सभी इन्फ्लुएंजा के मात्रात्मक विश्लेषण सरल स्लॉट ब्लाट Assays में एक वायरल Hemagglutinins और यूनिवर्सल एंटीबॉडी का उपयोग कर Neuraminidases प्रकार
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent or equipment | Company | Catalogue number | Comments |
|---|---|---|---|
| Bio-Dot SF Microfiltration Apparatus | Bio-Rad | 170-6542 | |
| Bio-Dot SF Filter paper | Bio-Rad | 162-0161 | |
| Immobilon-FL transfer membrane (PVDF) | Millipore | IPFL00010 | |
| Vacuum pump | Millipore | WP6111560 | |
| Chemiluminescence BioMax Light Film | Kodak | 178 8207 | |
| FluorChem Gel Documentation System | Alpha Innotech | 29-008-1896X | |
| Universal rabbit antibodies against HA and NA antigens | Uni-1 (HA) HCA-2, HCA-3 (NA) | Antibodies are available through MTA or can be generated by interested investigators according to the procedures previously described6,7. | |
| Influenza vaccine reference antigen | CBER/FDA or NIBSC | ||
| Influenza vaccine samples | Commonly available in most countries | ||
| Urea | Sigma-Aldrich | U1250 | |
| Zwittergent 3-14 Detergent | Calbiochem | 693017 | |
| Tween-20 | Fisher Scientific | BP337-500 | |
| Blotting Grade Blocker Non-fat dry milk | Bio-Rad | 170-6404 | |
| ImmunoPure Goat Anti-Rabbit IgG, (H+L), Peroxidase Conjugated | Thermo Scientific | 31460 | |
| SuperSignal West Dura Extended Duration Substrate | Thermo Scientific | 34075 |
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