VeriPlexTM Human Interferon 9-plex ELISA Kit
Contents
1. The recovery of high medium and low IL 1o spikes in normal human plasma with Na EDTA from donor B was particularly poor Poor recoveries of low IP 10 spikes in plasma are due to presence of 7110 pg ml of apparent endogenous IP 10 in the plasma lots 29 iii Intra assay and Inter assay CV a Intra assay 96 CV Normal Normal hu human man plasma Sample serum DMEM 10 with Na EDTA Analyte Diluent donor A donor B 3 2 5 6 6 7 4 3 0 f 4 i 30 b Inter assay CV Normal Normal human human plasma with Sample serum DMEM 10 Na EDTA Analyte Diluent or A S or B don FB don it ta 92 98 1499 ND 31 2 Cross Reactivity Studies i IFN subtypes Independant curves of recombinant human IFN 2 1 recombinant human IFN 42 and recombinant human IFN 23 were prepared in Sample Diluent A separate standard curve was prepared using the human IFN Multiplex Antigen Standard supplied in the product The recovery of those points with pixel intensitites withtin the range of pixel intensities of IFN 1 2 in the multiplex standard were averaged to estimate the Reactivity for each subtype Subtypes 96 Reactivity 32 ii Human IFN c subtypes Rhesus Monkey IFN c Cynomolgus monkey IFN a ILe16 and Cynomolgus monkey IFN B Independent curves of recombinant analytes listed in the table below were prepared in Sample Diluent A separate standard curve w2. noise reduction iv Set the exposure time to 6 min for the first time N Click on Acquire Image O Once the image is acquired save it and look at the pixel intensity of the high points on the standard curve On average most of the high points on the curve should be in the 45 000 50 000 pixel intensity range and on the second spot they should be in the 20 000 40 000 pixel intensity range i If there are spots where the PI pixel intensity on the high point of the curve is at 65 000 and the second point is 60 000 or higher then acquire another image of the plate for a shorter period of time ii If the spots on the high point of the curve in general fall below 40 000 PI then reacquire the image for a longer period of time 17 5 Acquiring an image using the Bio Rad VERSADOC 4000 Camera and Software Setup A File setup a Open the software on the computer b Click on File then select Versadoc c Make sure that only Channel 1 is enabled d Click on the Select button then select Custom and then Create e When the new window opens name this custom setup as Quansys 1X1 binning and change the settings to i Filter None ii Illumination None lii Gain 1X iv Binning 1X1 f Click on OK Now that this setting is saved you can use it again for future exposures Instead of selecting Create select Quansys 1X1 binning B Camera Setup and Focus a Open the imager door b Place a box or st
3. Press the Epi White button on the camera cabinet e Press the zoom buttons on the cabinet until the plate fills most of the screen on the computer f Replace the plate with the imaging target sheet or the mouse pad and close the door g Press the focus buttons on the cabinet until the targets or letters on the mouse pad are in focus on the computer screen h Replace the imaging target sheet mouse pad with the plate close the drawer and make sure the plate is in the center of the imaging screen and straight i Turn off the Epi White button on the cabinet and select freeze in the software C Image Acquisition a On Step Ill Acquire Image on the software change the exposure time to 30 seconds and select Manual Expose b When the exposure is complete convert the image into negative black background with white spots i Click Image and new menu appears li Select Transform and then check the box that says Invert Display lii Click OK c Save the image D Image Optimization a Once the image is acquired look at the pixel intensity of the high points on the standard curve On average most of the high points on 21 the curve should be in the 45 000 55 000 Pixel Intensity range and on the second spot they should be in the 20 000 40 000 pixel Intensity range i If there are spots where the PI pixel intensity on the high point of the curve is at 65 000 and the second point is 60 000 or higher
4. 2002 Dec 4 69 77 10 Human IFN lambda 1 modulates the Th1 Th2 response Gallagher G et al Genes amp Immunity 2007 March 8 254 261 11 Lambda Interferon inhibits Hepatitis B and Hepatitis C Virus Replication Chisar FV et af Journal of Virology 2005 March 79 6 3851 3854 12 Interferon Lambda as a potential new therapeutic for hepatitis C Williams DE et af Annals of the New York Academy of Sciences 2009 Dec 1182 80 87 36 NOTES 37 NOTES 38 NOTES 39 x terferon source
