Instruction Manual Optimiser™ Starter Kit
Contents
1. OBSERVATIONS e Al observations and conclusions listed for previous protocol e Note the difference in time required to load 12 columns with an electronic multi channel pipette Page 9 of 20 Frequently Asked Questions Variance If one well drains in say 1 minute and another in say 8 minutes how is it possible that they provide comparable results Although it may seem that difference of minutes may have an impact on the assay precision Siloam has demonstrated with multiple assays that well optimized assays on Optimiser easily achieve CV lt 6 10 The minimal effect of flow rate on precision is a combination of multiple factors 1 On the micro scale reaction kinetics are vastly different compared to the macro scale kinetics of conventional 96 well ELISA plate In microfluidic channels most surface binding reactions are saturated in 5 minutes Optimiser characterization data shows that up to 75 of peak adsorption is completed in only 10 seconds and assay binding reactions saturate in 5 minute This is a result of two factors a The diffusion distances in the microchannel are extremely small the channel has a cross section of only 200 um x 200 um hence diffusion is no longer a limiting factor b The surface area of the microchannel is 1 5 times the surface area at the base of a conventional 96 well ELISA plate The volume contained in the microchannel is 5 ul leading to 50x higher surface area to volume rat2. buffer in a clean plastic tube Add 8 ul of capture antibody stock solution to 0 5 mL of OptiBind b Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate OptiBlock OptiBlock is provided in ready to use form and is used to block the surfaces of the Optimiser s microfluidic reaction chambers following their incubation with the capture antibody solution OptiBlock is also used as the diluent for the standard detection antibody and SAv HRP in this experiment Recombinant IL 6 Standard a Stock Solution The IL 6 standard is provided in lyophilized form i Reconstitute the lyophilized standard by adding 420 uL OptiBlock blocking buffer ii Mix by gentle swirling until all of the lyophilized material has dissolved iii Vortex gently to ensure thorough mixing of the reconstituted standard iv Use freshly prepared material on the day of reconstitution b Working Solution The concentration of the reconstituted IL 6 standard is 4 ng mL Prepare a 3000 pg mL standard Standard 1 by mixing 120 uL IL 6 standard appropriately with 40 uL OptiBlock blocking buffer Vortex the 3000 pg mL standard briefly to mix c Standard Curve Prepare the remaining IL 6 standards by performing six serial three fold dilutions in OptiBlock beginning with the 3000 pg mL standard as follows i Dispense 120 uL of Standard 1 3000 pg mL to well A1 of the 96 well polypropylene v b
3. c ccecceesceseeeceeceaeeeeeeeeeeeeeeeeeaeeeeseeeeeaeeeaeesaeesaaeeaeenseeees 18 Rapid ASSaV ele El elo 18 Ultra sensitive Assay Gn Optimise eieiei e ueegeebegEee K EN detecivs ie REETA EAA EE des EEN 19 A Symbol indicates mandatory step required to ensure proper operation Q Symbol indicates helpful tips to achieve optimal performance INTRODUCTION Siloam Biosciences Optimiser technology offers a rapid and sensitive chemifluorescent based ELISA procedure that uses very small sample volumes The speed sensitivity and small sample requirements are enabled by the unique microfluidic design of the Optimiser microplate Standard immunoassay reactions such as analyte capture and detection occur within a 5 uL microfluidic reaction chamber The unique microchannel geometry and small reaction volumes favor rapid reaction kinetics The typical assay procedure utilizes a 5 uL sample and each reaction step is completed in 10 20 minutes With wash time substrate incubation time and read time accounted for a typical assay can be completed within approximately 2 hours Please refer to the Optimiser Technology page on Siloam s website for more details on the principles behind the Optimiser microplate platform Figure 1 Optimiser microplate The Optimiser microplate is a revolutionary new microplate format With an ANSI SBS compliant 96 well layout the Optimiser integrates the Power of Microfluidics to allow for low
4. all wells should be empty within 10 minutes If a well is not empty after 10 minutes please inspect under low power microscope and most likely a bubble will be evident near microchannel interface with well This bubble was accidentally injected due to incorrect pipetting technique Please refer to the pipetting guidelines and try again Note that as the dye reagent is changed in the well even a 5 uL volume will clear the previous reagent in the microchannel This demonstrates the efficiency of the flushing action instead of the traditional wash step Observe the wells as they drain out Note the variation in time to empty each well So far as each well drains out in 10 minutes this variation has NO EFFECT ON ASSAY PERFORMANCE gt Most assay protocols on Optimiser recommend a 10 minute incubation interval typically 20 minutes for sample standard The 20 minute incubation step with red dye shows that ALL incubations can be extended up to 20 minutes This may be useful for processing multiple Optimiser microplates in parallel gt Incubation steps should be at least 5 minutes and no more than 30 minutes Use at least 20 minutes incubation for sample standard Page 8 of 20 Assemble a new microplate 2 and pad on the holder Repeat the dispensing protocol shown above again in ALL 12 Columns of microplate 2 Time the dispensing cycles and check that all dispensing steps are completed within 1 minute CHECK THAT ALL WEL
