Yazarların Bayan Monika Tocchi yazının içerik incelemesi için teşekkür etmek istiyorum. AMH, Kanada, Suudi Arabistan Kültür Bürosu aracılığıyla, Kral Abdülaziz Üniversitesi Burs tarafından desteklenmektedir.

<ol>
	<li>Webster, R. G., Bean, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+influenza+virus.">Genetics of influenza virus.</a> Annu Rev Genet. 12, 415-431 (1978).</li><li>Skehel, J. J., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+and+membrane+fusion+in+virus+entry:+the+influenza+hemagglutinin.">Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.</a> Annu Rev Biochem. 69, 531-569 (2000).</li><li>Wood, J. 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M., Pervikov, Y., Palkonyay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+meeting+on+the+role+of+neuraminidase+in+inducing+protective+immunity+against+influenza+infection.">WHO meeting on the role of neuraminidase in inducing protective immunity against influenza infection.</a> Vaccine. 27, 6366-6369 (2008).</li><li>Chun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+antibodies+and+their+applications+to+the+quantitative+determination+of+virtually+all+subtypes+of+the+influenza+A+viral+hemagglutinins.">Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins.</a> Vaccine. 26, 6068-6076 (2008).</li><li>Gravel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+analyses+of+virtually+all+subtypes+of+influenza+A+and+B+viral+neuraminidases+using+antibodies+targeting+the+universally+conserved+sequences.">Qualitative and quantitative analyses of virtually all subtypes of influenza A and B viral neuraminidases using antibodies targeting the universally conserved sequences.</a> Vaccine. 28, 5774-5784 (2010).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+slot+blot+for+the+detection+of+virtually+all+subtypes+of+the+influenza+A+viral+hemagglutinins+using+universal+antibodies+targeting+the+fusion+peptide.">A simple slot blot for the detection of virtually all subtypes of the influenza A viral hemagglutinins using universal antibodies targeting the fusion peptide.</a> Nat Protoc. 5, 14-19 (2010).</li><li>Harvey, R., Wheeler, J. X., Wallis, C. L., Robertson, J. S., Engelhardt, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+haemagglutinin+in+H5N1+influenza+viruses+reveals+low+haemagglutinin+content+of+vaccine+virus+NIBRG-14+(H5N1).">Quantitation of haemagglutinin in H5N1 influenza viruses reveals low haemagglutinin content of vaccine virus NIBRG-14 (H5N1).</a> Vaccine. 26, 6550-6554 (2008).</li><li>Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+deglycosylation+and+electrophoresis+to+the+quantification+of+influenza+viral+hemagglutinins+facilitating+the+production+of+2009+pandemic+influenza+(H1N1)+vaccines+at+multiple+manufacturing+sites+in+China.">Application of deglycosylation and electrophoresis to the quantification of influenza viral hemagglutinins facilitating the production of 2009 pandemic influenza (H1N1) vaccines at multiple manufacturing sites in China.</a> Biologicals. 38, 284-289 (2010).</li><li>Johansson, B. E., Pokorny, B. A., Tiso, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supplementation+of+conventional+trivalent+influenza+vaccine+with+purified+viral+N1+and+N2+neuraminidases+induces+a+balanced+immune+response+without+antigenic+competition.">Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition.</a> Vaccine. 20, 1670-1674 (2002).</li><li>Hashem, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+antibodies+against+the+highly+conserved+influenza+fusion+peptide+cross-neutralize+several+subtypes+of+influenza+A+virus.">Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus.</a> Biochem Biophys Res Comm. , (2010).</li></ol>

Influenza virüs HA ve NA, bu iki yüzey proteinleri bağışıklık yanıtlarını 6-11 indükleyen en önemli viral bileşenler olduğundan kantitatif tayin aşı araştırma ve geliştirme için kritik öneme sahiptir . Daha önce bu proteinlerin tespiti için rapor immünolojik yöntemler, gerginlik spesifik antikorların gerektirir. Antikorlar, sadece evrensel korunmuş epitoplar 6-8 farkındayız beri HA ve NA antijenleri burada açıklanan ölçmek için, basit, tekrarlanabilir ve hızlı yu...