5. for 15 minutes After 15 minutes empty the contents of the plate and wash the wells 6 times with 300 ul working Wash Solution per well 4 Substrate Mix and Imaging Add 50 ul of the prepared Substrate Mix to each well Image the plate within 20 minutes of adding the Substrate Mix Refer to page 12 onwards for detailed instructions on imaging the plate using different imagers 10 D ASSAY PROCEDURE QUICK REFERENCE Total time 3 hours and 15 minutes 1 Add 50ul Assay Diluent 2 Add 50ul Test Sample Standard or Blank Incubate 2 hours with shaking medium to fast Aspirate and wash 3 x Add 50 ul reconstituted Detection Antibody Incubate 1 hour with shaking medium to fast Aspirate and wash 3 x Add 50 ul Streptavidin HRP with shaking medium to fast Incubate 15 minutes in the dark Aspirate and wash 6 x Add 50ul Mix of Substrate A and Substrate B Image plate within 20 minutes HHLA 11 E IMAGING PROCEDURE 1 Quansys O View Imager Acauiring an Image These are basic instructions for using the Q View Imager and Software to image your plate only A comprehensive software manual for use of Q View software is available A full version of the Quansys Q View Software Version 2 07 is available for free The download is available at http www quansysbio com pbl q view software The user manual for the software can be found under Support Help A Select New Project if
6. homology to one another with IFN A2 and IFN A3 sharing 96 amino acid identity and with IFN 2 1 sharing 81 homology with IFN A2 and IFN A3 Type Ill interferons are functionally similar to type interferons and are known to have similar downstream effects i e type Ill interferons promote the phosphorylation on STAT1 and STAT2 induce the ISRE3 complex elevate OAS and type IFN inducible Myxovirus resistance protein A MXA expression and exhibit anti viral activity 7 vitro 19 however type III interferons and their heterodimeric receptor subunits CRF2 12 IFN AR1 9 and CRF2 4 IL 10R2 9 are known to be more prominent in cells of the epithelial tissues 9 IFN 2 1 is known to modulate the development of Th1 Th2 cells 19 IFN A2 has been shown to inhibit the replication of hepatitis B and hepatitis C virus in murine hepatocyte cell lines and IFN A1 is being explored as a potential therapeutic for hepatitis C 12 The VerPlex Human Interferon 9 plex ELISA has been developed to simultaneously detect IFN a IFN B IFN y IFN w IFN A1 2 3 and other key pro inflammatory cytokines released upstream and downstream of interferon signaling including TNF a IP 10 IL 1c0 and IL 6 This assay has been developed using the Q Plex array spotting technology in which capture antibodies to the different analytes are spotted in a single well in a 3x3 array The functional format of the assay is as that of a sandwich ELISA with a chemiluminiscent
7. value of IL 6 in normal human serum was high due to presence of 1935 2 pg ml of IL 6 in serum from a particular donor Levels in serum from 17 donors were either close to LLOD or lt LLOD N A Not applicable because levels in serum or in plasma from all donors were lt LLOD 26 li Spike Recovery Low medium and high spikes were prepared using the Multiplex Antigen Standard in Normal human serum from a single donor Normal human plasma with different anti coagulants TCM DMEM 1096 FBS and Sample Diluent The concentration of spikes were extrapolated from a Standard Curve prepared in Sample Diluent The recoveries in Normal human serum and in Normal human plasmas were calculated after subtracting measured levels of endogenous analytes in the matrices from the recovered values a High Spike Normal Normal Normal Normal Normal human human human human human plasma plasma plasma plasma serum with Na with Na with Na with Na Sample DMEM 10 donor EDTA EDTA Heparin Citrate Diluen FBS A donor B or C donor C or D ENTM 7 1 6 38 87 don don IFN o z 9 3 oF n 82 8 92 5 0 27 b Medium Spike Normal Normal Normal human human Normal human Normal plasma plasma human plasma Analyte A B 12 C 63 28 c Low Spike Normal Normal Normal Sample DMEM 10 93 94 90 82 donor A donor B D C C
