Hercules, CA 94547 USA fiLe Set Up At 50% Size. …...96-Well Plate Uniformity Average % CV of MFI...
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96-Well Plate Uniformity Average % CV of MFI Ends* 96 Wells Columns 1–12 Rows A–H Columns Rows IS 4 3 4 3 4 IL-1β 3 3 3 3 4 IL-2 7 5 7 5 10 IL-4 6 5 5 6 5 IL-5 7 4 7 4 11 IL-6 4 4 3 5 3 IL-10 3 3 3 3 2 IL-12 4 3 3 3 3 IL-13 5 5 4 4 5 IFN-γ 4 4 4 5 5 TNF-α 4 4 4 5 5 * End wells defined as first and last two wells. Table 6. Summary of 96-well plate uniformity. MFI precision was determined across rows, across columns, and between the first and last two wells of each row and column. IS (internal standard) beads contain a covalently linked fluorophore to measure instrument-related variance. Cytokine Levels in Normal Human Serum Samples 100 Human Serum Samples (pg/ml) Range Mean Median Min Max IL-1β 0.30 0.03 0.00 12 IL-2 1.27 0.00 0.00 30 IL-4 0.67 0.00 0.00 18 IL-5 11.09 0.00 0.00 919 IL-6 5.24 0.50 0.00 179 IL-10 0.65 0.14 0.00 15 IL-12 12.52 0.27 0.00 1,053 IL-13 0.58 0.00 0.00 30 IFN-γ 3.66 0.25 0.00 76 TNF-α 1.38 0.16 0.00 41 Table 7. 100 individual normal off-clot human serum samples were assayed with the 10-plex cytokine assay using human serum standard diluent. Conclusions n Bio-Rad Laboratories has developed the first cytokine assay on novel magnetic 8.0 µm diameter beads for the Bio-Plex suspension array system n The magnetic beads are specially designed for automation and increased throughput, and will become available for future automated 96-well platforms n The magnetic beads are compatible with the current 96-well filter plate format for versatility in life science and clinical research n The Bio-Plex Precision Pro 10-plex cytokine magnetic bead assay panel (catalog #171-A1001P*) is highly precise, accurate, sensitive, specific, and robust — ideal for clinical research applications n Newly developed human serum and plasma standard diluents demonstrate similar binding characteristics (parallelism and linearity) to clinical samples, making them ideal for clinical research applications * This all-in-one assay does not require separate reagent and diluent kits. xMAP and Luminex are trademarks of Luminex Corporation. The Bio-Plex suspension array system includes fluorescently labeled microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation. Accuracy Human Serum Pooled Samples Spiked (Technical Specification 80–120% Recovery) % Standard Recovery* pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α 1,000 114 90 116 90 99 96 93 102 97 95 333 105 94 89 90 90 90 95 90 94 92 111 94 95 96 91 97 98 96 96 97 98 37 99 96 98 88 97 101 94 95 99 97 9.3 98 91 94 85 94 96 92 88 99 96 2.3 94 90 91 73 92 90 97 86 94 90 Human Plasma Pooled Samples Spiked (Technical Specification 80–120% Recovery) % Standard Recovery* pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α 1,000 125 99 103 101 104 104 94 102 106 111 333 92 97 101 93 97 95 98 95 99 93 111 98 96 104 94 100 100 99 97 100 102 37 103 99 103 95 97 103 99 95 101 103 9.3 106 100 107 96 100 106 98 100 103 107 2.3 