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1. for simultaneous quantification of extracellular cytokines Below is a table summarizing the advantages and disadvantages between various assays Types of Advantages Disadvantages Techniques ELISA High sensitivity Large sample volume Low equipment cost required Limited number of analytes and samples tested simultaneously Luminex High Sensitivity Expensive instrument Small sample volume Luminex required Technically demanding Batch testing possible More quality control Multiplexing Up to 100 needed analytes quantified per sample Flow High sensitivity Expensive instrument Cytometry Evaluates cytokine secretion Flow Cytometer on an individual cell basis Doesn t quantify secreted and correlates with cell cytokines phenotypes simultaneously Technically demanding B Criteria for Evaluation of Commercial Multiplexing Reagents The reagents used for multiplex assay in conjunction with the Luminex xMAP system are currently only available through commercial vendors The commercial vendors not only provide assay kits for human mouse and rat but also offer different panels of cytokine chemokine and growth factors etc to meet investigator s needs The different analytes may be prepackaged either as a single plex or mixed in combinations for multiplex kits Investigators may also order customized kits prepared and packaged by the vendors or purchase only analytes of interest individually to customize one2. must be set up in STarStation The number of analytes and standard points being tested must be entered The analyte information must be entered correctly This includes bead sets units of interest pg ml MFI calculation MFI minus blank curve fit data axes and expected concentration values In addition the percent acceptance criteria may be determined Refer to software manual for specific instructions 23 The work list must be created prior to reading The work list stores all sample information including the well location of each standard control and sample sample IDs and necessary dilution factors 24 Set the instrument as follows a 50 events bead some vendors may use 100 events bead b Sample Size 100uL c Refer to product insert or data sheet specific to the kit for bead regions 11 d Double Discriminator gate settings 8 000 to 15 000 This may vary among instruments with different software e g STarStation Bioplex LiquiChip Procedure Notes A There are assay protocol variations among reagents vendors concerning the multiplex bead reagents sample requirements preparation of standards matrix and incubation time etc In order to obtain consistent and reproducible test results one must read the protocol booklet from vendor of choice thoroughly to understand every technical aspect of the immunoassay procedures Protect the antibody immobilized beads from photobleaching by covering the bottle and the assay p
3. unbound reporter molecules in solution are not measured The amount of green fluorescence is directly proportional to the amount of analyte captured in the immunoassay Luminex software correlates each bead set to the assay reagent that has been coupled to it Extrapolating to a standard curve allows quantitation of each analyte in the sample Using the xMAP assay thousands of beads can be analyzed in seconds allowing up to 100 analytes to be measured in each well of a 96 well microplate in less than three hours In addition since the fluorescence from each bead is measured independently enough statistics are accumulated to allow for assaying each sample in one well and not in duplicate The xMAP technology is basically an ELISA assay on a bead As such the multiplex bead assays have all of the strengths of ELISA technology sensitivity specificity and reproducibility along with the added advantages of multiplexing and a significantly increased dynamic range of four logs 1pg 10ng whereas ELISA technology has at most a two log range Further enhancements to this technology will enable up to 500 analyte measurements using a magnetic bead format all accomplished in a much shorter read time These features make the Luminex xMAP technology a highly robust and cost effective alternative to ELISA Up to 100 individual assays can be conducted in a 5 25yul sample This greatly minimizes sampling error and provides a robust and cost effective assay
4. 2pg ml tube and mix well The tube labeled Opg ml which contains 200ul of assay buffer serves as blank control The diagram below outlines the serial dilution of the standard curve ranging from 3 2pg ml to 10 000pg ml Standard Concentration Into Number pg ml Assay Buffer Standard 1 10 000 250ul dH20 Standard 2 2 000 50ul of Standard 1 at 10 000pg ml Standard 3 50ul of Standard 2 at 2 000pg ml 50ul of Standard 3 at 400pg nl 50ul of Standard 4 at 80pg ml 50ul of Standard 5 at 16pg ml Reconstitute the Human Cytokine Control and Il each with 250ul Standard 4 Standard 5 Standard 6 5 Prepare Controls deionized water Invert each vial several times to mix then vortex Allow the proteins to re hydrate for 5 10 minutes and then transfer each control to an appropriately labeled polypropylene microfuge tube Unused portions may be stored at 20C for up to one month Include in house controls in each assay whenever possible 6 Prepare Serum Matrix Add 1 0ml of deionized water to the vial containing the lyophilized serum matrix Mix well and allow for complete re hydration The serum matrix is used for assaying serum or plasma specimens Store unused serum matrix at 20C or below for up to one month D Assay Steps Flow Chart in Appendix 1 Prepare worksheet in advance for the placement of standards controls and I and unknown samples in a vertical configuration on the well map Always run the standards in dupli
