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).

Hepatitis C virus antigens enzyme immunoassay for one-step diagnosis of hepatitis C virus coinfection in human immunodeficiency virus infected individuals

BACKGROUND Current diagnosis of hepatitis C virus(HCV)infection requires two sequential steps:testing for anti-HCV followed by HCV RNA PCR to confirm viremia.We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA)for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera:10 without HCV or HIV infection;54 with viremic HCV monoinfection;38 with viremic HCV/HIV coinfection;and 45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding,it was 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test.In five sera with HCV infection,HCV RNA was as low as 50-59 IU/mL,and four out of five tested positive for HCV-Ags EIA.Likewise,it was also 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection,regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL.It is highly sensitive and specific in the setting of HIV coinfection,regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.

Interaction between Aluminum and Zinc or Copper and Its Effects on the Pituitary-Testicular Axis Ⅱ. Testicular Enzyme and Serum Gonadotropin Assay

Sprague-Dawley male rats were divided into groups and maintained on a semipurified diet containing either 5 or 40 ppm of zinc or 2 or 8 ppm of copper. Half of the rats in each group received 1000 ppm aluminum in the diet. It was found that aluminum accumulated in the pituitary glands and testes when dietary copper levels were suboptimal. The ALP activity in testes was depressed by the added aluminum when the intake of zinc or copper was suboptimal. SDH, LDH, and LDH-X activities were inhibited and GRS and GGTP activities were elevated in rats fed either the suboptimal zinc or copper diet. However, the added aluminum in the diet reversed the changes to normal levels. The testosterone levels in plasma changed very little when the zinc or copper intake was suboptimal. An increase in plasma FSH was demonstrated in groups of both suboptimal zinc and copper intake. But the plasma LH was elevated only in the group receiving the suboptimal copper diet, and the added aluminum reversed plasma LH to control levels. A lower level of testosterone was demonstrated in the group given suboptimal copper with aluminum. It was concluded that dietary aluminum influenced the pituitary-testicular axis by interacting with certain essential trace metals, particularly zinc. 1990 Academic Press. Inc.

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.

Ultra-highly selective recognition of nucleosides over nucleotides by rational modification of tetralactam macrocycle and its application in enzyme assay

Artificial macrocycle with high binding selectivity in water is often challenging but urgently needed in various research and application areas.Herein,we report a new water-soluble biomimetic tetralactam macrocycle and realize the ultra-high selectivity to nucleosides over corresponding monophosphate nucleotides by rational modification.The introduction of charged groups at the periphery of endofunctionalized cavity makes the selectivity(guanosine to guanosine 5-monophosphate)increase remarkably from 100 to 1119.Based on the ultra-high selectivity of biomimetic tetralactam macrocycle,the sensitive CD73 enzyme activity assay was then achieved through product-selective fluorescence indicator displacement assay.Furthermore,the capability of the proposed method for inhibitor screening was successfully displayed.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity.Given that two C-10 modified camptothecin derivatives,topotecan and irinotecan,have been approved as potent anticancer agents,there is a critical need for methods to access other aromatic ringfunctionalized congeners(e.g.,C-9,C-10,etc.).However,contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold,thereby limiting the development of modified derivatives.Reported herein,we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa,via metabolomic and transcriptomic analysis.These consist of a cytochrome P450(Nt CPT9H)which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin,as well as two methyltransferases(Nt OMT1/2,converting 9-hydroxycamptothecin to 9-methoxycamptothecin),and a uridine diphosphate-glycosyltransferase(Nt UGT5,decorating 9-hydroxycamptothecin to9-β-D-glucosyloxycamptothecin).Importantly,the critical residues that contribute to the specific catalytic activity of Nt CPT9H have been elucidated through molecular docking and mutagenesis experiments.This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering.This will hasten the discovery of novel C-9 modified camptothecin derivatives,with profound implications for pharmaceutical manufacture.

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.

Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay

Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.

Development of a competitive format sorbent assay for the determination of parathion in water using molecular imprinted polymer as specific sorbent carrier

A rapid,simple,and reliable competitive molecular imprinted polymer sorbent assay（MIPSA） was developed and validated for measurement of parathion in water samples.This assay employed a molecular imprinted polymer（MIP） that was synthesized with non-covalent imprinting method as capture reagent and p-aminoparathion conjugate of horseradish peroxidase（para-HRP） as an enzyme label.The assay depended on a competitive binding reaction between the enzyme conjugate and analyte for the binding sites of the MIP.The optimized analysis conditions of 10 ng mL-1 para-HRP and 10 mg mL-1 polymer were found.The assay was acceptable to detect parathion in water samples under the optimized conditions,with a limit of detection of 50 ng mL-1.Mean analytical recoveries of added parathion in water samples ranged from 101.2%to 105%.The precision of the assay was satisfactory; relative standard deviation ranged from 4.3%to 6%.

