51500-1 Protocol
Contents
1. Epi White button on the camera cabinet Press the zoom buttons on the cabinet until the plate fills most of the screen on the computer Replace the plate with the imaging target sheet or the focusing plate and close the door Press the focus buttons on the cabinet until the targets or focusing plate are in focus on the computer screen Replace the imaging target sheet focusing plate with the plate to be read close the drawer and make sure the plate is in the center of the imaging screen and straight Turn off the Epi White button on the cabinet and select freeze in the software 20 Ima a C Ima a File a b C ge Acquisition On Step III Acquire Image on the software change the exposure time to 30 seconds and select Manual Expose 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 Save the image ge Optimization Once the image is acquired look at the pixel intensity of the high points on the standard cure 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 and th2. Interferon Alpha in malignant and viral diseases A review Dorr RT Drugs 1993 45 2 Interferon treatment for relapsing multiple sclerosis Zivadinov R et al Expert Opinion in Biological Therapeutics 2008 8 9 1435 37 Interferon y an overview of signals mechanism and functions Hume DA et al Journal of Leukocyte Biology 2004 75 163 189 IFN A Novel anti viral cytokines Paludan SR ef al Journal of Interferon amp Cytokine Research 2006 May 26 6 373 79 IL 28 IL 29 and their class II cytokine receptor IL 28R Sheppard P et al Nature Immunology 2002 Dec 4 63 68 Molecular characterization and Antiviral activities of Type lll interferons Blecha F et al Journal of Interferon amp Cytokine Research 2010 Nov 30 11 801 807 32 10 11 12 IFN As mediate antiviral protection through a distinct class Il cytokine receptor complex Donnolley RP et al Nature Immunology 2002 Dec 4 69 77 Human IFN lambda 1 modulates the Th1 Th2 response Gallagher G et al Genes amp Immunity 2007 March 8 254 261 Lambda Interferon inhibits Hepatitis B and Hepatitis C Virus Replication Chisari FV et al Journal of Virology 2005 March 79 6 3851 3854 Interferon Lambda as a potential new therapeutic for hepatitis C Williams DE et al Annals of the New York Academy of Sciences 2009 Dec 1182 80 87 33 111000000000000 a82 ZL W OL 6 PBL Assay Science 131 Ethe
3. b pbl assay science ASSAY SCIENCE VeriPlex Human Interferon 9 Plex ELISA Kit Catalog No 51500 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 23 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 Assay Science For research use only Not for diagnostic or clinical use in or administration to humans Not for resale in original or any modified form including inclusion in a kit for any purpose Not for use in the preparation of any commercial product Copyright 2013 Pestka Biomedical Laboratories Inc All rights reserved TABLE OF CONTENTS PART TITLE PAGE A introduction cose tacit ERR 3 B Assay Procedure Quick Reference 7 C Preparation of Reagents ssssssss 8 D Assay Procedure ioo ee eet 9 E Imaging Procedure NOOR WD Q View Imager Acquiring an Image 12 Q View Software Importing an Image 14 Alpha Innotech HD2 Camera 15 Alpha Innotech FC2 Camera 16 Bio Rad VERSADOC 4000 Camera 18 Bio Rad CHEMIDOC XRS Camera 20 FUJIFILM LAS 3000 Camera 22 Product Performance Characterization Matrix Studies sssssesessss
4. ASSAY PROCEDURE All incubations should be performed in a closed chamber at RT 22 25 C keeping the plate away from drafts and other temperature fluctuations Use a plate shaker at 500 rpm where indicated The shaking speed must be such that bubbles are not formed during shaking Use plate sealers to cover the plates as directed During all wash steps remove contents of plate by inverting and shaking over a sink and blotting the plate on lint free absorbent paper tap the plate It is recommended that the plate be soaked in Wash Solution for 30 seconds between washes Wash each well with a minimum of 300 ul of diluted Wash Solution for each wash step See Preparation of Reagents for details on dilution of concentrated solutions 1 Standards and Test Samples 1a Adding Assay Diluent Add 50 ul of Assay Diluent to each well We recommend running the standard blanks and test samples in duplicate Refer to the model plate setup in Figure 2 The highest dilution point of the standard S1 must be in wells A1 and A2 1b Adding Standards Blanks and Test Samples Add 50 yl of Standard to wells designated as Standard Add 50 yl of Blanks Sample Diluent or sample matrix to wells designated as Blanks Add 50 ul Test Samples to wells designated as Test Samples Cover with plate sealer and shake plate at 500 rpm at RT 22 25 C for 2 hours Figure 2 Example of a Typical Plate Setup A B 4 D E F G H
