Detection of Atrazine Residue in Food Samples by a Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay

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

Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

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.

Determnation of ochratoxin A in grain by monoclonal antibody－based enzyme－linked immunosorbent assay

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.

ENZYME-LINKED IMMUNOSORBENT ASSAY OF HUMAN PLACENTA TYPE GLUTATHIONE S-TRANSFERASE AND ITS APPLICATION IN THE DIAGNOSIS OF HEPATOCARCINOMA

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%.

Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

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.

SERODIAGNOSIS OF CLONORCHIASIS BY ENZYME—LINKED IMMUNOSORBENT ASSAY WITH HRP—SPA

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.

Establishment of enzyme-linked immunosorbent assay for beef and lamb contents in cooked meat

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.

Enzyme-free photothermally amplified fluorescent immunosorbent assay(PAFISA)for sensitive cytokine quantification

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.

Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay for the Analysis of Jasmonates in Plants

Methyl jasmonate （MeJA） and its free-acid form, jasmonic acid （JA） are naturally occurring plant growth regulators widely distributed in higher plants. In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples, a monoclonal antibody （MAb） （designated as MAb 3E5D7C4B6） against MeJA was derived from a JA- bovine serum albumin （BSA） conjugate as an immunogen. The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 ×10^-9 M. MAb3E5D7C4B6 is very specific to MeJA. It was used to develop a direct competitive enzyme-linked immunosorbent assay （dcELISA）, conventional and simplified indirect competitive ELISAs （icELISA）. JA was derivatized into MeJA for the ELISA analysis. The IC50 value and detection range for MeJA were, respectively, 34 and 4-257 nglmL by the conventional icELISA, 21 and 3-226 nglmL by the simplified icELISA and 5.0 and 0.7-97.0 nglmL by the dcELISA. The dcELISA was more sensitive than either the conventional or simplified icELISA. The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves. The increased jasmonates content indicated its role in response to the drought stress and pathogens.

Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol

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.

Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

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.

Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus

Background Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients’ sera.Methods Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. Results The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. Conclusion The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

A novel luciferase immunosorbent assay performs better than a commercial enzyme-linked immunosorbent assay to detect MERS-CoV specific IgG in humans and animals

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.

DETECTION OF LEUKEMIA INHIBITORY FACTOR (LIF) BY A ENZYME-LINKED IMMUNOSORBENT ASSAY

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-

Enzyme-Linked Immunosorbent Assay for Detecting Syphilis Dry Blood Spot Samples

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.

Tetra-substituted Amino Aluminum Phthalocyanine as a New Red-region Fluorescent Substrate for Horseradish Peroxidase Based Enzyme-linked Immunosorbent Assay

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.

A sensitive and convenient enzyme-linked immunosorbent assay method in serum MG7 antigen detection in gastric cancer

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-

Comparison of chemiluminescence enzyme immunoassay based on magnetic microparticles with traditional colorimetric ELISA for the detection of serum α-fetoprotein

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).

Von Willebrand Factor Antigen and ADAMTS13 Activity Assay in Pregnant Women and Severe Preeclamptic Patients

The present study examined von Willebrand factor (vWF) levels and ADAMTS13 activity in pregnant and severe preeclamptic women in order to shed light on the prothrombotic state in severe preeclampsia.Thirty healthy women of childbearing age,22 second trimester pregnant women,30 third trimester pregnant women and 10 severe preeclamptic patients were recruited in this study.ADAMTS13 activity was determined by the FRETS-vWF73 assay and vWF antigen (vWF:Ag) levels by an enzyme-linked immunosorbent assay.The results showed that there were statistically significant differences in plasma vWF antigen levels between the severe preeclamptic and third trimester pregnant women,between third and second trimester pregnant women (P【0.05).The third trimester pregnant women had significantly lower plasma ADAMTS13 activity than second trimester pregnant women (P【0.05).Nevertheless,no significant differences in plasma ADAMTS13 activity were found between severe preeclamptic patients and the third trimester pregnant women (P】0.05).In conclusion,plasma ADAMTS13 activity is normal in severe preeclampsia despite the increased vWF:Ag levels.Prothrombotic state is involved in the pathogenesis of severe preeclampsia,as a result of endothelial injury.

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.