8. 1 2 and 3 Human Tumor Necrosis Factor alpha TNF a Human Interleukin 6 IL 6 Human Interferon Gamma inducible protein IP 10 and Human Interleukin 1 alpha IL 10 in sera plasma and tissue culture media by sandwich enzyme linked immunosorbent assay ELISA using the Q Plex Multiplex technology Detection Ranges Refer to the supplied lot specific Certificate of Analysis Speed Incubation time 3 hr 15 min Specificity The FN A antigen in the Standard is an equal mix of IFN A 1 and A2 The IFN A antibody pair in the product detects IFN 2 1 22 and 23 although IFN A2 and 3 are detected less effectively Rhesus monkey IFN o Cynomolgus monkey IFN o and Cynomolgus monkey IFN D are detected by the product Mouse IFN p Mouse IFN y and Mouse IFN A3 do not cross react with the product 10 ug ml of Mouse IFN a demonstrated 0 004 cross reactivity Refer to pages 32 34 for details Storage Conditions Comments For retention of activity all reagents should be kept at 2 8 C in the dark when not in use Diluents and buffer reagents should be warmed to room temperature RT before use We have not fully evaluated the long term stability of reconstituted materials in liquid or frozen form Q Plex is a registered trademark of Quansys Biosciences 3 MATERIALS PROVIDED e Human IFN Multiplex 96 Well Microtiter plate e Plate Sealers e Wash Solution Concentrate e Human IFN Multiplex Antigen Standard e Sample Diluent e Assay D
9. a a c sa s3 sa salsa sa salsalsa s sa sa 2 5 A Standard Curve D S4 S4 Sa Sa Sa Sa Sa Sa Sa S Sa Sa a Sa Test Samples i a F S6 S6 Sa Sa Sa Sa Sa Sa Sa S Sa Sa a G S7 S7 Sa Sa Sa Sa Sa Sa Sa S Sa Sa 3 Figure 2 H Sa SalSa Sa Sa Sa Sa s sa sa Model a Plate Setup B K Blank Step 1B Adding Standards Blanks and Test Samples Add 50ul of Standards to wells designated as Standard Add 50ul of Blanks Sample Diluent to wells designated as Blanks Add 50pl Test samples to wells designated as Test Samples After 2 hours empty the contents of the plate and wash the wells 3 times with 300 ul of the working Wash Solution refer to Preparation of Reagents per well 2 Detection Antibody Add 50ul of reconstituted Detection Mix refer to Preparation of Reagents to each well Cover with Plate Sealer and shake plate at RT 22 25 C for 1 hour During the incubation prepare a Substrate Mix by mixing equal volumes of Substrate A and Substrate B Use full contents of Substrate A and Substrate B Store the mix at RT 22 25 C in the dark until use in Step 4 The mix must be prepared at least 10 minutes prior to use in Step 4 After 1 hour empty the contents of the plate and wash the wells 3 times with 300 ul working Wash Solution per well 3 Streptavidin HRP Add 50ul of supplied Streptavidin HRP to each well Cover with plate sealer and shake in the dark at RT 22 25 C
10. and in the cabinet below the camera to increase the imaging height between 4 and 6 inches c Place the imaging target sheet or the mouse pad in the cabinet on top of the box or stand d Open the aperture on the camera all the way to the lowest value e Leave the door slightly open to let in light while focusing 18 f Click Focus in the software and turn the focus on the camera until the imaging targets on the screen on the computer are in focus or the letters on the mouse pad are in focus Click Stop when in focus g Replace the imaging target sheet mouse pad with the plate and ensure the plate is centered in the imaging screen and is straight h Close the cabinet door C Image Acquisition a On Step Ill Set exposure time on the software change the exposure time to 30 seconds and select Acquire b When the exposure is complete convert the image into negative black background with white spots i Click Image and new menu appears ii Select Transform and then check the box that says Invert Display iii Click OK c Save the image D Image Optimization a Once the image is acquired look at the pixel intensity of the high points on the second dilution in the standard curve and make sure they are not saturated On average most of the high points on the curve should be in the 45 000 55 000 Pixel Intensity range and on the second spot they should be in the 20 000 40 000 pixel Intensity rang
11. as prepared using the Human IFN Multiplex Antigen Standard supplied in the product The 96 recovery of those points on the curves of the test analytes with pixel intensities within the range of pixel intensities of IFN o A 2a in the curve prepared using the Multiplex Antigen Standard were averaged to estimate the Reactivity for each analyte 33 2 3 ng ml of Cynomolgus IFN D was measured in tissue culture supernatant of a mammalian cell line expressing Cynomolgus IFN p No other antigen was detected in the supernatant iii Mouse IFN A Mouse IFN D Mouse IFN y and Mouse IFN A3 Independent curves starting at 10 ug ml of recombinant analytes listed below were prepared in Sample Diluent A separate standard curve was prepared using the Human IFN Multiplex Antigen Standard supplied in the product The recovery of those points with pixel intensities within the range of pixel intensities of corresponding human analytes in the curve prepared using the Human IFN Multiplex Antigen Standard were averaged to estimate the 96 cross reactivity for each analyte Catalog Number Cross reactivity 12100 1 Murine IFN a A 0 004 12400 1 Murine IFN B 12500 1 Murine IFN 12820 1 Murine IFN 3 34 3 Additional Studies Serum samples from 27 Rheumatoid Arthritis RA patients were tested Only 1 sample gave false positive The spike recovery was acceptable 20 human plasma samples with constituents known to interfere in immuno assay