98 94 102 67 104 100 88 97 99 105 * Mean of five assays, duplicates of each sample. Table 1. Human serum or plasma samples from individual donors were pooled (n = 10 each) and spiked with a known amount of 10-plex cytokines. The % spike recovery was calculated from the ratio of measured/expected cytokine concentration in each sample. The mean % spike recovery was determined from five independent assays. Inter-Assay Precision Human Serum Pooled Sample Spiked (Technical Specification ≤15%CV) Inter-Assay %CV* pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α 1,000 25.8 9.5 15.4 8.0 1.09 10.7 4.2 10.9 6.4 6.6 333 10.2 7.1 6.7 7.7 7.6 2.4 5.8 9.3 8.0 4.6 111 6.8 1.7 7.8 4.1 4.1 5.3 5.4 2.9 3.7 10.1 37 9.3 6.0 9.9 6.1 6.4 8.4 8.9 7.4 9.7 8.3 9.3 5.2 9.7 2.1 4.9 5.1 4.8 2.0 5.1 8.0 3.0 2.3 6.7 14.6 8.6 6.3 9.1 8.7 6.9 7.3 4.2 10.4 Human Plasma Pooled Sample Spiked (Technical Specification ≤15%CV) Inter-Assay %CV* pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α 1,000 12.3 3.6 7.8 10.7 7.3 7.3 3.9 5.0 6.3 14.8 333 5.3 6.8 8.2 5.5 5.5 6.5 5.2 3.3 3.4 6.4 111 4.9 5.6 10.1 1.6 2.7 2.8 4.4 1.7 2.3 1.9 37 4.9 2.6 5.3 2.8 1.8 4.1 1.7 0.8 3.9 3.0 9.3 6.1 5.7 9.6 10.0 9.6 6.1 5.4 6.4 4.7 9.1 2.3 9.2 17.0 7.2 33.6 10.0 3.7 8.9 10.3 12.9 7.9 * Mean of five assays, duplicates of each sample. Table 2. Human serum or plasma standard diluents were used to prepare standard curves. Human serum or plasma samples from individual donors were pooled (n = 10 each) and spiked with a known amount of 10-plex cytokines. Inter-assay precision (%CV) was determined from the measured concentrations of 5 independent assays. Assay Range Human Serum Standard (pg/ml) Human Plasma Standard (pg/ml) LLOQ* ULOQ** LLOQ* ULOQ** IL-1β 0.2 520 0.2 560 IL-2 1.9 3,200 2.3 3,200 IL-4 0.4 1,707 0.3 3,200 IL-5 2.7 3,200 3.7 3,200 IL-6 0.3 2,760 0.9 3,200 IL-10 0.2 2,320 0.3 3,200 IL-12 0.5 3,200 1.4 3,200 IL-13 0.2 2,320 0.4 3,200 IFN-γ 0.9 3,200 1.2 3,200 TNF-α 0.2 1,880 0.2 2,720 * LLOQ = lower limit of quantitation. ** ULOQ = upper limit of quantitation. Table 3. Assay range is the concentration range in which the assay is both precise (intra ≤10% CV; inter ≤15% CV) and accurate (80–120% standard and spike recovery). Sensitivity Human Serum Standard Human Plasma Standard (Technical Specifications ≤1 pg/ml) (Technical Specifications ≤1 pg/ml) Assay/LOD (pg/ml) Mean Assay/LOD (pg/ml) Mean Target 1 2 3 4 5 LOD Target 1 2 3 4 5 LOD IL-1β 0.11 0.13 0.05 0.07 0.07 0.08 IL-1β 0.11 0.12 0.10 0.06 0.13 0.10 IL-2 0.22 0.58 0.70 0.39 0.14 0.41 IL-2 0.75 0.78 0.12 0.84 0.69 0.64 IL-4 0.10 0.08 0.16 0.03 0.08 0.09 IL-4 0.20 0.26 0.12 0.10 0.19 0.17 IL-5 1.36 1.41 2.91 0.60 0.54 1.37 IL-5 1.53 0.18 0.20 2.42 0.09 0.88 IL-6 0.38 0.32 0.60 0.25 0.16 0.34 IL-6 0.11 0.42 0.62 0.28 0.04 0.29 IL-10 0.15 0.15 0.15 0.08 0.12 0.13 IL-10 0.08 0.29 0.21 0.14 0.12 0.17 IL-12 0.09 0.11 0.61 0.08 0.32 0.24 IL-12 0.45 0.36 0.52 0.31 0.20 0.37 IL-13 0.15 0.20 0.26 0.16 0.07 0.17 IL-13 0.20 0.18 0.16 0.27 0.14 0.19 IFN-γ 0.10 0.32 0.77 0.01 0.15 0.27 IFN-γ 0.33 0.58 0.48 0.16 0.19 0.35 