USER MANUAL
Contents
1. 1 2 Make sure all wells are empty before loading the next reagent Please ensure that all reagents for a given step in the assay sequence are loaded within 1 minute time span If a large number of samples or different concentration reagents during optimization are to be loaded please use a conical bottom 96 well plate or other reservoirs in 96 well format to prepare the solutions and transfer to the Optimiser using a multi channel pipette Reading the signal within 15 30 minutes after loading the substrate usually offers the best sensitivity and dynamic range Change the absorbent pad in following situations e After loading 120 140 uL total volume of solutions in each well o Please design the load sequence such that the pad change is done prior to a wash step e Do not wipe the bottom of the plate when changing the pad e Push the plate firmly onto the holder after changing the pad to ensure good contact between plate and absorbent pad e Changing the pad prior to the final wash of the two step wash sequence before adding substrate may give lower background e A pad change is recommended prior to second run if all 96 wells of the Optimiser are not used for a test run Since applications vary each investigator should titrate the reagent to obtain optimal results Following is the example concentration to start with o For indirect immunoassay Antigen concentration 5 times concentrated as used in conventional 96 well HRP con2. Add 30 ul of Wash Buffer into each well wait until all wells are empty 5 Add 10 ul of Detection Antibody Solution into each well and incubate at room temperature for 10 minutes 6 Repeat step 4 7 Add 10 ul of SAv HRP Solution into each well and incubate at room temperature for 10 minutes 8 Change the absorbent pad 9 Repeat step 4 twice 10 Add 10 ul of QuantaRed Working Solution in each well wait until all wells are empty and take off the plate from the holder Wipe off all residue from bottom of Optimiser plate with Kimwipe Measure the fluorescence at the time point 15 minutes after adding substrate Additional volume of sample may improve assay sensitivity Please discuss your application with Siloam tech support and we can offer more accurate guidance TYPICAL DATA Results of a typical standard run of a cytokine protein sandwich assay are shown below BioTek F1x800 Fluorescence reader 528 20 for excitation 590 35 for emission sensitivity at 45 plate type 96 WELL PLATE IL 2 pg ml Average Blank Subtracted oer 5498 5256 Optimiser Chemifluorescent 250 125 2978 2736 1654 1412 1000 63 31 1008 766 16 600 359 8 475 233 100 4 385 144 2 284 43 0 241 10 1 0 Antigen pe mlj 1000 Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 8 of 10 Note
3. binding to unblocked sites Prepare the detection antibody and enzyme conjugate in the blocking buffer Residue left on bottom of plate Wipe the bottom of plate thoroughly before placing it in reader Low signal noise ratio Reagent concentrations need to be fully optimized OR all possible causes listed that lead to high background signal Perform full optimization to determine the reagent concentrations especially for the detection antibody and enzyme conjugate See application notes on Siloam s website for example Unable to assemble the plate with the holder The plate is not in the same direction as holder Place the plate over the holder with correct orientation Optimiser microplates Products 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 one year 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 particular purpose or non infringement Buyers exclusive reme
4. diluted to 2 ug ml with Blocking Buffer 7 SAv HRP HRP conjugated streptavidin diluted to 0 125 ug ml with Blocking Buffer for Optimiser Sodium azide is excluded from all buffers as this interferes with HRP activity 8 Chemifluorescent Substrate Final Working Solution for Optimiser assay Equilibrate the QuantaRed substrate kit to room temperature for at least 10 minutes Mix 50 parts QuantaRed Enhancer Solution with 50 parts QuantaRed Stable Peroxide and 1 part QuantaRed ADHP Concentrate Use within 30 minutes of preparation 9 Optimiser priming Assemble Optimiser microplate with absorbent pad and holder load 10 ul of Opti Prime Solution into each well of the Optimiser plate and wait until all wells are empty Use the plate within 15 minutes Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 7 of 10 ASSAY PROCEDURE Ensure that the Optimiser priming procedure as described in Step 9 of Reagent and Plate Preparation section is completed before starting the assay procedure 1 Add 10 ul of Capture Antibody Solution into each well and incubate at room temperature for 5 minutes 2 Add 10 ul of Blocking Buffer into each well and incubate at room temperature for 5 minutes 3 Pipette 10 ul of each Assay Standard into appropriate wells in triplicate rows and incubate at room temperature for 10 minutes 4
