Manual - RayBiotech, Inc.
Contents
1. Altamirano Dimas et al Echinacea and anti inflammatory cytokine responses Results of a gene and protein array analysis Pharmacuetical Biology 2009 47 6 500 508 Cheung et al Cordysinocan a polysaccharide isolated from cultured Cordyceps activates immune responses in cultured T lymphocytes and macrophages Signaling cascade and induction of cytokines Journal of Ethonopharmacology 2009 124 1 61 68 Du et al P2 380 Identification and characterization of human autoantibodies that may be used for the treatment of prion diseases Alzheimer s and Dementia 2009 4 4 T484 T484 Van Rossum et al Granulocytosis and thrombocytosis in renal cell carcinoma a pro inflammatory cytokine response originating in the tumour Neth J Med 2009 67 5 191 4 10 Zhai et al Coordinated Changes in mRNA Turnover Translation and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation Molecular and Cellular Biology 2008 28 24 7414 7426 11 Gao et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal regulated kinase in cultured T lymphocytes FEBS Letters 2007 581 26 5087 5093 This reference validates mulitplex ELISA results for several analytes with standard ELISA test results 12 Piganelli et al Autoreactive T cell responses new technology in pursuit of an old nemesis Editorial Review Pediatric Diabetes 2007 8 249 251 Quantibody2. Buffer II Lyophilized cytokine standard mix Detection antibody cocktail Cy3 equivalent dye conjugated Streptavidin Slide Washer Dryer Adhesive device sealer Manual Te RR Re RK N RK 1 2 3 4 5 6 7 8 9 10 See Section VI for detailed cytokine concentrations after reconstitution Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5ml Polypropylene microcentrifuge tubes Quantibody Human Sepsis Biomarker Array 1 6 HI General Considerations A Preparation of Samples Use serum free conditioned media if possible If serum containing conditioned media is required it is highly recommended that complete medium be used as a control since many types of sera contains cytokines We recommend the following parameters for your samples 50 to 100 ul of original or diluted serum plasma cell culture media or other body fluids or 50 500 ug ml of protein for cell and tissue lysates If you experience high background or the readings exceed the detection range further dilution of your sample is recommended Handling glass slides Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only Handle all buffers and slides with latex free gloves Handle glass slide in clean environment Because there is no barcode on the slide transcribe the slide serial number from the slide
3. bag to the back of the slide with a permanent marker before discarding the slide bag Once the slide is disassembled this will enable you to distinguish one slide from the other C Incubation Completely cover the array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or lt 70 ul of sample or reagent is used Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4 C Make sure to cover the incubation chamber tightly to prevent evaporation Quantibody Human Sepsis Biomarker Array 1 7 IV Protocol READ ENTIRE PROTOCOL BEFORE STARTING A Completely air dry the glass slide 1 Take the glass slide from the box and let it equilibrate to room temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry at room temperature for another l 2 hours Note Incomplete drying of slides before use may cause the formation of streaks or comet tails on slide B Prepare Cytokine Standard Dilutions Note There is only one vial of standard provided in the two slide kit this is enough for making two stan