TNF-α 0.13 0.18 0.21 0.06 0.12 0.14 TNF-α 0.17 0.33 0.17 0.17 0.17 0.20 Table 4. Limit of detection (LOD) is defined as the cytokine concentration obtained at background median fluorescence intensity (MFI) plus 2 standard deviations for the human serum and plasma standard curves. Specificity % Cross-Reactivity (Technical Specification ≤1% Cross-Reactivity) Single Ag Multiplexed Capture Bead and Detection Ab 800 pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α IL-1β 100.00 0.02 0.00 0.27 0.05 0.00 0.06 0.00 0.03 0.01 IL-2 0.00 100 0.00 0.29 0.05 0.02 0.02 0.00 0.00 0.01 IL-4 0.00 0.09 100.00 0.28 0.13 0.03 0.07 0.01 0.00 0.02 IL-5 0.01 0.03 0.00 100.00 0.04 0.00 0.04 0.00 0.00 0.01 IL-6 0.00 0.04 0.00 0.24 100.00 0.03 0.06 0.01 0.00 0.01 IL-10 0.03 0.04 0.00 0.16 0.12 100.00 0.09 0.00 0.00 0.00 IL-12 0.00 0.02 0.00 0.15 0.06 0.00 100 0.00 0.00 0.00 IL-13 0.00 0.03 0.00 0.25 0.13 0.01 0.07 100.00 0.00 0.01 IFN-γ 0.01 0.07 0.02 0.22 0.08 0.01 0.10 0.01 100.00 0.01 TNF-α 0.00 0.00 0.00 0.14 0.00 0.00 0.00 0.00 0.00 100.00 % Cross-reactivity of Ab Y w /Ag X = Fl of Ab Y w /Ag X – bkgd Fl of Ab Y w /Ag Y – bkgd Table 5. Cross-reactivity was determined from median fluorescence intensity (MFI) of multiplexed capture bead and detection Abs in the presence of single antigens (Ag) at 800 pg/ml. Abstract Cytokines are key immunoregulatory proteins produced by white blood cells and other cell types. These low molecular weight proteins are involved in a wide variety of disease states, including inflammation and oncogenesis. We have chosen ten of the most widely publicized cytokines associated with the Th1/Th2 subsets (IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, IFN-γ) and inflammation (IL-1 β , IL-6, TNF-α) to develop a multiplex immunoassay on the Bio-Plex suspension array system (based on Luminex xMAP technology). The validation of this sandwich immunoassay is described. This assay is unique in that it incorporates the use of magnetic beads and uses newly designed human serum and plasma standard diluents. The 10-plex cytokine assay was run in five independent assays for each standard diluent, and validated for standard curve accuracy (80–120%), precision (intra ≤10% CV; inter ≤15% CV), sensitivity (≤1 pg/ml), specificity (≤1% cross-reactivity), and parallelism (<10% slope difference) using three independent donors for human serum and plasma samples, respectively. Assay robustness was determined by 96-well plate uniformity (3–7% CV) tests. For population analysis, cytokine levels were determined in 100 normal human samples. Overall, the ten cytokine assays were highly sensitive and precise — ideal for clinical research purposes. The future direction is to transfer this application onto an automated 96-well platform for increased throughput. Parallelism and Linearity 07-0109 0207 Standard curve Sample 1 r 2 = 0.9979 r 2 = 0.9975 r 2 = 0.9970 r 2 = 0.9936 r 2 = 0.9999 r 2 = 1.0000 r 2 = 0.9998 r 2 = 0.9996 IL-6 MFI 30,000 20,000 10,000 0 0.10 1 10 100 1,000 Concentration (pg/ml) 30,000 20,000 10,000 0 MFI IL-1β 0.10 1 10 100 1,000 30,000 20,000 10,000 0 MFI IL-6 0.10 1 10 100 1,000 Concentration (pg/ml) 30,000 20,000 10,000 0 MFI 0.10 1 10 100 1,000 IL-1β 4-PL curves for standard and spiked human serum samples. 