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Micropatterned assembly of silica nanoparticles for a protein microarray with enhanced detection sensitivity

BIOMEDICAL MICRODEVICES 2010年第3期12卷 457-464页

作者： Lee, Yeol Park, Sangphil Park, Jinwon Koh, Won-Gun Yonsei Univ Dept Chem & Biomol Engn Seoul 120749 South Korea

We used an assembly of silica nanoparticles (SNPs) as a three-dimensional template for protein immobilization to prepare a protein microarray with enhanced protein loading capacity and detection sensitivity. SNPs were first modified with 3-aminopropyltriethoxysilane (APTES) for covalent immobilization of protein and micropatterned on poly(ethylene glycol)(PEG)-coated glass slides using elastomeric membranes with an array of holes. proteins were selectively immobilized only on the SNP region, while the PEG regions served as an effective barrier to protein adsorption. Because of multi-layered SNPs that had curved surface, protein loading in the SNP micropattern was about six times greater than on a planar surface, as observed by fluorescence microscopy, which consequently improved the protein activity and reaction rate. GOX-catalyzed glucose oxidation and the molecular recognition mediated, specific binding between biotin and streptavidin were both successfully assayed using SNP microarrays, with better fluorescence signal and sensitivity than corresponding planar microarrays.

关键词： Silica nanoparticles Elastomeric membranes protein microarray Three-dimensional template Micropatterning

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An application of protein microarray in the screening of monoclonal antibodies against the oyster mushroom spherical virus

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ANALYTICAL BIOCHEMISTRY 2008年第2期374卷 313-317页

作者： Kim, Sang-Woo Kim, Min-Gon Jung, Hyo-Am Lee, Kyung-Hee Lee, Hyun-Sook Ro, Hyeon-Su Gyeongsang Natl Univ Dept Microbiol Chinju 660701 South Korea Gyeongsang Natl Univ Res Inst Life Sci Chinju 660701 South Korea Korea Res Inst Biosci & Biotechnol BioNanotechnol Res Ctr Taejon 305600 South Korea Gyeongsang Natl Univ Environm Biotechnol Natl Core Res Ctr Chinju 660701 South Korea

The oyster mushroom spherical virus (OMSV) is a causative agent of dieback disease in the oyster mushroom, Pleurotus ostreatus. Outbreaks of this virus occasionally result in serious disease that is associated with hefty economic losses. Thus, the detection and removal of OMSV-infected spawn is considered to be a crucial step for the stable production of P. ostreatus. For the detection of OMSV, we attempted to generate monoclonal antibodies (mAbs) against an RNA polymerase domain (RPD) of an OMSV protein. In an effort to simplify the laborious multistep mAb screening process, we developed a protein microarray on a slide glass that is chemically modified with the RPD protein. The culture supernatants of 87 hybridoma cells, which were prepared from the fusion of RPD-immunized mouse spleen cells with myeloma cells, were spotted onto the RPD-coated microarray. The binding of mAb to RPD was detected via Alexa 488 dye-labeled anti-mouse immunoglobulin G (IgG) as a secondary antibody. Of 87 samples, 13 evidenced a significant level of fluorescence signal intensity. Subsequent immunoblot analysis revealed that the specificity of each mAb against RPD coincided with the corresponding fluorescence signal intensity, thereby indicating the effectiveness of the protein microarray in mAb screening. (C) 2007 Elsevier Inc. All rights reserved.

关键词： protein microarray monoclonal antibody mycovirus screening fungi

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High-throughput screening of novel peptide inhibitors of an integrin receptor from the hexapeptide library by using a protein microarray chip

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JOURNAL OF BIOMOLECULAR SCREENING 2004年第8期9卷 687-694页

作者： Lee, Y Kang, DK Chang, SI Han, MH Kang, IC Chungbuk Natl Univ Prot Chip Res Ctr Biotechnol Res Inst Cheongju 361763 South Korea Chungbuk Natl Univ Dept Biochem Cheongju 361763 South Korea Proteogen Inc Seoul South Korea

protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, ProteoChip, in new drug discovery. Integrin alpha(v)beta(3) microarray immobilized on the ProteoChip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin alpha(v)beta(3)-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner and was inhibited not only by the synthetic RGD peptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and high-throughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the ProteoChip is a promising tool for high-throughput screening of lead molecules in new drug development.