12. d If not click on Refresh H Type the following settings in the ISO and F Stop fields ISO 400 F Stop 2 8 Recommended exposure times are 30 60 and 180 seconds Each exposure will have a different image The software will also display a stacked image J Enter the names of the image s For example Expt1 30 sec Expt 1 60 sec and Expt 1 180 sec K Place the plate in the plate housing slot close the plate housing door and select Capture Image The imaging should begin Once acquired the image will appear in the Q View Software main screen L Save the acquired image s by clicking Image Analysis Export Image Export the image s as TIFF file s 13 2 Q View Software Importing an Image File Q View Software can process images in the following formats CR2 raw image files from Canon cameras TIFF JPEG PNG and BMP Users should take images using supported imaging systems See Page 4 To acquire an image by importing an image file select Import Image Browse and select the image When prompted select the imager used to acquire the image If you imager is not listed select Other Also select Other if the image was acquired using the Q View Imager to override repeated vignette correction The time to upload the image will vary depending on the image file type and size Once imported the image will appear in the Q View Software main screen 14 Images acquired using the following imagers can be imp
13. e i If there are spots where the PI pixel intensity on the high point of the curve is at 65 000 and the second point is 60 000 or higher then re expose image of the plate for a shorter period of time 1 minute 19 ii If the spots on the high point of the curve in general fall below 40 000 PI then re expose the image for a longer period of time i e 3 minutes E File Conversion a After acquiring the images you need to convert them to TIFF files b Click on File then Export to Tiff image c Select Export raw data click on Export then click on Save 6 Acquiring an image using the Bio Rad CHEMIDOC XRS Camera and Software Setup A File setup a Open the software on the computer b Click on File then select ChemiDoc XRS c Under Step Select Application press the Select button then select Custom and then Create d When the new window opens name this custom setup as Quansys 1X1 binning Under Illumination select none and under gain amp binning select 2X 1X1 and click OK Now that this setting is saved you can use it again for future exposures Instead of selecting Create select Quansys 1X1 binning B Camera Setup and Focus a On the software select Live Focus b On the cabinet press the plus button to open the aperture or iris all the way the lowest number 20 c Open the camera s drawer place the plate in the middle of the drawer then close the drawer d
14. ers on the mouse pad are in focus d Remove the mouse pad from the imager and place the plate in the center of the tray Look on the computer screen to make sure the plate is centered and straight in the imaging screen Close the imager door when the plate is centered e Select the Return button on the software to close the focusing window C Image Acquisition a When the plate is ready to image press the Start button b After the plate has imaged invert the image to black with white spots by clicking on View then selecting Positive Gray in the drop down menu c Save the image by pressing the Save button In the new window select 16 bit linear tiff in the Save as type drop down menu Then type a name for the file and select Save d Press the Complete button to allow the imager to take another image 23 e Take multiple images at different exposure times to ensure you get the best reading possible Example exposure times are 20 seconds 45 seconds 60 seconds 90 seconds and 120 seconds D Image Optimization a Once the image is acquired look at the pixel intensity of the high points on the standard curve On average most of the high points on the curve should be in the 45 000 55 000 Pixel Intensity range and on the second spot they should be in the 20 000 40 000 pixel Intensity range i If there are spots where the PI pixel intensity on the high point of the curve is at 65 000 a