5. Adjustable multichannel pipette 50 300 pl Reagent reservoirs Wash bottle or plate washing system Distilled or deionized water Serological pipettes 1 5 10 or 25 ml Disposable pipette tips polypropylene Plate shaker One of the following cameras imagers Q View Imager recommended Alpha Innotech HD2 and FC2 Camera Bio Rad VERSADOC 4000 Camera Bio Rad CHEMIDOC XRS Camera Fujifilm LAS 3000 Camera KODAK 4000MM Camera e Q View software Specifications This kit quantitates Human Interferon Alpha IFN a Human Interferon Beta IFN 8 Human Interferon Gamma IFN y Human Interferon Omega IFN w Human Interferon Lambda IFN A 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 1a 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 IFN A antigen in the Standard is an equal mix of IFN A1 and A2 The IFN A antibody pair in the product detects IFN A1 A2 and A3 although IFN A2 and A3 are detected less effectively Rhesus monkey IFN a A Cynomolgus monkey IFN a and Cynomolgus monkey IFN B are detected by the product Mouse IFN B Mouse IFN y and Mouse IFN A3 do not cross rea
6. 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 focusing plate in the center of the tray for focusing D Open the aperture on the camera all the way to the lowest value 0 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 focusing plate can be seen on the computer screen H Adjust the focus on the actual camera lens until the focusing plate is in focus Remove the focusing plate J Place the plate to be read in the center of the tray and make sure it is in the center of the photo path on the computer screen 15 oz Close all doors on the camera and ensure there are no light leaks Ensure all cabinet lights are off and that the filter wheel is set to position 1 Set the software settings on the computer as follows i No lights on ii Resolution to Normal Ultra iii Select only noise reduction iv Setthe exposure time to 3 min for the first time Click on Acquire Image 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 pixel intensity range and on the second spot they should be in the 20 000 40 000 pixel intens
7. the imaging height between 4 6 inches Place the imaging target sheet or the focusing plate in the cabinet on top of the box or stand Open the aperture on the camera all the way to the lowest value Leave the door slightly open to let in light while focusing 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 focusing plate is in focus Click Stop when in focus Replace the imaging target sheet focusing plate with the plate to be read and ensure the plate is centered 18 in the imaging screen and is straight viii Close the cabinet door C Image Acquisition i On Step IlI Set exposure time on the software change the exposure time to 30 seconds and select Acquire ii When the exposure is compete convert the image into negative black background with white spots 1 Click image and new menu appears 2 Select Transform and then check the box that says Invert Display 3 Click OK iii Save the image Image Optimization i Once the image is acquired loot 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 range 1 If there are spots where the PI pixel inte
8. 1 S7 Standard Curve a Sample 10 After 2 hours empty the contents of the plate and wash the wells three times with at least 300 ul of working Wash Solution refer to Preparation of Reagents per well 30 second soak recommended between washes 2 Detection Antibody Add 50 yl of reconstituted Detection Mix refer to Preparation of Reagents to each well Cover with plate sealer and shake plate at 500 rpm at RT 22 25 C for 1 hour During this 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 three times with at least 300 ul of working Wash Solution per well 30 second soak recommended between washes 3 HRP Concentrate Add 50 ul of supplied HRP Concentrate to each well Cover with plate sealer and shake in the dark at 500 rpm at RT 22 25 C for 15 minutes After 15 minutes empty the contents of the plate and wash the wells six times with at least 300 ul of working Wash Solution per well 30 second soak recommended between washes 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 pg 12 and onwards for detailed instructions