5. 883 4 1 514 356 100 0 158 1 10 100 1000 10000 IL 6 pg mL Figure 6 IL 6 Standard Curve with Tabulated Data SIGNIFICANCE OF ASSAY BACKGROUND The reader setup in this example sets a value of 11 000 RFU as the high value for the 50 50 1 saturated 5 substrate signal Regardless of the substrate ratio used the background RFU readings blank signal should not exceed 350 RFU 3 of max value in reader setup e Background signals higher than 3 of RFU established during reader setup indicate that one or more steps of the assay was performed incorrectly and users should repeat the assay EN e Background signals higher than 6 of RFU max established during reader setup corresponding to l 700 RFU in current example indicate a failure and the assay must be repeated CAUSES am e High backgrounds are most commonly a result of pipetting errors and can be resolved with careful attention to the procedure and additional practice gt gt 4 e Another common cause for high background is use of alternate SAv HRP or direct HRP labeled A detection antibodies Optimiser based assays are exquisitely sensitive to HRP concentration and ef the SAv HRP provided by Siloam has been carefully optimized to achieve best performance Use of alternate buffers especially blocking buffers can also lead to high background signals Please consult with Siloam s Tech Support team before substituting any of the buffers provided with the start
6. OptiMax buffers are specially formulated to work with the Optimiser microplate and substitute buffers or reagents may lead to poor assay performance ENSURE THAT TIP CHANGES AS RECOMMENDED FOR STANDARD PREPARATION ARE FOLLOWED Continued use of same tip may lead to errors in dilution and consequent assay signals Page 13 of 20 Procedure 1 10 11 12 13 14 Assemble the Optimiser Microplate Optimiser Pad and Optimiser Microplate Holder as described on Page 4 Hint Optimiser incubation steps are from 10 to 20 minutes in length To achieve optimal assay performance all materials must be transferred to the Optimiser microplate within one minute at each step To accomplish this first place the materials to be transferred in the enclosed 96 well polypropylene v bottom plate or v shape reagent reservoir instructed in Reagent Preparation page 12 Then transfer the materials to the Optimiser wells using a multi channel pipette Dispense 5 uL capture antibody working solution to the required number of wells in the Optimiser microplate Incubate 10 minutes at room temperature RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL OptiBlock to the capture antibody coated wells Incubate 10 minutes at RT Dispense 5 uL of the standard and blank to the required number of replicate wells of the plate Incubate 20 minutes at RT Dispense 5 uL Op
7. for all Optimiser based assays Repeat Step 4 if a changing the reader or b changing the optical unit such as light bulb filters etc The Technical Support section on Siloam s website offers detailed guidance on set up of several major brand instruments as illustrative examples 1 The protocols refer to the Optimiser microplates with a numeric designation 1 through 5 to identify plates that are re used in certain experiments Optimiser 1 3 are required for completing the tutorials and the assay transfer protocol Optimiser 4 and 5 is provided as extra plates if required for pipetting practice The 5 Optimiser microplates are identical and are not labeled with numeric designator Page 7 of 20 TUTORIAL 1 PIPETTING TO THE OPTIMISER MICROPLATE Materials Required for Pipetting Tutorial and Supplied with Optimiser Starter kit Same Optimiser microplate 1 used for reader setup 3 or more unused columns will be used for pipetting One new Optimiser microplate 2 all 12 columns will be used for pipetting 1 One Optimiser holder 2 3 4 One Optimiser pad single use 5 OptiWash buffer 6 Green dye solution 6 Red dye solution Other Materials equipment Required hl ae Zei D Kimwipes or other laboratory tissue paper Reagent reservoirs V shape reservoir Single channel pipette capable of delivering in the ranges of 100 1000 uL Pipette tips for delivering i
8. repetitive loads with single aspiration step for rapid reagent transfers General setup for using an electronic multi channel pipette e Select pipette capable of delivery 5 uL amp 30 uL e g with volume range of 5 120 uL e Choose Reverse Pipetting in function setting e Use Multiple Dispensing mode to transfer the solution into the Optimiser microplate For example to transfer capture antibody solution in to a full Optimiser microplate set the program for 12 times dispensing 5 uL per dispensing Then the pipette will automatically aspirate 60 uL of solution and dispense 5 uL volumes 12 times Users will not need to move pipette back and forth to transfer solution Page 5 of 20 Multichannel pipette must be used for transferring solution into the Optimiser plate If the pipette tip is pushed inside the through hole the tip may cause the sealing tape at the base of the Optimiser to de laminate and lead to flow failure If the pipette tip does not touch the surface of well the solution may stick on the pipette tip end and not dispensed into the well OR may lead to air bubbles Small variations in flow rates time to empty well do not affect assay performance The incubation step smoothes out any flow variation differences An electronic multi channel pipette can allow for loading all reagents with a single aspiration step Ideally suited for processing multiple Optimiser
9. Instruction Manual Optimiser Starter Kit For training on use of the Optimiser Microplate System Catalogue Numbers OPS IL6WDR Manufactured by Siloam Biosciences Inc 413 Northland Blvd Cincinnati Ohio 45240 Intended Use The Starter kit provides a comprehensive overview to the Optimiser microplate system The user manual includes tutorials for pipetting to the Optimiser and running an IL 6 assay on the Optimiser After completing the procedures described in this manual a first time user shall be able to operate the Optimiser Microplate System effectively PLEASE USE WITHIN 1 MONTH OF RECEIPT FOR RESEARCH USE ONLY Not for use in clinical diagnostic procedures Read the Instruction Manual in its entirety before using the Optimiser Starter kit Optimiser microplates are warranted to perform in conformance with published product specifications in effect at the time of sale as set forth in product documentation and or package inserts Products are supplied for Research Use Only The use of this product for any clinical diagnostic applications is expressly prohibited The warranty provided herein is valid only when used by properly trained individuals and is limited to six months from the date of shipment and does not extend to anyone other than the original purchaser No other warranties express or implied are granted including without limitation implied warranties of merchantability fitness for any particu