4. of morbidity and mortality in the intensive care unit Sepsis results from a response to an infection bacterial viral fungal or parasitic and is an extremely complex chain of events involving inflammatory and anti inflammatory processes The infectious agent travels from the initial site infection to other organs via the bloodstream which in severe cases can lead to organ failure As a result septic shock can occur causing multiple organ dysfunction syndrome and death While anyone can develop sepsis infants children the elderly and people with weak immune systems are particularly susceptible The diagnosis of sepsis and evaluation of its severity is complicated due to the non specific nature of the signs and symptoms Biomarkers of sepsis can reflect the severity of sepsis and differentiate bacterial viral and fungal infections as well as systemic sepsis from local infection are undoubtedly useful for disease diagnosis prognosis and monitoring the effectiveness of antibiotic therapy Clinically C reactive protein CRP and procalcitonin PCT have been routinely used for monitoring infection However the limitation of CRP and PCT for assessing the severity and predicting prognosis prompts a continuous search for better biomarkers The most analyzed sepsis biomarkers include inflammatory factors cell markers receptors coagulation markers and biomarkers for vascular endothelial damage and organ dysfunction The traditional metho
5. spots Completely remove wash buffer in each wash step High Insufficient wash Increase wash time and use more wash buffer background Dus Minimize dust in work environment before starting experiment Slide is allowed to dry out Take additional precautions to prevent slides from dying out during experiment Quantibody Human Sepsis Biomarker Array 1 18 Select Quantibody Publications Stechova et al Influence of Maternal Hyperglycaemia on Cord Blood Mononuclear Cells in Response to Diabetes associated Autoantigens Scandinavian Journal of Immunology 2009 70 2 149 158 Willingham SB et al NLRP3 NALP3 Cryopyrin facilitates in vivo caspase 1 activation necrosis and HMGBI release via inflammasome dependent and independent pathways J Immunol 2009 183 3 2008 15 El Karim et al Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Journal of Endodontics 2009 35 6 829 833 Souqui re S et al T Cell tropism of simian T cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills Mandrillus sphinx J Med Primatol 2009 38 4 279 89 Sharma et al Induction of multiple pro inflammatory cytokines by respiratory viruses and reversal by standardized Echinacea a potent antiviral herbal extract Antiviral Research 2009 83 2 165 170
6. 0 0 721 72 33 683 65 FAS L 1 000 0 817 82 28 621 59 G CSF 1 000 394 1 091 70 2 659 66 ICAM 1 50 000 776 36 449 71 153 250 183 832 61 IL 1a 1 000 0 790 79 0 784 78 IL 1B 500 0 480 96 4 546 109 IL 2 1 000 0 845 84 2 541 54 IL 2 Ra 5 000 0 5 729 115 43 2 865 56 IL 4 1 000 0 857 86 2 804 80 IL 6 1 000 2 466 2 785 32 16 650 63 IL 8 200 172 304 66 17 161 72 IL 10 1 000 0 792 79 1 587 59 IL 12p70 500 0 453 91 0 266 53 IL 13 500 0 404 81 5 274 54 IL 18 2 000 0 1 448 72 113 1 618 75 Lipocalin 2 5 000 16 3 164 63 9 292 12 986 74 MCP 1 1 000 94 1 042 9596 62 589 5396 MCP 2 1 000 1 1 184 118 6 653 65 MIF 5 000 475 3 568 62 24 3 053 61 MIP 1a 5 000 47 7 462 14896 160 6 207 12196 MIP 1f 500 0 442 88 77 397 64 OPN 20 000 67 20 317 101 2 864 16 211 67 PAI I 50 000 31 317 62 417 62 19 620 45 102 51 PF4 10 000 16 5 195 52 7 897 8 357 5 PCT 50 000 251 53 979 107 49 26 359 53 RAGE 10 000 0 9 827 98 486 9 313 88 Resistin 5 000 0 5 582 112 4 796 9 962 103 ST2 2 000 0 1 995 100 8 1 955 97 TM 50 000 37 42 674 85 1 508 47 540 92 TNFa 5 000 31 4 507 90 92 4 056 79 TREM 1 50 000 8 61 870 124 59 36 345 73 Troponin 100 000 50 69 542 69 28 60 693 61 uPAR 10 000 2 296 10 587 83 730 5 903 52 VCAM 1 200 000 18 248 708 124 141 298 298 382 79 VEGF 5 000 538 3 990 69 0 3 237 65 Quantibody Human Sepsis Biomarker Array 1 16 VIII Quantibody Q Analyzer Quantibody Q Analyzer is an array spe