5. ASHI Lab Manual Volume 2 2 Serologic Assays Solid Phase Antibody Detection Assays include ELISA amp Microbead Luminex Platform amp Flow Cytometry Assays Section 3 Microbead Luminex Platform Module Name Quantification of Cytokines using xMAP Technology Authors Jonathan Barone American Red Cross Penn Jersey Baronej usa redcross org Susan H Hsu American Red Cross Penn Jersey Shsu usa redcross org Date Prepared 04 2010 Date Updated 09 30 2011 Date Updated 11 20 2012 Section 2 Comparison of Technical Alternatives A Advantages and Disadvantages Between Assays Recently many HLA laboratories have implemented various solid phase immunoassay systems for either HLA typing or antibody workups These immunoassays are all applicable for measuring human cytokines in both biological fluids and tissue culture specimen These assays include Enzyme Linked Immunosorbent Assay ELISA Mosaic ELISA Enzyme linked immunosorbent spot ELISPOT quantitative PCR and flow cytometry 1 6 Each of these techniques suffers one or more significant limitations ELISA is sensitive and specific however it only measures one cytokine at a time It requires large sample volume and is also time consuming when more cytokines need to be assayed Although Mosaic ELISA combines aspects of both the ELISA and multi analyte profiling assays it is still limited to detect up to 8 cytokines per sample In addition it requires the use of a digita
6. Control of Reagents and Equipment I The recommended storage for kit components is 2 8C Follow instructions on individual vials for temperature requirements for long term storage After the standards and controls are reconstituted transfer the re hydrated proteins immediately into properly labeled polypropylene tubes and store at or below 20C Do not freeze thaw these reagents more than twice Store the antibody immobilized beads the detection antibody and the streptavidin phycoerythrin at 2 8 C and do not freeze Perform QC of each new kit received using at least two previously quantified in house controls or controls included in the kit along with the standards in duplicate The concentrations obtained with the new kit for each cytokine for these controls must be comparable to those obtained with previous lot lt 20 CV The concentrations pg ml obtained for each standard excluding the lowest concentration point for each analyte must be within 20 CV of the expected values provided from the manufacture s insert If greater than 20 CVs are observed for 3 points excluding the lowest concentration point on the standard curve for a particular analyte repeat the test for the analyte in question Since this is a multiplexed system one needs to repeat the test for both the standards and the unknowns If repeat test of standards is still not acceptable for a particular analyte contact the vendor and request a new lot for furth
7. Incubate 1 hour Do not vacuum Add 25 uL Streptavidin phycoerythrin per well Incubate for 30 minutes at RT Vacuum and wash 2X with 200 uL Wash Buffer Add 150 uL Sheath fluid per well Agitate for 10 minutes at RT Read on Luminex 100 uL 50 beads per bead set 14 Procedure Limitations 1 One should not extrapolate the standard curve beyond the highest standard point The dose response is non linear beyond this point The influence of various drugs may not be thoroughly validated with the test kit Excessively hemolyzed hyperlipidemic and jaundiced sera are inappropriate specimen Biological fluids other than serum plasma tissue cultured supernatant and buffer may not have been validated by the test kits 15 Section 5 Interpretation Reporting Troubleshooting A Data Analysis Interpretation 1 The median fluorescence intensity MFI values are evaluated using curve fitting software Some of the software programs combine instrument controls data acquisition and analysis on the same program Generally the best curve fit is a five parameter logistic and the data axes set as Y linear and X linear or Y linear and X logarithmic The blank or zero standards should be subtracted from the MFI values giving each sample well a final net MFI The concentration of each standard point should match the expected values The coefficient of variation CV should not exceed 20 Samples run in duplicate s