10. LS DRAIN WITHIN 10 MINUTES FOR EACH DISPENSING STEP If any wells take longer than 10 minutes the most likely cause is an error in pipetting causing a visually evident or micro bubble Please refer to the pipetting instructions and repeat steps 6 and 7 User MUST complete dispensing protocol on to entire Optimiser plate with all wells on that microplate draining in 10 minutes for all steps in protocol Optimiser microplates are a powerful tool for ELISA s and require correct pipetting procedures to ensure repeatable results USERS MUST BE ABLE TO COMPLETE_THE DISPENSING PROTOCOLS FOR A COMPLETE Optimiser MICROPLATE WITH ALL WELLS DRAINING IN LESS THAN 10 MINUTES FOR ALL STEPS OF THE PROTOCOL THIS SIMPLE STEP IS CRITICAL TO ENSURE USERS CAN ACHIEVE EXCELLENT ASSAY RESULTS ON OPTIMISER Two additional Optimiser microplates are included with the package to allow users to further practice and perfect pipetting to the Optimiser Excess volumes of the red green dye and OptiWash are also included OPTIONAL Procedure with Electronic Multi channel Pipette 5 100 uL volume 1 2 3 Repeat the protocol described for Manual multi channel pipette with an Electronic multi channel pipette Choose Reverse Pipetting in function settings for Electronic pipette Choose Multiple Dispensing mode and program for 12 dispense cycles with 5 ul dispense volume per cycle for dispensing to all 12 columns
11. MS 0043 B1 Page 17 of 20 APPENDIX 1 ALTERNATIVE ASSAY PROCEDURES ON OPTIMISER Rapid Assay on Optimiser The standard Optimiser assay procedure as described on page 14 of this Instruction Manual requires approximately 2 hours 125 minutes to complete Most incubation steps are 10 minutes in length with the exceptions of sample incubation 20 minutes and substrate incubation 15 minutes Siloam Biosciences has developed an alternative method that can be completed in 90 minutes The sample incubation time 20 minutes final two washes 10 minutes and substrate incubation time 15 minutes are unchanged However the remaining incubation times can be reduced from 10 minutes to 5 minutes The plot in Figure 7 shows the adsorption kinetics on the Optimiser showing that in 5 minutes 92 of peak adsorption or binding is completed More importantly from 5 30 min next time point the adsorption only changes from 92 to 96 In doing so the total assay time is reduced from 125 minutes to 90 minutes with no change in method performance Siloam strongly recommends that only users proficient in the use of the Optimiser microplate system attempt the rapid test protocol It is especially important to ensure that pipetting for each step is completed within 30 seconds It is also critically important to maintain consistency in pipetting and incubation intervals when using the accelerated protocol Contact Siloam Biosc
12. annel pipette A multichannel pipette is essential to ensure that all dispense steps can be comfortably completed in 1 minute or less With a single channel pipette it is very difficult to complete pipetting to even 3 columns in 1 minute How critical is the accuracy of 5 ul dispense volume The Optimiser is designed such that the 5 ul volume represents a slight excess compared to the microchannel internal volume Provided that the dispense volume is greater than 4 5 ul slight even up to 10 dispense volume variations will not affect assay results Why has the recommended operating volume been changed to 5 uL remember seeing 10 uL as recommended volume in earlier version of the FAQ 1 Minimizing the volume helps with improving the precision When using the 10 ul protocol there is higher variation in the time to empty for different wells on each plate This is related to the flow rate of the microchannel and larger volume show more net effect on flow duration and variation of the duration 2 The new 5 ul protocol also reduces the incidences of slow or stopped flow With proper pipetting technique and by use of the new protocol our lab tests show that flow failure rate well does not empty after 10 minutes is now less than 0 2 3 We have verified through extensive assay tests that change from 10 ul to 5 ul does not affect the assay sensitivity This is partly owing to improvements made to the OptiMax buffer fo
13. cifications Please use 96 well standard or similar in plate type setting Step 3 Selecting the probe direction Please use top reading for probe direction Step 4 Selecting the sensitivity gain When defining reading parameters for fluorescence analysis setting the PMT sensitivity or gain in some types of fluorescence reader is important for obtaining useful measurements A manual sensitivity gain setting is recommended for reading Optimiser microplates The procedure is as described below 1 Ina clean plastic tube add 50 uL of OptiGlow A 50 uL of OptiGlow B 1 uL of OptiGlow C and Lut of supplied SAv HRP stock solution mix well and wait for 2 minutes The substrate will be fully developed and stable for hours 2 Load 4 ul of mixture into well A1 of Optimiser microplate 1 and wait until the well is empty do not use pad holder 3 Read that well in reader with various gain setting 4 Select the gain which gives the RFU reading closest to 11 000 5 Use the same gain setting read one blank well of Optimiser the readout should be less than 50 6 Save or record this gain setting 7 This defines the max reading RFU 3 that Optimiser based assays can reach with this reader gain sensitivity setting and the 50 50 1 substrate mix ratio Note that in the trial IL 6 assay RFU readings will be higher than 11 000 RFU owing to different substrate mix ratio The gain setting will be valid
