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.

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.

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.

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.

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%.

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.

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-

软骨藻酸检测方法研究进展

综述了软骨藻酸的检测方法,包括酶联免疫吸附法、高效液相色谱法以及高效液相色谱-串联质谱法等方法的原理及优缺点,同时介绍了软骨藻酸在贝类、拟菱形藻、血液、尿液等样本的检测方法及其应用,生物传感器法、分子印迹法等新型技术也被用于软骨藻酸的检测,近年来长期慢性低剂量摄入软骨藻酸对生物体的影响受到广泛关注,可作为今后研究重点。

早期推拿后大鼠24 h内疼痛变化特点的研究

目的通过机械、温度伤害感受变化评价早期推拿镇痛启动后,慢性压迫性神经损伤(minor CCI)模型大鼠24 h内疼痛变化情况及疗效最优时间点,为推拿镇痛启动疗效提供支撑。方法将49只SD大鼠随机分为7组,即假手术组、模型组、推拿后即刻组、推拿后6 h组、推拿后12 h组、推拿后18 h组与推拿后24 h组。模型组和推拿各亚组建立minor CCI模型。推拿各亚组在造模后7 d进行1次三法三穴干预;模型组与假手术组进行抓握束缚。干预结束后对推拿各亚组、模型组及假手术组先后进行冷敏阈值(CST)、机械缩足反射阈值(PWMT)和热缩足反射潜伏期(TWL)测试不同性质痛觉的变化;取血清进行ELISA测试白介素-10(IL-10)和肿瘤坏死因子(TNF-α)的变化;取造模侧背根神经节(DRG)进行qPCR检测;观察TNF-α、TRPV1和TRPA1基因表达的变化。结果行为学检测显示CST、PWMT、TWL均有不同程度的降低或延长;ELISA检测大鼠血清IL-10、TNF-α显示明显降低;qPCR检测显示TNF-α、TRPV1、TRPA1基因表达均减少,但三种检测方法结果显示指标变化显著时间点均不相同。结论对于CST、PWMT、TWL三种不同伤害性感受阈值,推拿干预后24 h内的镇痛疗效显著时间点不同;推拿可通过降低IL-10、TNF-α、TRPV1和TRPA1的表达发挥即刻镇痛和持续镇痛作用。

比较化学发光法与酶联免疫吸附试验(ELISA法)检测乙肝两对半的结果分析

目的探讨化学发光法与酶联免疫吸附试验(ELISA法)在乙肝两对半检测上的结果。方法选取2023年1月至2023年12月本院就诊的100例乙型肝炎病毒携带者,均对就诊患者进行血清标本采集,并分别实行化学发光法与ELISA法检测乙肝两对半。以病理结果为金标准,对比两种检查结果。结果ELISA法的乙肝两对半阳性率比化学发光法低,差异明显(P<0.05);ELISA法检测结果22例阳性,78例阴性;化学发光法检测结果69例阳性,31例阴性。化学发光法在乙肝两对半阳性抗体上的灵敏度、准确性和阳性预测值比ELISA法高(P<0.05),其特异度、阴性预测值较ELISA法差异无统计学意义(P>0.05)。结论对乙肝两对半检测上选择化学发光法比酶联免疫吸附试验法更具有良好优势,而且化学发光法作为一种半定量检验,更具临床检测价值和推广意义。

酶联免疫法和金标法对HIV抗体的检测结果比较

目的 探讨酶联免疫法和金标法对HIV抗体的检测结果,明确针对艾滋病更为有效的临床筛查手段。方法选取264例疑似艾滋病患者,采集入组患者静脉血并进行酶联免疫法与金标法检测HIV抗体。以临床诊断结果为准,比较酶联免疫法和金标法与临床检查结果的一致性;比较酶联免疫法和金标法对艾滋病的诊断效能。结果 264例疑似艾滋病患者中,经临床筛查试验和确证试验确诊225例。κ检验分析结果显示,酶联免疫法检查结果与临床诊断结果具有高度一致性(κ=0.897, P <0.05);金标法检查结果与临床诊断结果具有高度一致性(κ=0.902, P <0.05)。酶联免疫法与金标法诊断艾滋病的灵敏度、特异性与准确率比较,差异均无统计学意义(P>0.05)。结论 酶联免疫法和金标法对HIV抗体均具有较好的检出效果,可为艾滋病临床诊断提供较为准确的数据参考,临床检测中可根据患者自身情况选择相应的检测方法,必要时还可进行联合检测。