7. 33 40 000 PAI I 0 137 412 1 235 3 704 11 111 33 333 100 000 PF4 0 27 82 247 741 2 222 6 667 20 000 Procalcitonin 0 137 412 1 235 3 704 11 111 33 333 100 000 RAGE 0 27 82 247 741 2 222 6 667 20 000 Resistin 0 14 41 123 370 1 111 3 333 10 000 ST2 0 5 16 49 148 444 1 333 4 000 Thrombomodulin 0 137 412 1 235 3 704 11 111 33 333 100 000 TNFa 0 14 41 123 370 1 111 3 333 10 000 TREM 1 0 137 412 1 235 3 704 11 111 33 333 100 000 Troponin I 0 274 823 2 469 7 407 22 222 66 667 200 000 uPAR 0 27 82 247 741 2 222 6 667 20 000 VCAM 1 0 549 1 646 4 938 14 815 44 444 133 333 400 000 VEGF 0 14 41 123 370 1 111 3 333 10 000 Quantibodv Human Sepsis Biomarker Arrav 1 15 VII System Recovery The antibody pairs used in the kit have been tested to recognize their specific antigen The spiking recovery rate of the cytokines by the kit in 2x diluted Human serum SR and 2x diluted Human cell culture media CM are reported in the following table The spiking recovery rate for culture media and serum pg ml Spiking CM CM Ag CM SR SR Ag SR CD14 10 000 0 6 030 60 3 442 10 229 68 CD40 10 000 0 9 383 94 24 5 489 55 CD163 100 000 0 63 773 64 18 652 42 092 23 CRP 10 000 0 7 437 74 3 594 8 608 50 E Selectin 20 000 183 24 763 123 9 428 20 020 53 Fas 1 00
8. 5 min each with 150 ul of Ix Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step Note Incomplete removal of the wash buffer in each wash step may cause dark spots Background signal is higher than that of the spot D Incubation with detection antibody cocktail and wash 9 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 10 Add 80 ul of the detection antibody cocktail to each well Incubate at room temperature for l 2 hour Longer incubation time is preferable for higher signals and backgrounds 11 Decant the samples from each well and wash 5 times with 150 ul of 1x Wash Buffer I and then 2 times with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer after each wash step Quantibody Human Sepsis Biomarker Array 1 10 E Incubation with Cy3 equivalent dye Streptavidin and wash 12 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 13 Add 80 ul of Cy3 equivalent dye conjugated streptavidin to each well Cover the device with aluminum foil to avoid exposure to light or incubate in dark room Incubate at room temperature for hour 14 Decant the samples from each well and wash 5 times with 150 ul of 1x Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each was
9. 60 human or 160 mouse cytokines in a single experiment This is not only one of the most efficient products on the market for cytokine quantification but makes it more affordable for quantification of large number of proteins Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool for drug and biomarker discovery Quantibody Human Sepsis Biomarker Array 1 4 How It Works Array support YY VA l 0 9 Incubation of Sample FISS With arraved antibodv l 2 hr Supports Cocktail of kie a a d OY K KX Incubation with YYY Biotinylated Ab Labeled mama 4 streptavidin l l ig i Incubation with Yey Cy3 equivalent dye l hr Labeled streptavidin ee Detection of signals Samples l 2 hr gt Data analysis and graph Quantibody Human Sepsis Biomarker Array 1 5 II Materials Provided Upon receipt all components of the Quantibody Array kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 6 months Once thawed the glass slide cytokine standard mix detection antibody cocktail and Cy3 equivalent dye conjugated Streptavidin should be kept at 20 C and all other components may be stored at 4 C The entire kit should be used within 6 months of purchase Components Item Description 1 Slide kit 2 Slide kit Quantibodv Array Glass Slide Sample Diluent 20X Wash Buffer I 20X Wash
