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

Quantitative determination of erlotinib in human serum using competitive enzyme-linked immunosorbent assay

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.

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.

Development of a competitive enzyme-linked immunosorbent assay for therapeutic drug monitoring of afatinib

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.

Enzyme-linked Immunosorbent Assay for Detection of Anti-idiotype Antibodies to Antibodies to Ligand of Nicotinic Acetylcholine Receptor in Sera of Patients with Myasthenia Gravis

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.

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.

A PRELIMINARY STUDY OF SERUM GLYCOCONIUGATES IN PATIENTS WITH CANCER USING THE ENZYME-LINKED LECTIN ASSAY

After primary analyses on the serum glycocon-jugates of lung cancer and normal individul using the enzyme-linked lectin assay (ELLA) with 12 kinds of lectins, PHA and LCA were selected to further study in the sera of 8 kinds of cancers, 4 kinds of non-malignant disease and 1 kind of postoperative cancer. It was found that the test values of 7 kinds of cancers with PHA or LCA were significantly higher than that of the normal (P<0.01); the values of 4 kinds of non-malignant diseases with PHA were not higher (P>0.05); the values of the postoperative cancer with PHA were obviously lower than that of the preoperative (P<0.05). The results showed that the serum glycoconjugates which can bind to PHA seemed related to the cancerous existence in human bodies. The significance of the findings was discussed.

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%.

A Comparison of Enzyme-Linked Immunosorbent Assay versus Multiplex Methodology Using an <i>in Vitro</i>Model of Pulmonary Hypertension and Inflammation

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.

Immunoregulatory Effect and Mechanism of Epigallocatechin-3-Gallate in A Mouse Oral Cancer Model

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.

Location-based prediction model for Crohn’s disease regarding a novel serological marker,anti-chitinase 3-like 1 autoantibodies

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.

Expression of Bt Protein in Transgenic Pest-resistant Rice

[Objective] The aim of this study was to study on expression of Bt protein in transgenic pest-resistant rice. [Method] Enzyme-linked immunosorbent assay （ELISA） was used to measure Bt protein expression in different tissues of transgenic pest-resistant rice at same growth stage. [Result] Absolute content of Bt protein from high to low was as follows： leaves 〉 immature seeds and glumes 〉 roots 〉 stems in different tissues of transgenic rice in grain-filling stage; Bt protein content of trans- genic rice changed a little in different growth stages （including tillering stage, booting stage, and grain-filling stage）; in general, its level declined a little in later growth stage, but the resistibility would not be influenced significantly. [Conclusion] The ex- periment is significant for pest prevention and transgenic rice breeding.

食品中丙烯酰胺检测方法的研究进展

丙烯酰胺是在食品高温加工过程中产生的小分子有机化合物,具有致癌性。从样品提取、衍生化、净化、富集等前处理过程以及对检测器的选择出发,总结国内外近年来用于检测丙烯酰胺的方法,如从传统的气相色谱、液相色谱及其联用技术,到新兴开发的分子印迹技术、酶联免疫吸附和生物传感器等新检测技术。并根据其适用范围和操作条件,对各分析方法的优点和不足进行讲述,最后对将来丙烯酰胺检测方法发展新思路提供策略和依据。

Preparation and Application of Monoclonal Antibodies Specific for Salicylic Acid

Salicylic acid (SA) is widely distributed in many monocots and dicots and has many physiological effects. It can induce heat production in the thermogenic inflorescences of Arum lily([1]), block the biosynthesis of ethylene, and more attractively, it seems to be an important natural signal molecule in the induction of systemic acquired resistance (SAR) in tobacco, cucumber and other plants([2,3]). Studies in recent years showed that SA was also intimately related to the resistance of plants to aboitic stress, for example, SA increased chilling resistance of maize seedlings. Hence, SA has been accepted as a kind of new plant hormones. Up to date, the quantification of SA usually has been performed by HPLC[4,5], which often needs a large quantity of sample and a verbose pretreatment. Compared to HPLC, immunoassays, including radio-immunoassays (RIA) and enzyme-immunoassays (EIA), are easy to perform and have been widely used in the quantification of other plant hormones, such as IAA([6]), ABA([7,8]), GAs([9]), cytokinins et al([10]), and jasmonic acid (JA)([11]), and other low-molecular-weight, none-immunogenic compounds in plants([12]). Till now, only an indirect enzyme-linked immunosorbent assay (ELISA) for SA based on polyclonal antibodies (PAbs) has been developed by our group([13]), although Bennett et al([14]) had prepared SA PAbs using 4-aminosalicylic acid linked to KLH as immunogen in goat. However, the sensitivity of the ELISA we established formerly was relatively low, and also relatively larger quantity of sample is needed than other ELISAs for plant hormones. In this paper, an ELISA for SA based on monoclonal antibody raised against SA-NH-CH2-NH-KLH was introduced, and the fluctuation of SA content in cucumber leaves after inoculated with Pseudomonas syringae pv. syringae was determined.

