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
The control and prevention of pasteurellosis in rabbits which makes the hosts unsuitable for experimental use raised the needs to improve and simplify the procedures of Enzyme-Linked Immunosorbent Assay (ELISA) for de...The control and prevention of pasteurellosis in rabbits which makes the hosts unsuitable for experimental use raised the needs to improve and simplify the procedures of Enzyme-Linked Immunosorbent Assay (ELISA) for detect of antibody against P.multocida. A comparison on the sensitivity and specificity of bacterial culture of antemortem and postmortem samples, complement fixation test and enzyme-linked immunosorbent assay of 11 apparently healthy adult rabbits was conducted. The incidence rates showed 45.45%,54.54% and 72.73% respectively. The sensitivity for the three methods were 0.63, 0.67,and 1.00,and specificity for them were 1.00, 0.67 and 1.00 respectively. Somatic serotypes of isolates of P.multocida from rabbits of three groups (rabbits of group 2 were with clinic signs, those of groups 1 and 3 were apparently healthy) revealed no remarkable differences,and the predominant types were type 3 and type 3, 4. This was somewhat different from the reports derived from other states. As the antigen of different serotype used in ELISA may have different sensitivity and specificity, which is affected also by different preparation method, a type non-specific antigen should be selected to meet such request. The trial of accomplishment of ELISA without positive and negative controls was presented for discussion.展开更多
Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra f...Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.展开更多
Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneousl...Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.展开更多
In thes paper the authors used the Horseradish peroxidase labelledstaphylococcal protein A(HRP—SPA)in ELISA,for the detection of Clo-norchis sinensis infection.Serum tests were made on 116 confirmed cases ofclonorchi...In thes paper the authors used the Horseradish peroxidase labelledstaphylococcal protein A(HRP—SPA)in ELISA,for the detection of Clo-norchis sinensis infection.Serum tests were made on 116 confirmed cases ofclonorchiasis,103(88.8%)of them showed positive,while only 6(4.4%)werepositive among 138 healthy people.Samples were collected on filter paperstrips,111(95.7%)cases were positive among 116 comfirmed cases tested,but only 2(1.5%)were positive out of 138 healthy persons.The resultswere similar to those obtained by sheep antihuman IgG.Animal experimentalso showed that the SPA—ELISA can be used for the diagnosis ofclonorchiasis.In an endemic area,stool egg positive rate was 8.8%(62/703).whenchecked with SPA—ELISA,the rate of conformity in both filter paperstrips and stool examinations was 90.3(56/62).Among 641 serum testsfrom individuals negative in stool examinations,only 35(5.5%)reactedpositively.The authors suggested—that SPA—ELISA with soluble Clo-norchis antigens could be used in a large scale seroepidemiological surveyin endemic areas.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
Cytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases,such as tumors,microbial infections,and immunological diseases.Enzyme-linked immunosorbent assa...Cytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases,such as tumors,microbial infections,and immunological diseases.Enzyme-linked immunosorbent assay(ELISA)is one of the most popular methods in cytokine detection,ascribing to the lavish signal amplification methods in the ELISA platform.In addition to classical enzymes,other signal amplifiers such as fluorescent probes,artificial nano-enzymes,and photothermal reagents have been applied to reduce the detection limit and produce more sensitive ELISA kits.Due to the accumulative effect of heat,photothermal reagents are promising materials in the signal amplification of ELISA.However,the lack of efficient photothermal generation material at an aggregate scale may delay the further development of this area.In this contribution,based on an efficient organic photothermal aggregate material,an enzyme-free photothermally amplified fluorescent immunosorbent assay system consisting of an assay microfluidic chip and detecting platform was developed.The photothermal nanoparticles with highly efficient photothermal conversion by harvesting energy via excited-state intramolecular motions and enlarging molar absorptivity were successfully prepared.The detection concentration at 50 pg/mL of interleukin-2 was achieved,realizing a signal improvement of detection limits by 20-fold compared to that of previously reported photothermal ELISA.The microscopic imaging integrated with plane sweeping technology provided high spatial resolution and precision,indicating the potential of achieving high throughput profiling at the microscale.Moreover,as an alternative excitation source,light-emitting diode not only provided a more affordable and miniaturized detection system but also revealed the great feasibility of intramolecular motion-induced photothermy nanoparticles for biological analyses.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
The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for ...The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.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
Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive met...Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay(LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase(NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization(MN) assays.Specificity and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus(DENV), Japanese encephalitis virus(JEV), and hepatitis C virus(HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172–352 amino acids(aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV Ig G detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV Ig G in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.展开更多
Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory int...Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory interference dial,and laboratory precision dial were constructed.The linear range,sensitivity,specificity,precision and other performances of ELISA for detecting syphilis dry blood spot samples were comprehensively evaluated,and the stability of dry blood spot samples at 37℃was detected.In addition,112 suspected syphilis antibody-positive plasma samples were selected as the control,and dry blood spot samples were prepared accordingly.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples and plasma samples in ELISA for syphilis detection were compared,and the consistency and correlation between the two samples were analyzed by the Kappa consistency test and Spearman rank correlation analysis.Results:The results of the linear analysis showed that the serial dilution of dry blood spot samples in ELISA for syphilis antibody ranged from 23to 27,and there was a good linear range[R2=0.9862(P<0.05)].Sensitivity and specificity analysis showed that the positive coincidence rate and negative coincidence rate of the two detection methods were 100%(15/15).The results of the interference dial test showed that ELISA based on dry blood spot samples could accurately detect 6 syphilis antibody samples from 18samples,and the detection accuracy rate was 100.00%(6/6).The results of the precision test showed that the RSD of syphilis antibody detection in dry blood spot samples with different dilution times(23,25and 27)was 0.24%to 3.87%between spots,0.06%to4.07%between batches and 0.49%to 3.88%between days.Within 7 days,the inter-day RSD of dry blood spots with different dilution times(23,25,27)were 0.27%,0.65%and 0.95%,respectively.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples in ELISA detection of syphilis antibody were 96.00%(72/75),100.00%(37/37),100.00%(72/72)and 92.50%,respectively.The results of the Kappa consistency test and Spearman rank correlation analysis showed that the Kappa value of the two methods was 0.941(P<0.01),and the correlation coefficientρbetween the S/CO ratios of the two methods was 0.211(P<0.01).The comparison of S/CO ratio results showed that the distribution characteristics of the S/CO ratio between the two methods were similar,and the ratio distribution was relatively concentrated.Conclusion:using dry blood spot samples to perform ELISA for syphilis detection has good precision,strong anti-interference ability and excellent stability.Although false-positive results appear in weak positive samples,it still has a high application value in ELISA for syphilis antibody detection,which can provide an important reference for disease diagnosis.展开更多
The use of tetra-substituted amino aluminum phthalocyanine (TAAlPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAlPc displayed...The use of tetra-substituted amino aluminum phthalocyanine (TAAlPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAlPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H 2O 2 could rapidly and significantly react with TAAlPc, thus quenching the fluorescence of TAAlPc. The Michaelis-Menten parameters K m and V max were measured to be 2.82×10 -6 mol/L -1 and 6.0×10 -9 mol·L -1·s -1, respectively. In this paper, TAAlPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5-200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.展开更多
Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent...Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-展开更多
BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by th...BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.展开更多
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The control and prevention of pasteurellosis in rabbits which makes the hosts unsuitable for experimental use raised the needs to improve and simplify the procedures of Enzyme-Linked Immunosorbent Assay (ELISA) for detect of antibody against P.multocida. A comparison on the sensitivity and specificity of bacterial culture of antemortem and postmortem samples, complement fixation test and enzyme-linked immunosorbent assay of 11 apparently healthy adult rabbits was conducted. The incidence rates showed 45.45%,54.54% and 72.73% respectively. The sensitivity for the three methods were 0.63, 0.67,and 1.00,and specificity for them were 1.00, 0.67 and 1.00 respectively. Somatic serotypes of isolates of P.multocida from rabbits of three groups (rabbits of group 2 were with clinic signs, those of groups 1 and 3 were apparently healthy) revealed no remarkable differences,and the predominant types were type 3 and type 3, 4. This was somewhat different from the reports derived from other states. As the antigen of different serotype used in ELISA may have different sensitivity and specificity, which is affected also by different preparation method, a type non-specific antigen should be selected to meet such request. The trial of accomplishment of ELISA without positive and negative controls was presented for discussion.
文摘Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.