8. Provided by the Multiplex Kit 1 NOOR WD amp 10 11 12 Antibody Immobilized Beads 50X concentrated Antibody conjugated beads are supplied either premixed or in individual vials for customization Human Cytokine Chemokine Standard lyophilized Human Cytokine Chemokine Quality Controls I and II Serum Matrix lyophilized Bead Diluent Mixing Bottle Human Cytokine Chemokine Detection Antibodies diluted in assay buffer ready for use Streptavidin Phycoerythrin diluted in assay buffer ready for use Assay Buffer 10X Wash Buffer Microtiter Filter Plate Plate Sealers B Reagents and Supplies Required but not Provided oN BO es Q O Luminex Instrument 100 Luminex Sheath Fluid Adjustable Pipettes with tips to deliver 25ul to 1000ul Multichannel Pipettes to deliver 5ul to 200ul Reagent Reservoirs Polypropylene Microfuge Tubes Aluminum Foil Absorbent Pads or Paper Towels Vortex Sonicator Microplate Shaker 12 13 14 Vacuum Filtration Unit Using a vacuum regulator the vacuum pressure should be adjusted such that 200uL of buffer is removed in 3 to 5 seconds approximately 100 mmHg Vacuum Regulator Software for data analysis with the Luminex 100 STarStation v 2 0 Applied Cytometry Systems or similar application software C Reagent Preparation for Immunoassay 1 Prepare the 1x wash solution from 10x wash buffer The working wash solution may be pr
9. at proficiency testing PT is currently not available from any PT agency For the time being labs that are actively engaged in assaying cytokines using XMAP technology may want to organize inter lab cytokine panel comparison on a mutually agreed time interval Labs can also follow ASHI Standard C 1 1 5 when a graded external PT program is not available The future direction of the clinical applications of cytokines and cytokine receptors is expected to continue to advance in the major areas of academic and clinical medicine biotech pharma and cancer research discussed in Section 6 23 9 References Maecker Holden T Measuring Human Cytokines Ch17 from Handbook of Human Immunology 2 Edition CRC Press 2008 Prabhakar U Eirikis E Miller B E et al Multiplexed cytokine sandwich immunoassays clinical applications Methods Mol Med 114 223 232 2005 Kalyuzhny A E Chemistry and biology of the ELISPOT assay Method Mol Biol 302 15 31 2005 Tassignon J Burny W Dahmani S et al Monitoring of cellular responses after vaccination against tetanus toxoid comparison of the measurement of IFN gamma production by ELISA ELISPT flow cytometry and real time PCR J Immunol Methods 305 188 198 2005 Hill H R and Martins T B The flow cytometric analysis of cytokines using multi analyte fluorescence microarray technology Methods 38 312 316 2006 Elshal M F and McCoy J P Multipl
10. cate and the unknowns singly or in duplicate as desired attachment 1 2 Allow reagents to warm to room temperature 20 25C before use in the assay 3 Pre wet the filter plate by pipetting 200ul of assay buffer into each designated well Seal and agitate on a plate shaker for 10 minutes at room temperature 20 250 4 Remove assay buffer with the vacuum manifold Blot the residual liquid from the bottom of the plate on an absorbent pad or paper towels 5 Add 25ul of assay buffer to the unknown sample wells 6 Add 25ul of each blank standard and controls into the appropriate wells 7 Add 25ul of appropriate matrix to the blank standard and control wells When assaying serum or plasma use serum matrix provided in the kit When assaying tissue culture or other supernatant use appropriate culture medium as the matrix 8 Add 25ul of each unknown sample into the appropriate wells for steps 3 9 see attachment 1 9 Vortex bead bottle for 30 60 seconds Add 25ul of the mixed beads into each well while intermittently shaking the bead mix to avoid settling 10 Gently seal plate and cover with aluminum foil without pressing down in order to avoid liquid leaking through bottom of filter plate which will cause inaccurate reaction volume Incubate with agitation on a plate shaker for one hour at room temperature 20 25C Tray may be incubated overnight 16 to 18 hours to improve assay sensitivity Note If assay is incubated overnight same serie
11. each with its own unique signature Each of the 100 spectrally addressed bead sets can be covalently conjugated with a capture antibody specific for an analyte i e target protein of interest In a multiplexed assay antibody conjugated beads are allowed to react with an unknown sample plasma serum or cell culture supernatant along with standards of known analyte and controls in a 96 well microtiter plate During this first incubation the specific analyte binds to the primary captured antibodies on the beads After removing the unbound sample or analyte the secondary analyte specific biotinylated detection antibody is added which binds to a different epitope on the appropriate immobilized analyte This is followed by addition of the fluorescently labeled reporter molecule streptavidin phycoerythrin SA PE to form a capture four member sandwich After removing the unbound SA PE the resuspended beads are analyzed by the Luminex fluorometric array reader which obtains two fluorescence readings for every single bead The red classification laser identifies each bead in the suspension in each well as a member of one of the 100 possible sets and thus the analyte being assayed The green reporter laser measures the amount of fluorescent dye phycoerythrin PE bound to each of these beads thus quantifying the amount of analyte bound to each bead Since the reporter fluorescence is measured only in conjunction with the classification florescence