乙型肝炎表面抗原不同检测方法的优势比较分析

目的：比较化学发光微粒子免疫分析法（CMIA）和酶联免疫吸附法（ELISA）用于乙型肝炎（乙肝）表面抗原检测的优势效果。方法选取2011年3月～2013年4月采用ELISA法与CMIA法对386例患者进行了乙肝表面抗原检测的对比研究。结果 CMIA法检测乙肝表面抗原的阳性率为51.3%，ELISA法阳性率为43.5%，CMIA法阳性率明显更高，与ELISA法比较差异有统计学意义（P〈0.05）；CMIA法的灵敏度与特异性均明显高于ELISA法（P〈0.05）。结论与ELISA法比较，CMIA法对乙肝表面抗原检测在阳性率、灵敏度以及特异性等方面均存在有比较明显的优势，故建议将CMIA法作为临床检测乙肝表面抗原的首选方法而推广应用。

Primary Study of Egg Yolk Antibody for Detection of King Cobra Venom Antigens

Aim To study whether antivenom from laying hens can be used for the detection of venom antigens, Methods Chickens （white Leghorn） were immunized with detoxicated king cobra venom by formaldehyde and egg yolk immunoglobulin （IgY） isolated from yolk; IgY was labelled with the horseradish peroxidase （HRP）. Experimental condition and parameters were determined by chessboard test. The specificity, sensitivity, precision, and stability of this method were assayed in the experiment. Results This method could detect as low as 32 μg· L^-1 of the king cobra antigens. A good linear relation was found within 32 ～ 750 μg· L^-1 of king cobra venom concentrations （ r = 0. 963）. There was no cross reactivity for the reagents with Agkistrodon acutus Guenther venom or Vipera russelli siamensis Smith venom;slight cross reactivity .with Bungarus multicinctus Blyth venom or Bungarus fasciatus Chmeider venom; and notable cross reactivity with cobra venom. The average intra-assay relative standard deviation （RSD） was 1% - 3%, and the inter-assay RSD was less than 8%. The reagents （including IgY and HRP-IgY） were stable; no differences （P 〉 0.05） were observed for the detection of venom antigens when the reagents were stored at 37 ℃ up to 6 d. Conclusion IgY is a good reagent for diagnosis of snakebite after eliminating the genus cross reactivity.

鼻息肉组织中T淋巴细胞功能的研究

鼻息肉是多因素引发和介导的一种炎症性疾病（[1]）,多种炎性细胞浸润是其主要病理学特征（[2]）。我们前期的研究发现,鼻息肉组织中可见大量树突状细胞浸润[3],树突状细胞将抗原递呈给T淋巴细胞后,两者通过一系列的相互作用,介导免疫反应,而T细胞的分化失衡在鼻息肉发病过程中起到关键性作用（[4]）。然而,鼻息肉组织中T淋巴细胞的功能状态目前尚不明确。

Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma

Glypican-3(GPC3)is a cell surface oncofetal proteoglycan that is anchored by glycosylphosphatidylinositol.Whereas GPC3 is abundant in fetal liver,its expression is hardly detectable in adult liver.Importantly,GPC3is overexpressed in hepatocellular carcinoma(HCC),and several immunohistochemical studies reported that overexpression predicts a poorer prognosis for HCC patients.Therefore,GPC3 would serve as a useful molecular marker for HCC diagnosis and also as a target for therapeutic intervention in HCC.Indeed,some immunotherapy protocols targeting GPC3 are under investigations;those include humanized anti-GPC3cytotoxic antibody,peptide vaccine and immunotoxin therapies.When considering the clinical requirements for GPC3-targeting therapy,companion diagnostics to select the appropriate HCC patients are critical,and both immunohistochemical analysis of tissue sections and measurement of serum GPC3 level have been suggested for this purpose.This review summarizes current knowledge regarding the clinical implication of GPC3detection and targeting in the management of patients with HCC.

Clinical value of fecal calprotectin in determining disease activity of ulcerative colitis

AIM： To investigate possibility and clinical application of fecal calprotectin in determining disease activity of ulcerative colitis （UC）. METHODS： The enzyme-linked immunosorbent assay （ELISA） was used to measure the concentrations of calprotectin in feces obtained from 66 patients with UC and 20 controls. C-reactive protein （CRP）, erythrocyte sedimentation rate （ESR）, acid glycoprotein （AGP） were also measured and were compared with calprotectin in determining disease activity of UC. The disease activity of UC was also determined by the Sutherland criteria. RESULTS： The fecal calprotectin concentration in the patients with active UC was significantly higher than that in the inactive UC and in the controls （402.16 ± 48.0 μg/g vs 35.93 ± 3.39 μg/g, 11.5 ± 3.42 μg/g, P 〈 0.01）. The fecal calprotectin concentration in the inactive UC group was significantly higher than that in the control group （P 〈 0.05）. A significant difference was also found in the patients with active UC of mild, moderate and severe degrees. The area under the curve of the receiver operating characteristics （AUCR^c） was 0.975, 0.740, 0.692 and 0.737 for fecal calprotectin, CRP, ESR and AGP, respectively. There was a strong correlation between the fecal calprotectin concentration and the endoscopic gradings for UC （r = 0.866, P 〈 0.001）. CONCLUSION： Calprotectin in the patient＇s feces can reflect the disease activity of UC and can be used as a rational fecal marker for intestinal inflammation in clinical practice. This kind of marker is relatively precise, simple and noninvasive when compared with other commonlyused markers such as CRP, ESR and AGP.