14. commendations for pipetting small volumes Page 5 Variance lt 10 and background lt 3 of RFU established during reader setup are expected Do not substitute provided assay buffers or Sav HRP Signal of lower standard s are lt 0 following Degraded standard e Use standard on the day of its reconstitution or e Thaw single use aliquots fresh on each test day e Avoid repeated freeze thaws background subtraction Degraded capture antibody e Use within specified expiration period e Store according to recommended storage temperature Page 16 of 20 Technical Assistance If you require assistance please contact Siloam Biosciences Inc Technical Support at 513 429 2976 or techsupport siloambio com Additional technical assistance is available under the Technical Support tab on the Siloam Biosciences web site http siloambio com e Using Optimiser Immunoassay Microplate Video Optimiser User s Guide Reader Settings Quick Reference Guide Frequently Asked Questions Application Notes Two additional videos appear under the Technology tab of the web site e Optimiser Principles of Operation e Running an Assay with Optimiser Siloam Biosciences Inc 413 Northland Blvd Cincinnati OH 45240 USA Phone 1 513 429 2976 Fax 1 513 429 2946 http www siloambio com SILOAM biosciences rough Innovative Microfluidics Better Immunoassays DOC ID OPTI 2
15. ction chambers using a laboratory sample processor Each additional sample incubation is 5 minutes in length Thus with 95 additional minutes of assay time the total assay time is approximately 3 hours with a corresponding increase in assay sensitivity of 20 fold The repeat sample loading methods is a reliable and simple method to tune the sensitivity of the assay to the desired range simply by adjusting the number of sample addition and incubation steps 100000 5 uL sample E5 uLsample repeatedly load 20 times 10000 RFU 1000 100 0 1 1 10 100 1000 Human IL 6 picogram mL Figure 8 Ultra sensitive assay using repeat sample loading technique with the OptiMax Human IL 6 ELISA kit with an automated pipetting station Contact Siloam Biosciences for additional details Page 19 of 20 PLEASE CONTACT TECHNICAL SUPPORT FOR ASSISTANCE WITH THIS PROTOCOL The description provided here should not be used a formal protocol
16. er evaluation kits or as part of the OptiMax assay buffer reagent sets TUTORIAL 2 TARGETED OUTCOME First time users can run a complete assay on the Optimiser and confirm that they can generate similar data as listed in the User Manual This Tutorial is intended to serve two purposes a to familiarize users with the assay operation sequence on the Optimiser and ensure performance matches with Siloam s data and b to provide users an introduction to the capabilities of the Optimiser to deliver high sensitivity assay data even when using only 5 ul sample volumes and a 2 hour assay protocol Page 15 of 20 TROUBLESHOOTING The Optimiser technology and OptiMax ELISA kits have been designed and manufactured to ensure problem free sample analysis However Siloam Biosciences has prepared the following guidance for trouble shooting problems that might be encountered due to the unique features of the Optimiser technology as well as problems that can be encountered with immunoassays in general Problem Possible Cause Solution Liquid does not drain from the Optimiser well or does not drain within 10 minutes A bubble is in the well Disrupt the bubble with a clean 26 gauge needle Follow recommended pipetting guidelines Prepare excess reagent to avoid aspirating air Do not use detergents Sample contains particulates Centrifuge sample for 10 min at 13 000 RPM or Filter the sample usi
17. form practice makes perfect most users see noticeable improvement after running a few Optimiser plates The most common pipetting related issues that are resolved with careful attention to details and practice include 1 Tips do not touch well surface leading to incomplete dispensing This can lead to very high signal e g if block buffer is not properly dispensed or very low signals e g if SAV HRP is not properly dispensed 2 Inconsistent load times as users learn pipetting procedures With significant differences between loading intervals across the plate there is higher variance with practice users can establish a steady rhythm with improved precision 3 Failure to change tips in reagent preparations and or between dispensing steps for assay sequence 4 Use of inappropriate pipette tips and or pipettes Most users after completing 10 12 Optimiser based assays can achieve a background signal lt 2 3 of peak signal see Page 15 and b variance lt 10 for all points on standard curve These can be used as metrics to determine user proficiency on the Optimiser system Page 10 of 20 TUTORIAL 2 IL 6 DEMONSTRATION ASSAY ON THE OPTIMISER The Optimiser starter kit also contains necessary reagents of an IL 6 sandwich ELISA Assay to demonstrate the capabilities of Optimiser based assays under controlled conditions by a user The representation of the expected data produced from Optimiser starter ki
18. h assay well to be used a Prepare a 1 150 dilution of the detection antibody stock in OptiBlock in a clean plastic tube add 4 uL of SAv HRP stock solution to 0 6 mL of OptiBlock b Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate 7 Substrate solution The procedure requires 10 pL of the working substrate solution for each assay well to be used a Prepare the working substrate solution no more than 30 minutes before the anticipated time for reading the completed assay b To create the substrate working solution combine OptiGlow A OptiGlow B and OptiGlow C in a ratio of 50 50 5 parts respectively in a clean plastic tube and vortex gently to mix add 250 ul of OptiGlow A 250 ul of OptiGlow B and 25 ul of OptiGlow C OptiGlow C must be thoroughly thawed to function effectively Warm the reagent in a 37 C incubator oven heater or by holding the vial gently in your hands c Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate 8 OptiWash OptiWash is provided in ready to use form No further preparation is required The procedure requires 75 uL of OptiWash for each assay well to be used Dispense 4 mL OptiWash buffer to a v shaped reagent reservoir and use for all wash steps in the assay DO NOT SUBSTITUTE OTHER BUFFERS OR REAGENTS FOR THOSE PROVIDED WITH THE KIT