12. epared ahead by mixing 1 part of the 10x wash buffer and 9 parts of deionized water and stored at 2 8C for up to one month 2 Prepare Antibody Immobilized Beads Sonicate the bottle containing premixed beads for 30 seconds then vortex for 1 minute before use For customized kit sonicate individual antibody bead bottle for 30 seconds and then vortex for one minute Pipette 90ul from each antibody bead bottle into the mixing bottle and then bring the final volume to 3 0ml with bead diluent Prior to use vortex the mixed beads for 30 60 seconds Unused beads maybe stored at 2 8C for up to one month 3 Prepare and Reconstitute Cytokine Standards a b Prepare the standards within one hour of performing the assay Pre label 6 polypropylene tubes at 2000 400 80 16 3 2 and Opg ml Pipette 200ul of assay buffer into each tube Reconstitute the lyophilized human cytokine standard with 250ul deionized water to give a 10 000pg ml stock standard Invert the stock standard vial several times then vortex for 10 seconds and allow the proteins to re hydrate for 10 minutes Prepare serial dilutions by transferring 50ul of the 10 000pg ml stock standard to the 2000pg ml tube mix well and transfer 50ul of the 2000 standard to the 400pg ml tube mix well and transfer 50ul of the 400 standard to the 80pg ml tube mix well and transfer 50ul of the 80 standard to 16pg ml tube mix well and transfer 50ul of the 16 standard to the 3
13. er testing When each point excluding the lowest concentration point along the standard curve obtained for each analyte is comparable CV within 20 to the expected value provided by the manufacture s insert one can then proceed to analyze the unknown samples For preventive maintenance and QC of the Luminex 100 instrument refer to the users manual and section on Trouble Shooting in this Teaching Module 10 Consult and follow ASHI Standards for additional QA QC required 19 Section 7 Clinical Considerations 20 Since cytokines and their receptors are intimately involved in nearly all facets of host immunity as well as the pathogenesis of a variety of disease conditions tremendous progress in the clinical applications of cytokines has recently been made in the following major areas 12 1 To use select panels of cytokines as biomakers of disease 13 and provide clues for the underlying mechanisms of disease To measure cytokine production in vivo or in vitro for monitoring immune status 14 To use recombinant cytokines as therapeutic anti viral neoplastic and anti cancer agents The most well known example is the IFNs which are licensed in more than 40 countries for their proven efficacies in many disease conditions 12 15 To use cytokine antagonists and cytokine receptor antagonists as therapeutic targets to treat a variety of acute and chronic conditions or to downregulate pathogenic responses
14. ex bead array assays performance evaluation and comparison of sensitivity to ELISA Methods 38 312 316 2006 Khan S S Smith M S Reda D et al Multiplex bead array assays for detection of soluble cytokines comparisons of sensitivity and quantitative values among kits from multiple manufacturers Cytometry B Clin Cytom 61 35 39 2004 De Jager W and Rijkers G T Solid Phase and bead based cytokine immunoassays a comparison Methods 38 294 303 2006 ASHI Standards 2013 10 Human Cytokine 30 Plex Panel Invitrogen User Manual Rev 0 2 June 2009 11 Human Cytokine Chemokine Kit 96 Well Plate Assay Millipore User Manual Dec 2009 12 Detrick B Chandrasekharam N and Hooks J Cytokines Regulators of Immune Responses and Key Therapeutic Targets Ch16 from Handbook of Human Immunology 2 edition 2008 24 13 Lokshin A Multiplexed Serum Assay Ch18 from Measuring Immunity Elsevier Science 2005 14 Djoba Siawaya J F Chegou N N Van Den Heuvel M M et al Differential cytokine chemokines and KL 6 profiles in patients with different forms of tuberculosis Cytokine 47 132 136 2009 15 Vilcek J and Feldmann M Historical review Cytokines as therapeutics and targets of therapeutics Trends Pharmacol Sci 25 201 209 2004 16 Lori F Weiner D B Calarota S A et al Cytokine adjuvanted HIV DNA vaccination stragies Springer Semin Immunopathol 28 231 238 2006 17 Gonza
