OMP-50 - Siloam Biosciences
Contents
1. Em 11 Ultra sensitive Assay on OptiMax c ccccccccsssssssececccecsssesseaeceeecsseeseeeaeeesecsseeeeseseeeseceseesesaeseeeeecuseeseaaeaeeseeesssuseaaeaeeeeeeesees 11 APPENDIX 3 COMPARISON OF BACKGROUND SIGNAL IN OPTIMAX AND OPTIMISER PLATES eene 12 APPENDIX 4 DETAILED PROCEDURE FOR READER GAIN SETTING VERIFICATION sees enne 13 The user manual for OptiMax microplates also includes references to Optimiser microplate Note that Optimiser microplate is designed for manual use and detailed Assay Transfer Methods have been developed using the Optimiser microplate These methods are also applicable to the OptiMax microplate with changes as noted in this User Manual Page 1 INTRODUCTION Siloam Biosciences Optimiser technology platform 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 amp OptiMax microplates 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 Typical Optimiser assay procedures utilize 5 uL sample volumes and each reaction step is completed within 10 20 minutes Most standard Optimiser technology based ELISAs2. Page 13
3. are completed within approximately 2 hours Please refer to the Optimiser Technology page on Siloam s website www siloambio com for more details regarding the principles behind the Optimiser amp OptiMax 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 volume rapid and sensitive immunoassay protocols Figure 1 shows the Optimiser microplate schematic with a magnified view of one cell of the Optimiser Each cell of the Optimiser has a loading well only used to add reagents 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 The automation compatible OptiMax microplate is built on the same principles as the Optimiser microplate The OptiMax plate integrates the holder and absorbance pads within the plate body The resulting footprint is identical to the standard ANSI SBS
4. necessary during sample preparation Position of Tips While Dispensing While dispensing the liquid into the OptiMax plate the pipettor tips must contact the well surface Solutions must be dispensed onto the surface of the conical well shown as the red area in figure below Well surface Through hole Once the liquid touches the well surface it will flow into the microchannel beneath the loading well via the through hole The basic structure of an OptiMax well is shown below Please see Appendix 1 at Page X for detailed plate drawing Ww o Top 2 65 mm Bottom Siloam recommends positioning pipettor tips relative to the loading well as illustrated below With the programmed setting illustrated on right the tip will bend a little as it comes in contact with the well surface The resulting tip to well Actual contact will ensure proper delivery of position solutions This setting has been used x successfully with various automatic systems Please see complete list on page 3 Programmed setting IMPORTANT e DO NOT POSITION THE TIP INSIDE THE THROUGH HOLE e TIP MUST CONTACT WELL SURFACE DURING DISPENSE Page 3 Liquid Class Configurations The following recommendations for precisely dispensing small volume of solution into the OptiMax plate can ensure correct dispense operation without introducing bubbles 1 Dispensing rate Use low dispense rate 20 50 ul sec to dispense small volume of liquid
5. 96 well layout and fits on the corresponding hardware of all robotic liquid handling systems The built in pads provide adequate absorbent capacity for all anticipated ELISA procedures and do not need to be removed prior to plate reading The opaque sealing layer of the OptiMax plate prevents excitation of liquids absorbed within the pad and allows the plate to be read without disassembly Microfluidic Microplate lt Absorbent Pads Holder Figure 2 Assembled OptiMax plate left and illustration of plate components right NOTE Optimiser and OptiMax are virtually identical in performance Optimiser microplates are limited to manual use external holder must be manually detached prior to reading OptiMax plates are full automation compatible with no need for disassembly Page 2 Automatic Pipetting System Setup System Requirements e The system must be capable of precisely dispensing 5uL and 30 uL volumes e The system must have carrier s capable of holding SBS standard 96 well plates e The system should be equipped with 8 or 12 channel pipettor and or 96 channel pipettor head e OptiMax plate is compatible with both disposable and fixed tips Do not use low retention tips e Minimize dust in the working environment Reagent and Sample Preparation e Solutions must be free of visible insoluble material e g particulates precipitate e Centrifuge for 10 minutes at 13 000 g is
6. Instruction Manual OptiMax Microplate For Use with Automated Liquid Handling Systems Catalogue Numbers OMP 2 OMP 10 OMP 50 Manufactured by Siloam Biosciences Inc 413 Northland Blvd Cincinnati Ohio 45240 FOR RESEARCH USE ONLY Not for use in clinical diagnostic procedures Read the Instruction Manual in its entirety before using the OptiMax microplate Please refer to the Assay Transfer Guide for detailed assay development procedures The assay transfer guide refers to the Optimiser manual use plate model OptiMax plate may be substituted with differences as noted in this Manual OptiMax 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 particular 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 conformi