文摘Enzyme-linked immunosorbent assay (ELISA) is the most widely used method for measuring a single cytokine. Recent developments in cytokine quantification such as multiple arrays measure multiple cytokines simultaneously. Although good correlations between ELISA and multiplex methods have been observed, side by side comparisons are limited. In the present study we hypothesized that ELISA and Luminex techniques are comparable in detecting cytokines in culture medium when pulmonary artery smooth muscle cells (PASMC) are exposed to stress. Primary human PASMC were cultured in modular chambers and exposed to 21% FiO2 and peak inspiratory and positive end expiratory pressure of 24 and 8 cmH2O respectively, and 95% FiO2. At 24 hours, culture medium was collected and assayed for interleukin-6 (IL-6) and IL-8 by quantitative ELISA and by Human Cytokine 25-Plex Panel using a Luminex 200 analyzer. A comparative analysis of agreement between our ELISA and Luminex data was detailed for control and stress conditions using the Bland-Altman plot analysis. Each assay resulted in comparable increased (p < 0.001) levels of IL-6 and IL-8 as compared to control in response to oxidative and biophysical stress. The Bland-Altman analysis demonstrated that 95% of the differences between ELISA and Luminex values were within ±1.96 SD from the mean difference indicated by the 95% limits of agreement for the measurements of IL-6 and IL-8. There was no systematic bias as a function of inflammation level. We conclude that in this cell culture model, ELISA and Luminex are comparable in detecting the levels of IL-6 and IL-8 in the culture medium. If measurements of multiple cytokines are demanded and the amount of sample is limited, Luminex multi-analyte profiling technology is accurate and sensitive.
文摘In thes paper the authors used the Horseradish peroxidase labelledstaphylococcal protein A(HRP—SPA)in ELISA,for the detection of Clo-norchis sinensis infection.Serum tests were made on 116 confirmed cases ofclonorchiasis,103(88.8%)of them showed positive,while only 6(4.4%)werepositive among 138 healthy people.Samples were collected on filter paperstrips,111(95.7%)cases were positive among 116 comfirmed cases tested,but only 2(1.5%)were positive out of 138 healthy persons.The resultswere similar to those obtained by sheep antihuman IgG.Animal experimentalso showed that the SPA—ELISA can be used for the diagnosis ofclonorchiasis.In an endemic area,stool egg positive rate was 8.8%(62/703).whenchecked with SPA—ELISA,the rate of conformity in both filter paperstrips and stool examinations was 90.3(56/62).Among 641 serum testsfrom individuals negative in stool examinations,only 35(5.5%)reactedpositively.The authors suggested—that SPA—ELISA with soluble Clo-norchis antigens could be used in a large scale seroepidemiological surveyin endemic areas.
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.
基金Basic and Applied Basic Research Foundation of Guangdong Province,Grant/Award Number:2023A1515010702National Natural Science Foundation of China,Grant/Award Numbers:31870981,82020108016+2 种基金Innovation and Technology Commission,Grant/Award Number:ITC-CNERC14SC01Research Grants Council,University Grants Committee,Grant/Award Numbers:16306620,GRF 16209820STU Scientific Research Initiation Grant,Grant/Award Number:NTF22023。
文摘Cytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases,such as tumors,microbial infections,and immunological diseases.Enzyme-linked immunosorbent assay(ELISA)is one of the most popular methods in cytokine detection,ascribing to the lavish signal amplification methods in the ELISA platform.In addition to classical enzymes,other signal amplifiers such as fluorescent probes,artificial nano-enzymes,and photothermal reagents have been applied to reduce the detection limit and produce more sensitive ELISA kits.Due to the accumulative effect of heat,photothermal reagents are promising materials in the signal amplification of ELISA.However,the lack of efficient photothermal generation material at an aggregate scale may delay the further development of this area.In this contribution,based on an efficient organic photothermal aggregate material,an enzyme-free photothermally amplified fluorescent immunosorbent assay system consisting of an assay microfluidic chip and detecting platform was developed.The photothermal nanoparticles with highly efficient photothermal conversion by harvesting energy via excited-state intramolecular motions and enlarging molar absorptivity were successfully prepared.The detection concentration at 50 pg/mL of interleukin-2 was achieved,realizing a signal improvement of detection limits by 20-fold compared to that of previously reported photothermal ELISA.The microscopic imaging integrated with plane sweeping technology provided high spatial resolution and precision,indicating the potential of achieving high throughput profiling at the microscale.Moreover,as an alternative excitation source,light-emitting diode not only provided a more affordable and miniaturized detection system but also revealed the great feasibility of intramolecular motion-induced photothermy nanoparticles for biological analyses.