15. hould be calibrated against National Institute for Biological Standards and Control NIBSC standard whenever it is available Most importantly the standard for each analyte must be standardized with fixed ranges from lot to lot The standards provided should not vary more than 20 between lots in order to minimize large variations when studies are going to be conducted over a period of time Section 3 Specimen Requirements Collect serum specimen in red top tube and plasma sample in an ethylenediaminetetraacetic acid EDTA tube Centrifuge serum or plasma sample within 30 minutes of collection at 1000xg for 10 minutes To avoid more than two freeze thaw cycles aliquot appropriate volume of serum or plasma into each pre labeled polypropylene microfuge tube and store at 20C or below After tissue culture supernatant is collected spin it down immediately at 1000xg to remove debris and aliquot appropriate volume into pre labeled polypropylene tubes and store at 20C or below Do not use grossly hemolyzed or lipemic samples Section 4 Procedures The multiplex kits from one manufacturer are used here as an example to outline the general procedure steps for the Material and Methods section Discussions relating to data analysis interpretation quality assurance quality control troubleshooting procedure notes and limitations are also applicable to all commercial multiplex reagent kits using xMAP technology 10 11 A Reagents and Supplies
16. hould not exceed 20 CV Prior to becoming proficient with the assay system all samples should be run in duplicate or triplicate to exclude any outliers Check all controls to confirm that the expected values were obtained R squared values for each curve should be close to 1 Samples below the lowest point of the standard curve cannot be measured using this assay The lowest standard point is the lowest concentration that can be measured accurately and precisely If samples are below detectable levels one may want to re test those samples using a higher sample volume Samples above the highest point on the curve must be diluted and re tested Check that each sample is acceptable Users can reject unacceptable or outlier samples The percent acceptance criteria are different for standards and unknown samples For standards it is the percent deviation allowed between observed and expected concentrations i e lt 20 CV For unknowns it is the CV for replicates Both standards and unknown samples may need to be repeated if CV exceeds 20 16 7 When all data is evaluated a final report can be printed and should include the plate layout the standard curve for each analyte being tested summary of results and acceptance criteria B Trouble Shooting 1 When removing fluid or wash buffer with the vacuum manifold the liquid cannot be aspirated This is due to clogging of the filter plate Use the closed end of a latex rubber bulb
17. ht at room temperature for subsequent down stream assay steps Prior to using the protocol and depending on the sample types plasma serum and tissue culture supernatant etc the protocol must be validated first on untested samples by performing spike recovery and linearity experiments to determine whether sample results obtained from the un validated sample types are accurate It is important to use properly selected diluent matrix to reconstitute and dilute the standards to reflect the sample type being measured For example diluent used to quantify tissue culture supernatant samples is not the appropriate diluent to quantify plasma or serum samples In the spike recovery assay a known quantity of ELISA standard is spiked into a sample The recovery of the spiked material verifies whether a sample component interferes with the ELISA test The spiked and un spiked samples need to be serially diluted to test for linearity to determine if sample component interferes with the accurate determination of a specific analyte at a given dilution For both the spike recovery and linearity experiments the recovery should be in the range of 80 120 When the range of recovery is consistently achieved in the repeated validation tests one can then use the development kit proficiently to quantify a particular cytokine of interest for a specific sample type 22 Another immediate challenge facing the lab interested in implementing a cytokine assay is th
18. l imaging system ELISPOT is an assay which will quantify the secreted cytokines at the single cell level While quantitative PCR offers high sensitivity and specificity as well as the capability of multiplexing it does not however quantify proteins Flow cytometry evaluates cytokine secretion on an individual cell basis and correlates with cell phenotypes simultaneously but it does not quantify the amount of secreted cytokines Other platforms that enable multiple analyte measurements are based on planar modules as in slide arrays or plate spotting However these methodologies are limited by the number of analytes or samples that can be tested simultaneously Consequently a technique that is capable of quantifying multiple cytokines simultaneously in biological fluids including serum lavage urine tears tissue culture supernatant or cell and tissue homogenates will not only be useful in any laboratory setting but also further enhance our understanding of immune responses disease pathogenesis and the outcomes of therapeutic targets LUMINEX Corporation introduced a novel protein array system xMAP for Multi Analyte Profiling and x for variable e g cytokines ligands or enzymes etc which allows for simultaneous quantification of up to 100 soluble analytes in one sample 2 5 xMAP technology uses polystyrene microspheres internally dyed with differing ratios of two spectrally distinct fluorophores to create a family of 100 bead sets
