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
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
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
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
In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentra...In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.展开更多
The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on i...The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.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
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:This investigation delineates the anti-cancer potency of epigallocatechin-3-gallate(EGCG)in an oral cancer mouse model,with a focus on its effect on T-cell activation.Methods:An oral cancer model was establi...Objective:This investigation delineates the anti-cancer potency of epigallocatechin-3-gallate(EGCG)in an oral cancer mouse model,with a focus on its effect on T-cell activation.Methods:An oral cancer model was established in male Balb/c mice using 4-nitroquinoline 1-oxide(4-NQO).The mice were systematically grouped and administered graded concentrations of EGCG.Key parameters such as body weight,hydration levels,tumor volume,and mass were meticulously tracked.T-cell activity and cytokine expression profiles,focusing on interleukin-2(IL-2),interferon-gamma(IFN-γ),and tumor necrosis factor-alpha(TNF-α),were quantified using ELISA.A comprehensive statistical evaluation included one-way ANOVA,Tukey’s HSD multiple comparison test,and the Kruskal-Wallis non-parametric assessment.Results:EGCG-administered cohorts exhibited a pronounced reduction in tumor size and mass,with the high-dose group showing the greatest efficacy.ELISA findings corroborated a significant increase in T-cell activity and concomitant upregulation of key cytokines,including IL-2,IFN-γ,and TNF-α(P<0.05).Conclusion:This investigation confirms the tumor-suppressive efficacy of EGCG in a murine oral squamous cell carcinoma model.The therapeutic effects of EGCG are mediated through T-cell activation and the upregulation of pivotal cytokine expression,highlighting its potential immunomodulatory role in oral cancer treatment.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
Objective: Survivin as a tumor marker in the diagnosis of bladder cancer has not been completely confirmed yet and there are few reports about using Survivin enzyme-linked immunosorbent assay (ELISA) kit to detect ...Objective: Survivin as a tumor marker in the diagnosis of bladder cancer has not been completely confirmed yet and there are few reports about using Survivin enzyme-linked immunosorbent assay (ELISA) kit to detect the urine of bladder cancer patients. This study aimed to develop a Survivin ELISA and validate its value in the detection of bladder cancer. Methods: Through square matrix titration, different combinations of coating antibody and detecting antibody, a Survivin ELISA was constructed. This assay was evaluated according to intra-assay precision, inter-assay precision and minimum detectable dose (MDD). Survivin levels were detected and analyzed in 102 bladder cancer patients and 102 healthy people by established ELISA. Then cutoff value was defined according to the analysis of receiver operating characteristic (ROC) curve. The sensitivity and specificity of detection were calculated on the basis of cutoff value to diagnose bladder cancer patients. Furthermore, the value of Survivin expression detected by ELISA among different clinicopathological characteristics of patients was also compared. Results: Through optimization of different conditions, intra-assay precision was 8.39%, inter-assay precision 8.57% and MDD 0.0625 ng/mL in this assay. When the optical density at 450 nm (OD 450 ) was 0.09, it could get the optimized diagnostic cutoff value. According to this value, the sensitivity and specificity of diagnosis in bladder cancer patients were 70.6% and 89.2%, respectively. The associations between patients' clinical variables and OD 450 were not significant except tumor numbers in patients. Conclusions: This experiment has preliminarily developed a Survivin ELISA and confirmed Survivin as a biomarker which owned a practical and significant value in the diagnosis of bladder cancer.展开更多
Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). Methods Full length and two trunca...Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R^2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.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This research was funded by Hebei Provincial Department of Science and Technology(21375501D)the Hebei Academy of Sciences(2019Q01).
文摘In this study,an enzyme 1linked immunosorbent assay(ELISA)was established to detect beef and 1amb components,and its performance was tested.Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4000,a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase(HRP)of 1:1000,a sample incubation time of 60 min and a detection antibody reaction time of 60 min.The specificity,sensitivity,repeatability and stability of this assay were detemmined.The limit of detection for beef and 1amb skeleta1 muscle troponin I was 45 mg/kg,the inter-assay and intra-assay recovery rates ranged from 80.4%to 115.7%,the coefficients of variation were below 13.6%,and the cIoss reaction rates of the tissue components of chicken,duck and fish were below 13.4%.The sandwich ELISA method established in this study is stable and has high accuracy.The test results were consistent with the polymerase chain reaction(PCR)method at 50 and 100 g/kg-Therefore,this ELISA method can be used to quantitatively detect beef and 1amb components in meat products.
