User Manual
Contents
1. application Uses other than the labeled intended use may be a violation of applicable law Components supplied with this kit romp Control Bead Mix 50X 192 uL 60681 Assay Buffer E 2x12 mL 60682 Wash Bufer 20 a ane peed 50X 60684 r For Research Use Only www origene com Page 6 Rev N5K06G13A1 ORIGENE Your Gene Company Storage Instructions e The kit is shipped on blue ice TruePLEX Human Antibody Profiling Array Detection Kit e Store individual kit components as listed above or on the label Materials required or recommended but not provided e Spin X Centrifuge Tube filters Costar 8160 highly recommended e Calibrated adjustable micropipettors with disposable plastic tips e An 8 well multichannel pipettor is recommended but not required e De ionized or molecular biology grade water e Propylene tubes 1 5 ml e Absorbent paper towels for example Wypall X60 from Kimberly Clark Equipment and software required Description Luminex 100 200 or equivalent Microplate vacuum manifold with pressure gauge Luminex Data Acquisition Software Vortex Mixer Mini centrifuge Orbital Shaker for Microplates Water Bath Sonicator Data Analysis Software For Research Use Only Rev N5N8413A1 www origene com Recommended Supplier Cat No Luminex Millipore Hitachi Bio Rad Pall Multi well plate vacuum manifold PN 5017 XPONENT 3 1 Luminex IS 2 3 Luminex2. of lipemic of hemolyzed samples 4 Collect samples according to standard protocols immediately mix and separate and then aliquot the samples into polypropylene tubes If not tested immediately store samples at 80 C Analyze fresh samples as quickly as possible after collection Allow frozen samples to thaw on ice and then mix well Avoid multiple freeze thaw cycles of frozen samples Optimum results are obtained when samples are clarified with a Spin X column Costar 8160 Add serum or plasma to the sample cup Centrifuge at 12 14 000 x g for 5 20 minutes depending upon the sample volume and the amount of particulate material in the samples Recover the clarified sample from the bottom of the tube the filtrate and use immediately Turbid samples may also be clarified by centrifugation Centrifuge samples at 14 000 x g for 10 minutes at 4 C in a refrigerated microcentrifuge immediately prior to analysis Remove an aliquot from the supernatant taking care not to disturb any pellet and avoiding any lipid that may be floating on the top Run Controls and samples in duplicate For Research Use Only www origene com Page 10 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Reagent Preparation 1X Wash Buffer Prepare 1X Wash Buffer by diluting the entire contents of the 20X Wash Buffer bottle with 950 ml of sterile deionized water For long term storage add 0 5 g sodium azid
3. 47 161 Page 18 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Normal Sera Twenty five normal human sera were tested with a bead mix containing multiple beads coupled to well known autoimmune antigens The serum was diluted 1 200 and the assay used the high stringency Sample Diluent and one hour IgG binding The following results were obtained These values can be used for guidance in developing algorithms for sample data analysis Median Fluorescent Intensity Statistic TRIM21 SNRPA SSB TROVE2 SNRNP70 SNRPC Min 12 16 20 16 24 38 Max 197 41 57 54 215 332 Average 32 27 33 25 62 123 Median 25 26 31 23 49 97 Std Dev 36 8 8 9 47 77 MFI Distribution Histogram Number of Samples MFI Range TRIM21 SNRPA SSB TROVE2 SNRNP70 SNRPC 0 25 13 12 i i a 25 50 10 13 23 6 ie 7 50 75 1 0 1 75 100 0 0 0 0 1 5 100 150 0 0 0 5 150 200 1 0 0 o 7 f 200 250 0 0 0 r I 250 0 0 0 0 0 r For Research Use Only www origene com Page 19 Rev N5K06G13A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Plate Template Layout For Research Use Only www origene com Page 20 Rev N5K06G13A1 ORIGENE Your Gene Company Troubleshooting TruePLEX Human Antibody Profiling Array Detection Kit To troubleshoot problems with the Luminex instrument consult the appropriate Luminex manuals contact Luminex technical support http www luminexcorp com sup
4. HEK 293T Purified proteins are then coupled to Luminex beads available separately and by special order Each protein is coupled to a different type or color of Luminex beads A bead mix is prepared by combining antigen coupled beads with the control beads supplied in this kit Diluted serum or plasma is mixed with the beads and autoantibodies if present will bind to the specific protein coupled beads After washing an anti human phycoerythrin conjugate is added to detect the bound human IgG After a final wash the samples are read in the Luminex 100 or Luminex 200 instrument The median fluorescent intensity measured for each bead reflects the amount of human IgG bound to the bead Protein source and characterization The proteins coupled to the Luminex beads are recombinant human proteins expressed in human cells Thus antibodies in serum or plasma samples may react differently to these proteins than those expressed in E coli or insect cells due to the presence of true human post translational modifications For Research Use Only www origene com Page 4 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company OriGene s recombinant human proteins are expressed with a fusion tag on the C terminus Protein TRTRPLEQKLISEEDLAANDILDYKDDDDKV The sequence EQKLISEEDL is known as the myc tag The sequence DYKDDDDK is known as the DDK or FLAG tag The tag sequence provides for eff