19. iences for additional details and specific guidance on running this alternate protocol Direct Coating of FITC Labeled Protein In Optimizer Microfluidic Chamber zc N O Q O a gt 90 in 5 minutes gt 75 in 10 seconds Normalized Adsorption D a O O N 0 10 20 30 40 50 60 70 Residence Time in Minutes Y axis normalized to 60 minute signal Figure 7 Adsorption characteristics of capture antibody on the Optimiser microchannel surface Page 18 of 20 PLEASE CONTACT TECHNICAL SUPPORT FOR ASSISTANCE WITH THIS PROTOCOL The description provided here should not be used a formal protocol Ultra sensitive Assay on Optimiser Because of the unique features of the Optimiser plate and OptiMax ELISA procedures users can apply sample to individual microfluidic reaction chambers multiple times The result is a significant improvement in assay sensitivity when ultralow sensitivity is required The additional sample applications can be performed manually for a limited number of repeat sample loads but Siloam strongly recommends use of a laboratory sample processor for the ultra high sensitive protocol The data in the figure below illustrates the sensitivity and dynamic range obtained using the standard OptiMax ELISA procedure a single 5 uL sample addition and the improvement in sensitivity that is gained by performing 20 consecutive 5 uL sample applications to individual rea
20. io which allows for extremely efficient binding reactions 2 Even for the well that drains in 8 minute the initial section of the microchannel towards the center is filled up in 2 3 minutes Optimiser characterization data shows that the first few loops of the microchannel contribute 95 of the optical signal hence even if the last 1 2 loops take significantly longer to fill their contribution to the signal is almost negligible Consequently variations in signal from the last loops have little impact on overall assay signal variation 3 For most reaction steps in the assay sequence except for sample standard loading step the biomolecules are present in vast abundance and the binding reactions are completed extremely quickly To ensure good precision it IS recommended that the sample standard incubation should be 20 min 4 Finally the incubation interval when there is no liquid left in the well smooths out the effect of flow rate variances How does the variance CV of Optimiser microplates compare to conventional plates In most assays conventional plates raw signal variance for triplicates is lt 10 which is also true for Optimiser microplates Please see Siloam s website for a Technical Note detailing variance studies on the Optimiser platform For first time users it is common to see variances at 15 and even up to 20 In almost all cases this is related to pipetting techniques and as any other plat
21. lar purpose or non infringement Buyers exclusive remedy for non conforming product during the warranty period is limited to replacement of or refund for the non conforming product Table of Contents INTRODUCTION DE 1 MATERIALS PROVIDED AND REQUIRED ou eececeesseceenceceeeeeeaeeeeaaeseaeeceeeeesaaeeeeaaesaaeeessaeeeeaaesssaeesaeeesaeeesaaeseeaeesnaeeessaeeeaaaeeaes 2 UNIQUE CONSIDERATIONS FOR OPTIMISER MICRORLATE 4 Optimiser Microplate and Assembhy tnnnnetnnkeneas kee iia ateena keo aanne din en aea ARDE Baaren ose Ee 4 Optimiser Microplate Pipetting Instruction 4 Avoiding Bubbles While Dipetting 4 Accurate and Precise Delivery of 5 UL Volumes ntt 5 Additional Technical Considerations 0 ce cescecesceeesneceeceseeeeeeeeeecaeeeeaaeeseaeeceeaeeeeaaeceeaaeseaeeecsaeeesaeeseaaeseeeeeseaeeeeaaeeeeeaeeeea 5 Using Electronic Multi channel Pipette ccccccsccccecessessseececcesseseeaeseceeeceseeseaeseaeeeeeceseeseeaeaeeeeeeesesaeaeseseessessecaaeeeaeeeeens 5 READER SETUP Sir ee ee eebe H TUTORIAL 1 PIPETTING TO THE OPTIMISER MICRORLATE 8 TUTORIAL 2 IL 6 DEMONSTRATION ASSAY ON THE OPTIMISER cccscesscessceeseeceseeeeeeeseneseneeceeeeeneseneeeaeeeaeesaeesaeseaeeneeenes 11 TROUBLESHOOTING es ssccssdsssncsusceticestsdocecnstanccccnsbscdiensaudacevsdaceceuh eia a E bes sstvehsvente vite cdaerihacaueaanessel edicesstbcandvaseaacdeatadeceevetans 16 APPENDIX 1 ALTERNATIVE ASSAY PROCEDURES ON OPTIMISER
22. le of accurately and precisely delivering 5 uL Multichannel pipette capable of delivery of 30 uL Vortex mixer Fluorescence plate reader and control software Analytical software Microcentrifuge Timer CON OTR WN Page 3 of 20 UNIQUE CONSIDERATIONS FOR OPTIMISER MICROPLATE Optimiser Microplate and Assembly L Optimiser Microplate Optimiser Pad i imi lt _ Optimiser Pa Figure 2 Optimiser microplate assembly Position absorbent pad on holder align the Optimiser lt Optimiser Holder microplate and press down gently to click lock the plate in holder Optimiser Microplate Pipetting Instruction Tutorial 1 provides hands on training for first time users to practice pipetting with Optimiser Please read the entire Pipetting Instruction section before attempting Tutorial 1 Avoiding Bubbles While Pipetting 1 Bubbles will compromise the performance of assays on Optimiser by interfering with the flow of liquid within the microchannels 2 OptiBlock reagent may form bubbles readily with standard pipetting techniques 3 To avoid complications due to bubbles Siloam Biosciences recommends the use of the Reverse Pipetting technique during all pipetting steps a To aspirate liquid press the operating button of the pipette to the second stop refer to illustration below b Immerse the pipette tip in the liquid to a depth of about 2 mm and steadily release the operating but
23. microplates in parallel Frequently Asked Questions Pipetting Almost all pipetting protocols specify users NOT to touch the well surface during pipetting Why does the Optimiser user guide suggest the exact opposite In conventional 96 well ELISA plates if the pipette tip touches the bottom surface of the well it may physically disrupt some of the bound bio molecules In the Optimiser all the assay reactions occur within the microchannel Hence touching the pipette tip on the loading well of the Optimiser has absolutely no effect on the assay performance For most dispensing steps in Optimiser based assays users are dispensing only 5 ul volumes If the pipette tip does NOT touch the well surface the dispensed well volume may bead and stick to the end of the tip The well geometry of the Optimiser is engineered to ensure smooth filling of well microchannel provided the liquid is dispensed steadily and directly on the well surface See the Optimiser Technology page on Siloam s website for instructional videos on pipetting techniques Why must all materials be transferred to the Optimiser plate within one minute at each step in the assay procedure Optimiser incubation steps are from 10 to 20 minutes in length Longer time to transfer material will cause time difference between each well in incubation which may affect the assay accuracy don t have a multichannel pipette can try the kit with a single ch