文摘The simple rapid and sensitive enzyme-linked immunosorbent assay (ELISA) methods, di-rect and indirect ELISA, for quantitation of ochratoxin A in cereal had been developed by theutilization of monoclonal antibody on immunomicroplate. Direct FLIAS was found to be less timeconsuming than indirect ELISA. For direct FLISA, recovery of 1 -500 ppb OA added to wheat was78.9-100.0% and rice was 88.9- 120.0%. For indirect EI.IAS, recovery of 1-500 ppb OA addedto wheat was 79.0- 110.0% and rice was 82.0 120.0%. The minimal detection level for OA was Ippb. Analyses of 31 samples that caused humanintoxicant for OA showed that the ELISA resultsagreed wtll with those obtained by thin-layer chromatogrdphy.
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-
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Innovation and Entrepreneurship Project for College Students in Changsha Medical University,Changsha Medical Education 2022(Project No.41-149)。
文摘Objective:This investigation delineates the anti-cancer potency of epigallocatechin-3-gallate(EGCG)in an oral cancer mouse model,with a focus on its effect on T-cell activation.Methods:An oral cancer model was established in male Balb/c mice using 4-nitroquinoline 1-oxide(4-NQO).The mice were systematically grouped and administered graded concentrations of EGCG.Key parameters such as body weight,hydration levels,tumor volume,and mass were meticulously tracked.T-cell activity and cytokine expression profiles,focusing on interleukin-2(IL-2),interferon-gamma(IFN-γ),and tumor necrosis factor-alpha(TNF-α),were quantified using ELISA.A comprehensive statistical evaluation included one-way ANOVA,Tukey’s HSD multiple comparison test,and the Kruskal-Wallis non-parametric assessment.Results:EGCG-administered cohorts exhibited a pronounced reduction in tumor size and mass,with the high-dose group showing the greatest efficacy.ELISA findings corroborated a significant increase in T-cell activity and concomitant upregulation of key cytokines,including IL-2,IFN-γ,and TNF-α(P<0.05).Conclusion:This investigation confirms the tumor-suppressive efficacy of EGCG in a murine oral squamous cell carcinoma model.The therapeutic effects of EGCG are mediated through T-cell activation and the upregulation of pivotal cytokine expression,highlighting its potential immunomodulatory role in oral cancer treatment.
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 the National High Technology Research Development Plan (No.2012AA02A504)the Capital Healthy Development Special Fund (No.2011-1009-03)the Capital Laboratory Medicine Clinical Characteristic Fund (No.Z121107005112004)
文摘Objective: Survivin as a tumor marker in the diagnosis of bladder cancer has not been completely confirmed yet and there are few reports about using Survivin enzyme-linked immunosorbent assay (ELISA) kit to detect the urine of bladder cancer patients. This study aimed to develop a Survivin ELISA and validate its value in the detection of bladder cancer. Methods: Through square matrix titration, different combinations of coating antibody and detecting antibody, a Survivin ELISA was constructed. This assay was evaluated according to intra-assay precision, inter-assay precision and minimum detectable dose (MDD). Survivin levels were detected and analyzed in 102 bladder cancer patients and 102 healthy people by established ELISA. Then cutoff value was defined according to the analysis of receiver operating characteristic (ROC) curve. The sensitivity and specificity of detection were calculated on the basis of cutoff value to diagnose bladder cancer patients. Furthermore, the value of Survivin expression detected by ELISA among different clinicopathological characteristics of patients was also compared. Results: Through optimization of different conditions, intra-assay precision was 8.39%, inter-assay precision 8.57% and MDD 0.0625 ng/mL in this assay. When the optical density at 450 nm (OD 450 ) was 0.09, it could get the optimized diagnostic cutoff value. According to this value, the sensitivity and specificity of diagnosis in bladder cancer patients were 70.6% and 89.2%, respectively. The associations between patients' clinical variables and OD 450 were not significant except tumor numbers in patients. Conclusions: This experiment has preliminarily developed a Survivin ELISA and confirmed Survivin as a biomarker which owned a practical and significant value in the diagnosis of bladder cancer.
基金National High-tech Development Project (863 Project) (No. 2002AA215021)
文摘Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor (VEGF) in sera of population and the patients with hepatocellular carcinoma (HCC). Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length (His-Trx-VEGF1-165), N-terminal (His-Trx-VEGF1-100) and C-terminal (GST-VEGF100-165) human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R^2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.