10. Human Sepsis Biomarker Array 1 19 XI Experiment Record Form PMT Well No Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 a 2 4 Std5 aj aj 5 Std4 6 Std3 5 6 7 Std2 8 Std J IL 11 L IL IL 14 15 16 Quantibody Human Sepsis Biomarker Array 1 20 XII How to Choose Quantibody Products Species based selection Human QAH Mouse QAM Rat QAR CYT 1 QAR CXT 2 QAR CYT 3 QAR INF 1 Porcine QAP CYT 1 Non Human Primates NHP QAN CYT 1 Canine QAC CYT 1 Feline QAF CYT 1 Equine QAE CYT 1 Function based selection TH1 TH2 TH17 Array QAH TH 1 QAH TH17 QAM TH17 Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 QAM INF 1 QAR INF 1 Angiogenesis Arrays QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 Chemokine Arrays QAH CHE 1 QAM CHE 1 MMP Array QAH MMP 1 Immunoglobin Isotype Array QAH ISO 1 AAM ISO G1 Cytokine Number based selection 320 cytokines QAH CAA 7000 280 cytokines QAH CAA 6000 240 cytokines QAH CAA 5000 200 cytokines QAH CAA 4000 160 cytokines QAH CAA 3000 QAM CAA 3000 120 cytokines QAH CAA 2000 QAM CAA 2000 80 cytokines QAH CAA 1000 QAM CAA 1000 60 cytokines QAH ANG 1000 QAM CYT Q2000 40 cytokines QAH INF 3 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CYT 4 QAH CYT 5 QAH CYT 6 QAH CYT 7 QAM INF 1 QAM CYT 4 QAM CYT 5 QAM CYT 6 30 cytokines QAH ANG 2 QAH ANG 3 QAM INT 1000 QAR C
11. Quantibody Human Sepsis Biomarker Array 1 Quantitative measurement of 40 human Sepsis associated cytokines Patent Pending Technology User Manual Version Oct 2013 Cat QAH SEP 1 RayBiotech Inc We Provide You With Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com Cytokine Detected 40 CD14 CD40 CD163 CRP E Selectin Fas FasL G CSF ICAM 1 IL 1a IL 1B IL 1R4 IL 2 IL 2Ra IL 4 IL 6 IL 8 IL 10 IL 12p70 IL 13 IL 18 Lipocalin 2 MCP 1 MCP 2 MIF MIP 1a MIP 1f OPN PAI 1 PF4 PCT RAGE Resistin TM TNFa TREM 1 Troponin 1 uPAR VCAM 1 VEGF Format One standard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Detection Method Fluorescence with laser scanner Cy3 equivalent dye Sample Volume 50 100 ul per array Reproducibility CV lt 20 Assay duration 6 hrs 00000000 00000000 00000000 90000000 00000000 20000000 00000000 20000000 20000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 EEE See Section V For Array Map gt Fluor dye cy3 equivalent Biotin Streptavidin
12. YT 3 QAM CHE 1 20 cytokines QAH CYT 1 QAH CYT 2 QAM CYT 1 QAM CYT 2 QAM CYT 3 QAM INT 1 QAH TH17 1 QAM THI7 1 10 cytokines QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 QAH ADI 1 QAM INT 2 QAR CYT 1 QAR CYT 2 QAR INF 1 QAN CYT 1 QAP CYT 1 QAH IGF 1 less than 10 cytokines QAH ISO 1 QAH ADI 2 QAP CYT 1 QAM ISO G1 Purpose based selection Custom Arrays Choose from over 500 cytokine pool Any kind Any number Order slide only or full service in house Desired marker not in our pool No problem For certain developmental fee we may be able to add the marker to your panel if the paired antibodies are available on the market Quantibody Human Sepsis Biomarker Array 1 21 Note Quantibody is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for three months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase pri
13. ce This product is for research use only 2013 RayBiotech Inc Quantibody Human Sepsis Biomarker Array 1 22
14. cific Excel based program However it is not a simple calculation macro as it contains sophisticated data analysis Key features e Simplicity Easy to operate and requires no professional training With a simple copy and paste process the cytokine concentration is determined e Outlier Marking amp Removing The software can automatically mark and remove the outlier spots for more accurate data analysis e Normalization The program allows for intra and inter slide normalization for large numbers of samples e Two Positive Controls The program utilizes the two positive controls in each array for normalization e Two Analytical Algorithms Users can choose either linear regression or log log algorithms to meet their analytical needs e Two Data Outputs standard curves and digital concentration e User Intervention The program allows for user manual handling of outliers and other analytical data e Lower and Upper Limits Determination The program automatically marks out the values below or above the detection range e Standard Deviation The program outputs the standard deviations of the quadruplicate spots for data accuracy e Analytical Tips Q Analyzer analysis tips are included in the program Quantibody Human Sepsis Biomarker Array 1 17 IX Troubleshooting guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper dilution C