4-PL curves for standard and spiked human plasma samples. 1,400 1,200 1,000 800 600 400 200 0 0 200 400 600 800 1,000 1,200 Expected concentration (pg/ml) 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0 0 200 400 600 800 1,000 1,200 450 400 350 300 250 200 150 100 50 0 0 50 100 150 200 250 300 350 1,600 1,400 1,200 1,000 800 600 400 200 0 0 200 400 600 800 1,000 1,200 Expected concentration (pg/ml) Fig. 3. Examples of parallelism between 8-point standard (blue square) and 6-point spiked serum sample (orange circle) dilutions using 4-PL curve fitting. Slopes were <10% different for all cytokines. Linearity was demonstrated in three individual samples with r 2 > 0.99 for all ten cytokines. Observed concentration (pg/ml) Observed concentration (pg/ml) Observed concentration (pg/ml) Observed concentration (pg/ml) r 2 = 1.0000 r 2 = 0.9969 r 2 = 0.9993 r 2 = 0.9993 r 2 = 0.9981 r 2 = 0.9999 r 2 = 0.9996 r 2 = 0.9998 Sample 1 Sample 2 Sample 3 Spiked control Fig. 2. Five-parameter logistic (5-PL) curve fitting with Bio-Plex Manager ™ 4.1 standard software. Shown are median fluorescence intensity (MFI) data for a Bio-Plex Precision Pro ™ 10-plex human cytokine assay using human serum and human plasma standard diluents. 10-Plex Human Cytokine Standard Curve Profiles 30,000 25,000 20,000 15,000 10,000 5,000 0 30,000 25,000 20,000 15,000 10,000 5,000 0 Human Serum Standard Diluent MFI 0.1 1 10 100 1,000 10,000 Concentration, pg/ml Human Plasma Standard Diluent MFI 0.1 1 10 100 1,000 10,000 Concentration, pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α 19 71 71 19 Sandwich assay Bead Coupled antibody Analyte Detection antibody Reporter A. 25 color codes = 25 simultaneous tests. Using a two-dye method, xMAP technology produces 25 distinct magnetic bead sets with magnetic properties. B. Tagging the reaction. Fluorescently labeled reporter tags bind to the sample molecules. C. Beads in a fluid stream. Precision fluidics align the beads in single file and pass them through the lasers one at a time. Bio-Plex Suspension Array Technology Fig. 1. The Bio-Plex system is a complete suspension array system that comprises a flow-based 96-well fluorescent microplate assay reader integrated with specialized software, automated validation and calibration protocols, and assay kits. Based on Luminex xMAP technology, Bio-Plex magnetic assays can detect up to 25 distinct analytes in as little as 12 μl of sample. Color-coded beads Bio-Plex system reads the assay Development and Validation of a Novel Multiplex Cytokine Immunoassay on Magnetic Beads Using the Bio-Plex ® Suspension Array System Joyce Eldering, Woei Tan, Joe Fedynyshyn, Li Ma, and Sophie Allauzen Life Science Group 2000 Alfred Nobel Drive Hercules, CA 94547 USA
Transcript of Hercules, CA 94547 USA fiLe Set Up At 50% Size. …...96-Well Plate Uniformity Average % CV of MFI...