9. ce 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 File setup A i ii iii iv V vi Open the software on the computer Click on File then select Versadoc Make sure that only Channel 1 is enabled Click on the Select button then select Custom and then Create When the new window opens name this custom setup as Quansys 1x1 binning and change the settings to 1 Filter None 2 Illumination None 3 Gain 1X 4 Binning 1X1 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 Camera Setup and Focus iv V vi vii Open the imager door Place a box or stand in the cabinet below the camera to increase
10. centrate to 950 ml of distilled or deionized water and mix thoroughly Diluted Wash Solution can be stored at RT 22 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 dissolved and store on wet ice until use Do not vortex Do not introduce bubbles Standard Curve Preparation Label seven polypropylene tubes S1 S7 Prepare a 3 fold dilution series using the reconstituted Antigen Standard and Sample Diluent as per Figure 1 Mix thoroughly between each dilution by pipeting 5x The high point S1 in the series is the reconstituted Antigen Standard Assay Diluent Reconstitute the Assay Diluent as per instructions in the lot specific Certificate of Analysis COA using distilled or deionized water Diluent Additive Ill and Diluent Additive IIl Do not vortex Store at RT 22 25 C until use in step 1 of the Assay Procedure Note Solution may appear cloudy and contain bubbles Figure 1 7 Point Standard Curve Prepared in Sample Diluent 6Oul 6Opl GOpl 60pl GOp GOUT CVOVOVONVOVOY 180 ul Reconstituted Antigen Standard Detection Mix Reconstitute the Human IFN Multiplex Detection Mix in 6 ml distilled or deionized water Mix gently until the mix is completely reconstituted Store on ice until use in step 2 of the Assay Procedure D
11. ct with the product 10 ug ml of Mouse IFN a demonstrated 0 004 cross reactivity Refer to pages 29 31 for details Storage Conditions Comments For retention of full 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 22 25 C before use We have not fully evaluated the long term stability of reconstituted materials in liquid or frozen form B ASSAY PROCEDURE QUICK REFERENCE Total Time 3 hr 15 min 1 Add 50 pl Assay Diluent 2 Add 50 pl Standard Test Sample or Blank l Incubate 2 hrs shake at 500 rpm Aspirate and wash 3x 30 second soak between washes Add 50 ul Reconstituted Detection Mix b Incubate 1 hr shake at 500 rpm Aspirate and wash 3x 30 second soak between washes Add 50 ul HRP Concentrate M Incubate 15 min in the dark shake at 500 rpm Aspirate and wash 6x 30 second soak between washes Add 50 ul Mix of Substrate A and B Image plate within 20 min b Note ALL incubations are at room temperature 22 25 C C 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 The Wash Solution Concentrate may contain crystals Place the bottle in a warm water bath and gently mix until completely dissolved Prepare a 1 20 working wash solution e g Add 50 ml of Wash Solution Con
12. e E Optional If the imager has not been focused previously place a Quansys focusing plate in the imager do not close 12 the plate housing door and adjust the focus under Settings gt Administration gt Manage Imagers until the image is clear Remove the focusing plate Close the Manage Imagers section Plate 1 Exposure Time s second 2 40 180 recommended fer Plex 3040 80 Image Nemets 30 sec 60 tec 180 see Q View Version 3 01 Select Acquire Image Ensure that the Q View Imager is recognized If not follow step B and click on Refresh Select Image Processing method either Legacy similar to previous versions of Q View or Standard new feature in Q View V Enter Exposure Time s Recommended exposure times are 30 60 and 180 seconds for the Legacy setting or 270 seconds for Standard Image Processing When using Legacy each exposure will have a different image The software will also display a stacked image Enter the names of the image s For example Expt1 30 sec Expt1 60 sec and Expt1 180 sec Place the plate to be read in the plate housing slot close 13 the plate housing door and select Capture Image s The imaging should begin Once acquired the image will appear in the Q View Software main screen L If the image is needed for use in other programs save the acquired image s by clicking Export Export the image s as TIFF file s Exposure Times These times can be modi