电化学发光法和酶联免疫法在乙型肝炎病毒血清学检验中的应用效果分析

目的:探究电化学发光法(ECLIA)和酶联免疫法(ELISA)在乙型肝炎病毒(HBV)血清学检验中的应用效果。方法:选取2022年3月—2023年3月西宁市第三人民医院收治的108例疑似乙型肝炎(简称乙肝)患者作为研究对象。所有研究对象行ECLIA、ELISA检验,以实时荧光定量聚合酶链式反应(qPCR)为“金标准”,比较两种检验方法的诊断效能。结果:108例疑似乙肝患者经qPCR确诊79例。ECLIA灵敏度与准确率高于ELISA,差异有统计学意义(P<0.05);两种检验方法特异度比较,差异无统计学意义(P=0.640)。ECLIA乙肝E抗体(HBeAb)、乙肝表面抗原(HBsAg)、乙肝E抗原(HBeAg)、乙肝核心抗体(HBcAb)阳性检出率高于ELISA,差异有统计学意义(P<0.05);两种检验方法乙肝表面抗体(HBsAb)阳性检出率比较,差异无统计学意义(P=0.524)。79例确诊病例中HBsAg阳性42例。HBsAg 1.01~3.00 IU/mL时,两种检验方法HBsAg检出率比较,差异无统计学意义(P>0.05);HBsAg 0.06~1.00 IU/mL时,ECLIA HBsAg检出率高于ELISA,差异有统计学意义(P<0.05)。结论:ECLIA在HBV血清学检验中的应用效果优于ELISA,诊断效能及低水平HBsAg检出率更高。

酶联免疫法测定甘精胰岛素中单链前体残留研究

目的 建立酶联免疫法检测甘精胰岛素中单链前体残留,为甘精胰岛素药物质量控制和安全性评价提供新方法。方法采用双抗夹心酶联免疫法,以甘精胰岛素原为标准品,建立单链前体残留检测方法,验证方法的重复性、准确度、范围和精密度,并应用该方法检测9批甘精胰岛素原料药。结果在0.156~10.00 ng·mL^(-1)浓度范围内,对甘精胰岛素原标准品进行四参数曲线拟合,曲线相关系数R~2大于0.99,方法准确度较高、重复性良好。重复检测3批甘精胰岛素原料药,平均加标回收率在104%~113%范围内,RSD%小于10%,方法精密度良好。9批样品中单链前体残留量均小于1 ng·mg^(-1)。结论建立的酶联免疫法可用于甘精胰岛素及其他重组人胰岛素类似物中单链前体残留量的质量控制和安全性评价。

福林酚法高通量测定牛奶中蛋白酶活力的优化

[目的]优化福林酚法测定牛奶中蛋白酶活力的试验条件,解决检测结果重复性差、检测效率不高等问题。[方法]用酶标仪测定牛奶中蛋白酶活力,同步对比各试验条件对测定结果的影响。[结果]酶标仪测定时,显色保温时间15 min以上、取样350μL、福林试剂浓度0.33~0.5 mol/L时,牛奶中蛋白酶活力测定结果重复性好,检测效率高。[结论]酶标仪测定牛奶中蛋白酶活力是一种检测效率高、结果重复性好的方法,更适合乳品企业大批量筛查牛奶中蛋白酶活力。

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.

Establishment of a Sandwich ELISA Method for Detection of Vascular Endothelial Growth Factor in Serum Samples of Hepatocellular Carcinoma Patients

Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor （VEGF） in sera of population and the patients with hepatocellular carcinoma （HCC）. Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length （His-Trx-VEGF1-165）, N-terminal （His-Trx-VEGF1-100） and C-terminal （GST-VEGF100-165） human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R^2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.