15. complex Detect antibody Cytokine Capture antibody Glass Slide Support Quantibody Human Sepsis Biomarker Array 1 1 TABLE OF CONTENTS We HOV CL ACW sal a A 1 TNO GUC MOM L id sar fr dana l A vacate 3 HOW LU W OLKS ia i e tana ars ne enacts 5 IH Materials Provided ii2 sce senuttin aciiecinakoed 6 Additional Materials Required seen 6 MI General Considerations nn 7 A Preparation of Samples cccceeeeee ees 7 B Handling Glass Slides ne 7 CCD AO a WET E E E E N 7 IVe PROG GO l sa E T 8 A Complete Air Dry the Glass Slide 8 B Prepare Cytokine Standard Dilutions 8 C Blocking and Incubation nn 9 D Incubation with Detection Antibody Cocktail 10 E Incubation with Cy3 Equivalent Dye Streptavidin 10 F Fluorescence Detection c cee esee eee eens 11 G Data Analysis eine eran E EA aes 12 V Cytokine Array Map Standard Curves 13 VI 8 Point Standards sci25oes6 c0t os er gccivaeccmeaimeianeien 14 VII System Recovery tas ww u ean ta ee odd 15 VIII Quantibody Q Analyzer cccccccececeeececeeseeeees 16 IX Troubleshooting Guide 17 X Select Quantibody Publications 000 seen 18 XI Experimental Record Form eee 19 XII How to Choose Quantibody Products tess ji seen ik 20 Quantibody Human Sepsis Biomarker Array 1 2 I Introduction Sepsis is the most common cause
16. d for cytokine detection and quantification is through the use of an enzyme linked immunosorbent assay ELISA While this traditional method works well for a single protein the overall procedure is time consuming and requires a relatively high volume of sample Thus conservation of precious small sample quantities becomes a difficult task To solve this problem take advantage of the innovations in microarray technology over the last decade A long standing leader in the field Raybiotech has pioneered the development of cytokine antibody arrays which have now been widely applied in the research community with hundreds of peer reviewed publications including top tier journals such as in Cell and Nature Quantibody Human Sepsis Biomarker Array 1 3 The Quantibody array Our quantitative array platform uses the multiplexed sandwich ELISA based technology and enables researchers to accurately determine the concentration of multiple cytokines simultaneously It combines the advantages of the high detection sensitivity amp specificity of ELISA and the high throughput of arrays Like a traditional sandwich based ELISA it uses a pair of cytokine specific antibodies for detection A capture antibody is first bound to the glass surface After incubation with the sample the target cytokine is trapped on the solid surface A second biotin labeled detection antibody is then added which can recognize a different epitope of the target cytokine The cyto
17. dard curves Reconstitute the lyophilized standard within one hour of usage If you must use the standard for two different days store only the Std dilution at 80 C for future use Prepare serial dilution of cytokine standards 100ul 100ul 100ul 100ul 100ul 100ul TS OSOS TSO DY Add 500u1 Sample Diluent 200ul 200ul 200ul 200ul 200ul 200u1 100ul Vial Labels Stdi Std2 Std3 Std4 Std5 Std6 Std7 CNTRL 2 Reconstitute the Cytokine Standard Mix lyophilized by adding 500ul Sample Diluent to the tube For best recovery always quick spin vial Quantibody Human Sepsis Biomarker Array 1 8 prior to opening Dissolve the powder thoroughly by gentle mixing Label the tube as Std1 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200ul Sample Diluent to each of the tubes A Pipette 100ul of Std1 into tube Std2 and mix gently Perform 5 more serial dilutions by adding 100ul of Std2 to tube Std3 and so on 5 Add 10Oul Sample Diluent to another tube labeled as CNTRL Do not add standard cytokines or samples to the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Note Since the starting concentration of each cytokine are different the serial concentrations from Stdl to Std7 for each cytokine are varied and can be found in section VI C Blocking and Incubation 6 Add 100ul Sample Diluent into each well and incubate at room temperature for 30 min to block slide