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.
基金This work was supported by the following grants:the National Major Project for Control and Prevention of Infectious Disease in China(No.2018ZX10101002 and 2018ZX10732401)the National Key Research and Development Program of China(No.2016YFD0500301 and 2017YFC1200503)。
文摘The Middle East respiratory syndrome(MERS)is a lethal zoonosis caused by MERS coronavirus(MERS-CoV)and poses a significant threat to public health worldwide.Therefore,a rapid,sensitive,and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness.Here,we evaluated various novel luciferase immunosorbent assays(LISA)based on nucleocapsid protein(NP)as well as fragments derived from spike protein(S)including subunit 1(S1),N terminal domain(NTD),receptorbinding domain(RBD)and subunit 2(S2)of S for the detection of MERS-CoV-specific IgG.Fusion proteins,including nanoluciferase(NLuc)and various fragments derived from the NP or S protein of MERS-CoV,were expressed in human embryonic kidney 293 T cells.LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins.Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR,commercial S1-based ELISA,and pseudovirus particle neutralization test(ppNT).Our results showed that the S1-,RBD-,and NP-LISAs were more sensitive than the NTD-and S2-LISAs for the detection of anti-MERS-CoV IgG.Furthermore,the S1-,RBD-,and NP-LISAs were more sensitive(by at least 16-fold)than the commercially available S1-ELISA.Moreover,the S1-,RBD-,and NPLISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV(OC43,229E,HKU1,NL63)-infected patients.More importantly,these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels,monkeys,and mice,among which the RBD-LISA exhibited excellent performance.The results of this study suggest that the novel MERS-CoV RBD-and S1-LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples.These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.
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the National Key Research and Development Program of China(2016YFD0500301)the National Major Project for Control and Prevention of Infectious Disease in China(2018ZX10101002 and 2017YFC1200503)+1 种基金the Bureau of Science and Information Technology of Guangzhou Municipality,China(201604020011,201704020219)National Key R&D Program of China(2018ZX10732401).
文摘Zika virus(ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications;therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay(LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase(NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization(MN) assays.Specificity and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus(DENV), Japanese encephalitis virus(JEV), and hepatitis C virus(HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172–352 amino acids(aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV Ig G detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV Ig G in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.
文摘Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood spots.Methods:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory interference dial,and laboratory precision dial were constructed.The linear range,sensitivity,specificity,precision and other performances of ELISA for detecting syphilis dry blood spot samples were comprehensively evaluated,and the stability of dry blood spot samples at 37℃was detected.In addition,112 suspected syphilis antibody-positive plasma samples were selected as the control,and dry blood spot samples were prepared accordingly.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples and plasma samples in ELISA for syphilis detection were compared,and the consistency and correlation between the two samples were analyzed by the Kappa consistency test and Spearman rank correlation analysis.Results:The results of the linear analysis showed that the serial dilution of dry blood spot samples in ELISA for syphilis antibody ranged from 23to 27,and there was a good linear range[R2=0.9862(P<0.05)].Sensitivity and specificity analysis showed that the positive coincidence rate and negative coincidence rate of the two detection methods were 100%(15/15).The results of the interference dial test showed that ELISA based on dry blood spot samples could accurately detect 6 syphilis antibody samples from 18samples,and the detection accuracy rate was 100.00%(6/6).The results of the precision test showed that the RSD of syphilis antibody detection in dry blood spot samples with different dilution times(23,25and 27)was 0.24%to 3.87%between spots,0.06%to4.07%between batches and 0.49%to 3.88%between days.Within 7 days,the inter-day RSD of dry blood spots with different dilution times(23,25,27)were 0.27%,0.65%and 0.95%,respectively.The clinical sensitivity,clinical specificity,positive predictive value and negative predictive value of dry blood spot samples in ELISA detection of syphilis antibody were 96.00%(72/75),100.00%(37/37),100.00%(72/72)and 92.50%,respectively.The results of the Kappa consistency test and Spearman rank correlation analysis showed that the Kappa value of the two methods was 0.941(P<0.01),and the correlation coefficientρbetween the S/CO ratios of the two methods was 0.211(P<0.01).The comparison of S/CO ratio results showed that the distribution characteristics of the S/CO ratio between the two methods were similar,and the ratio distribution was relatively concentrated.Conclusion:using dry blood spot samples to perform ELISA for syphilis detection has good precision,strong anti-interference ability and excellent stability.Although false-positive results appear in weak positive samples,it still has a high application value in ELISA for syphilis antibody detection,which can provide an important reference for disease diagnosis.