5. LDS1 7 or BioPlex Manager VWR Analog Vortex Mixer 58816 121 120V 58816 123 230V VWR Minituge 93000 196 120V 93000 198 230V Eppendorf Mix Mate 022674200 120V 60HZz 022674226 230V 50Hz Bransonic Model B3 B5 000 951 005 Model B3 115V 000 951 103 Model B5 230V Microsoft Excel Page 7 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Control Bead Mix The Control Bead Mix contains a mixture of beads that have been coupled with certain proteins to provide assurance that the assay is working correctly The Control Bead Mix contains beads have been coupled with the following e Anti human IgG This bead binds to human IgG in serum samples and should be positive for all human serum samples e Recombinant Human Serum Albumin Recombinant human serum albumin is bound to Luminex beads This bead serves as a non specific binding control for samples and the detection antibody This protein does not contain the myc DDK tag e BSA myc DDK Bovine serum albumin coupled to the complete OriGene myc DDK fusion tag and then coupled to Luminex beads This bead serves as a control for binding to the myc DDK fusion tag sequence that is present on OriGene s recombinant human proteins e Donkey IgG Donkey IgG is bound to Luminex beads This bead serves as a non specific binding control for samples and the detection antibody Bead Assignments and Expected Results for Control Beads Be
6. ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Low Stringency Sample Diluent Optional Volume per Number of Final Buffer A Som Buffer C Som Add 15 mg of Blocking Reagent per ml e Mix Sample Diluent thoroughly by gently vortexing and place on a tube rotator while preparing the remaining reagents Sample Diluent should appear clear before use If Blocking Reagent is not completely dissolved clogging of the filter plate may occur e Do not store Sample Diluent Prepare a fresh batch for each assay Controls e Dilute the Positive Control 50X into the Sample Diluent as follows Component Volume per Number Final p Reaction of Wells Volume e Use Sample Diluent as the Negative Control Alternatively dilute a known negative serum or plasma sample into Sample Diluent to create a negative control Filter Plate Before starting the assay cover the wells that will not be used in the assay with an aluminum plate sealer supplied Press sealer down so that all wells are tightly sealed Trim edges with a razor blade 1X Bead Mix e Multiple Bead mixes can be combined to prepare a larger multiplex However ensure that none of the bead regions overlap e Briefly vortex 5 seconds the vial of Bead Mix 50X e Sonicate the Bead Mix 50X for 30 seconds e Prepare 1X Bead Mix according to the table below Adjust volumes to account for the addition of additional beads or Bead Mixes Pre
7. ad D E Epitope Expected Results escription E Anti Human IgG E Positive Positive DiS human T m n i Donkey IgG Additional beads added to the bead mix that have been coupled with OriGene s recombinant proteins will be positive with the Positive Control Additional beads that have been added to the bead mix that have been coupled to native proteins or proteins without a DDK fusion tag will give negative results with the Positive Control For Research Use Only www origene com Page 8 Rev N5K06G13A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Note To achieve optimal results this kit requires the use of the High Gain of High PMT setting on the Luminex 100 or 200 Some instruments may use this setting as the default Check with your instrument supplier for more information Setting the Luminex 100 or 200 for High Gain of High PMT Reading 1 Create a new lot number for Cal 2 and enter it into the Luminex software as a new Cal 2 lot number use the actual lot number with an HG at the end to designate High Gain 2 Record the Cal 2 target value which is usually around 3800 3 Multiply the Cal 2 target value by 4 55 to get a new Target value of approximately 17 290 4 Enter the new Target Value as the value for your New Cal 2 lot 5 Run the Cal 2 Calibration Procedure Notes e For preparation of all reagents prepare enough for several extra wel