An author's affiliation was omitted from the publication of <a href="http://www.jove.com/video/2784">Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays</a>.

Erratum: Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

<table border="1"><tbody><tr><th> Reaktif veya ekipman Adı </th><th> Şirket </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> Bio-Dot SF Mikrofiltrasyon Aparatı </td><td> Bio-Rad </td><td> 170-6542 </td><td></td></tr><tr><td> Bio-Nokta SF Filtre kağıdı </td><td> Bio-Rad </td><td> 162-0161 </td><td></td></tr><tr><td> Immobilon-FL transfer membran (PVDF) </td><td> Millipore </td><td> IPFL00010 </td><td></td></tr><tr><td> Vakum pompası </td><td> Millipore </td><td> WP6111560 </td><td></td></tr><tr><td> Kemilüminesans BioMax Işık Film </td><td> Kodak </td><td> 178 8207 </td><td></td></tr><tr><td> FluorChem Jel Dökümantasyon Sistemi </td><td> Alpha Innotech </td><td> 29-008-1896X </td><td></td></tr><tr><td> Evrensel tavşan HA ve NA antijenlere karşı antikorlar </td><td></td><td> Uni-1 (HA) HCA-2, HCA-3 (NA) </td><td> Antikorlar MTA mevcuttur ya da daha önce 6,7 açıklanan prosedürlere göre ilgilenen araştırmacılar tarafından oluşturulmuş olabilir. </td></tr><tr><td> Grip aşısı referans antijen </td><td> CBER / FDA veya NIBSC </td><td></td><td></td></tr><tr><td> Grip aşısı örnekleri </td><td></td><td></td><td> Pek çok ülkede yaygın olarak bulunan </td></tr><tr><td> Üre </td><td> Sigma-Aldrich </td><td> U1250 </td><td></td></tr><tr><td> Zwittergent 3-14 Deterjan </td><td> Calbiochem </td><td> 693017 </td><td></td></tr><tr><td> Tween-20 </td><td> Fisher Scientific </td><td> BP337-500 </td><td></td></tr><tr><td> Blot Sınıf Engelleyicisi Yağsız kuru süt </td><td> Bio-Rad </td><td> 170-6404 </td><td></td></tr><tr><td> ImmunoPure Keçi anti-tavşan IgG, (H + L), Konjuge Peroksidaz </td><td> Thermo Scientific </td><td> 31460 </td><td></td></tr><tr><td> SuperSignal Batı Dura Genişletilmiş Süre Yüzey </td><td> Thermo Scientific </td><td> 34075 </td><td></td></tr></tbody></table>

quantitative analyses of all influenza type a viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays

Hemaglutinin (HA) ve nöraminidaz (NA), viral bir yaşam döngüsü ve koruyucu immün yanıtları 1,2 indüksiyonu önemli rol oynadığı bilinmektedir grip virüsleri iki yüzey proteinleri. HA nötralize edici antikorlar için ana hedef olarak, şu anda grip aşısı potens belirteci olarak kullanılır ve tek radyal immunodiffüzyon (SRID) 3 ile ölçülür . Ancak, ilgili alt tip spesifik antiserumlar durumu SRID bağımlılığı her yeni aşı serbest bırakılması için en az 2-3 ay gecikme neden olur. Ayrıca, NA da, koruyucu bağışıklığı 4, influenza aşılarının NA miktarını uygun reaktifler analitik yöntem 5 eksikliği nedeniyle henüz standart değil uyardığı kanıtlara rağmen . Böylece, HA ve NA antijenleri nicel kapasitesine sahip basit bir alternatif yöntemler hızlı serbest bırakılması ve influenza aşıları daha iyi kalite kontrol için arzu edilir. Mevcut tüm influenza A HA ve NA dizileri Evrensel korunmuş bölgelerde 6-7 biyoinformatik analizleri ile tespit edilmiştir. Bir dizisi (Uni-1 olarak belirlenen), iki korunmuş dizileri, enzimatik aktif sitesine yakın (HCA-2 olarak belirlenmiş) ve nöraminidaz tespit ise, füzyon peptid 6 HA, sadece evrensel korunmuş epitop tespit edildi N-terminalindeki diğer yakın (HCA-3 olarak belirlenmiş) 7. Bu amino asit dizileri ile Peptitler sentezlenen ve antikor üretimi için tavşan bağışıklık kullanılır. HA Uni-1 epitop karşı antikor HCA-2 ve HCA-3 bölge (NA) karşı antikorlar 9 NA alt tipi bağlama kapasitesine sahip iken 13 alt tipi influenza A HA (H1-H13) bağlamak mümkün oldu. Tüm antikorlar allantoik proteinlere karşı çapraz reaktivite tespit edildiğini gözlem kanıtladığı gibi viral dizileri karşı olağanüstü özgüllük gösterdi. Bu evrensel antikorlar daha sonra spesifik antiserumlar 7,8 gerek kalmadan aşılar influenza A HA ve NA ölçmek için slot blot testleri geliştirmek için kullanılmıştır. Aşı örnekleri çeşitli konsantrasyonlarda seyreltilmiş referans standartları ile birlikte bir yuva leke aparatı kullanarak PVDF membran üzerine uygulandı. HA tespiti için, örnekler ve standart ilk seyreltilmiş edildi Tris tamponlu salin (TBS), NA ölçümü için% 0,01 olarak Zwittergent bu koşulları içeren TBS seyreltilmiş iken 4M üre içeren algılama hassasiyetini önemli ölçüde geliştirilmiş. HA ve NA antijenleri kendi evrensel antikorlar ile immün tespiti sonrasında, sinyal intensitesi dansitometrisi tarafından ölçüldü. HA ve NA aşıların miktarları daha sonra çeşitli konsantrasyonlarda kullanılan referanslar sinyal intensitesi ile kurulmuş bir standart eğrisi kullanılarak hesaplanmıştır. Bu antikorların, HA ve NA evrensel epitoplar bağlamak göz önüne alındığında, ilgilenen araştırmacılar sadece slot leke dışında immunoassay araştırma araçları olarak kullanabilirsiniz.

Watch this Scientific Journal Video about Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays at JoVE.com

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

En korunmuş dizileri biyoinformatik analizleri ile tespit hedefleyen evrensel bir antikor kullanarak influenza virüs hemaglutinin ve nöraminidaz miktarının tayini için basit bir yuva blot yöntemi geliştirilmiştir. Bu yenilikçi yaklaşım, tüm viral hemaglutinin ve nöraminidaz kantitatif tayini için kullanışlı bir alternatif sunabilir.

Tüm Influenza Kantitatif Analiz Basit Slot Blot Tahliller Evrensel Antikorlar kullanarak Viral Hemagglutinins ve nöraminidaz yazın

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

quantitative-analyses-all-influenza-type-viral-hemagglutinins

Research

JoVE Journal

Immunology and Infection

18.1K Görüntüleme.  Health canada. En korunmuş dizileri biyoinformatik analizleri ile tespit hedefleyen evrensel bir antikor kullanarak influenza virüs hemaglutinin ve nöraminidaz miktarının tayini için basit bir yuva blot yöntemi geliştirilmiştir. Bu yenilikçi yaklaşım, tüm viral hemaglutinin ve nöraminidaz kantitatif tayini için kullanışlı bir alternatif sunabilir.

Video: Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

İmmünoloji ve Enfeksiyon

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Gen İfade Tüm Dönemlerinde Viral Fonksiyonu sayısallaştırabilmek için vaksiniya Reporter Virüsler

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Kullanımı Geleneksel Hikaye ve Roman Yerleşimi Sistemleri: Plak Tahliller sayesinde viral Konsantrasyon Tayini

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A Virüsü hemaglutininler aviditesi ve özgünlüğünün değerlendirilmesi için Minyatür Glycan Mikroarray Testi

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Enzim-linked Lektin Testi tarafından Grip Nöraminidaz İnhibisyon antikor titreleri Ölçme

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Grip A virüs enfeksiyonu bir fare modeli çalışmalarda

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Grip Hemagglutinin özel antikorlar tarafından indüklenen Fc-aracılı efektör fonksiyonları değerlendirmek için bir yöntem

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...