18. h step F Fluorescence Detection 15 Disassemble the device by pushing clips outward from the slide side Carefully remove the slide from the gasket Be careful not to touch the surface of the array side 16 Place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube add enough Ix Wash Buffer I about 30 ml to cover the entire slide and then gently shake at room temperature for 15 minutes Decant Wash Buffer I Wash with 1x Wash Buffer II about 30 ml with gentle shaking at room temperature for 5 minutes then decant Wash Buffer II 17 Remove buffer droplets completely by one of the following ways e Put the glass slide into the Slide Washer Dryer and dry the glass slide by centrifuge at 1 000 rpm for 3 minutes without cap e Or dry the glass slide by a compressed N stream e Or gently apply suction with a pipette to remove buffer droplets Do not touch the array only the sides Quantibody Human Sepsis Biomarker Array 1 11 18 Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength such as Axon GenePix Make sure that the signal from the well containing the highest standard concentration Std1 receives the highest possible reading yet remains unsaturated Note If the signal intensity for different cytokines vary greatly in the same array we recommend using multiple scans with a higher PMT for low signal cytokines and a low PMT for high signal cytokine
19. heck pipettes and ensure correct preparation Short incubation time Weak Signal Ensure sufficient incubation time or change sample incubation step to overnight Too low protein concentration in sample Dilute starting sample less or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Bubble formed during incubation Handle and pipette solutions more gently De gas solutions prior to use Atrays are not completed covered by Uneven signal reagent Prepare more reagent and completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Cross contamination from neighboring wells Avoid overflowing wash buffer between wells Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard reconstitution or Improper dilution Poor standard curve Reconstitute the lyophilized standard at the room temperature before making serial dilutions Check pipettes and ensure proper serial dilutions Inadequate detection Increase laser power so the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed cytokine standards Always use new cytokine standard vial for new set of experiment Discard any leftover Overexposure Lower the laser power Dark
20. kine antibody biotin complex can then be visualized through the addition of the streptavidin labeled Cy3 equivalent dye using a laser scanner Unlike the traditional ELISA Quantibody products use array format By arraying multiple cytokine specific capture antibodies onto a glass support quantitative multiplex detection of cytokines in one experiment is made possible In detail one standard glass slide is divided into 16 wells of identical cytokine antibody arrays Each antibody together with the positive controls is arrayed in quadruplicate The slide comes with a 16 well removable gasket which allows for the process of 16 samples on one slide Four slides can be nested into a tray which matches a standard microplate footprint and allows for automated robotic high throughput process of 64 arrays simultaneously For cytokine quantification the array specific cytokine standards whose concentration has been predetermined are provided to generate a standard curve for each cytokine In a real experiment standard cytokines and samples will be assayed in each array simultaneously through a sandwich ELISA procedure By comparing signals from unknown samples to the standard curve the cytokine concentration in the samples will be determined Quantibody array kits have been confirmed to have similar detection sensitivity as traditional ELISA Our current high density Quantibody kits allow scientists to quantitatively determine the concentration of 3