15. iluent e Human IFN Multiplex Detection Mix e HRP Concentrate e Substrate A e Substrate B e Diluent Additive ll e Diluent Additive III ADDITIONAL MATERIALS REQUIRED WOT PROVIDED e Variable volume micro titer pipettes e Adjustable multi channel pipette 50 300ul e Reagent reservoirs e Wash bottle or plate washing system e Distilled or deionized water e Serological pipettes 1 5 10 or 25m1 e Disposable pipette tips polypropylene e Plate shaker One of the following cameras imagers e Q View Imager recommended e Alpha Innotech HD2 and FC2 Camera e Bio Rad VERSADOC 4000 Camera e Bio Rad CHEMIDOC XRS Camera e Fujifilm LAS 3000 Camera e KODAK 4000MM Camera e Q View software Version 2 0 INTRODUCTION Interferons IFNs are a group of cytokines which exhibit pleiotropic activities that play major roles in both innate and adaptive immunity There are three types of interferons namely type I Il and Ill Type IFNs consist of multiple Interferon Alpha IFN o genes at least one Interferon Beta IFN D gene and one Interferon Omega IFN 0 gene in most vertebrates IFN a IFN B and IFN W are released by a host of mammalian cells on exposure to viruses or double stranded RNAs 2 and on triggering of Toll like receptors TLR3 4 7 8 9 by CPG DNAs and lipopolysaccharide LPS Upon binding to their cellular receptor chains IFN o Rc1 and IFN c Rc2 type interferons signal through the Jak Stat pathway to further elicit a ho
16. ixel intensity range and on the second spot they should be in the 20 000 40 000 pixel intensity range i If there are spots where the PI pixel intensity on the high point of the curve is at 65 000 and the second point is 60 000 or higher then acquire another image of the plate for a shorter period of time ii If the spots on the high point of the curve in general fall below 40 000 PI then reacquire the image for a longer period of time 4 Acquiring an image using the Alpha Innotech FC2 Camera and Software Setup A Open the camera door B Setthe adjustable tray on the top shelf C Place the mouse pad in the center of the tray for focusing D Open the aperture on the camera all the way to the lowest value 1 8 E Open the software on the computer F Click the Acquire button 16 G Close the door on the camera slightly so some light can get in and the letters on the mouse pad can be seen on the computer screen H Adjust the focus on the actual camera lens until the letters on the mouse pad are in focus Remove the mouse pad J Place the plate in the center of the tray and make sure it is in the center of the photo path on the computer screen K Close all doors on the camera and ensure there are no light leaks L Ensure all cabinet lights are turned off and that the filter wheel is set to T M Set the software settings on the computer i No lights on ii Resolution to Normal Ultra lii Select only
17. nd the second point is 60 000 or higher then re expose image of the plate for a shorter period of time i e 20 seconds ii If the spots on the high point of the curve in general fall below 40 000 PI then re expose the image for a longer period of time i e 2 minutes 24 F PRODUCT PERFORMANCE CHARACTERIZATION 1 Matrix studies i Levels of analytes in Normal human serum and Normal human plasma Normal human serum from 20 individual donors and Normal human plasma with different anti coagulants Na EDTA Na Citrate and Na Heparin from 13 other individual donors were tested in the assay The levels of analytes in the samples were extrapolated from a Standard Curve prepared in Sample Diluent Serum and plasma from all donors had detectable levels of IP 10 The average concentration of IP 10 in serum was 55 6 pg ml while in plasma was 86 9 pg ml Serum from one donor had 16 0 pg ml of IFN c serum from a second donor had 6 4 pg ml of IL 1 o and 40 pg ml of IL 6 serum from a third donor had 9 1 pg ml of IL 1 1935 2 pg ml of IL 6 and 27 0 pg ml of TNF c and serum from a fourth donor had 43 4 pg ml of IL 6 Serum from remaining 16 donors had undetectable levels of analytes other than IP 10 Plasma from all 13 donors had undetectable levels of analytes other than IP 10 25 Normal human serum Normal human plasma 20 donors 13 donors Analyte pvetaee Range pg ml Average Range pg ml pg ml pg ml Average