8. an orbital plate shaker at 600 800 rpm Read plate in the Luminex instrument lf using a partial plate with unused wells remaining remove the liquid by gentle vacuum and let the used wells dry Mark used wells so that they are not inadvertenly used again Data Analysis 1 Upload or transfer the Luminex data to an Excel Spreadsheet 2 Use the Median Fluorescent Intensity MFI for analysis Establish a working cutoff for the specific sample population and sample dilution using the values of the controls and a set of Known negative samples Protocol Variations The following protocol variations can be utilized to increase the sensitivity or specificity of detection Increase the IgG binding time from 60 minutes to 90 or 120 minutes Increase or decrease the dilution of the sample Alter the stringency of the binding step by using the moderate or low stringency sample Diluent Dilute the Detection Reagent anti human phycoerythin conjugate to 0 5X 1 100 dilution or 0 25X 1 200 instead of 1X 1 50 For Research Use Only www origene com Page 15 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Limitations of the Procedure 1 Plasma samples collected in anticoagulants other than sodium heparin have not been investigated The influence of drugs or other substances not usually found in serum or plasma have not been investigated The influence of abnormal jaundice
9. assay used the high stringency Sample Diluent and one hour IgG binding 100000 i Pos Control E Neg Control E Samples 15 anti Human HS Albumin BSA myc DDK Donkey IgG Control Bead Autoimmune Sera Four autoimmune sera A1 A4 were tested with a bead mix that included six beads coupled to well known autoimmune antigens The sera were diluted 1 200 and the assay used the high stringency Sample Diluent and one hour IgG binding For Research Use Only www origene com Page 17 Rev N5N8413A1 ORIGENE Your Gene Company Autoimmune Sera Dilution TruePLEX Human Antibody Profiling Array Detection Kit One autoimmune serum was tested with a bead mix containing multiple beads coupled to well known autoimmune antigens The serum sample was tested at a wide range of dilutions The assay used the high stringency Sample Diluent and one hour IgG binding For some antigens a strong signal gt 1000 MFI is observed even for very high dilutions Dilution 1 100 1 200 1 400 1 800 1 1600 1 3200 1 6400 1 12800 For Research Use Only Rev N5N8413A1 SSB 29240 28927 28269 26488 29316 20234 13619 9640 SNRPB 31492 31080 30095 2 081 21517 11622 6707 4156 SNRPC 2 256 23069 14371 6696 3937 1860 978 516 www origene com TROVE2 15216 10703 6945 4680 2588 1536 861 516 SNRNP70 25684 17202 10442 5490 3438 1982 849 572 SNRPA 13107 8324 4044 1825 1253 486 3
10. d hemolyzed lipemic sera or plasma samples has not been investigated Samples with unusually low or unusually high IgG levels have not been investigated The rate of degradation of antibodies in human serum or plasma has not been investigated The affect of heterophilic antibodies on the assay has not been investigated Protocol Summary 1 Prepare Sample Diluent 1X Wash Buffer and 1X Bead Mix 2 Dilute Samples in Sample Diluent 1 Add 100 ul 1X Bead Mix to each well 4 Remove liquid from wells by gentle vacuum Blot thoroughly 5 Add 100 ul of controls and diluted samples to the designated wells 6 Cover and incubate for 1 2 hours with shaking 600 800 rpm 7 Prepare 1X Detection Reagent 8 Wash three times with 100 ul 1X Wash Buffer 9 Add 100 ul of 1X Detection Reagent to each well 10 Cover plate and incubate for 1 hour with shaking 600 800 rpm 11 Prepare Luminex instrument for reading 12 Wash three times with 100 ul 1X Wash Buffer Blot thoroughly 13 Add 100 ul of 1X Wash Buffer to each well 14 Shake plate for 2 minutes 600 800 rpm 15 Read in Luminex instrument For Research Use Only www origene com Page 16 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Example Data Normal Sera The Positive and Negative Controls and fifteen normal human sera were tested with the four beads contained in the Control Bead Mix The sera were diluted 1 200 and the
11. d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company APPLICATION GUIDE TruePLEX Human Antibody Profiling Array Detection Kit Part Number AP100001 For Research Use Only For Research Use Only www origene com Page 1 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company For Research Use Only www origene com Page 2 Rev N5K06G13A1 ORIGENE Your Gene Company TruePLEX Human Antibody Profiling Array Detection Kit TABLE OF CONTENTS Overview and Intended Use ccseeceeeeeeeneeeensesensenenseseasesensenensesensenennesones 4 Detection of human autoantibodies with OriGene recombinant proteins 4 Protein Source and characterization cccscccessecceseeeeseeeseeeeeeeeeseeeeseeeeseeeeeeees 4 Se glie SAMOS S EAN E A E A E AA E A E 5 Important Information sssssnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmn 5 Components supplied with this Kit ccccsssesssseeseeseeseeseeseeneesenseesenseesensees 6 Storage Instructions sosicacncsweseatesuceressemeeuatesmevsnteuns sguaseusdesseawasaatemasiscemadeatemetanl 7 Materials required or recommended but not provided ccssscceeseeeeeeeeeeeees 7 Equipment and software required cccscscsesseceeseceeeeenseeenseeeaseseaseseeeenensenes 7 Bead Assignments and Expected Results for Control Beads 000 8 Setting the L