关键词： ProteoChip protein microarray ProLinker integrin PS-SPCL

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Analysis of serum alpha-fetoprotein (AFP) and AFP-L3 levels by protein microarray

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JOURNAL OF INTERNATIONAL MEDICAL RESEARCH 2018年第10期46卷 4297-4305页

作者： Wu, Min Liu, Haidong Liu, Zhaobo Liu, Chao Zhang, Aiying Li, Ning Capital Med Univ Dept Gen Surg Beijing YouAn Hosp Beijing Peoples R China Capital Med Univ Beijing Inst Hepatol Beijing YouAn Hosp 8 Xitoutiao Beijing 100069 Peoples R China

Objectives: This study aimed to examine a simple, effective, time-saving, and low-cost protein microarray method for detecting serum alpha-fetoprotein (AFP) and AFP-L3 levels. Methods: Serum samples from patients with hepatocellular carcinoma (HCC) (n = 33) and control subjects (n = 39) were collected and evaluated for the presence of AFP using a novel protein microarray. Glycoprotein (including AFP-L3) was enriched from crude samples by a Hotgen Biotech glycosyl capture spin column and then detected by protein microarray. An electrochemiluminescence immunoassay (ECLIA) was used to validate the measured values. Results: Neither AFP levels lower than 20 ng/mL in the HCC group nor AFP levels higher than 20 ng/mL in the control group were found when tested by the ECLIA and protein microarray. The kappa test showed good consistency in the diagnostic performance of measuring serum AFP levels and the percentage of AFP-L3 in total AFP by the ECLIA and protein microarray. protein microarray had advantages of smaller sample size required, low cost, and convenience compared with the ECLIA. Conclusion: The protein microarray assay that was developed in the present study shows potential as an economic and convenient technique for detecting AFP and AFP-L3 levels in serum samples from patients with HCC.

关键词： protein microarray alpha-fetoprotein (AFP) AFP-L3 hepatocellular carcinoma electrochemiluminescence immunoassay glycoprotein Lens culinaris agglutinin

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Multiplex approaches in protein microarray technology

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EXPERT REVIEW OF PROTEOMICS 2005年第4期2卷 499-510页

作者： Kersten, B Wanker, EE Hoheisel, JD Angenendt, P German Canc Res Ctr D-69120 Heidelberg Germany Max Delbruck Ctr Mol Med D-13125 Berlin Germany

The success of genome sequencing projects has provided the basis for systematic analysis of protein function and has led to a shift from the description of single molecules to the characterization of complex samples. Such a task would not be possible without the provision of appropriate high-throughput technologies, such as protein microarray technology. In addition, the increasing number of samples necessitates the adaptation of such technologies to a multiplex format. This review will discuss protein microarray technology in the context of multiplex analysis and highlight its current prospects and limitations.

关键词： antibody chip high throughput multiplex protein microarray

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Discovery of novel Schistosoma japonicum antigens using a targeted protein microarray approach

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PARASITES & VECTORS 2014年第1期7卷 1-11页

作者： McWilliam, Hamish E. G. Driguez, Patrick Piedrafita, David McManus, Donald P. Meeusen, Els N. T. Univ Melbourne Dept Microbiol & Immunol Peter Doherty Inst Infect & Immun Melbourne Vic Australia QIMR Berghofer Med Res Inst Mol Parasitol Lab Brisbane Qld Australia Federat Univ Sch Engn & Appl Sci Churchill Vic Australia Monash Univ Dept Microbiol Sch Biomed Sci Melbourne Vic 3004 Australia