文摘The use of tetra-substituted amino aluminum phthalocyanine (TAAlPc) as a new red-region fluorescent substrate for horseradish peroxidase (HRP)-based enzyme-linked immunosorbent assay was investigated. TAAlPc displayed an excitation maximum at 610 nm and emission maximum at 678 nm in a strong acidic medium. In the presence of HRP, trace amounts of H 2O 2 could rapidly and significantly react with TAAlPc, thus quenching the fluorescence of TAAlPc. The Michaelis-Menten parameters K m and V max were measured to be 2.82×10 -6 mol/L -1 and 6.0×10 -9 mol·L -1·s -1, respectively. In this paper, TAAlPc was used in an HRP-based enzyme-linked immunosorbent assay (ELISA) of α-fetoprotein (AFP) in human serum with satisfactory results. AFP could be determined in the concentration range of 0.5-200 ng/mL with a detection limit of 0.2 ng/mL, which was close to that of radioimmunoassay. The advantage of proposed method was strongly minimizing the interference resulting from background fluorescence or scattering light and had a high analytical sensitivity.
文摘Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-
基金Supported by the German Federal Ministry of Education and Research(BMBF-Wachstumskern-PRAEMED.BIO),03WKDB2Csupported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences,BO/00232/17/5+1 种基金Research Grants of National Research Development and Innovation Office,K115818/2015/1New National Excellence Program of the Ministry of Human Capacities,ÚNKP-18-4 Bolyai Plus.
文摘BACKGROUND Defective neutrophil regulation in inflammatory bowel disease(IBD)is thought to play an important role in the onset or manifestation of IBD,as it could lead to damage of the intestinal mucosal barrier by the infiltration of neutrophils in the inflamed mucosa and the accumulation of pathogens.Like neutrophils in the context of innate immune responses,immunoglobulin A(IgA)as an acquired immune response partakes in the defense of the intestinal epithelium.Under normal conditions,IgA contributes to the elimination of microbes,but in connection with the loss of tolerance to chitinase 3-like 1(CHI3L1)in IBD,IgA could participate in CHI3L1-mediated improved adhesion and invasion of potentially pathogenic microorganisms.The tolerance brake to CHI3L1 and the occurrence of IgA autoantibodies to this particular target,the exact role and underlying mechanisms of CHI3L1 in the pathogenesis of IBD are still unclear.AIM To determine the predictive potential of Ig subtypes of a novel serological marker,anti-CHI3L1 autoantibodies(aCHI3L1)in determining the disease phenotype,therapeutic strategy and long-term disease course in a prospective referral cohort of adult IBD patients.METHODS Sera of 257 Crohn’s disease(CD)and 180 ulcerative colitis(UC)patients from a tertiary IBD referral center of Hungary(Division of Gastroenterology,Department of Internal Medicine,Faculty of Medicine,University of Debrecen)were assayed for IgG,IgA,and secretory IgA(sIgA)type aCHI3L1 by enzyme-linked immunosorbent assay using recombinant CHI3L1,along with 86 healthy controls(HCONT).RESULTS The IgA type was more prevalent in CD than in UC(29.2%vs 11.1%)or HCONT(2.83%;P<0.0001 for both).However,sIgA subtype aCHI3L1 positivity was higher in both CD and UC patients than in HCONT(39.3%and 32.8%vs 4.65%,respectively;P<0.0001).The presence of both IgA and sIgA aCHI3L1 antibodies was associated with colonic involvement(P<0.0001 and P=0.038,respectively)in patients with CD.Complicated disease behavior at sample procurement was associated with aCHI3L1 sIgA positivity(57.1%vs 36.0%,P=0.009).IgA type aCH3L1 was more prevalent in patients with frequent relapse during the disease course in the CD group(46.9%vs 25.7%,P=0.005).In a group of patients with concomitant presence of pure inflammatory luminal disease and colon involvement at the time of diagnosis,positivity for IgA or sIgA type aCH3L1 predicted faster progression towards a complicated disease course in time-dependent models.This association disappeared after merging subgroups of different disease locations.CONCLUSION CHI3L1 is a novel neutrophil autoantigenic target in IBD.The consideration of antibody classes along with location-based prediction may transform the future of serology in IBD.