18. orted into the Q View software for analysis Refer to page 14 for details on importing images to Q View software 3 Acquiring an image using the Alpha Innotech HD2 Camera and Software Setup A Open the camera door B Set the adjustable tray on the lowest level C Place the place the mouse pad in the center of the tray for focusing D Open the aperture on the camera all the way to the lowest value 95 E Open the software on the computer F Click the acquire button G Close the door on the camera slightly so some light can get in and the letters on the mouse pad can be seen on the computer screen H Adjustthe focus on the actual camera lens until the letters on the mouse pad are in focus Remove the mouse pad J Place the plate in the center of the tray and make sure it is in the center of the photo path on the computer screen K Close all doors on the camera and ensure there are no light leaks L Ensure all cabinet lights are off and that the filter wheel is set to position us 15 M Set the software settings on the computer as follows i No lights on ii Resolution to Normal Ultra lii Select only noise reduction iv Set the exposure time to 3 min for the first time N Click on Acquire Image O Once the image is acquired save it and look at the pixel intensity of the high points on the standard curve On average most of the high points on the curve should be in the 45 000 55 000 p
19. output The assay is compatible with multiple matrices including tissue culture media human serum human plasma and buffers B PREPARATION OF REAGENTS Supplied Human IFN Multiplex Antigen Standard Human IFN Multiplex Detection Mix and HRP Concentrate should be kept on wet ice Wash Solution Prepare a 1 20 working wash solution Add 5Oml of the Wash Solution Concentrate to 950ml of distilled or deionized water mix thoroughly The working Wash Solution can be stored at 2 25 C when not in use Standard Reconstitute the supplied Human IFN Multiplex Antigen Standard by adding the volume of Sample Diluent indicated in the lot specific Certificate of Analysis Mix gently until the Antigen Mix is completely reconstituted and store on wet ice until use Do not vortex Do not introduce bubbles Standard Curve Preparation Label 7 polypropylene tubes as S2 S7 Prepare a 3 fold dilution series using the reconstituted Antigen Standard and Sample Diluent as per figure 1 below Mix thoroughly between each dilution by pipeting 5X The high point S1 in the series is the reconstituted Antigen Standard 0 06 ml 0 06mi 0 06mi 0 06mi 0 06mi 0 06mi Recon Antigen Blank STD S1 0 12mi 0 12mi 0 12mi 0 12mi 0 12mi 0 12mi 0 12 ml e Pm Y Pn Y 77 h S2 S3 S4 S5 S6 S7 Volume of Sample Diluent Figure 1 Standard curve 1 2 3 4 5 6 7 8 9 1 11 12 0 1 Sa Sa Sa Sa Sa Sa Sa S Sa Sa a S2 Sa Sa Sa Sa Sa Sa Sa S Sa S
20. phi Ms source VeriPlex Human Interferon 9 plex ELISA Kit Product 51500 1 Store all components at 2 8 C We recommend reading the protocol in its entirety prior to use First time users must pay particular attention to pages 12 24 and read the manual of the Q View imager and software available for download at www quansysbio com Sold under license from Pestka Biomedical Laboratories Inc d b a PBL InterferonSource For research use only Not for diagnostic or clinical use in or administration to humans Not for resale in original or any moditied form including inclusion in a kit for any purpose Not for use in the preparation of any commercial product Copyright 2012 Pestka Biomedical Laboratories Inc All rights reserved Table of Contents a Tee 51500 1 Rev 03 PRODUCT INFORMATION PREPARATION OF REAGENTS ASSAY PROCEDURE ASSAY PROCEDURE QUICK REFERENCE IMAGING PROCEDURE Q View Imager Acquiring Q View Software Importing an Image Alpha Innotech HD2 Camera Alpha Innotech FC2 Camera Bio Rad VERSADOC 4000 Camera Bio Rad CHEMIDOC XRS Camera FUJIFILM LAS 3000 Camera PRODUCT PERFORMANCE CHARACTERIZATION Matrix Studies Cross Reactivity Studies Additional REFERENCES A PRODUCT INFORMATION Specifications This kit quantitates Human Interferon Alpha IFN o Human Interferon Beta IFN B Human Interferon Gamma IFN y Human Interferon Omega IFN w Human Interferon Lambda IFN A
21. s were tested No sample gave false positive Please note that detection of analytes in serum and plasma from patients on certain therapeutics can be affected due to the presence of antibodies against analytes from the multiplex in such samples 35 G REFERENCES 1 Evolution of the class 2 cytokines and receptors and discovery of new friends and relatives Pestka S et ak Pharmacology amp Therapeutics 2005 June 106 3 299 346 2 Interferons Interferon like cytokines and their receptors Pestka S et ak Immunological Reviews 2004 202 8 32 3 Interferon Alpha in malignant and viral diseases A review Dorr RT Drugs 1993 45 2 4 Interferon treatment for relapsing multiple sclerosis Z vadinov R et af Expert Opinion in Biological Therapeutics 2008 8 9 1435 37 5 Interferon g an overview of signals mechanism and functions Hume DA et af Journal of Leukocyte Biology 2004 75 163 189 6 IFN Novel anti viral cytokines Paludan SR et af Journal of Interferon amp Cytokine Research 2006 May 26 6 373 79 T IL 28 IL 29 and their class II cytokine receptor IL 28R Sheppard P et al Nature Immunology 2002 Dec 4 63 68 8 Molecular characterization and Antiviral activities of Type IIl interferons Blecha F et af Journal of Interferon amp Cytokine Research 2010 Nov 30 11 801 807 9 IFN As mediate antiviral protection through a distinct class II cytokine receptor complex Donnolley RP et al Nature Immunology