Drosophila melanogaster as an indispensable model to decipher the mode of action of neurotoxic compounds

Exposure to some toxic compounds causes structural and behavioral anomalies associated with the neurons in the later stage of life.Those toxic compounds are termed as a neurotoxicant,which can be a physical factor,a toxin,an infection,radiation,or maybe a drug.The incongruities caused due to a neurotoxicant further depend on the toxicity of the compound.More importantly,the neurotoxicity of the compound is associated with the concentration and the time point of exposure.The neurodevelopmental defect appears depending on the toxicity of the compound.A neurodevelopmental defect may be associated with a delay in developmental time,defective growth,structural abnormality of many organs,including sensory organs,behavioral abnormalities,or death in the fetus stage.Numerous model organisms are employed to assess the effect of neurotoxicants.The current review summarizes several methods used to check the effect of neurotoxicant and their effect using the model organism Drosophila melanogaster.

Serological diagnosis of nasopharyngeal carcinoma by enzyme linked immunosorbant assay:optimization,standardization and diagnostic criteria

Objectives To produce an enzyme linked immunosorbant assay (ELISA) to detect antibodies against Epstein Barr virus (EBV) specified nuclear antigen 1 (EBNA 1) and the 18kD EBV matrix protein, and to determine and optimize its sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma (NPC).Methods We used a combination of highly purified glutathione transferase fusion proteins of the 40kD carboxy domain of EBNA1 and the 18kD EBV matrix protein for coating ELISA plates. In three separate studies, we tested for IgA antibodies in serum specimens from 28 EBV seronegative donors, 284 EBV seropositive donors and 160 newly diagnosed NPC patients. By comparing the sensitivity and specificity of diagnosis obtained for different cutoff values, we derived several quantitative parameters to evaluate assay performance, establish objective diagnostic criteria which optimize the intrinsic diagnostic capability of the assay and assess the significance of individual test results, respectively. Optimum cutoff optical density (OD) is defined as the cutoff OD where sensitivity of the assay equals its specificity, and resolution of the assay is indicated by the value of sensitivity (or specificity) determined at the optimum cutoff OD. Diagnosis of NPC was achieved by setting a cutoff zone at +/-20% of this value.Results All the EBV seronegative donors tested were not reactive, and most of the EBV seropositive donors were weakly reactive, while the majority of NPC patients were moderately or strongly reactive. While the assay was thus shown to be specific for EBV, there was an overlap in the level of these serum antibodies between few individuals of the two latter groups. It was shown that the assay performed equally well in two separate studies conducted under different testing conditions and using different collections of sera in that assay resolution determined on these occasions were 86% and 87% respectively. Diagnosis of NPC can be achieved at the same expected sensitivity of 89% and 83% determined at the lower and upper limits of the cutoff zones, with the corresponding values of specificity being 78% and 91%. It was further shown in the third study that resolution of the assay can be increased to 90% using an assay produced with a higher concentration of the same antigens, and that diagnosis of NPC can be achieved at a higher sensitivity ranging between 86% and 95% at a corresponding specificity of 93% and 86%.Conclusions After optimization and standardization, the ELISA can achieve a sensitivity ranging from 86% to 95%, with corresponding specificities of 93% and 86% respectively for the diagnosis of NPC.

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen(SCCA;cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction(HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle(AuNP). Then, the HCR reaction was readily executed between two glucose oxidase(GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.

Target-induced mimic enzyme deactivation based on mixed-node metal-organic frameworks for colorimetric assay of hydrogen sulfide

Accurate detection of hydrogen sulfide(H_(2)S)is of great significance for environmental monitoring and protection.We propose a colorimetric method for the detection of H_(2)S by the use of mixed-node Cu-Fe metal organic frameworks(Cu-Fe MOFs)as highly efficient mimic enzymes for target-induced deactivation.The Cu-Fe MOFs were synthesized by a simple solvothermal method and could catalyze the H_(2)O_(2)mediated oxidation of 3,30,5,50-tetramethylbenzidine(TMB)to oxTMB with a blue color.The presence of dissolved H_(2)S would deactivate the mimic enzymes,and then the blue color disappeared.The mechanism of the sensor was discussed by steady-state kinetic analysis.The designed assay was highly sensitive for H_(2)S detection with a linear range of 0à80 mmol/L and a detection limit of 1.6 mmol/L.Moreover,some potential substances in the water samples had no interference.This method with the advantages of low cost,high sensitivity,selectivity,and visual readout with the naked eye was successfully applied to the determination of H_(2)S in industrial wastewater samples.

银杏外种皮对钉螺的杀灭机理

To study the toxicity of extracts of Ginkgo biloba sarcotesta to Oncomelania hupensis,snails were exposed to 40% and 80% of 24 h LC 50 of the extract of Ginkgo bilba for 24 h,choline esterase(ChE),alanine aminotransferase(ALT),alkaline phosphatase(ALP),lactate dehydrogenase(LDH),succino dehydrogenase(SDH),malic dehydrogenase(MDH)activities in cephalopodium and liver were determined by enzyme kinetic assay.Arecoline and niclosamide were used as reference molluscicides.The results showed that sarcotesta of Ginkgo biloba could inhibit ChE,ALT,ALP and MDH activities both in cephalopodium and liver;arecoline could inhibit ChE,ALP,SDH and MDH activities in cephalopodium and ChE,ALT,ALP,SDH and MDH activities in liver.Niclosamide had inhibitory effects upon ChE,ALT,ALP,SDH and MDH activities in cephalopodium,and ChE,ALT,ALP and SDH activities in liver.All three molluscicides did not inhibite LDH activity in cephalopodium and liver.These results indicate that lethal effects of extracts of sarcotesta of Ginkgo biloba are mediated via inhibition of MDH activitiy,and interference with the NADH respiratory chains.Inhibition of vital enzymic mechanisms causes snails to death.