13. e pg ml Average Range pg ml Y pg mi 4795 PSN pgiml 72092 P9 IFN B N A N A IFN y N A N A NA NA NA LLOD to 102 7 Nase lt LLOD N A IP 10 9 14 to 130 3 21 3 to 182 2 lt LLOD LLOD to 27 0 lt LLOD N A Average 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 LLOD N A Not applicable because levels in serum or in plasma from all donors were LLOD 25 ii 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 human human human human plasma plasma plasma plasma with Na with Na with Na with Na Heparin Citrate xm 8 2 ENA N2 8 E 3 The recovery of high medium and low IL 1a spikes in normal human plasma with Na EDTA from donor B was particularly poor 26 b Medium Spike Normal Normal Normal Normal Normal h
14. e second point is 60 000 or higher 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 Conversion After acquiring the images you need to convert them to TIFF files Click on File then Export to TIFF image Select Export raw data click on Export then click on Save 21 7 Acquiring an image using the Fujifilm LAS 3000 Camera and Software Setup A File Setup a b C Open the software on the computer Under Exposure Type select Precision in the drop down menu 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 in the second box Under Sensitivity select Standard in the drop down menu Ensure the box next to Image Acquire amp Digitize is checked 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 Camera Setup and Focus a b C Open the camera box and make sure the tray is in position 2 Place the focusing plate or other imaging target sheet on the tray and close the door On the software select Focus and a new window appears In the Adjust area click up or do
15. erferon 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 subunits thereby generating binding sites for two IFN y R2 chains a process that subsequently triggers intracellular signaling and activation of Jak1 Jak2 and Stat1 that in turn induce genes with the y activation sequence in the promoter IFN y plays a role in several immunomodulatory functions including up regulation of pathogen recognition antiviral action activation of microbicidal functions in immune cells and leukocyte trafficking The newly characterized Type III IFNs consist of Interferon Lambda 1 IFN M or IL 29 Interferon Lambda 2 IFN A2 or IL 28A and Interferon Lambda 3 IFN A3 or IL 28B The members of the Type III family share close homology to one another with IFN A2 and IFN A3 sharing 96 amino acid identity and with IFN A1 sharing 81 homology with IFN A2 and IFN A3 Type III IFNs are functionally similar to Type IFNs and are known to have similar downstream effects i e Type Ill IFNs promote the phosphorylation on STAT1 and STAT2 induce the ISRE3 complex elevate OAS and Type IFNs induce Myxovirus resistance protein A MxA expression and exhibit antiviral activity in vitro however Type III IFNs and their heterodimeric receptor
16. fied to meet your specific assay but 30 60 and 180 second exposure times Legacy or 270 second exposure time Standard are recommended for most assays Once set one can exit the Exposure Times dialog by mousing outside of the dialog box Imager Imager Image Processing Image Processing Standard Legacy G View 2 Exposure Time s seconds Exposure Time s seconds 270 recommended for Q Plex 30 60 180 recommended for Q Plex 270 30 60 180 Image Namef s Image Name s 270 sec 30sec 60sec 180see capture mageti Standard Image Acquisition Option Legacy Image Acquisition Option 2 Q View Software Importing an Image File Q View Software can open images in the following formats TIFF CR2 raw image files from Canon cameras JPEG PNG and BMP However it should be noted that lossy or low bit depth images JPEG PNG and BMP are insufficient to use for analysis and therefore should be imported for the purpose of 14 display only Users should take images using supported imaging systems See Page 5 To acquire an image by importing an image file select Import Image Browse and select the desired image 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 Images acquired using the following imagers can be imported into the Q View software for analysis Refer above for details on
