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Tetra-substituted Amino Aluminum Phthalocyanine as a New Red-region Fluorescent Substrate for Horseradish Peroxidase Based enzyme-linked immunosorbent assay

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Chinese Journal of Chemistry 2002年第12期20卷 1573-1578,1463页

作者：杨黄浩李东辉陈小兰曲会英丁马太许金钩 Key Laboratory of Analytical Science of Ministry of Education Department of Chemistry Xiamen University Xiamen Fujian 361005 China Department of Material Science Xiamen University Xiamen Fujian 361005 China Cancer Research Center Xiamen University Xiamen Fujian 361005 China Email: Jin-Gou Xu (jgxu@***)

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.

关键词： tetra-substituted amino aluminum phthalocyanine red-region fluorescent substrate horseradish peroxidase enzyme-linked immunosorbent assay

enzyme-linked immunosorbent assay for Detecting Syphilis Dry Blood Spot Samples

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Chinese Journal of Biomedical Engineering(English Edition) 2022年第2期31卷 64-71页

作者： CUI Zhi-gang DU Yan-li JIANG Yu-shan ZHENG Lu Department of Clinical Laboratory Harbin Blood CenterHarbin Heilongjiang 150056China

Objective:To explore the feasibility of enzyme-linked immunosorbent assay(ELISA)in detecting syphilis dry blood ***:Based on dry blood spot samples,laboratory linear dial,laboratory basic dial,laboratory interference dial,and laboratory precision dial were *** 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 *** addition,112 suspected syphilis antibody-positive plasma samples were selected as the control,and dry blood spot samples were prepared *** 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 ***: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 *** 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%,*** clinical sensitivity,clinical specificity,positive predictive value a

关键词： syphilis blood spot sample enzyme-linked immunosorbent assay

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

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Neural Regeneration Research 2011年第32期6卷 2490-2494页

作者： Xiaoli Wu Zhangyuan Liao Jing Ye Huiqing Dong ChaodongWang Piu Chan Department of Neurology and Key Laboratory Ministry of Education on Neurodegenerative Diseases Xuanwu Hospital of Capital MedicalUniversity Beijing 100053 China Department of Neurology Xuanwu Hospital of Capital Medical University Beijing 100053 China

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.

关键词： neuromyelitis optica cell-based immunofluorescence assay anti-aquaporin 4 antibody enzyme-linked immunosorbent assay long and extended spinal cord lesions neural regeneration

Monoclonal Antibody-Based enzyme linked immunosorbent assay for the Analysis of Jasmonates in Plants

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Journal of Integrative Plant Biology 2008年第8期50卷 1046-1052页

作者： Aixing Deng Weiming Tan Suping He Wei Liu Tiegui Nan Zhaohu Li Baomin Wang Qing X. Li College of Agronomy and Biotechnology China Agricultural University Beijing 100094 China Department of Molecular Biosciences and Bioengineering University of Hawaii Honolulu Hawaii 96822 USA

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.

关键词： enzyme-linked immunosorbent assay immunoassay methyl jasmonate plant growth regulators

Anti-epitope antibody, a novel site-directed antibody against human acetylcholinesterase

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Acta Pharmacologica Sinica 2004年第4期25卷 33-37页

作者： Xing-mei ZHANG, GangLIU, Man-ji SUN2 Institute of Pharmacology and Toxicology, Academyof MilitaryMedical Sciences, Beijing 100850, China Institute of Pharmacology and Toxicology Academy of Military Medical Sciences

AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induc- tion and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody. METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChEwas chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE) polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

关键词： acetylcholinesterase butyrylcholinesterase epitopes antibodies enzyme-linked immunosorbent assay Western blotting

Induction of systemic lupus erythematosus syndrome in BALB/c mice by immunization with active chromatin

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Acta Pharmacologica Sinica 2004年第6期25卷 105-109页

作者： Hong LI, Yun-yi ZHANG, Ya-nan SUN, Xi-yi HUANG, Yong-feng JIA, Duan LI2 Department of Pharmacology, Schoolof Pharmacy, Fudan University, Shanghai 200032, China Department of Pharmacology School of Pharmacy Fudan University Shanghai 200032 China