5. precipitates and bubbles All buffers particularly blocking buffer should be filtered using a 0 2 um vacuum filtration system and stored at 2 8 C If any precipitate appears or if any well flow issues arise the buffers should be re filtered It is necessary to prepare biological samples such as serum cell lysates for analysis on the Optimiser by centrifuging samples at 13 000 g for 10 minutes and then using the supernatant This process will ensure efficient flow through the microchannels Horseradish peroxidase SAv HRP is inactivated by sodium azide Therefore sodium azide should not be included in any buffers used for the biotinylated detection antibody the washes or the SAv HRP itself Don t let the pipette tip touch the top ring surface flat portion at top of each well Avoid any cross contamination particularly of SAv HRP solution Always change the pipette tips when handling different buffers reagents or when changing from high to low concentration solutions Detection antibody and enzyme conjugates should be prepared in the blocking buffer employed in the assay to minimize nonspecific binding Please pay extra attention to avoid air bubble in loading reagents with surfactant The air bubble will impede reagent flow through the microchannels For same reason detergent e g Tween 20 is NOT recommended for use in any solutions Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3
6. www siloambio com Release date February 9 2011 Page 4 of 10 Example Procedure Optimiser versus Standard 96 well Assay The operation in ELISA includes four major steps 1 reagent loading and incubation 2 dilution 3 washing and 4 substrate loading followed by plate reading The operation with the Optimiser is as simple as that with standard 96 well plates as described below Operation step Standard 96 well plate Optimiser Reagent loading and incubation Load 100 ul of solution into each well seal the plate with film and incubate for 2 hours in room temperature Load 10 uL of solution into each well incubate for 5 10 minutes in room temperature ONLY See example protocols below No sealing film is required Dilution Dilute in tubes then transfer All dilution must be done in tubes reservoirs to plate serial dilution in then transfer to plate plate Washing After each incubation load Washing in Optimiser Load 30 ul of and aspirate dump 300 ul wash buffer in each well 3 5 times washing buffer into the well and wait until it is empty no aspiration dumping required When washing is required 1 Two wash steps are required before loading substrate 3 One wash step is required after loading the 3 reagent and every reagent thereafter Please check the examples in following pages Substrate loading and plate reading Load 100 ul substrate into each well read wit
7. PTIMISER USER MANUAL Optimiser Microplate Description Optimiser Microplate System for Immunoassay ELISA Optimser Microplate System Through hole Optimiser Microplates Absorbent Pads Holder Microchannel Introduction The Optimiser integrates the Power of Microfluidics within the traditional microplate architecture to offer up to 10x saving on reagent consumption and total assay time The Optimiser is SBS ANSI compliant can be used without any specialized liquid handling equipment and can be read by conventional microplate fluorescence readers For operation reagents are sequentially added to the loading well drawn into the channel by capillary forces and excess drawn out by absorbent pad The microfluidic design ensures that the channel is not emptied by absorbent pad allowing for static incubation step Subsequent liquid addition breaks capillary barrier at inlet and flow resumes Please visit www siloambio com for more details All assay reactions occur in the microfluidic channel at the bottom surface under each well The high surface area to volume ratio and short diffusion distances of the microfluidic channels allow rapid reactions 5 to 10 minutes incubation step for 30 to 60 minutes immunoassays The Optimiser provides significant reagent savings time savings and elimination of traditional wash requirements Siloam Biosciences Inc
8. Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 1 of 10 Optimiser Assembly 1 Place the holder on a flat work surface with the Optimiser logo embossed on the front of the holder facing to the laboratory technician 2 Place an absorbent pad on the top of the holder platform The absorbent pad has a smooth surface and a rough surface Position the absorbent pad with the smooth surface facing down on holder and rough surface facing up 3 Place an Optimiser plate over the holder and pad with the well position markers A through H facing to the left 4 Push the Optimiser microplate on the holder until it clicks in place Ensure that the plate is locked in position plate should not move more than I mm within holder Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 2 of 10 Pipetting into the Optimiser Liquid handling on the Optimiser is very similar to a conventional microplate Please follow the guidelines below for repeatable operation Pipetting guidelines Air bubble in solution will impede the flow of reagents from the well to the microfluidic chamber Pipette carefully to prevent creation of air bubbles in samples and reagents o Make sure the end of tip is submerged into the solution when drawing the liquid up into the pipette tip o Push the pi
9. dy for non conforming product during the warranty period is limited to replacement of or refund for the non conforming product Siloam Biosciences Inc 513 429 2976 www siloambio com Page 10 of 10 Patent pending Document ID OPTI 2 MS 0002 B3 Release date February 9 2011
10. e matrices 6 Secondary Antibody Solution HRP conjugated anti mouse IgG antibody diluted to 0 1 ug ml with Blocking Buffer Sodium azide is excluded from all buffers as this interferes with HRP activity 7 Chemifluorescent Substrate Final Working Solution Equilibrate the QuantaRed substrate kit to room temperature for at least 10 minutes Mix 50 parts QuantaRed Enhancer Solution with 50 parts QuantaRed Stable Peroxide and 1 part QuantaRed ADHP Concentrate Use within 30 minutes of preparation 8 Optimiser priming Assemble Optimiser microplate with absorbent pad and holder load 10 ul of Opti Prime Solution into each well of the Optimiser plate and wait until all wells are empty Use the plate within 15 minutes ASSAY PROCEDURE Ensure that the Optimiser priming procedure as described in Step 8 of Reagent and Plate Preparation section is completed before starting the assay procedure 1 Add 10 ul of Antigen Solution into each well and incubate at room temperature for 5 minutes 2 Add 10 ul of Blocking Buffer into each well and incubate at room temperature for 5 minutes 3 Pipette 10 ul of each Primary Antibody Standard into appropriate wells in triplicate rows and incubate at room temperature for 10 minutes 4 Add 30 ul of Wash Buffer into each well wait until all wells are empty 5 Add 10 ul of Secondary Antibody Solution into each well and incubate at room temperature for 10 minutes 6 Re
11. h 96 well microplate reader Load 10 ul chemifluorescence substrate into each well wait until all wells empty take the plate off the holder wipe off the liquid residue on the bottom with Kimwipe or tissue paper read with 96 well microplate fluorescence reader Note that the substrate is still present in the microchannel and signal is generated from substrate volume in the microchannel Reader set up Specify a wavelength b gain sensitivity setting and c plate type Please refer to TN0002 https www siloambio com optimiser reader settings pdf for reader set up procedure Siloam Biosciences Inc 513 429 2976 www siloambio com Patent pending Document ID OPTI 2 MS 0002 B3 Release date February 9 2011 Page 5 of 10 The following protocols are example applications for Optimiser Microplate System Specific applications require optimization for reagent concentrations See examples on Siloam s website reference to application notes for optimization process Example 1 Indirect ELISA Procedure using the Optimiser microplate REAGENT AND PLATE PREPARATION 1 Coating Buffer OptiBind Coating Buffer 2 Blocking Buffer OptiBlock Blocking Buffer 3 Wash Buffer OptiWash Wash Buffer 4 Antigen Solution Purified antigen diluted to 1 ug ml with Coating Buffer 5 Primary Antibody Standards Primary antibody mouse IgG diluted to various concentrations with appropriat
12. jugated secondary antibody concentration 0 1 ug ml o For sandwich assay Capture antibody concentration same as that used in conventional 96 well Detection antibody concentration same as that used in conventional 96 well SAv HRP 0 125 ug mL In the rare instance where the liquid in the well does not flow through the microchannel within 10 minutes after loading wipe off the residue and discard the reading from that well Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 9 of 10 Troubleshooting Problem Possible Cause Solution Flow failure the well did not empty in 10 minutes Bubble in the well Follow recommended pipetting guidelines always prepare a little extra amount of reagent to avoid bubble in pipetting avoid detergent Do not dispense beyond first stop on the pipette Solution contains precipitate Centrifuge at 13 000g for 10 minutes or filter with 0 2 um membrane Plate loses contact with absorbent pad Make sure the plate is firmly assembled with pad and holder The plate is left too long in last incubation Ensure that all incubation steps are no more than 20 minutes High Background Signal Free enzyme left in the microchannel or well Wash at least twice with 30 uL before loading substrate Low quality blocking Use OptiBlock blocking buffer Nonspecific
