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.

甲氧基有机磷杀虫剂广谱特异性抗体的制备

以通用结构O,O-二甲基硫代磷酸酯为目标检测基团,制备针对甲氧基有机磷杀虫剂的广谱特异性抗体。利用O,O-二甲基硫代磷酸钠和氯乙酸合成半抗原S-羧甲基-O,O-二甲基二硫代磷酸酯(CMP),通过混合酸酐法(MA)和活性酯法(AE)分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联。CMP-MA-BSA、CMP-AE-BSA作为免疫原均获得了免疫应答。其中,CMP-AE-BSA所获得的抗血清效价最高,为256000。研究了有机溶剂种类及含量、pH因素对ELISA曲线的影响,确定CMP酶联免疫分析方法(ELISA)的最佳工作条件,CMP的最低检测浓度为0.076μg/L,IC50为93.97μg/L。以14种常见有机磷杀虫剂为对象,检测抗体对其交叉反应,测定结果表明:抗体对马拉硫磷、稻丰散、乐果、亚胺硫磷、倍硫磷、甲基嘧啶磷、甲基对硫磷、杀螟硫磷及杀扑磷等均有识别作用。IC50分别为69.92、136.90、230.39、416.84、508.57、510.38、607.21、835.30和850.21μg/L。该技术可用于多种甲氧基有机磷杀虫剂的快速定性或半定量检测。

人参皂苷Rb1单克隆抗体的制备与鉴定

用人参皂苷Rb1分别与牛血清白蛋白（BSA）和卵清蛋白（OVA）反应合成了交联抗原GRb1-BSA（GRb1与BSA的结合比为10：1）和GRb1—OVA（GRb1与OVA的结合比为8：1）。以GRb1—BSA为免疫原，分5次免疫3只BAuyC小鼠，取免疫脾细胞与小鼠骨髓瘤细胞进行融合，Hat筛选，有限稀释法克隆。建立分泌单克隆抗体的杂交瘤细胞系。采用ELISA（酶联免疫吸附测定）间接法和竞争法检测抗体的特异性。结果获得了一只小鼠分泌抗体效价达1：16000，并获得两株分泌GRb1的单克隆抗体的细胞系，分别命名为1F7和1G3，单抗检测显示在0．01～1μg／mL呈线性关系，GRb1的检测范围达50～350ng／mL，两株单抗针对相同的抗原决定簇，制备的单克隆抗体可用于GRb1含量的检测和分离。

4种偶联率的苦马豆素-HSA合成及其对小鼠免疫原性研究

研究不同偶联率的SW-HSA对小鼠免疫原性的影响,为获得理想的SW人工抗原奠定基础。首先将SW与活性酯在70℃条件下反应制备季铵盐,然后将季铵盐与HSA冰浴搅拌反应12 h,通过调节季铵盐与HSA的摩尔比制备SW-HSA,透析并冷冻干燥,采用紫外分光光度法测定SW-HSA偶联率。将20只昆系小白鼠随机分为A、B、C、D和E组,每组4只。用4种不同偶联率的SW-HSA对前4组小鼠进行免疫,首次免疫抗原量为0.050 mg/只,第30天进行第2次免疫,抗原量与首免相同,以后每隔20 d进行1次,抗原量均为前一次免疫所用抗原量的1.5倍,E组为对照,注射等量生理盐水。从第3次免疫后ELISA检测血清中抗SW抗体效价。紫外光谱测定结果显示,合成的SW-HSA偶联率分别为18.6、12.5、13.5和32.6。ELISA测定结果显示,从第3次免疫后,A、B、C、D组试验小鼠血清的抗SW抗体效价均呈上升趋势,在第5次免疫后,测得其抗SW抗体效价分别为28、29、29、29,达到最高峰。其中D组的抗SW抗体效价在第4次免疫后首先达到29,并在第5次免疫后维持在相同水平。各组试验小鼠血清的抗SW抗体效价于第6次免疫时开始逐渐下降。试验结果证实,不同偶联率的SW-HSA均可诱导小鼠产生抗体,较大偶联率的SW-HSA可以更好地提高抗SW抗体水平。

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.