Background: Novel vaccine candidates against Schistosoma japonicum are required, and antigens present in the vulnerable larval developmental stage are attractive targets. Post-genomic technologies are now available which can contribute to such antigen discovery. Methods: A schistosome-specific protein microarray was probed using the local antibody response against migrating larvae. Antigens were assessed for their novelty and predicted larval expression and host-exposed features. One antigen was further characterised and its sequence and structure were analysed in silico. Real-time polymerase chain reaction was used to analyse transcript expression throughout development, and immunoblotting and enzyme-linked immunosorbent assays employed to determine antigen recognition by antibody samples. Results: Several known and novel antigens were discovered, two of which showed up-regulated transcription in schistosomula. One novel antigen, termed S. japonicum Ly-6-like protein 1 (Sj-L6L-1), was further characterised and shown to share structural and sequence features with the Ly-6 protein family. It was found to be present in the worm tegument and expressed in both the larval and adult worms, but was found to be antigenic only in the lungs that the larvae migrate to and traverse. Conclusions: This study represents a novel approach to vaccine antigen discovery and may contribute to schistosome vaccine development against this important group of human and veterinary pathogens.

关键词： Schistosoma japonicum Vaccine development Ly-6 proteins protein microarray Immunomics

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Plasmodium falciparum protein microarray Antibody Profiles Correlate With Protection From Symptomatic Malaria in Kenya

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JOURNAL OF INFECTIOUS DISEASES 2015年第9期212卷 1429-1438页

作者： Dent, Arlene E. Nakajima, Rie Liang, Li Baum, Elisabeth Moormann, Ann M. Sumba, Peter Odada Vulule, John Babineau, Denise Randall, Arlo Davies, D. Huw Felgner, Philip L. Kazura, James W. Case Western Reserve Univ Ctr Global Hlth & Dis Cleveland OH 44106 USA Rainbow Babies & Childrens Hosp Cleveland OH 44106 USA Univ Calif Irvine Irvine CA USA Univ Massachusetts Sch Med Ctr Global Hlth Res Worcester MA USA Rho Inc Chapel Hill NC USA Kenya Govt Med Res Ctr Kisumu Kenya Antigen Discovery Inc Irvine CA USA

Background. Immunoglobulin G antibodies (Abs) to Plasmodium falciparum antigens have been associated with naturally acquired immunity to symptomatic malaria. Methods. We probed protein microarrays covering 824 unique P. falciparum protein features with plasma from residents of a community in Kenya monitored for 12 weeks for (re) infection and symptomatic malaria after administration of antimalarial drugs. P. falciparum proteins recognized by Abs from 88 children (aged 1-14 years) and 86 adults (aged >= 18 years), measured at the beginning of the observation period, were ranked by Ab signal intensity. Results. Abs from immune adults reacted with a total 163 of 824 P. falciparum proteins. Children gradually acquired Abs to the full repertoire of antigens recognized by adults. Abs to some antigens showed high seroconversion rates, reaching maximal levels early in childhood, whereas others did not reach adult levels until adolescence. No correlation between Ab signal intensity and time to (re) infection was observed. In contrast, Ab levels to 106 antigens were significantly higher in children who were protected from symptomatic malaria compared with those who were not. Abs to antigens predictive of protection included P. falciparum erythrocyte membrane protein 1, merozoite surface protein (MSP) 10, MSP2, liver-stage antigen 3, PF70, MSP7, and Plasmodium helical interspersed subtelomeric domain protein. Conclusions. protein microarrays may be useful in the search for malaria antigens associated with protective immunity.

关键词： malaria antibody antigen protein microarray protective immunity

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Antibody profiling in plague patients by protein microarray

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MICROBES AND INFECTION 2008年第1期10卷 45-51页

作者： Li, Bei Zhou, Dongsheng Wang, Zuyun Song, Zhizhong Wang, Hu Li, Min Dong, Xingqi Wu, Mingshou Guo, Zhaobiao Yang, Ruifu Inst Microbiol & Epidemiol State Key Lab Pathogen & Biosecur Lab Analyt Microbiol Beijing 100071 Peoples R China Qinghai Inst Endem Dis Prevent & Control Xining Qinghai Prov Peoples R China Yunnan Inst Epidem Dis Control & Res Dali Yunnan Prov Peoples R China

A protein microarray containing 144 known or putative virulence-related proteins of Yersinia pestis was used to evaluate the antibody responses of plague patients. Forty-two proteins were found to be expressed in vivo and antibodies against 14 of them were detected in all patients analyzed, providing potential candidates for novel protective antigens and novel serodiagnostic markers in Y. pestis. Moreover, the lack of antibody to LcrV in the five patients in Focus F might be a challenge to our understanding of the pathogenesis of Y. pestis. (c) 2007 Elsevier Masson SAS. All rights reserved.

