Name: quantitative analyses of all influenza type a viral hemagglutinins and neuraminidases using universal antibodies in simple slot blot assays
Uploaded: 2011-04-04T13:00:00
Description: 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

लेखकों पांडुलिपि के संपादकीय समीक्षा के लिए श्रीमती मोनिका Tocchi धन्यवाद देना चाहूंगा. AMH किंग अब्दुलअ विश्वविद्यालय से एक छात्रवृत्ति के द्वारा समर्थित है, कनाडा में सऊदी अरब सांस्कृतिक ब्यूरो के माध्यम से.

1. अभिकर्मकों और उपकरणों की तैयारी <ol><li> स्लॉट दाग प्रक्रिया शुरू करने से पहले, Tris-buffered खारा (20 मिमी Tris, 137 मिमी NaCl, पीएच 7.6) Zwittergent (टीबीएस), या hemagglutinin हा, स्लॉट दाग, या 0.01% की 20_mls के लिए में 4M यूरिया समाधान के 20_mls तैयार neuraminidase क?...

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+the+host+cell+on+standardisation+of+influenza+vaccine+potency.">The influence of the host cell on standardisation of influenza vaccine potency.</a> Dev Biol Stand. 98, 183-188 (1999).</li><li>Sylte, M. J., Suarez, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+neuraminidase+as+a+vaccine+antigen.">Influenza neuraminidase as a vaccine antigen.</a> Curr Top Microbiol Immunol. 333, 227-241 (2009).</li><li>Bright, R. A., Neuzil, K. 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>

इन्फ्लूएंजा वायरल हा और एनए के मात्रात्मक निर्धारण के टीके के अनुसंधान और विकास के लिए महत्वपूर्ण हैं के बाद से इन दो सतह प्रोटीन सबसे महत्वपूर्ण वायरल प्रतिरक्षा प्रतिक्रिया 6-11 उत्प्रेरण घटक है...

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> अभिकर्मक या उपकरण के नाम </th><th> कंपनी </th><th> सूचीपत्र संख्या </th><th> टिप्पणियाँ </th></tr><tr><td> जैव डॉट एस एफ Microfiltration उपकरण </td><td> जैव - रेड </td><td> 170-6542 </td><td></td></tr><tr><td> जैव डॉट एस एफ फ़िल्टर कागज </td><td> जैव - रेड </td><td> 162-0161 </td><td></td></tr><tr><td> Immobilon FL हस्तांतरण झिल्ली (PVDF) </td><td> Millipore </td><td> IPFL00010 </td><td></td></tr><tr><td> वेक्यूम - पंप </td><td> Millipore </td><td> WP6111560 </td><td></td></tr><tr><td> Chemiluminescence Biomax लाइट फिल्म </td><td> कोडक </td><td> 178 8207 </td><td></td></tr><tr><td> FluorChem जेल प्रलेखन प्रणाली </td><td> अल्फा Innotech </td><td> 29-008-1896X </td><td></td></tr><tr><td> हा और एनए प्रतिजनों के खिलाफ यूनिवर्सल खरगोश एंटीबॉडी </td><td></td><td> विश्वविद्यालय 1 (हेक्टेयर) HCA-2, HCA-3 (एनए) </td><td> एंटीबॉडी MTA के माध्यम से उपलब्ध हैं या रुचि जांचकर्ताओं द्वारा पहले 6,7 वर्णित प्रक्रियाओं के अनुसार उत्पन्न किया जा सकता. </td></tr><tr><td> इन्फ्लुएंजा टीका संदर्भ प्रतिजन </td><td> CBER / एफडीए या NIBSC </td><td></td><td></td></tr><tr><td> इन्फ्लूएंजा टीका के नमूने </td><td></td><td></td><td> सामान्यतः अधिकांश देशों में उपलब्ध </td></tr><tr><td> यूरिया </td><td> सिग्मा Aldrich </td><td> U1250 </td><td></td></tr><tr><td> Zwittergent 3-14 डिटर्जेंट </td><td> Calbiochem </td><td> 693017 </td><td></td></tr><tr><td> बीच - 20 </td><td> फिशर साइंटिफिक </td><td> BP337 - 500 </td><td></td></tr><tr><td> सोख्ता ग्रेड अवरोधक गैर वसा शुष्क दूध </td><td> जैव - रेड </td><td> 170-6404 </td><td></td></tr><tr><td> ImmunoPure विरोधी - खरगोश बकरी आईजीजी (एच एल +), संयुग्मित peroxidase </td><td> थर्मो वैज्ञानिक </td><td> 31460 </td><td></td></tr><tr><td> SuperSignal पश्चिम Dura अवध्ि सब्सट्रेट विस्तारित </td><td> थर्मो वैज्ञानिक </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

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

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

एक सरल स्लॉट दाग विधि इन्फ्लूएंजा वायरल hemagglutinin और neuraminidase सार्वभौमिक उनके सबसे संरक्षित जैव सूचना विज्ञान विश्लेषण के माध्यम से की पहचान दृश्यों लक्ष्यीकरण एंटीबॉडी का उपयोग कर की मात्रा का ठहराव के लिए विकसित किया गया था. इस नए दृष्टिकोण सभी वायरल hemagglutinin और neuraminidase के मात्रात्मक निर्धारण के लिए एक उपयोगी विकल्प प्रदान कर सकता है.

सभी इन्फ्लुएंजा के मात्रात्मक विश्लेषण सरल स्लॉट ब्लाट Assays में एक वायरल Hemagglutinins और यूनिवर्सल एंटीबॉडी का उपयोग कर Neuraminidases प्रकार

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

Research

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