山萸肉饮片中黄曲霉毒素B_1含量及限度的研究

目的测定山萸肉饮片中黄曲霉毒素B1(AFB1)的含量并建立限度标准。方法间接竞争酶联免疫吸附法(ELISA)。结果山萸肉饮片均有不同程度的污染黄曲霉毒素B1(AFB1),炮制后降低,但都在国家规定的食品类AFB1限度范围之内。结论山萸肉饮片中黄曲霉毒素B1的含量限度标准初步确定为≤5μg/kg,与国家食品类限度标准相同。

赭曲霉毒素A模拟抗原表位及噬菌体展示酶联免疫吸附分析法的建立

以驴抗鼠二抗包被微孔板,以捕获方式包被抗赭曲霉毒素A（OTA）单克隆抗体,利用噬菌体随机七肽库筛选OTA模拟抗原表位,并以其替代检测抗原,建立了基于噬菌体展示技术的酶联免疫吸附分析（Phage ELISA）检测OTA的方法。结果表明,筛选获得的模拟OTA抗原表位七肽氨基酸序列为GMSWMMA。基于模拟表位噬菌体建立的Phage ELISA方法半数抑制浓度（IC50值）为（0.15±0.02）ng/m L,检测OTA的线性范围为0.03~0.50 ng/m L,OTA的检出限为0.03 ng/m L,且Phage ELISA与其它4种常见真菌毒素（黄曲霉毒素B1、伏马毒素B1、脱氧雪腐镰刀菌烯醇及玉米赤霉烯酮）无交叉反应。大米样品OTA加标实验表明,批內加标回收率为97.0%~115.2%,批间加标回收率为107.2%~123.1%,与ELISA试剂盒检测结果比较,无显著性差异。

多剂量皮下注射重组人促红细胞生成素在健康人体的药代动力学

目的观察不同剂量国产重组人促红细胞生成素(rhEPO)皮下注射在健康人体的药代动力学.方法12名健康志愿者随机分为2个剂量组(50和150 IU·kg-1),每组6例.分别皮下注射rhEPO,共7次.用酶联免疫吸附实验法测定血清rhEPO浓度.结果给药前12名健康志愿者血清EPO基础水平波动于3.75～13.75 mU·ml-1.2组谷浓度(Cmin)在给药4～6次后,达稳态水平.高剂量组首次和末次给药后的AUC(0-96h)分别是低剂量组的2.8倍和3.0倍,Cmax分别是3.1倍和3.6倍.第1次给药后,每个受试者的tmax和t1/2等药代动力学参数与末次给药后比较无明显差异.结论剂量在50～150 IU·kg-1,多次皮下注射rhEPO,血药浓度水平与给药剂量呈正相关;药物吸收和清除基本上无时间依赖性;在给药7次内,未观察到体内蓄积倾向.

化学发光成像分析法定量检测人尿液中β-人绒毛膜促性腺激素

采用一种灵敏、简单的方法, 可实现对β-人绒毛膜促性腺激素(β-HCG)的高通量检测.本法具有酶联免疫吸附(ELISA)法的特异性、增强化学发光法(ECL)的高灵敏度及化学发光成像分析高样品通量等优点.冷却电感耦合 (CCD)成像系统被用于检测增强化学发光信号.发光强度值与β-HCG在12.5 ～ 400 mIU/mL范围内呈线性关系;检出限为4 mIU/mL.测定了妇女尿液中β-HCG的含量,分析结果与医院的诊断结果一致.

罗丹明123人工抗原的合成及抗体的酶联免疫检测

为建立罗丹明B(rhodamine B)的免疫分析方法,采用戊二醛法,将半抗原罗丹明123与载体牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联制备免疫抗原R123-B S A和包被抗原R123-OVA。经动物免疫实验证实人工抗原合成,并制备抗罗丹明123的多克隆抗体,此抗体同时能够检测食品中的罗丹明B,且最低检测限为0.001ng/mL。