12. e to bring the sodium azide concentration to 0 05 7 5 mM Store diluted Wash Buffer at 2 8 C Alternatively a portion of the 20X Wash Buffer can be diluted Mix 1 volume 20X Wash Buffer with 19 volumes of deionized water Sample Diluent Sample Diluent can be prepared at different stringencies Low stringency diluent may give higher signals but non specific signals from some serum samples may be observed High stringency Sample Diluent may generate lower specific signals but non specific signals will be minimized Test samples first with the High stringency Sample Diluent Use Moderate stringency or Low stringency Sample Diluent for higher sample dilutions 1 500 or greater for samples with low antibody levels or for samples in which the antibody affinity may be low e Calculate the volume of Sample Diluent required Each serum sample requires 100 uL of Sample Diluent Additional Sample Diluent may be required to prepare intermediate dilutions and to account for pipetting losses e Prepare Sample Diluents according to the table below High Stringency Sample Diluent Recommended for initial testing Volume per Number of Final Buffer B Som Buffer C Som Add 15 mg of Blocking Reagent per ml Moderate Stringency Sample Diluent Optional Volume per Number of Final Buffer A oo o BM fo Buffer B e a Buffer C a ee o Add 15 mg of Blocking Reagent per ml For Research Use Only www origene com Page 11 Rev N5N8413A1 d
13. he newer MagPlex magnetic beads Human serum or plasma samples containing IgG are reacted with beads that have been covalently coupled to a native or recombinant protein peptide hormone or small molecule If the sample contains antibodies specific for the coupled entity then these antibodies will bind to the coupled bead After washing away the unbound material the human IgG antibodies are detected with an anti human IgG phycoerythrin conjugate and the bound phycoerythrin is detected in the Luminex instrument An optimal use for this kit is for the detection and profiling of human autoantibodies Human autoantibodies are known to play a pivotal role in many diseases including over 170 human autoimmune diseases and many cancers Many autoantibody antigens have been identified but others remain elusive or difficult to confirm In addition many questions remain unanswered regarding the role of autoantibodies in the pathology and natural history of the disease OriGene has purified over 7000 recombinant human proteins from human cells that can be used for autoantibody testing This kit may be used as a basic research tool for the detection of human autoimmune antibodies in serum and plasma for basic and clinical research studies It is not intended for use in clinical diagnostics Detection of human autoantibodies with OriGene recombinant proteins OriGene s recombinant human proteins have been expressed and purified from a human cell line
14. icient purification from the HEK cells and allows for verification that sufficient protein has been coupled to the beads The tag sequence also provides a method for a positive control for the assay performance Control Samples The kit contains a positive control e The Positive Control PC is a concentrated antibody solution that binds to the fusion tag on OriGene s recombinant human proteins produced from OriGene s TrueOrf cDNA clones The anti tag antibody has been conjugated to human IgG so that it can be detected with the anti human antibody conjugate Important Information e Store the reagents at the temperature and condition specified on the labels e Read the entire protocol before use e Research Use Only The product you have received is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application Uses other than the labeled intended use may be a violation of applicable law e Hazards It is the end user s responsibility to consult the applicable MSDS s before using this product Disposal of waste materials must comply with all appropriate federal state and local regulations If you have any questions concerning the hazards associated with this product please call OriGene Technologies Inc at 1 888 267 4436 e Terms and Conditions By opening the packaging containing this Assay Product which contains fluorescent