新霉素ELISA检测方法的建立

目的:比较直接和间接竞争酶联免疫法(enzyme linked immunosorbent assay,ELISA)的优缺点,建立新霉素残留ELISA检测方法。方法:利用自制的新霉素多克隆抗体,采用直接竞争和间接竞争ELISA方法检测新霉素残留,并比较两种方法的优缺点。结果:新霉素抗血清和庆大霉素的交叉反应率为2.04%,和卡那霉素的交叉反应率为0.02%,和氨苄青霉素、红霉素、四环素的交叉反应率均小于0.01%。初步测试新霉素间接竞争ELISA法的准确性和回收率。板内误差小于4%,板间误差小于11%,回收率为135.5%～191.3%。直接竞争和间接竞争ELISA方法的检测极限分别为28.58ng/mL和51.74ng/mL,达到了国家对新霉素规定的500μg/kg MRL检测限。结论:建立了直接竞争和间接ELISA吸附检测方法,条件优化更成功的间接竞争ELISA可用于开发新霉素检测试剂盒。

重症大疱性类天疱疮患者血清抗体变化规律与病情相关性的研究

目的：研究重症大疱性类天疱疮（ bullous pemphigoid ， BP ）患者血清中抗体BP180NC16a的酶联免疫吸附试验（ELISA）指数变化情况，观察其与病情变化的相关性，并分析用于病情监测和指导治疗的临床意义。方法对12例皮损面积＞50％的重症大疱性类天疱疮患者血清抗体BP180 NC16 a水平在不同时期进行监测及评分，并分析之间的关系。结果12例患者，平均年龄65岁，皮损面积均大于全身体表面积的50％以上。皮疹主要表现为疱壁紧张的大疱、水疱，部分有口腔黏膜损害。患者皮损面积和病情评分与血清抗体BP180NC16a-ELISA指数具有显著性关联（P＜0．05），患者疾病活动期和临床缓解期抗体BP180NC16a-ELISA指数几乎与病情呈平行变化，并且该指数可以预测病情，从而指导治疗。结论重症大疱性类天疱疮多为老年患者，病情危重，在发病早期不易诊断，因而延误治疗导致死亡。血清中抗体BP180 NC16 a-ELISA 指数可反映疾病的活动程度，用于病情监测，为治疗时根据个体差异选用适量的糖皮质激素快速控制病情提供了有利的实验室证据。

三甲氧苄氨嘧啶单克隆抗体制备以及酶联免疫试剂盒的研究

[目的]探讨系统地检测水质中三甲氧苄氨嘧啶残留量的方法。[方法]通过三甲氧苄氨嘧啶与马来酸酐反应，得到三甲氧苄氨嘧啶半抗原，再通过免疫动物得到抗三甲氧苄氨嘧啶单克隆抗体，并将其应用于能够检测水质中三甲氧苄氨嘧啶残留量的ELISA试剂盒。[结果]试验表明，该试剂盒对水质中三甲氧苄氨嘧啶的检测限为2．34μg／kg，IC50（50％抑制浓度）为4．8μg／L，回收率为60．5％～79．7％，试剂盒的标准曲线范围为0～80μg／L，批内、批间的相对标准偏差均小于10％，三甲氧苄氨嘧啶单克隆抗体与二甲氧苄氨嘧啶的交叉反应率小于1％，4℃下能够保存12个月，稳定性较好。[结论]研究可为监管三甲氧苄氨嘧啶的滥用提供参考。

单克隆抗体技术在小麦贮藏蛋白研究及其遗传改良中的应用进展

近20年来,人们制备了许多小麦种子贮藏蛋白的单克隆抗体(Monoclonal Antibody,McAb),一方面作为有效工具研究胚乳贮藏蛋白(主要是麦谷蛋白聚集体、特定的谷蛋白亚基及醇溶蛋白)的结构与功能关系;另一方面用作诊断试剂(diagnostic tools),为筛选具有合适加工品质的小麦品种提供依据。本文综述了国内外单克隆抗体技术在小麦贮藏蛋白研究及其遗传改良中的应用进展,并展望其应用前景。

Changes of p53 protein blood level in esophageal cancer patients and normal subjects from a high incidence area in Henan,China

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Serological prevalence and risk factor analysis of hepatitis G virus infection in Hubei Province of China

INTRODUCTIONHepatitis G virus(HGV),also known as GB virus C,is arecently cloned virus which may be associated with humannon A-E hepatitis.It is parenterally transmitted andusually coinfected or superinfected with hepatitis B orhepatitis C virus.Some investigations have