17. inant human IFN A1 recombinant human IFN A2 and recombinant human IFN A3 were prepared in Sample Diluent A separate standard curve was prepared using the Human IFN Multiplex Antigen Standard supplied in the product The 96 recovery of those points with pixel intensities within the range of pixel intensities of IFN A1 2 in the multiplex standard were averaged to estimate the Reactivity for each subtype Subtypes Reactivity IFN A1 167 4 IFN A2 39 5 IFN A3 41 7 i Human IFN a subtypes Rhesus Monkey IFN a Cynomolgus Monkey IFN a lle16 and Cynomolgus Monkey IFN B Independent curves of recombinant analytes listed in the table on the next page 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 on the curves of the test analytes with pixel intensities within the range of pixel intensities of IFN a A 2a in the curve prepared using the Multiplex Antigen Standard were averaged to estimate the Reactivity for each analyte 29 Catalog No Analyte Reactivity Human IFN a 1 a D Ala 114 14110 1 Rhesus Monkey IFN a 10 6 16100 1 Cynomolgus Monkey IFN a A lle16 2 3 ng ml of Cynomolgus IFN B was measured in tissue culture supernatant of a mammalian cell line expressing Cynomolgus IFN B No other antigen was detected in the supernatant iii Mouse IFN a A Mouse IFN 8 Mouse IFN
18. ity 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 B C D Open the camera door Set the adjustable tray on the top shelf Place the focusing plate in the center of the tray for focusing Open the aperture on the camera all the way to the lowest value 1 8 16 omm oz Open the software on the computer Click the Acquire button Close the door on the camera slightly so some light can get in and the focusing plate can be seen on the computer Screen Adjust the focus on the actual camera lens until the focusing plate is in focus Remove the focusing plate Place the plate to be read in the center of the tray and make sure it is in the center of the photo path on the computer screen Close all doors on the camera and ensure there are no light leaks Ensure all cabinet lights are turned off and that the filter wheel is set to 1 Set the software settings on the computer i No lights on ii Resolution to Normal Ultra iii Select only noise reduction iv Setthe exposure time to 6 min for the first time Click on Acquire Image On
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20. nsity 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 2 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 File Conversion i After acquiring the images you need to convert them to TIFF files ii Click on File then Export to TIFF image iii Select Export raw data click on Export then click on Save 19 6 Acquiring an image using the Bio Rad CHEMIDOC XRS Camera and Software Setup A File Setup iv Open the software on the computer Click on File then select ChemiDoc XRS Under Step I Select Application press the Select button then select Custom and then Create 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 Camera Setup and Focus a b C 2 On the software select Live Focus On the cabinet press the plus button to open the aperture or iris all the way the lowest number Open the camera s drawer place the plate in the middle of the drawer then close the drawer Press the
21. on imaging the plate using different imagers 11 E IMAGING PROCEDURE 1 Quansys Q View Imager Acquiring 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 is available for free The download is available at http www quansysbio com q view software The user manual for the software can be found under Manuals in the Support section A Select New Project if starting a new project and save under a new name 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 The connection status can be confirmed under Settings gt Administration gt Manage Imagers If needed click on Refresh C Optional Uncheck the box Discard sub images after stacking is complete under Settings gt Preferences in order to see images of different exposure times after the imaging process is 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 Settings gt Administration gt Manage Imagers Ensure that there is no plate in the plate housing slot in the imager and that the housing door is closed Select Calibrat
22. sity 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 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 23 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 a serum from a second donor had 6 4 pg ml of IL 1 a and 40 pg ml of IL 6 serum from a third donor had 9 1 pg ml of IL 1 a 1935 2 pg ml of IL 6 and 27 0 pg ml of TNF a 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 24 a Normal human serum Normal human plasma 20 donors 13 donors Analyte Average Rang