22. st of anti viral actions including production of protein kinase A and 2 5 Oligoadenylate Sythetase OAS 2 Type interferons are used therapeutically to treat viral infections cancers and auto immune disorders IFN is used therapeutically to treat hepatitis B and hepatitis C infections Additionally IFN o is known to have significant biological activity in inhibition of proliferation of multiple cancers IFN D is used therapeutically to treat multiple sclerosis Type II Interferon consists of Interferon gamma IFN y IFN y is produced by a host of immune cells lymphocytes CD4 T cells NK cells and such antigen presenting cells APCs as macrophages monocytes and dendritic cells IFN y uses receptor chains IFN y R1 and IFN y R2 IFN y a homodimer binds two IFN y R1 sub units thereby generating binding sites for two IFN y R2 chains a process that subsequently triggers intra cellular signaling and activation of Jak1 Jak2 and Stat1 that in turn induce genes with the y activation sequence in the promoter 9 IFN y plays a role in several immunomodulatory functions including up regulation of pathogen recognition anti viral action activation of microbicidal functions in immune cells and leukocyte trafficking The newly characterized type III interferons consist of Interferon lambda1 IFN 2 1 or IL 29 Interferon lambda2 IFN A2 or IL 28A and Interferon lambda3 IFN A3 or IL 28B The members of Type Ill family share close
23. starting a new project Otherwise select Open Project to browse and select a previous project The new image will not overwrite prior images in the project B Ensure that the Q View Imager is connected to the computer C Optional Uncheck the box Discard sub images after stacking is complete under Preferences under Settings to see images of different exposure times after the imaging process in complete Otherwise a stacked image will be displayed D Optional It is recommended to periodically calibrate the Q View Imager To calibrate the imager select Manage Imagers under Administration under Settings Ensure that there is no plate in the plate housing slot in the imager Select Calibrate E Optional If the imager has not been focused previously place the mouse pad in the imager do not close the plate housing door and adjust the focus under Manage Imagers under Administration under Settings until the letters on the mouse pad are in focus Remove the mouse pad Close the Manage Imagers section Q View is a registered trademark of Quansys Biosciences 12 File Project Settings Support 9811 ecce QUANSYS spas 38 Acquire Image lt gt Import Image Ell Image Option li Plate Optio BIOSCIENCES eece Product Plate 1 e Image Processing Well Assignment Data Analysis Images F Select Acquire Image G Ensure that the Q View Imager is recognize
24. then re expose image of the plate for a shorter period of time i e 1 minute ii If the spots on the high point of the curve in general fall below 40 000 PI then re expose the image for a longer period of time i e 3 minutes E File Conversion a After acquiring the images you need to convert them to TIFF files b Click on File then Export to Tiff image c Select Export raw data click on Export then click on Save T Acquiring an image using the Fujifilm LAS 3000 Camera and Software Setup A File setup a Open the software on the computer b Under Exposure Type select Precision in the drop down menu c Under Exposure Time set the imager to take a 30 second image by selecting Manual then entering 30 in the first box and selecting sec inthe second box d Under Sensitivity select Standard in the drop down menu e Ensure the box next to Image Acquire amp Digitize is checked 22 f Click on the Method Tray Position button In the window that appears select Chemiluminescence and under tray position select 2 Then select OK to close the window B Camera Setup and Focus a Open the camera box and make sure the tray is in position 2 b Place the mouse pad on the tray or other imaging target sheet and close the door c On the software select Focus and a new window appears In the Adjust area click up or down on the arrows until the lett
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