24. n the ranges of 100 1000 uL Multichannel pipette capable of accurately and precisely delivering 5 uL Multichannel pipette capable of delivery of 30 uL Procedure with Manual Multi channel Pipette Assemble the Optimiser Microplate Optimiser Pad and Optimiser Microplate Holder as described on Page Transfer 1 5 mL of green dye solution into a V shape reagent reservoir Transfer 1 5 mL of red dye solution into another V shape reagent reservoir Transfer 3 mL of OptiWash solution into a V shape reagent reservoir To aspirate liquid hold the multi channel pipette nearly vertical and immerse the pipette tip in the liquid to a depth of approximately 2 mm in the liquid Withdraw the operating button steadily Wait 1 second Withdraw 1 4 2 3 the tip from the liquid 4 Dispense into columns 2 4 3 columns for Optimiser microplate 1 per the sequence illustrated below a To dispense liquid hold the multi channel pipette nearly vertical With ALL the pipette tips touching the surface of the Optimiser wells depress the operating button steadily until the liquid is dispensed DO NOT position pipette tips into the hole at the bottom of the surface 5 ul Green dye wait for 10 min 5 ul OptiWash wait for 10 min 5 ul Red dye wait for 10 min 5 ul OptiWash wait for 10 min 5 ul Red dye wait for 20 min 30 ul OptiWash wait for 10 min OBSERVATIONS AND CONCLUSIONS In each step
25. ng a 0 2 um filter Plate has lost contact with the absorbent pad or is positioned incorrectly Ensure that the absorbent side rough of the pad is in contact with Optimiser and the tape side smooth is facing down to touch holder Ensure the topside of the pad is touching the bottom of Optimiser plate by pushing down firmly on the 4 corners of the plate Ensure the plate and pad are securely aligned in the holder No signal or unexpectedly low signal Standard has degraded Use standard on the day of its reconstitution or Thaw single use aliquots fresh on each test day Avoid repeated freeze thaws Incorrect reader filters Confirm filters meet requirements for substrate Antibodies or SAv HRP are degraded Use within specified expiration period Store according to recommended storage temperature Substrate was prepared Thaw OptiGlow C thoroughly before preparing incorrectly substrate working solution Substrate working solution has e Prepare substrate no more than 30 minutes degraded before plate is read Unexpectedly high signal Incorrect reader filters with overlapped wavelength bandwidth Confirm filters meet requirements for substrate Reagent contamination Avoid cross contamination in reagents Always change the pipet tips when handling different buffers reagents Poor precision Pipetting errors use of alternate assay buffers or SAv HRP Follow re
26. onventional 96 well ELISA plates by eliminating the traditional wash step Tutorial 2 also shows the capabilities of the Optimiser to deliver equivalent sensitivity to conventional 96 well ELISA plates while using only 5 uL sample volume Page 1 of 20 MATERIALS PROVIDED AND REQUIRED Materials Provided Optimiser Starter kit provides the critical materials and reagents necessary for the Tutorials described in this manual Table 1 identifies the kit contents their function and their required storage temperature It is recommended that the package be opened and various components stored separately as listed in Table 1 to conserve refrigerator shelf space Table 1 Materials Provided with the Optimiser Starter kit Quantity Storage Handling before and after opening Optimiser Holder 1 Holds Optimiser Microplate and Optimiser Pad in proper alignment Contains microfluidic reaction chambers Optimiser 5 Four Optimiser microplates for pipetting practice Microplate described in tutorial 1 One for IL 6 demonstration assay Room described in tutorial 2 Temperature Optimiser Pad 10 Absorbs used reagent volume single use 26 wel polypropylene 1 For dilutions and reagent reservoir v bottom plate MN OptiBind H 1 vial 10 mL Coating buffer for model IL 6 assay a Blocking buffer and diluent for detection antibody and OptiBlock 1 vial 30 mL SAV HRP OptiWash 1 vial 60 mL Wash buffer Op
27. ottom plate ii Dispense 80 uL OptiBlock to each of the seven wells of the same column immediately below the 3000 pg mL containing well wells B1 H1 iii Transfer 40 uL of the 3000 pg mL standard from well A1 to well B1 immediately below it Mix the contents of well B1 gently Then transfer 40 uL from well B1 to well C1 change tips and titrate iv Continue serial dilutions while changing tips after each 40 ul transfer and before mixing until the 4 1 pg mL standard has been created in the seventh well well G1 of the column v Do not transfer IL 6 solution to the eighth well H1 It contains OptiBlock only and will provide material for the blank wells 1 1 A 120 ul Std 1 A 3000 B 80 uL Blocking B 1000 C 80 uL Blocking C 333 3 D 80 uL Blocking D 111 1 E 80 uL Blocking E 37 0 F 80 uL Blocking F 12 3 G 80 uL Blocking G 4 1 H 80 uL Blocking H 0 Page 12 of 20 5 Detection Antibody The procedure requires 5 uL of the detection antibody working solution for each assay well to be used a Prepare a 1 25 dilution of the detection antibody stock in OptiBlock in a clean plastic tube add 20 uL of detection antibody stock solution to 480 uL of OptiBlock b Dispense 60 uL of the working solution into each well of a single column in the polypropylene 96 well v bottom plate 6 SAv HRP The procedure requires 5 uL of the SAv HRP working solution for eac