AIM: To establish an animal model for systemic lupus erythematosus (SLE)-like syndrome in mice. METHODS: BALB/c mice were immunized with active chromatin isolated from ConA-actived syngeneic spleno-lymphocytes. Plasma samples of mice were tested by enzyme-linked immunosorbent assays (ELISA) for the presence of IgG anti-dsDNA, -ssDNA, and anti-histone antibodies. Tumor necrosis factor-α (TNF-α) in serum was measured by ELISA. Spleno-lymphocyte proliferation assays and the levels of interferon-γ (IFN-γ) in supernatants were tested respectively. Proteinuria was measured. Kidneys were examined by direct immunohistochemical method and light microscopy. RESULTS: Anti-ds DNA, ssDNA, and histone antibodies were induced in active chromatin-immu- nized mice, the proliferation response of splenocytes to ConA and LPS were reduced, levels of interferon-γ in supernatants and TNF-α in serum were lowered. Lupus nephritis was assessed by the presence of Ig deposits, glomerular pathology and proteinuria. CONCLUSION: The active chromatin-induced SLE-like mouse model was similar to idiopathic SLE in human.

关键词： systemic lupus erythematosus chromatin autoantibody tumor necrosis factor interferon type II B-lymphocytes T-lymphocytes female enzyme-linked immunosorbent assay

Preparation and Immunogenicity of Serogroup B Meningococcal OS-OMPC Conjugates

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Biomedical and Environmental Sciences 1999年第4期12卷 296-303页

作者： SUNYIN－YAN HUXU－JING InstituteofEpidemiologyandMicrobiology ChineseAcademyofPreventiveMedicineBeijing102206 InstituteofEp

The objective of the present study is to purify oligosaccharides (OSs) from serogroup B meningococci (3407 or 542852) with common lipooligosaccharide (LOS) immunotypes prevalent in China and to successtully conjugate them to their outer membrane protein complexes (OMPCs).Conjugates possessing broad cross-reactivity with different serogroup B meningococci were *** LOSs from the above two strains were purified on a Sephghex G75 column and hydrolyzed with acetic acid, and then the pare OSs were obtained by using a Sephadex G50 chromatographic column. The pure OSs were conjugated to corresponding OMPCs by carbodiimide mediated condensation. Mice were immunized with the OS-OMPC conjugates to produce antibodies. Immunognicities of the conjugates were tested by EUSA, bactericidal test and Western-blotting. The titers of antibodies against OS and LOS elicited by the two OS-OMPC conjugates increased respectively by 2. 5and 2 to 10 times as compared with those of antibodies against unconjugated OS and LOS. The sera elicited by the conjugates possessed a stronger bactericidal activity to the stndus (3407, 542852,29021) with the same LOS immunotype than to the strain with different LOS immunotype (86155).The results determined by ELISA indicated that the sera against the conjugaes showed strong response to the strains with the same or different LOS immunotypes. It was confirmed by westem-blotting analysis that the sera elicited by the two conjugates reacted with 42, 39 and 26 kDa proteins from the OMPC. Among the reactive bands, the 42kDa proteins were class 1 OMPs. The two serogroup B meningococcal OMPCs were strong immunogens and good carriers coupling with OSs as well. The immunogencity of the conjugaed OS was increased and the OS-OMPC conjugates of the serogroup B meogingococci showed a broad antigenic response to the strains tested.

关键词： Bacterial Outer Membrane Proteins Animals Antibodies Bacterial Blotting Western enzyme-linked immunosorbent assay Immunoconjugates Meningitis Meningococcal Mice Neisseria meningitidis Oligosaccharides Research Support Non-U.S. Gov't

Expression of Human Vascular Endothelial Growth Factor Receptor Flt-1 Extracellular Domain 1-3 Loop cDNA in Pichia pastoris, Purification of the Expressed Product and Detection of Its Biological Activity

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Biomedical and Environmental Sciences 2001年第4期14卷 292-301页

作者： MALI ZHANGZHI－QING 等 InstituteofMolecularImmunology ThefirstMilitaryMedicalUniversityGuangzhou510515China StateKeyLaboratoryofMolecularVirologyandGeneticEngineering Beijing100052China