7. RAWING OF OPTIMAX MICROPLATE Customer Drawing OptiMax Microplate Valid for Item Number OMP 02 OMP 10 OMP 50 Y Y A A O JT J um 34 3x d aa HOODOO EOOOOD OOOOOO0O0O0O Bete ee eee ced un 1817 3 46 9 9 9 96 i OO OD OOOOOOO ODOOOOOOOGOO K L K 39 39 39449 OOOO DON HOOCOOOCOOOG soa E Prepared Approved Released Wiedcuien dq o in an pm p pem TI RID i m Kauffung iis Lee ie Lee Semen e Argon Page 10 APPENDIX 2 ALTERNATIVE ASSAY PROCEDURES ON OPTIMAX Rapid Assay on OptiMax The standard OptiMax assay procedure as described on page 6 of this Instruction Manual requires approximately 100 minutes to complete Adsorption kinetics study on the OptiMax shows that for most assay steps in 5 minutes 9296 of peak adsorption or binding is completed More importantly from 5 30 min the adsorption only changes from 9296 to 96 This offers the potential to reduce to the assay time significantly In some cases reducing incubation times may reduce assay sensitivity and or assay precision slightly CAUTION Users should empirically determine through a structured DOE approach if the changes suggested below have a noticeable effect on assay performance The following suggestions should be used as a guide and should be verified for each assay PLEA
8. SE CONSULT SILOAM TECHNICAL SUPPORT FOR FURTHER ASSISTANCE Suggestions for reducing assay duration 1 In most cases incubation time for capture antibody can be reduced to 5 minutes 2 In most case incubation time for blocking can be reduced to 5 minutes 3 Ifthe expected molar concentration of antigen will not be higher than 1096 of detection antibody concentration users may e Skip the wash step after antigen incubation e Mix the detection antibody with antigen and then transfer to OptiMax plate for incubation Please note that antibody concentration optimization procedure described in the Assay transfer guide may need to be repeated if the antigen detection antibody mixture method is used 4 Capture antibody pre coated OptiMax plate can be used to shorten the assay procedure Ultra sensitive Assay on OptiMax Because of the unique features of the OptiMax plate and ELISA procedures users can apply sample to individual microfluidic reaction chambers multiple times The result is a significant improvement in assay sensitivity when ultralow detection limit 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 ad
9. d incubation to increase sensitivity ee Transfer 5 uL of OptiWash wait 5 minutes E Transfer 5 uL of detection antibody incubate 10 minutes E Transfer 5 uL of OptiWash wait5 minutes JLL Transfer 5 uL of SAv HRP incubate 10 minutes AL Transfer 5 uL of OptiWash wait 5 minutes E Transfer 30 uL of OptiWash wait 10 minutes h Again transfer 30 uL OptiWash wait 10 minutes h Transfer 5uL OptiGlow working solution develop 15 minutes h Transfer plate to a plate reader and measure the fluorescence bi Optimiser and OptiMax based assays use the optimal OptiBind coating buffer OptiBind coating buffer is available in 12 compositions A L For assays using OptiBind A E use Protein free blocking buffer Pierce Cat 37572 following the capture antibody load step For assays using OptiBind F L Use OptiBlock buffer following capture antibody load step DO NOT use OptiWash buffer following capture antibody load step nis For the repeat loading procedure the antigen volume can be reduced to 2 uL per dispense cycle without compromising assay sensitivity The integrated pad of the OptiMax plate can hold up to 300 uL of solution for each well Up to 200 uL of sample standard solution can be transferred into the OptiMax plate when using the repeat loading procedure Page 6 KEEPING INCUBATION TIMES CONSISTENT FOR EACH WELL Kinetic studies on the OptiMax plate
10. d the 50 50 1 substrate mix ratio Note that the RFU readings will be higher than 11 000 RFU if different substrate mix ratios are used The gain setting will be valid for all OptiMax based assays Repeat Step 4 if a using a different reader or b if changing elements of the reader s optical unit such as light bulb or filters NOTE The signal intensity from OptiMax and Optimiser plates will be different In order to compare results between the two platforms the instrument gain needs to be adjusted independently as described below for both the OptiMax and the Optimiser plates APPENDIX 4 describes a detailed method to troubleshoot reader gain settings Page 5 Sandwich Assay Procedure Using OptiMax Plates Assay procedures using OptiMax plates are similar to those using Optimiser plates with key differences shown in the workflow below Example Assay Workflow for OptiMax Plates Place tip boxes reagent reservoirs and the OptiMax plate on the robot s work table Transfer 5 uL of capture antibody to each well incubate 10 minutes ll Transfer 5 uL of OptiBlock OR Protein free Block buffer wait 5 minutes m Transfer 5 uL of OptiBlock incubate 10 minutes m Standard Procedure Single 5 uL dispense Repeat Load Procedure Multiple 5 uL dispenses Transfer 5uL of sample standard incubate 5 minutes Transfer 5 uL of sample standard incubate 10 minutes j HMM H ple Repeat transfer an