28. rmulations Page 6 of 20 READER SETUP Optimiser based assays are compatible with standard fluorescence plate readers and multi mode plate readers with fluorescence reading capability Below is the general guidance for setting up the readers For further assistance please contact Siloam s technical support Step 1 Selecting the wavelength for excitation and emission light Assays on Optimiser uses OptiGlow substrate which can be detected using the appropriate excitation and emission settings Figure 5 Quantitation does not require filters that precisely match the excitation emission maxima However a non overlapping filter set with a bandpass that includes the excitation emission spectra is required Wavelengths at 530 575 nm for excitation and 585 630 nm for emission can be used for detection Below are examples for different types of readers Filter based readers install 528 20 nm or similar filter for excitation 450 500 550 600 650 700 OS i W Wavelength nm and 590 35 nm or similar filter for emission Figure 5 Normalized absorption Monochromator based readers in wavelength setting set excitation left and emission right spectra of at 528 20 nm and emission at 590 35 nm Readers with pre configured optical set select the wavelength setting OptiGlow chemifluorescent substrate for Rhodamine or Cy3 Step 2 Selecting the plate type Optimiser microplate fits 96 well SBS standard in all spe
29. slight differences in the time required for different wells to empty This difference has no impact on assay performance To facilitate work flow incubations designated as 10 minutes may be extended to 20 minutes with no impact on method performance Optimiser washes are performed by simply dispensing OptiWash to the wells Wipe the plate bottom thoroughly Any liquid residue on the bottom surface will cause false positive signal In rare cases lt 0 2 a well may not empty in 10 min If so blot the reagent from the well with a tissue Do not include data from this well in calculations Calculations 1 Calculate the mean background signal from the blank wells wells containing OptiBlock only at the sample incubation step 2 Subtract the mean background signal from the signal of individual standard 3 Create a standard curve by plotting the standard concentration x axis vs the background adjusted signal y axis A five parameter logistic curve fit with appropriate software is recommended Typical Data The IL 6 standard curve ranges from 4 1 to 3000 pg mL Concentration x axis and signal y axis are plotted on Log scales A typical standard curve is presented below 100000 IL 6 pg ml Average Blank Subtracted 5 3000 36665 36506 10000 1000 31722 31564 g 333 3 15764 15606 111 1 6254 6096 1000 37 0 2572 2414 a 12 3 1041
30. t is not intended to be used as a routine use commercial assay kit Siloam offers an IL 6 assay kit under catalog OMA H IL6 please contact customer support to order Materials Required for Demonstration Assay and Supplied with Optimiser Starter kit 1 One Optimiser holder Optimiser microplate 3 One new Optimiser pad single use One 96 well v bottom plate OptiBind H buffer OptiBlock buffer OptiWash buffer OptiGlow substrate kit contains component A B and C IL 6 capture antibody 10 Lyophilized IL 6 standard 11 IL 6 detection antibody biotinylated 12 Streptavidin HRP CONDO RWHN Materials Required for Assay But Not Supplied with Optimiser Starter kit 1 Eppendorf or similar tubes for centrifugation and dilutions 2 Kimwipes or other laboratory tissue paper 3 Reagent reservoirs V shape reservoir 4 Pipet tips for delivering in the ranges of 1 10 10 100 and 100 1000 uL Equipment Required 1 Pipettes capable of accurately and precisely delivering liquids in the ranges of 1 10 10 100 and 100 1000 uL Multichannel pipette capable of accurately and precisely delivering 5 uL Multichannel pipette capable of delivery of 30 uL Vortex mixer Microplate fluorescence reader and control software Analytical software Ou See I Assay Layout The plate layout of IL 6 standard concentration is shown in below Each concentration will repeat 3 times 3 columns of one Optimiser microplate
31. tiGlow A 1 vial 5 mL Refrigerated o OptiGlow B 1 vial 5mL Components of chemifluorescent substrate 2 8 OptiGlow C 1 vial 1 mL Red dye solution 1 vial 7 mL Dyed blocking buffer solution for pipetting exercise Green dye solution 1vial 7mL Dyed wash buffer solution for pipetting exercise a IL 6 standard 1 vial Lyophilized recombinant IL 6 protein for model assay REES standard curve 2 8 C After 1 vial IL 6 Capture Antibody IL 6 Detection 1 vial Antibody 1 vial Captures IL 6 on solid phase Binds captured IL 6 biotin conjugated Binds detection antibody interacts with substrate to yield chemifluorescence signal 1 150 diluted with OptiBlock to make working solution reconstitution standard must be aliquoted and stored at lt 20 C Avoid repeated freeze thaw cycles for standard Material Safety Data Sheets MSDS are available on the Siloam Biosciences web site http www siloambio com Page 2 of 20 Materials Required for Testing but Not Supplied With Optimiser Starter kit 1 Eppendorf or similar tubes for centrifugation and dilutions 2 Kimwipes or other laboratory tissue paper 3 Reagent reservoirs V shape reservoir 4 Pipette tips for delivering in the ranges of 1 10 10 100 and 100 1000 uL Equipment Required 1 Pipette capable of accurately and precisely delivering liquids in the ranges of 1 10 10 100 and 100 1000 uL Multichannel pipette capab
32. tiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL detection antibody working solution to each well Incubate 10 minutes at RT Dispense 5 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 5 uL SAv HRP to each well Incubate 10 minutes at RT Dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step Again dispense 30 uL OptiWash to each well Wait 10 minutes to proceed to the next step Dispense 10 ul OptiGlow working solution to each well Incubate for 15 minutes at RT a Caution Observe the wells during the incubation When the substrate has completely drained from all wells remove the plate and pad from the holder Discard the pad Wipe the bottom of the plate with a Kimwipe to remove any liquid on the bottom surface of the plate Step 13a will be completed within the 15 minute substrate incubation time Place the plate in the reading chamber of a fluorescence plate reader Promptly at the conclusion of the 15 minute incubation read the plate If all the assay reagent preparation steps and protocol are followed correctly the wells microfluidic channels corresponding to top 2 up to top 3 standards clearly appear pink owing to developed substrate If the pink color is not evident even for topmost standard one or more reagent preparation steps or assay steps was not performed correctly Page 14 of 20 It is common to see