21. rd curve of a given antigen are listed below The detection sensitivity of each protein in one experiment is user dependent Try our array specific Quantibody Q Analyzer to see your Limit of Detection LOD Section VIII Serial standard concentration pg ml pg ml Cntrl Std7 Std6 Std5 Std4 Std3 Std2 Std1 CD14 0 27 82 247 741 2 222 6 667 20 000 CD40 0 27 82 247 741 2 222 6 667 20 000 CD163 0 274 823 2 469 7 407 22 222 66 667 200 000 CRP 0 27 82 247 741 2 222 6 667 20 000 E Selectin 0 55 165 494 1 481 4 444 13 333 40 000 Fas 0 5 16 49 148 444 1 333 4 000 FAS L 0 3 8 25 74 222 667 2 000 G CSF 0 3 8 25 74 222 667 2 000 ICAM 1 0 137 412 1 235 3 704 11 111 33 333 100 000 IL 1a 0 3 8 25 74 222 667 2 000 IL 1B 0 4 12 37 111 333 1 000 IL 2 0 3 8 25 74 222 667 2 000 IL 2 Ra 0 14 41 123 370 1 111 3 333 10 000 IL 4 0 3 8 25 74 222 667 2 000 IL 6 0 3 8 25 74 222 667 2 000 IL 8 0 1 2 5 15 44 133 400 IL 10 0 3 8 25 74 222 667 2 000 IL 12p70 0 1 4 12 37 111 333 1 000 IL 13 0 1 4 12 37 111 333 1 000 IL 18 0 5 16 49 148 444 1 333 4 000 Lipocalin 2 0 14 41 123 370 1 111 3 333 10 000 MCP 1 0 3 8 25 74 222 667 2 000 MCP 2 0 3 8 25 74 222 667 2 000 MIF 0 14 41 123 370 1 111 3 333 10 000 MIP 1a 0 14 41 123 370 1 111 3 333 10 000 MIP 1f 0 1 4 12 37 111 333 1 000 OPN 0 55 165 494 1 481 4 444 13 3
22. s 7 Decant buffer from each well Add 100ul standard cytokines or samples to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals Note We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation if less than 70 ul of sample or reagent is used Note This step may be done overnight at 4 C for best results Quantibody Human Sepsis Biomarker Array 1 9 8 Wash Calculate the volumes of Wash Buffers required based on the number of samples being processed and the entire remaining protocol described below Dilute 20x Wash Buffer I and 20x Wash Buffer I separately with ddH O to generate the required volume of 1x Wash Buffer I and Ix Wash Buffer II For example 100 ul of 20x Wash Buffer I would be diluted to a final volume of 2 000 ul Decant the samples from each well and wash 5 times 5 min each with 150 ul of Ix Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer after each wash step Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with Ix Wash Buffer I cover the entire glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min Decant the 1x Wash Buffer I from each well wash 2 times
23. s Quantibody Human Sepsis Biomarker Array 1 12 G Data Analysis 19 Data extraction can be done with most of the microarray analysis software GenePix ScanArray Express ArrayVision or MicroVigene For quantitative data analysis our Quantibody Q Analyzer software is available It gives visual output as well as digital values More information can be found in section VIII Experiments U Image scan laser scanner U Data extraction EM 2 1 3 2 89 88 90 91 GenePix etc 21 22 21 23 222 223 232 213 55 54 57 56 188 178 189 190 Data computation Q Analyzer U Final Result pg ml Quantibody Human Sepsis Biomarker Array 1 13 V Cytokine Array Map Standard Curves 106 10 104 10 Signal Intensity 10 10 10 10 10 POS1 POS2 CD14 CD40 CD163 CRP E Selectin Fas FAS L G CSF ICAM 1 IL la IL 1f IL 2 IL 2 Ra IL 4 IL 6 IL 8 IL 10 IL 12p70 IL 13 IL 18 Lipocalin 2 MCP 1 MCP 2 MIF MIP la MIP 1f OPN PAI I PF4 PCT RAGE Resistin ST2 TNFa TREM 1 Troponin I uPAR VCAM 1 VEGF QAH SEP 1 Standard Curves 10 10 10 104 10 Cytokine Concentration pg ml Quantibody Human Sepsis Biomarker Array 1 14 VI 8 Point Standards After reconstitution and serial dilution of the lyophilized cytokine standard mixes the concentrations used for generating the 8 point cytokine standa
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