96-Well Plate Uniformity
Average % CV of MFI Ends* 96Wells Columns1–12 RowsA–H Columns RowsIS 4 3 4 3 4IL-1β 3 3 3 3 4IL-2 7 5 7 5 10IL-4 6 5 5 6 5IL-5 7 4 7 4 11IL-6 4 4 3 5 3IL-10 3 3 3 3 2IL-12 4 3 3 3 3IL-13 5 5 4 4 5IFN-γ 4 4 4 5 5TNF-α 4 4 4 5 5
*Endwellsdefinedasfirstandlasttwowells.
Table6.Summaryof96-wellplateuniformity.MFIprecisionwasdeterminedacrossrows,acrosscolumns,andbetweenthefirstandlasttwowellsofeachrowandcolumn.IS(internalstandard)beadscontainacovalentlylinkedfluorophoretomeasureinstrument-relatedvariance.
Cytokine Levels in Normal Human Serum Samples
100 Human Serum Samples (pg/ml) Range Mean Median Min MaxIL-1β 0.30 0.03 0.00 12IL-2 1.27 0.00 0.00 30IL-4 0.67 0.00 0.00 18IL-5 11.09 0.00 0.00 919IL-6 5.24 0.50 0.00 179IL-10 0.65 0.14 0.00 15IL-12 12.52 0.27 0.00 1,053IL-13 0.58 0.00 0.00 30IFN-γ 3.66 0.25 0.00 76TNF-α 1.38 0.16 0.00 41
Table7.100individualnormaloff-clothumanserumsampleswereassayedwiththe10-plexcytokineassayusinghumanserumstandarddiluent.
Conclusionsn Bio-RadLaboratorieshasdevelopedthefirstcytokineassayonnovel
magnetic8.0µmdiameterbeadsfortheBio-Plexsuspensionarraysystem
n Themagneticbeadsarespeciallydesignedforautomationandincreasedthroughput,andwillbecomeavailableforfutureautomated96-wellplatforms
n Themagneticbeadsarecompatiblewiththecurrent96-wellfilterplateformatforversatilityinlifescienceandclinicalresearch
n TheBio-PlexPrecisionPro10-plexcytokinemagneticbeadassaypanel(catalog#171-A1001P*)ishighlyprecise,accurate,sensitive,specific,androbust—idealforclinicalresearchapplications
n Newlydevelopedhumanserumandplasmastandarddiluentsdemonstratesimilarbindingcharacteristics(parallelismandlinearity)toclinicalsamples,makingthemidealforclinicalresearchapplications
*Thisall-in-oneassaydoesnotrequireseparatereagentanddiluentkits.
xMAPandLuminexaretrademarksofLuminexCorporation.TheBio-PlexsuspensionarraysystemincludesfluorescentlylabeledmicrospheresandinstrumentationlicensedtoBio-RadLaboratories,Inc.bytheLuminexCorporation.
Accuracy
Human Serum Pooled Samples Spiked(TechnicalSpecification80–120%Recovery)
%StandardRecovery*
pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α
1,000 114 90 116 90 99 96 93 102 97 95 333 105 94 89 90 90 90 95 90 94 92 111 94 95 96 91 97 98 96 96 97 98 37 99 96 98 88 97 101 94 95 99 97 9.3 98 91 94 85 94 96 92 88 99 96 2.3 94 90 91 73 92 90 97 86 94 90
Human Plasma Pooled Samples Spiked(TechnicalSpecification80–120%Recovery)
%StandardRecovery*
pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α
1,000 125 99 103 101 104 104 94 102 106 111 333 92 97 101 93 97 95 98 95 99 93 111 98 96 104 94 100 100 99 97 100 102 37 103 99 103 95 97 103 99 95 101 103 9.3 106 100 107 96 100 106 98 100 103 107 2.3 98 94 102 67 104 100 88 97 99 105
*Meanoffiveassays,duplicatesofeachsample.
Table1.Humanserumorplasmasamplesfromindividualdonorswerepooled(n=10each)andspikedwithaknownamountof10-plexcytokines.The%spikerecoverywascalculatedfromtheratioofmeasured/expectedcytokineconcentrationineachsample.Themean%spikerecoverywasdeterminedfromfiveindependentassays.