19. late containing beads with aluminum foil during all incubation steps Place the filter plate on the non flat side of the plate cover to avoid direct contact of the bottom of the filter plate with an absorbent material during assay steps Pipette reagents with care to prevent tearing of the filter plate Maintain a consistent order of adding reagents from well to well to ensure equal incubation time for all wells Do not invert the filter plates during assay The filter plate should always be used in conjunction with a vacuum manifold to remove fluid or wash buffer Keep the vacuum suction as low as possible empty 200ul in about 3 seconds on the plate Use a vacuum regulator to monitor the pressure After each washing step ensure complete removal of excess liquid from bottom of plate by blotting the bottom of the plate on clean paper towels Excess liquid will cause inaccurate reaction volumes due to capillary action through the pores in the bottom of the filter plate wells Excessive wetness may indicate tearing of the filter bottom plate Standards prepared by serial dilution should be used within one hour Discard any unused standards and store the standard stock 10ng ml at 80C up to several months It is recommended to assay standards controls and unknown samples in duplicate until one becomes highly proficient with the assay system 12 J Keep the microplate shaker at a speed between 500 600 rpm to p
20. les G Crombet T Neninger E et al Therapeutic vaccination with epidermal growth factor EGF in advanced lung cancer analysis of pooled data from three clinical trials Hum Vaccin 3 8 13 2007 25
21. rovide strong agitation without causing liquid splashing from the wells K The plate should be read immediately after the assay is completed The plate may be read within 24 hours if sealed covered with aluminum foil and stored at 2 8C Before reading return the plate to the shaker for 10 minutes at room temperature L Delay in plate reading may result in decreased sensitivity for certain cytokines chemokines M Depending on unknown specimen to be assayed serum plasma or tissue culture supernatant use the appropriate matrix for blank standard curve and controls For example for tissue culture supernatants the culture medium supplemented with 5 FCS used for culture should be used as matrix diluent N If controls are not provided by the reagent vendor in house controls must be included for each batch of assay O Select proper equipment settings ensure needle probe is clean and adjust probe height to the filter plate prior to reading the assay Protocol Flow Chart for Human Cytokine Panel Add 200 uL Assay Buffer per well Shake 10 min RT Vacuum Add 25 uL Standard or control to appropriate wells Add 25 uL Assay buffer to background and sample wells Add 25 uL Sample to sample wells Add 25 uL Matrix to background standards and control wells Add 25 uL Beads to each well Incubate 1 hr or overnight at 4 C with agitation Vacuum and wash 2X with 200 uL wash buffer Add 25 uL Detection antibody per well 13
22. s own kit Each vendor provides protocol booklets for the assay kits manufactured There are however protocol variations accompanying each vendor s assay kits Most commercially available cytokine measuring immunoassay kits are comparable robust and have intra and inter assay coefficients of variation CV under 20 1 7 8 Nevertheless it is up to the users to evaluate kits from multiple manufacturers critically thoroughly and make comparisons of sensitivity and specificity obtained 9 before selecting the kits from vendor of choice The major considerations that should be assessed when evaluating and selecting the commercial multiplexing kits are analytical sensitivity specificity precision simplicity and cost The vendors should provide data on the performance characteristics of the test kits with respect to the above listed criteria The analytical specificity sensitivity must be comparable to ELISA technology with a correlation coefficient r above 0 98 from the vendor s own internal comparison and validation studies There must be no cross reactivity with any other analyte included in a panel The inter assay variability precision between assays and the intra assay variability within an assay should be within 10 S E to ensure accurate and reproducible results The assay kits must be validated for serum plasma and tissue culture supernatant as sample matrix for both in vitro and in vivo models The standard for each analyte s