15. ls to account for pipetting losses e This kit provides sufficient reagents to run the assay at least three times However with careful attention to reagent preparation additional uses may be possible e The vacuum pressure on the vacuum manifold should not exceed 5 mm Hg Optimal filtration occurs between 2 and 4 mm Hg e The fluorescent beads are light sensitive and are especially sensitive to direct sunlight and incandescent light Use aluminum foil to cover plates and tubes Store plates in a dark location during incubations e After vacuum filtration blot plates on clean absorbent material such as WYPALL X60 towels from Kimberly Clark e During loading or incubations do not place the filter plate on absorbent material as liquid will be drawn through the plate by wicking For Research Use Only www origene com Page 9 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Recommended Plate Layout Before starting it is recommended that a plate plan be designed Such a plan will assist in assay workflow and data analysis A suggested plate plan is shown below A plate plan template is provided on page 18 Sample Collection and Preparation 1 Serum is the preferred sample type for use in this assay Plasma is also Suitable but non specific binding may be increased Additional sample types may also be suitable but have not been validated with this assay Avoid the use
16. ly labeled microsphere beads authorized by Luminex Corporation or using this Assay Product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this Assay Product for a full refund prior to using it in any manner You the customer acquire the right under Luminex Corporation s patent rights if any to use this Assay Product or any portion of this Assay Product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser For Research Use Only www origene com Page 5 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company based fluorescent analytical test instrumentation marketed under the name Luminex Instrument e Safety and Use All biological materials should be handled as potentially hazardous Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing of potentially infectious or hazardous agents This product is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic
17. pare enough for several extra wells to account for losses during pipetting For Research Use Only www origene com Page 12 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Volume per Number of Final e Mix thoroughly by gentle vortexing Protect from light by covering tube in aluminum foil Preparation of Samples 1 Clarify samples with a Costar Spin X column Costar 8160 Add serum or plasma to the sample cup Centrifuge at 12 14 000 x g at 4 C for 5 20 minutes depending upon the sample volume and the amount of particulate material in the samples Recover the clarified sample from the bottom of the tube the filtrate and use immediately Store excess at 20 to 80 C Alternatively samples may be clarified by centrifugation Centrifuge samples at 14000 x g for 10 minutes at 4 C in a refrigerated microcentrifuge immediately prior to analysis Remove an aliquot from the supernatant taking care not to disturb any pellet and avoiding any lipid that may be floating on the top Dilute clarified sera or plasma samples into Sample Diluent An initial dilution of 1 200 to 1 500 is recommended However dilutions from 1 50 to 1 2000 may be appropriate depending upon the antibody levels in the samples Assay Protocol lgG Binding 1 Briefly vortex the 1X Bead Mix 2 Add 100 ul of 1X Bead Mix to each well of the filter plate Remove liquid from the plate by gently vacuum Blot
18. plate thoroughly on absorbent paper Add 100 ul Sample Diluent into the Negative blank wells Add 100 ul of the diluted Positive Control into the PC wells Add 100 ul diluted samples into the designated sample wells Or Pr Cover and incubate the plate for 1 hour at room temperature on an orbital plate shaker 600 800 rpm 7 Fifteen minutes before the end of the incubation prepare 1X Detection Reagent anti human streptavidin phycoerythrin conjugate Preparation of 1X Detection Reagent e Prepare 1X Detection reagent according to the table below Prepare enough for several extra wells to account for losses during pipetting For Research Use Only www origene com Page 13 Rev N5K06G13A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Component Volume per Number of Final p Reaction Wells Volume Detection Reagent 50X Peak fe Detection of Bound IgG 1 Remove the liquid from the wells by gentle vacuum If any of the wells have become clogged and contain residual liquid pipette the liquid in each clogged well up and down ten times Then continue with the washing steps below 2 Wash beads three times by adding 100 ul 1X Wash Buffer to the wells even if they contain residual liquid Remove liquid with gentle vacuum After washing blot plate thoroughly on absorbent material 3 Add 100 ul Detection Reagent into each well 4 Cover and incubate the plate for 1 hour at room tempe