Inter-Assay Precision
Human Serum Pooled Sample Spiked (TechnicalSpecification≤15%CV)
Inter-Assay%CV*
pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α
1,000 25.8 9.5 15.4 8.0 1.09 10.7 4.2 10.9 6.4 6.6 333 10.2 7.1 6.7 7.7 7.6 2.4 5.8 9.3 8.0 4.6 111 6.8 1.7 7.8 4.1 4.1 5.3 5.4 2.9 3.7 10.1 37 9.3 6.0 9.9 6.1 6.4 8.4 8.9 7.4 9.7 8.3 9.3 5.2 9.7 2.1 4.9 5.1 4.8 2.0 5.1 8.0 3.0 2.3 6.7 14.6 8.6 6.3 9.1 8.7 6.9 7.3 4.2 10.4
Human Plasma Pooled Sample Spiked (TechnicalSpecification≤15%CV)
Inter-Assay%CV*
pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α
1,000 12.3 3.6 7.8 10.7 7.3 7.3 3.9 5.0 6.3 14.8 333 5.3 6.8 8.2 5.5 5.5 6.5 5.2 3.3 3.4 6.4 111 4.9 5.6 10.1 1.6 2.7 2.8 4.4 1.7 2.3 1.9 37 4.9 2.6 5.3 2.8 1.8 4.1 1.7 0.8 3.9 3.0 9.3 6.1 5.7 9.6 10.0 9.6 6.1 5.4 6.4 4.7 9.1 2.3 9.2 17.0 7.2 33.6 10.0 3.7 8.9 10.3 12.9 7.9
*Meanoffiveassays,duplicatesofeachsample.
Table2.Humanserumorplasmastandarddiluentswereusedtopreparestandardcurves.Humanserumorplasmasamplesfromindividualdonorswerepooled(n=10each)andspikedwithaknownamountof10-plexcytokines.Inter-assayprecision(%CV)wasdeterminedfromthemeasuredconcentrationsof5independentassays.
Assay Range
HumanSerumStandard(pg/ml) HumanPlasmaStandard(pg/ml)
LLOQ* ULOQ** LLOQ* ULOQ**
IL-1β 0.2 520 0.2 560IL-2 1.9 3,200 2.3 3,200IL-4 0.4 1,707 0.3 3,200IL-5 2.7 3,200 3.7 3,200IL-6 0.3 2,760 0.9 3,200IL-10 0.2 2,320 0.3 3,200IL-12 0.5 3,200 1.4 3,200IL-13 0.2 2,320 0.4 3,200IFN-γ 0.9 3,200 1.2 3,200TNF-α 0.2 1,880 0.2 2,720
*LLOQ=lowerlimitofquantitation.**ULOQ=upperlimitofquantitation.
Table3.Assayrangeistheconcentrationrangeinwhichtheassayisbothprecise(intra≤10%CV;inter≤15%CV)andaccurate(80–120%standardandspikerecovery).