23. s of assays must be incubated overnight 11 Gently remove fluid with vacuum manifold 10 12 Wash plate 2 times with 200ul well of wash buffer Remove wash buffer by vacuum filtration between each wash Blot the residual liquid from the bottom of the plate on an absorbent pad or paper towels 13 Add 25ul of detection antibody into each well Note allow the detection antibody to warm to room temperature prior to addition 14 Seal cover with aluminum foil and incubate with agitation on a plate shaker for 30 minutes at room temperature 20 25C Note If first incubation was overnight incubate this step for 1 hour at room temperature 15 Add 25ul streptavidin phycoerythrin to each well 16 Seal cover with aluminum foil and incubate with agitation on a plate shaker for 30 minutes at room temperature 20 25C 17 Pre warm the Luminex 100 instrument during the above incubation step 18 Gently remove all fluid with the vacuum manifold 19 Wash plate 2 times with 200ul well of wash buffer Remove wash buffer by vacuum filtration between each wash Blot the residual liquid from the bottom of the plate on an absorbent pad or paper towels 20 Add 150ul of sheath fluid to all wells Seal cover with aluminum foil and re suspend the beads on a plate shaker for 5 minutes 21 Uncover the plate insert the plate into the XY platform of the Luminex 100 instrument and analyze the samples 22 Prior to data acquisition the multiplex assay
24. the instrument To prevent this problem always aliquot 150 of sheath fluid into each well or adjust the probe height appropriately Keep the instrument needle probe clean to avoid crystal formation from the sheath fluid A clogged probe may also contribute to this problem 5 If the background is too high this indicates that the wash steps were not properly performed after each incubation step It is important to follow proper washing and removing of fluid with the vacuum manifold 6 During data analysis the beads appear as a diagonal line on the bead map instead of in the appropriate map region This is due to beads forming aggregates The assay needs to be repeated To prevent such problem one must always sonicate the beads for 30 seconds followed by vortexing for 30 60 seconds prior to performing the assay 7 Discrepancies between observed and expected standard concentrations for the analytes could be due to inaccurate pipetting while performing the serial dilutions If the percent CV exceeds 20 for more than 3 points of the standards the test needs to be repeated Inappropriate matrix diluent used may also contribute to the discrepancies 8 The Luminex 100 instrument fails the calibration test or other malfunctions Contact an equipment service representative for diagnosis and repair if referring to the user s manual does not help 18 Section 6 Quality Control Quality Assurance Validation Quality Assurance and Quality
25. to endogenous cytokine production 12 15 To use cytokines as a new class of adjuvants in vaccine formulations to enhance protection or to stimulate immunity 12 15 17 The continued expanding applications of cytokines and their receptors in both health and disease states will undoubtedly increase the need for monitoring cytokine levels or gene expressions in research and clinical laboratories There are three basic types of methodologies currently in use to measure cytokines molecular assays immunoassays and bioassays 1 Section 8 Challenges Future Direction 21 Due to the intensive research conducted in the cytokine field coupled with the continued expanding applications of cytokines and their receptors in health and diseases many new cytokines and receptors are expected to be identified in the future Commercial reagent kits may not always be readily available to assay these new cytokines on the xMAP platform If one s project dictates the need to quantify newly identified cytokines one may have to use the developmental ELISA kits available through some commercial vendors In order to use such developmental kits appropriately investigators must have prior expertise in the ELISA method The developmental kit typically contains the capture and detection antibodies standards streptavidin HRP and the basic solutions used in an ELISA test The investigator will have to coat the plate first with diluted capture antibody overnig
26. to gently push the bottom of the plate under the clogged well To prevent plate clogging centrifuge specimen at 10 000xg for 10 minutes to remove debris or fine particles prior to assay 2 Excessive wetness at the bottom of the filter plate is most likely due to piercing the filter membrane with the pipette during dispensing reagents for the assay In order to prevent this from happening always pipette to the side of well Applying excessive vacuum pressure may also cause tearing of the filter bottom plate Adjust vacuum pressure such that 200uL of buffer can be removed in 3 to 5 seconds To check if the plate is pierced place the plate containing reagents or wash buffer on a stack of dry paper towels 3 If the bead count is low this could be due to photobleaching of the embedded dyes Always store beads in the dark protected from light Other possibilities include that the beads may form aggregates during storage or settle while dispensing for the assay The test may need to be repeated Sonicate the beads in a sonicating water bath for at least 30 seconds followed by vortex for 30 60 seconds to prevent the aggregates When dispensing the beads for assay one should intermittently shake the bead bottle to avoid settling of the beads 4 Erratic bead count during sample analysis may be due to an insufficient volume of sheath fluid delivered into each well or the height 17 of the probe is incorrectly adjusted to allow the entry of air into
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