23. sss 24 Cross Reactivity Studies 29 Additional or eio 31 Ref r riCes cde cst ed ete ease deed aed 32 Q Plex is a registered trademark of Quansys Biosciences 51500 Rev 05 A 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 III Type IFNs consist of multiple Interferon Alpha IFN a genes at least one Interferon Beta IFN B gene and one Interferon Omega IFN w 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 and on triggering of Toll like receptors TLR3 4 7 8 9 by CPG DNAs and lipopolysaccharides LPS Upon binding to their cellular receptor chains IFN a Rc1 and IFN a Rc2 Type IFNs signal through the Jak Stat pathway to further elicit a host of antiviral actions including production of protein kinase A and 2 5 Oligoadenylate Synthetase OAS Type interferons are used therapeutically to treat viral infections cancers and auto immune disorders IFN a is used therapeutically to treat hepatitis B and hepatitis C infections Additionally IFN a is known to have significant biological activity in inhibition of proliferation of multiple cancers IFN B is used therapeutically to treat multiple sclerosis Type II IFN consists of Int
24. subunits CRF2 12 IFN AR1 and CRF2 4 IL 10R2 are known to be more prominent in cells of the epithelial tissues IFN A1 is known to modulate the development of Th1 Th2 cells 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 The VeriPlex 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 1a 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 output The assay is compatible with multiple matrices including tissue culture media human serum human plasma and buffers MATERIALS PROVIDED Human Interferon Multiplex 96 Well microtiter plate Plate sealers Wash Solution Concentrate Human Interferon Multiplex Antigen Standard Sample Diluent Assay Diluent Human Interferon Multiplex Detection Mix HRP Concentrate Substrate A Substrate B Diluent Additive II Diluent Additive III ADDITIONAL MATERIALS REQUIRED NOT PROVIDED Variable volume microtiter pipettes
25. uman human human human Analvte Sample DMEM human plasma plasma plasma plasma yte Diluent HO FBS serum with Na with Na with Na with Na Heparin Citrate IFN a 100 095 121 896 107 1 106 5 73 396 IFN B 82 6 97 9 138 4 103 3 98 7 100 2 98 1 5 3 TNF a c Low Spike Normal Normal Normal Normal human Normal human human human plasma Sample DMEM human plasma plasma plasma R Analyte Diluent 10 FBS serum with Na with Na with Na t Na 00 21 3 IFN y 125 5 97 5 110 6 104 9 89 4 94 4 77 3 FN A 1 2 114 995 118 096 115 5 104 2 84 2 87 7 72 696 7 3 Citrate D 91 2 35 I 81 096 98 3 EXT 54 4 705 95 2 8 65 50x 1 3 9 Pao 70 8 TNFa 350 41 9 Poor recoveries of low IP 10 spikes in plasma are due to presence of 110 pg ml of apparent endogenous IP 10 in the plasma lots 27 E iii Intra assay and Inter assay CV a Intra assay 9o CV Normal hu Normal human Analyte olli man serum ys plasma with Na donor A V EDTA donor B 0 IFN A 1 2 11 4 15 7 l 7 3 5 b Inter assay CV Normal hu Normal human Analyte man serum plasma with Na donor A EDTA donor B 12 8 19 0 19 8 25 1 IP 10 13 9 21 5 13 2 12 4 12 9 12 7 0 28 2 Cross Reactivity Studies i IFN A subtypes Independent curves of recomb
26. wn on the arrows until the focusing plate is in focus Remove the focusing plate from the imager and place the plate to be read 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 Select the Return button on the software to close the focusing window 22 C Image Acquisition a b When the plate is ready to image press the Start button 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 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 Press the Complete button to allow the imager to take another image 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 inten
27. y and Mouse IFN A3 Independent curves starting at 10 ug ml of recombinant analytes listed on the next page 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 30 Antigen Standard were averaged to estimate the cross reactivity for each analyte Catalog No Cross reactivity 12100 1 Murine IFN a A 0 004 12400 1 Murine IFN B 12500 1 Murine IFN y 12820 1 Murine IFN A3 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 immunoassays 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 31 REFERENCES Evolution of the class 2 cytokines and receptors and discovery of new friends and relatives Pestka S et al Pharmacology amp Therapeutics 2005 June 106 3 299 346 Interferons Interferon like cytokines and their receptors Pestka S et al Immunological Reviews 2004 202 8 32
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