Sensitivity
Human Serum Standard Human Plasma Standard(TechnicalSpecifications≤1pg/ml) (TechnicalSpecifications≤1pg/ml)
Assay/LOD(pg/ml) Mean Assay/LOD(pg/ml) MeanTarget 1 2 3 4 5 LOD Target 1 2 3 4 5 LOD
IL-1β 0.11 0.13 0.05 0.07 0.07 0.08 IL-1β 0.11 0.12 0.10 0.06 0.13 0.10IL-2 0.22 0.58 0.70 0.39 0.14 0.41 IL-2 0.75 0.78 0.12 0.84 0.69 0.64IL-4 0.10 0.08 0.16 0.03 0.08 0.09 IL-4 0.20 0.26 0.12 0.10 0.19 0.17IL-5 1.36 1.41 2.91 0.60 0.54 1.37 IL-5 1.53 0.18 0.20 2.42 0.09 0.88IL-6 0.38 0.32 0.60 0.25 0.16 0.34 IL-6 0.11 0.42 0.62 0.28 0.04 0.29IL-10 0.15 0.15 0.15 0.08 0.12 0.13 IL-10 0.08 0.29 0.21 0.14 0.12 0.17IL-12 0.09 0.11 0.61 0.08 0.32 0.24 IL-12 0.45 0.36 0.52 0.31 0.20 0.37IL-13 0.15 0.20 0.26 0.16 0.07 0.17 IL-13 0.20 0.18 0.16 0.27 0.14 0.19IFN-γ 0.10 0.32 0.77 0.01 0.15 0.27 IFN-γ 0.33 0.58 0.48 0.16 0.19 0.35TNF-α 0.13 0.18 0.21 0.06 0.12 0.14 TNF-α 0.17 0.33 0.17 0.17 0.17 0.20
Table4.Limitofdetection(LOD)isdefinedasthecytokineconcentrationobtainedatbackgroundmedianfluorescenceintensity(MFI)plus2standarddeviationsforthehumanserumandplasmastandardcurves.
Specificity
% Cross-Reactivity(TechnicalSpecification≤1%Cross-Reactivity)
SingleAg MultiplexedCaptureBeadandDetectionAb
800pg/ml IL-1β IL-2 IL-4 IL-5 IL-6 IL-10 IL-12 IL-13 IFN-γ TNF-α
IL-1β 100.00 0.02 0.00 0.27 0.05 0.00 0.06 0.00 0.03 0.01IL-2 0.00 100 0.00 0.29 0.05 0.02 0.02 0.00 0.00 0.01IL-4 0.00 0.09 100.00 0.28 0.13 0.03 0.07 0.01 0.00 0.02IL-5 0.01 0.03 0.00 100.00 0.04 0.00 0.04 0.00 0.00 0.01IL-6 0.00 0.04 0.00 0.24 100.00 0.03 0.06 0.01 0.00 0.01IL-10 0.03 0.04 0.00 0.16 0.12 100.00 0.09 0.00 0.00 0.00IL-12 0.00 0.02 0.00 0.15 0.06 0.00 100 0.00 0.00 0.00IL-13 0.00 0.03 0.00 0.25 0.13 0.01 0.07 100.00 0.00 0.01IFN-γ 0.01 0.07 0.02 0.22 0.08 0.01 0.10 0.01 100.00 0.01TNF-α 0.00 0.00 0.00 0.14 0.00 0.00 0.00 0.00 0.00 100.00
%Cross-reactivityofAbYw/AgX=FlofAbYw/AgX–bkgd
FlofAbYw/AgY–bkgd
Table5.Cross-reactivitywasdeterminedfrommedianfluorescenceintensity(MFI)ofmultiplexedcapturebeadanddetectionAbsinthepresenceofsingleantigens(Ag)at800pg/ml.
AbstractCytokinesarekeyimmunoregulatoryproteinsproducedbywhitebloodcellsand
othercelltypes.Theselowmolecularweightproteinsareinvolvedinawidevariety
ofdiseasestates,includinginflammationandoncogenesis.Wehavechosentenof
themostwidelypublicizedcytokinesassociatedwiththeTh1/Th2subsets(IL-2,IL-4,
IL-5,IL-10,IL-12(p70),IL-13,IFN-γ)andinflammation(IL-1β,IL-6,TNF-α)todevelopa
multipleximmunoassayontheBio-Plexsuspensionarraysystem(basedonLuminex
xMAPtechnology).Thevalidationofthissandwichimmunoassayisdescribed.This
assayisuniqueinthatitincorporatestheuseofmagneticbeadsandusesnewly
designedhumanserumandplasmastandarddiluents.The10-plexcytokineassay
wasruninfiveindependentassaysforeachstandarddiluent,andvalidatedforstandard
curveaccuracy(80–120%),precision(intra≤10%CV;inter≤15%CV),sensitivity
(≤1pg/ml),specificity(≤1%cross-reactivity),andparallelism(<10%slopedifference)
usingthreeindependentdonorsforhumanserumandplasmasamples,respectively.