19. port or contact your instrument supplier Problem Insufficient bead count Wells in filter plate will not vacuum Leaking plate High Background For Research Use Only Rev N5N8413A1 Cause Bead mix not prepared correctly Vacuum pressure too high Plate leaked in the Luminex instrument Clogged sample probe Clogged wells Clogged wells Plate has not made a tight seal with the vacuum manifold Probe Height not adjusted correctly Insufficient blotting of filter plate Vacuum pressure too high Cross contamination of wells Ineffective or omitted wash steps www origene com Solution Prepare new bead mix and re run samples Sonicate and vortex vial containing 50X Bead Mix Adjust vacuum pressure to 2 5 mm Hg during filtration See section below on Leaking plate Clean sample probe Remove and sonicate if necessary Clarify samples before removing aliquot for testing Pipette liquid in well up and down 10 times The add 100 ul Wash Buffer to all wells and continue with next wash step Replace gasket on vacuum manifold Adjust probe height with two alignment disks using the filter plate in the kit Blot filter plate thoroughly onto paper towels after each washing cycle Ensure that the vacuum pressure is less than 5 mm Hg Pipette carefully to ensure that no material from the control or sample wells reaches the blanks Neg wells Follow wash instructions carefully Inc
20. rature on an orbital plate shaker at 600 800 rpm 5 During this incubation prepare the Luminex instrument for reading see below Prepare the Luminex Instrument for Reading 1 Set up the instrument as described in the user s manual Instruments from different vendors may have different set up procedures 2 Warm up the instrument This may take up to 30 minutes 3 Enter the analyte names and bead numbers from the Bead Mix datasheet s 4 Analysis Parameters e Set the instrument to count the Total number of events e The number of events to count should be 100 x the number of bead regions For a mix containing 10 bead regions the count should be set to 1000 e The minimum events setting should be 30 e The sample size should be set to 50 uL and the flow rate should be set to Fast 5 Check the probe height and adjust it if necessary to accommodate the filter plate 6 Perform 1 prime with sheath fluid 1 alcohol flush and 2 sheath fluid washes 7 Calibrate to High Gain setting if necessary See page 9 Washing and Reading 1 Wash beads three times by adding 100 ul Wash Buffer to all wells and then removing liquid with gentle vacuum For Research Use Only www origene com Page 14 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company oS oe 6 IN Blot plate thoroughly on absorbent material Add 100 ul 1X Wash Buffer to each well Shake plate for 2 minutes on
21. rease number of washes if necessary Page 21 ORIGENE Your Gene Company Problem Low signal in the controls Beads not in region For Research Use Only Rev N5N8413A1 Cause Luminex instrument gain setting is incorrect 1X Detection Antibody prepared incorrectly Incubations were too short or shaking was insufficient Instrument is out of calibration Beads are photo bleached Incorrect bead regions entered www origene com TruePLEX Human Antibody Profiling Array Detection Kit Solution Make sure that the reporter channel is calibrated to the High Gain or High PMT setting Prepare new 1X Detection Antibody Check assay conditions and remedy as necessary Recalibrate instrument Protect beads from light at all times Use amber colored tubes Check protocol template Correct if necessary Page 22
22. uminex 100 or 200 for High Gain of High PMT Reading 9 Procedure NOTOS cicero ce se tes ects EEEE E esse easiest eos seston EE EEEE EEEE 9 Recommended Plate Layout ccscccseeecseseeceseeeeseeeeseeeeseeeenseeeneeeeseeeenesnenees 10 Sample Collection and Preparation ccccsseesssseeccsseeseeneeseeseesenseesensesseaes 10 Me agen rie Dal allON coiii aaen a i E EE E E E EE E E EE EE EEE 11 Preparation of Samples sannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnm nnna 13 Assay Protocol ssnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn mnnn 13 POLO ON Vy AN VAC ONS o REAR 15 Protocol SU iY aoeteasseso iene eo aeste wen emedeeesectewnneuhewueendeanuneedtewaneeaieensennteceneunn 16 Exampie D ta sirsa a aasa eaaa EuS ESEESE 17 Plate Template Layout wesccscccciecnieesdewsieesieecde cede scceencsencesncesniecsieceicenbevsiewsdescaeceds 20 Plate Template Layout ccccccesesesseseseseseenseenseoesseeaseoeseoeaeeoaseonseneaeenaeeneeeneas 20 PPOUDIE SIO ONG srisicsnisonniniun niina aaa aaa aaa 21 For Research Use Only www origene com Page 3 Rev N5N8413A1 d ORIGENE TruePLEX Human Antibody Profiling Array Detection Kit Your Gene Company Overview and intended Use This kit is designed for the detection and profiling of human IgG antibodies bound to Luminex xMAP beads The kit should work with both the traditional polystyrene MicroPlex beads as well as t
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