关键词： protein microarray yersinia pestis antibody profile

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Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma

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JOURNAL OF VETERINARY INTERNAL MEDICINE 2019年第5期33卷 2327-2335页

作者： White, Samuel Moore-Colyer, Meriel Marti, Eliane Couetil, Laurent Hannant, Duncan Richard, Eric A. Alcocer, Marcos Royal Agr Univ Sch Equine Management & Sci Cirencester Glos England Univ Nottingham Sch Biosci Loughborough Leics England Nottingham Trent Univ Anim & Equine Sci Brackenhurst Lane Nottingham NG25 0QF Notts England Univ Bern Dept Clin Res & Vet Publ Hlth Bern Switzerland Purdue Univ Coll Vet Med Vet Clin Sci W Lafayette IN 47907 USA Univ Nottingham Sch Vet Med & Sci Loughborough Leics England LABEO Frank Duncombe Caen France Normandie Univ UniCaen BIOTARGEN St Contest France

Background Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. Objective To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. Animals Six SEA-affected and 6 clinically healthy Warmblood performance horses. Methods Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. Results This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti-horse IgE 3H10 source (R-2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R-2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40-fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R-2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. Conclusions and Clinical Importance We have established that protein microarrays can be used for large-scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.

关键词： allergen horse IgE protein microarray severe equine asthma

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LABORATORY INVESTIGATION 2004年第2期84卷 235-244页

作者： Zha, HB Raffeld, M Charboneau, L Pittaluga, S Kwak, LW Petricoin, E Liotta, LA Jaffe, ES NCI Pathol Lab Ctr Canc Res NIH Bethesda MD 20892 USA NCI Expt Transplantat & Immunol Branch Bethesda MD 20892 USA US FDA Ctr Biol Evaluat & Res Bethesda MD 20892 USA

Overexpression of Bcl-2 protein has been known to play a role in the pathogenesis of follicular lymphoma (FL). However, 10-15% of FLs are negative for Bcl-2 by immunohistochemistry, raising the possibility that another gene product(s) may provide prosurvival signal(s). We used reverse phase protein microarray to analyze lysates of follicle center cells isolated by laser capture microdissection from: Bcl-2+ FL, Bcl-2- FL and reactive follicular hyperplasia (FH) (nine cases each group). TUNEL assay confirmed similar and reduced levels of apoptosis in Bcl-2+ FL and Bcl-2- FL, indicating the likelihood of Bcl-2-independent inhibition of apoptosis. Arrays were quantitatively analyzed with antibodies to proteins involved in the apoptotic pathway. As expected, Bcl-2 levels were up to eight-fold higher in Bcl-2+ FL than in FH and Bcl-2- FL. However, there was no difference in levels of Mcl-1 and survivin among these three groups. Bcl-X-L showed a trend for increased expression in Bcl-2- FL as compared with Bcl-2+ FL, although the differences did not reach statistical significance (P>0.1). The increase in Bcl-X-L may provide an alternative antiapoptotic signal in FL negative for Ill protein. Interestingly, Bax expression was higher in FL (Bcl-2+ or -) than in FH (P=0.001). Notably, phospho-Akt (Ser-473) was increased in FL (Bcl-2+ or -) (P<0.03) with increased phospho-Bad (Ser-136), as compared with levels in FH. The activation of the Akt/Bad pathway provides further evidence of prosurvival signals in FL, independent of Bcl-2 alone. These data suggest that nodal FL represents a single disease with a final common biochemical pathway.

关键词： apoptosis follicular lymphoma Bcl-2 protein microarray proteomics

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