Objective To express human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop cDNA in Pichia. pastroris, and to purify the expressed product and detect its biological activity. Methods By inserting human Flt-1 (1-3 loop) cDNA coding 316 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of α secreting signal peptides, a recombinant expression plasmid pPIC9K/Flt-1 (1-3) was constructed and transformed to yeast host strain GS115, then His + Mut s phenotype transformant was screened out and cultured in flasks, and Flt-1 (1-3) was expressed under the induction of 1% methanol. Results SDS-PAGE showed that after being induced with 1% methanol for 4d, the expressed product existed in supernatant in the form of soluble molecule and contained 60% of total protein expressed. Western blot showed good antigenicity and specificity of expressed product. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, the purity of the expressed product reached above 90%. Biological assay proved that the expressed product could bind to hVEGF 165 and inhibit the proliferation of HUVEC stimulated by hVEGF 165. Conclusion Human vascular endothelial growth factor receptor Flt-1 extracellular domain 1-3 loop was successfully expressed. The study lays a foundation for further application of the expressed product in the treatment of vasoformation related diseases, such as tumor and diabetic retinopathy.

关键词： Base Sequence Blotting Western Cells Cultured Cloning Molecular DNA Primers DNA Complementary Electrophoresis Polyacrylamide Gel enzyme-linked immunosorbent assay Humans Phenotype Pichia Plasmids Polymerase Chain Reaction Proto-Oncogene Proteins Receptor Protein-Tyrosine Kinases Recombinant Proteins Research Support Non-U.S. Gov't Vascular Endothelial Growth Factor Receptor-1

Efficacy of catgut embedding in Baihui(GV20)and Feishu(BL13)and Pishu(BL20)on lung tissue,brain tissue and blood related indexes in rats with allergic rhinitis of lung deficiency type

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Journal of Traditional Chinese Medicine 2023年第5期43卷 973-982页

作者： HUANG Heyin WANG Yichen ZHANG Qinxiu WU Xiaojuan WANG Jiang Li Xiangyu PANG Rui FAN Li CAO Liu TANG Luo Department of ENT Sichuan Integrative Medicine HospitalChengdu 610041China Department of orthopaedics Sichuan Province Orthopaedic HospitalChengdu 610000China Department of ENT Affiliated Hospital of Chengdu University of Traditional Chinese MedicineChengdu 610000China Department of ENT Quxian Traditional Chinese Medicine HospitalQuxian 635299China

OBJECTIVE:To investigate the effects of acupoint catgut embedding for 3 weeks on lung tissue,blood immunoglobulin E(IgE)and interleutin-4(IL-4),brain tissue microglia x-42(OX-42)and toll-like receptor-2(TLR-2)in rats with allergic rhinitis of lung deficiency ***:Forty-five female Sprague-Dawley rats were randomly divided for two *** first time,they were randomly divided into model group and blank group(Group C)according to 2∶1,and the second time,the model group were randomly divided into model control group(Group B)and intervention treatment group(Group A)according to 1∶1.15 in each *** Group A and Group B,the lung deficiency model was made by"sulfur–moxa fumigation",and then the allergic rhinitis model was established by"ovalbumin(OVA)sensitization".Then catgut embedding was performed at acupoints in Group A and not in Group *** 3 weeks,collect lung tissue samples for hematoxylin-eosin staining,then take blood to observe the concentration of IgE and IL-4,and finally take brain tissue to observe the results of OX-42 and ***:IgE level(μg/m L)was(3.11±0.20)in the Group A,(4.19±0.44)in the Group B,and(2.29±0.30)in the Group C(all P<0.001).IL-4 level(pg/mL)was(14.2±0.7)in the Group A,(18.6±2.4)in the Group B,and(11.4±1.2)for the Group C(all P<0.001).The mean OD for OX-42 is(0.1728±0.0016)in the Group A,(0.1810±0.0046)in the Group B and(0.1674±0.0025)in the Group C(all P<0.001).CONCLUSION:Although 3 weeks of acupoint catgut embedding already showed obvious efficacy on rats with allergic rhinitis,the allergic reaction in the body still *** achieve further treatment,prolonging the catgut embedding time is necessary.

关键词： rhinitis,allergic acupoint catgut embedding enzyme-linked immunosorbent assay immunoglobulin E interleukin-4 hippocampus toll-like receptor 2