Assayrobustnesswasdeterminedby96-wellplateuniformity(3–7%CV)tests.For
populationanalysis,cytokinelevelsweredeterminedin100normalhumansamples.
Overall,thetencytokineassayswerehighlysensitiveandprecise—idealforclinical
researchpurposes.Thefuturedirectionistotransferthisapplicationontoanautomated
96-wellplatformforincreasedthroughput.
Parallelism and Linearity
07-0109 0207
Standardcurve
Sample1
r2=0.9979r2=0.9975r2=0.9970r2=0.9936
r2=0.9999r2=1.0000r2=0.9998r2=0.9996
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4-PLcurvesforstandardandspikedhumanplasmasamples.
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Fig.3.Examplesofparallelismbetween8-pointstandard(bluesquare)and6-pointspikedserumsample(orangecircle)dilutionsusing4-PLcurvefitting.Slopeswere<10%differentforallcytokines.Linearitywasdemonstratedinthreeindividualsampleswithr2>0.99foralltencytokines.
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trat
ion
(pg/
ml)
Obs
erve
dco
ncen
trat
ion
(pg/
ml)
r2=1.0000r2=0.9969r2=0.9993r2=0.9993
r2=0.9981r2=0.9999r2=0.9996r2=0.9998
Sample1Sample2Sample3Spikedcontrol
Fig.2.Five-parameterlogistic(5-PL)curvefittingwithBio-PlexManager™4.1standardsoftware.Shownaremedianfluorescenceintensity(MFI)dataforaBio-PlexPrecisionPro™10-plexhumancytokineassayusinghumanserumandhumanplasmastandarddiluents.
10-Plex Human Cytokine Standard Curve Profiles
30,000
25,000
20,000
15,000
10,000
5,000
0
30,000
25,000
20,000
15,000
10,000
5,000
0
HumanSerumStandardDiluent
MFI
0.1 1 10 100 1,000 10,000
Concentration,pg/ml
HumanPlasmaStandardDiluent
MFI
0.1 1 10 100 1,000 10,000
Concentration,pg/ml
IL-1β
IL-2
IL-4
IL-5
IL-6
IL-10
IL-12
IL-13
IFN-γ
TNF-α
19
71
71 19
Sandwichassay
Bead
CoupledantibodyAnalyte
DetectionantibodyReporter
A.25colorcodes=25simultaneoustests.Usingatwo-dyemethod,xMAPtechnologyproduces25distinctmagneticbeadsetswithmagneticproperties.
B.Taggingthereaction.Fluorescentlylabeledreportertagsbindtothesamplemolecules.
C.Beadsinafluidstream.Precisionfluidicsalignthebeadsinsinglefileandpassthemthroughthelasersoneatatime.
Bio-Plex Suspension Array Technology
Fig.1.TheBio-Plexsystemisacompletesuspensionarraysystemthatcomprisesaflow-based96-wellfluorescentmicroplateassayreaderintegratedwithspecializedsoftware,automatedvalidationandcalibrationprotocols,andassaykits.BasedonLuminexxMAPtechnology,Bio-Plexmagneticassayscandetectupto25distinctanalytesinaslittleas12μlofsample.
Color-codedbeads Bio-Plexsystemreadstheassay
Development and Validation of a Novel Multiplex Cytokine Immunoassay on Magnetic Beads Using the Bio-Plex® Suspension Array System
JoyceEldering,WoeiTan,JoeFedynyshyn,LiMa,andSophieAllauzen
Life Science Group2000 Alfred Nobel Drive
Hercules, CA 94547 USA
fiLe Set Up At 50% Size. fiNAL Size iS 36” x 48”
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