33. ton completely c Withdraw the tip from the liquid touching it against the edge of the reservoir to remove excess liquid d Dispense the liquid into the loading well of Optimiser microplate by gently and steadily pressing the pipette s operating button to the first stop Briefly hold the operating button in this position e With the button in this position move the tip from the loading well to the reagent reservoir immerse the tip in the liquid and aspirate Pipetting step Ready position 1 2 st stop HF lt Second Stop Figure 3 Reverse Pipetting procedure THE USE OF PROPER PIPETTING TECHNIQUE IS CRITICAL TO AVOID AIR BUBBLES Page 4 of 20 A The pad must be oriented correctly with the smooth surface tape side facing the holder and absorbent surface touching the microplate A THE USE OF PROPER PIPETTING TECHNIQUE IS CRITICAL TO AVOID AIR BUBBLES Air bubbles will occlude the microfluidic channel and stop the flow of the Optimiser Accurate and Precise Delivery of 5 uL Volumes Assays on Optimiser require the accurate and precise delivery of 5 uL volumes The following guidance is offered to users 1 Use pipette for which the upper limit of their operating range is lt 10 UL 2 Use pipette tips appropriate for 5 uL pipetting 3 To aspirate liquid hold the pipette near vertical and immerse the pipette tip in the liquid to a depth of approximately 2 mm in the liquid Withdraw the operating b
34. utton steadily Wait 1 second Withdraw the tip from the liquid 4 To dispense liquid hold the pipette nearly vertical With the pipette tips touching the surface of the Optimiser well depress the operating button steadily until the liquid is dispensed 5 Note The pipette tip must make contact with the well surface for proper dispensing see RIGHT frame below Do not pipet directly into the hole at the bottom of the well see WRONG frame RIGHT 7 WRONG b A V Se Kia Ka Figure 4 Pipette tip positioning for dispensing in the Optimiser Additional Technical Considerations 1 The Optimiser system has been qualified with aqueous liquids only Do not use solvent containing samples 2 The buffer reagents provided with the assay kit have been developed and validated for the Optimiser microplate Do not substitute alternate buffers or reagents 3 The presence of particulates in liquids dispensed to Optimiser wells may block liquid flow through the microchannels a Centrifuge serum samples and serum containing tissue culture supernates for 10 minutes at 13 000 rpm prior to testing 4 Small flow rate variations time to empty well do not affect assay results Using Electronic Multi channel Pipette An electronic multi channel pipette is ideally suited for use with Optimiser microplates since a it eliminates possibility of injecting bubbles and b can be used for convenient
35. volume rapid and sensitive immunoassay protocols Figure 1 shows the Optimiser microplate schematic with magnified view of one cell of the Optimiser Each cell of the Optimiser has a loading well only used to add reagents S and a microfluidic reaction chamber Reagents samples are added to the well and transported via capillary action to an absorbent pad not shown The unique design of the Optimiser allows the well to be drained but each liquid is trapped in the channel by capillary forces As the next liquid volume is added the capillary barrier is broken and the liquid within the microchannel is drawn out by the absorbent pad and replaced by the new reagent All assay reactions occur within the microfluidic reaction chamber Siloam Biosciences Optimiser starter kit is designed to provide a first time user a comprehensive introduction the methods of use and the capabilities of the Optimiser platform Specifically e The Pipetting Instruction Section and Tutorial 1 are designed to guide users through the correct method for pipetting to the Optimiser microplate Although very similar to the conventional 96 well ELISA plate pipetting to the Optimiser requires careful attention to a few key details for reliable performance e Tutorial 2 is designed to allow users to complete a model IL 6 assay Tutorial 2 illustrates that the workflow for Optimiser based assays is similar but much simplified when compared to c
36. will be used Table 2 Plate layout of IL 6 concentration pg mL for demonstration assay 1 2 3 A 3000 3000 3000 B 1000 1000 1000 c 333 3 333 3 333 3 D 111 1 111 1 111 1 E 37 0 37 0 37 0 F 12 3 12 3 12 3 G 4 1 4 1 4 1 H 0 0 0 Page 11 of 20 A Very small volumes of assay reagents are required and provided for Optimiser based assays A quick spin mini centrifuge is CRUCIAL to recover all material in Items 9 12 Use a spin each time the assay reagents are to be used Reagent Preparation The incubation times for Optimiser are only 10 20 minutes Preparing all the reagents samples standards in advance will allow for proper timing especially for first time users Always prepare extra volume of solution for easy transferring Siloam suggest to prepare 30 uL extra volume each well in 96 well v bottom plate This volume can be reduced with careful pipetting if sample is very limited or precious Bring all reagents to room temperature before use and prepare all necessary dilutions before beginning the test procedure 1 3 OptiBind OptiBind H is provided in a ready to use form No further preparation is required Do not substitute other coating buffers for OptiBind H Capture Antibody The procedure requires 5 uL of capture antibody working solution for each assay well to be used a Prepare a 1 62 5 dilution of the capture antibody stock in OptiBind
Download Pdf Manuals
Related Search