. 08B004)
文摘A cyanidin-based horseradish peroxidase(HRP)-catalyzed reaction system was established in this work.In B-R buffer solutions(pH 6.8),a UV-visible absorbance peak of cyanidin(CAG) at 540 nm(Ap1) appeared.After the oxidation reaction of CAG catalyzed by HRP in the presence of H2O2,a significant absorbance peak at 482 nm(Ap2) occurred.The ratio R(AP2/AP1)was proportional to the HRP concentration.The application of CAG in the enzyme-linked immunosensing assays was investigated using food and mouth disease virus antigen(FMDVAg) as a model analyte.In sandwich immunoreaction,the analyte FMDVAg and food and mouth disease virus antibody(FMDVAb)-modified magnetic nanoparticles bound the supported conconvalina(Con A) bound with HRP-FMDVAb.After de-absorbing and separating,the HRP-FMDVAb-FMDVAg-FMDVAb-magnetic nanoparticles complexes were subject to enzymatic reaction and UV-visible absorbance measurements.The HRP moiety of the immunoreaction complexes can catalyze the oxidation reaction of CAG by H2O2,and the substrate CAG is converted to products.Based on this principle,a sandwich assay model has been employed to determine FMDVAg in rabbit serum samples with the aid of FMDVAb-Fe3O4 magnetic nanoparticles.The linear range of the FMDVAg determination is 1.5×10-8-2.7×10-6 g/mL with the relatively standard deviation of 3.7%(n = 11).The detection limit is 3.1×10-9 g/mL.Additional advantages of the typical substrate such as OPD,OAP and TMB are good water-solubility and stability.
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 ammo aluminum phthalocyanine (TAAIPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAIPc 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 H2O2 could rapidly and significantly react with TAAIPc, thus quenching the fluorescence of TAAIPc. The Michaelis-Menten parameters Km and Vmax were measured to be 2.82 × 10?6 mol/L?1 and 6.0 × 10?9 mol·L?1, respectively. In this paper, TAAIPc 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-
基金supported by the National Natural Science Foundation of China(22067016)the Project of Qinghai Outstanding Youth Fund Project(2023-ZJ-964J).
文摘Objective:To explore the mechanism of action of Zangjiangzhi capsule(ZJZC)in treating hyperlipidemia(HLP).Methods:The components of ZJZC were analyzed and identified using ultra-high performance liquid chromatography with Q-Exactive Orbitrap tandem mass spectrometry(UHPLC-Q-Exactive-Orbitrap-MS/MS).Network pharmacology analysis was used to explore the mechanism of action of ZJZC in HLP treatment.The SwissTargetPrediction database was used to predict compound targets,and GeneCards,DisGeNet,OMIM,and DRUGBANK databases were used to identify HLP-related targets.Proteineprotein interaction diagrams were constructed using the STRING database.The targets were subjected to gene ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The“herbingredient-target”network was visualized using Cytoscape.Preliminary validation was performed using molecular docking and enzyme-linked immunosorbent assay.Results:Ninety compounds were identified in ZJZC,including 34 flavonoids,12 phenols,10 terpenoids,10 alkaloids,8 organic acids,8 anthraquinones,and 9 other compounds.In total,904 targets were identified for these compounds.Among them,158 targets intersected with the HLP target network.Network pharmacology analysis showed that MAPK1,PPAR-a,RXRA,HSP90AA1,PIK3R1,AKT1,PIK3CA,IL6,TNF,and ESR1 are the key targets of action.KEGG enrichment analysis identified 164 pathways.Among these,the AGE-RAGE signaling pathway in diabetic complications,lipid and atherosclerosis pathways,regulation of lipids in adipocytes,and insulin resistance are related to HLP.Molecular docking showed good affinity between the key targets and ingredients.Further,ZJZC treatment in mice resulted in lower expression of MAPK1 protein and increased expression of PPAR-a protein,which have been shown to be strongly associated with HLP.Conclusions:This study showed that ZJZC contains various active ingredients and can modulate multiple targets and pathways associated with HLP,providing evidence at the molecular level for its clinical application in the treatment of HLP.
基金supported by the National Natural Science Foundation of China(32272339)National Key R&D Program of China(2021YFD2100104)。
文摘The role of S-layer proteins(SLP),which form the outermost layer of cell walls in lactic acid bacteria(LAB),plays a crucial role in regulating immune-stimulating activity,thereby closely influencing LAB's ability to boost host immunity.In this study,the heterologous expression,purification,and characterization of Lactobacillus acidophilus CICC6074-derived slpX protein and the molecular mechanism of its anti-inflammatory effect on LPS-induced RAW264.7 cells were investigated.Initially,the PCR results were shown to successfully clone the slpX DNA sequence by homologous recombination to obtain the pet-32a-slpX recombinant plasmid.SDS-PAGE results revealed that slpX protein with a molecular weight of 54 kDa was successfully obtained under 0.7 mmol/L IPTG-induced conditions,which was further demonstrated by Western blot(WB)and LC-MS/MS.ELISA,WB and molecular docking results indicated that slpX protein can inhibit LPS-induced cellular inflammatory responses by linking to TLR4 and MD2 via hydrogen bonding and increasing the levels of anti-inflammatory factor IL-10 and decreasing the levels of inflammatory factors(TNF-α,IL-6,NO)and ROS via MAPK and NF-κB signaling pathways.The study is essential for the preparation of pure slpX protein and revealing its anti-inflammatory molecular mechanism.