13. ntific Finnpipette Finntip http www pipettecalibration net pipette calibration files Guide To Pipetting 2 pdf Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 3 of 10 Additional Equipment Material and Reagents Required not included OptiMax Reagent Pack for Optimser Microplate contains o OptiPrime Pre Wetting Solution o OptiBind Coating Buffer o OptiWash Wash Buffer o OptiBlock Blocking Buffer QuantaRed Enhanced Chemifluorescent HRP Substrate Kit Fluorescence 96 well microplate reader with appropriate filters See example at https www siloambio com optimiser reader settings pdf Centrifuge Centrifuge tubes Precision micropipette set 5 50 ul multichannel micropipette 96 well polypropylene conical bottom plate or other 96 well format reservoirs Sample reservoirs Kimwipe or tissue paper DI water For Alkaline Phosphatase based assay use AttoPhos AP Fluorescent Substrate for detection Please contact Siloam for detail information Related Product OMR 5 OptiMax 5 Plate Reagent Pack OMR 10 OptiMax 10 Plate Reagent Pack OMR 50 OptiMax 50 Plate Reagent Pack CRITICAL CONSIDERATIONS FOR SUCCESSFUL ASSAY PROCEDURE Only aqueous solutions have been verified in Optimiser It is unknown whether solvent based solutions are compatible with the Optimiser All solutions must be free of particulates
14. peat step 4 7 Change the absorbent pad 8 Repeat step 4 9 Add 10 ul of QuantaRed Working Solution in each well wait until all wells are empty and take off the plate from the holder Wipe off all residue from bottom of Optimiser plate with Kimwipe Measure the fluorescence at the time point 15 minutes after adding substrate Siloam Biosciences Inc Patent pending 513 429 2976 Document ID OPTI 2 MS 0002 B3 www siloambio com Release date February 9 2011 Page 6 of 10 TYPICAL DATA Results of a typical standard run of a cytokine protein indirect assay are shown below BioTek F1x800 Fluorescence reader 528 20 for excitation 590 35 for emission sensitivity at 45 plate type 96 WELL PLATE FLU 14000 Standard ug ml FLU oe 10 12265 10000 5 12453 at 2 5 12459 1 25 12258 6000 0 63 11270 idi 0 31 10862 0 16 7955 2000 0 08 4915 0 0 04 3156 0 2 4 6 8 10 12 0 171 Primary antibody ug ml Example 2 Sandwich ELISA Procedure for Optimiser REAGENT AND PLATE PREPARATION 1 Coating Buffer OptiBind Coating Buffer 2 Blocking Buffer OptiBlock Blocking Buffer 3 Wash Buffer OptiWash Wash Buffer 4 Capture Antibody Solution Purified anti mouse IL 2 antibody diluted to 2 ug ml with Coating Buffer 5 Assay Standards Recombinant protein diluted to various concentration with appropriate matrices 6 Detection Antibody Solution Biotinylated antibody
15. pette button to first stop to dispense the liquid into the well DO NOT blow out the residual liquid by pushing to second stop from the tip Please review a short instructional video at https www siloambio com optimiser video php to review the pipetting procedure If there is bubble after loading please use small 26 gauge syringe needle without syringe to remove the bubble by gently poking it out Recommended pipettes for samples and reagents 0 1 10 ul Recommended pipettes for wash buffer 1 100 ul The protocol described in the user manual suggests 10 ul of working volume for all liquid handling steps except washing buffer It is possible to use as low as 5 ul of volume but it is necessary to ensure that all dispense volume is transferred to the well not retained on the pipette tip outside surface It is mandatory to use the 0 1 10 ul tips when dispensing lower than 10 ul Please ensure that all reagents for a given step in the assay sequence are loaded within 1 minute time span If a large number of samples or different concentration reagents during optimization are to be loaded please use a V shape 96 well plate or other reservoirs in 96 well format to prepare the solutions and transfer to the Optimiser using a multi channel pipette The tip should NOT be placed into the through hole as shown below RIGHT WRONG k a a a b 4 1 Good Laboratory Pipetting Guide Thermo Scie
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