11. dition and the improvement in sensitivity that is gained by performing 20 consecutive 5 uL sample applications to individual reaction 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 199099 9 5 uL sample E5 uL sample repeatedly load 20 times 10000 RFU 1000 Figure 4 Ultra sensitive assay using repeat sample loading technique with the OptiMax Human IL 6 ELISA kit with an automated 0 1 1 10 100 1000 pipetting station Human IL 6 picogram mL 100 Please contact Siloam s technical support for additional details Page 11 APPENDIX 3 COMPARISON OF BACKGROUND SIGNAL IN OPTIMAX AND OPTIMISER PLATES Siloam has compared the background signal observed in OptiMax based and Optimiser based ELISAs Briefly a mouse IL 2 standard was prepared at 3 pg mL The standard and its diluent were dispensed to 48 capture antibody coated and blocked wells of an OptiMax and an Optimiser plate The plates were processed in a side by side comparison using the same materials and the standard sandwich ELISA procedure The backgrou
12. eloped to a red fluorescence dye solution and stable for hours 2 Prepare 1 2 serial diluted solutions with DI water to prepare 15 fluorescence solutions with 1 zero a Load 50 uL of DI water to well B1 H1 and A2 H2 b Transfer 50 uL of solution from well A1 to well B1 and mix well c Change the tip repeat same procedure till well H1 then continue to well B2 and repeat till well G2 leaving well H2 as zero 1 2 A no dilution 1 256 B 1 2 1 512 C 1 4 1 1024 D 1 8 1 2048 E 1 16 1 4096 F 1 32 1 8192 G 1 64 1 16384 H 1 128 0 Transfer to OptiMax and reading Transfer 4 pL solution of each solution to corresponding well on OptiMax plate Wait for all the wells to empty Read with different gain settings e For capability to run wide dynamic range assay 729 folds in OptiMax the reader should have o Clear discrimination in fluorescence signal intensity from wells A1 D2 AND o Clearly distinguish well D2 to well H2 e For capability to run standard dynamic range assay 64 folds in OptiMax the reader should have o Clear discrimination in fluorescence signal intensity from wells C1 D2 o Clearly distinguish well D2 to well H2 The following results show typical data on a BioTek FIx800 Fluorescence reader A1 22794 A2 278 B1 17528 B2 159 C1 10957 C2 93 D1 6126 D2 54 E1 3503 E2 34 F1 1747 F2 23 G1 907 G2 16 H1 461 H2 10
13. into OptiMax plate 2 Donot blow out the liquid in dispensing step 3 Aspirate extra liquid Aspirate the volume that you intend to dispense e g 5 uL plus an additional 2 5 uL of extra solution to avoid inadvertently dispensing air into the well and creating a bubble 4 Foraspiration Small quantities of assay reagents will be used in most of steps Siloam suggests using reservoirs with v shaped bottoms for most reagents and always aspirating from near the bottom of the v shaped reservoir 5 Dispensing sample volume less than 5 uL Sample volumes can be reduced to as low as 2 uL for OptiMax based assay procedures without compromising assay sensitivity Please consult with Siloam technical support for appropriate settings for precisely dispensing less than 5 uL solutions onto the OptiMax well surfaces Siloam has tested OptiMax plates on the following automation platforms Siloam can provide more detailed settings and programs for the systems listed below Please contact Siloam technical support for more information Table 1 Automation systems verified with OptiMax Plates Manufacturer Model System configuration Tip BioTek Precision 8 channel pipettor Disposable tip Tecan EVO100 8 channel pipettor RoMa Arm and M200 reader Disposable amp Fixed Hamilton MicroLab Star 12 channel pipettor and 96 channel head Disposable Beckman Coulter BioMek FXP 8 channel pipettor and 96 channel head Disp
14. keep consistent incubation time for each well Poor dispensing precision of the pipettor e Please check the precision of the pipettor Signal of lower standard s are lt 0 following background subtraction 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 Degraded capture antibody e Use within specified expiration period e Store according to recommended storage temperature Page 8 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 e Optimiser User s Guide e Reader Settings e Quick Reference Guide e Frequently Asked Questions e 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 T o Siloam Biosciences Inc p s NL 413 Northland Blvd i S LOA j NS Cincinnati OH 45240 biosciences USA Phone 1 513 429 2976 Fax 1 513 429 2946 Better Immunoassays Through Innovative Microfluidics T eens E dd DOC ID OPTI 2 MS 0072 A1 Page 9 APPENDIX 1 CUSTOMER D
15. mine or Cy3 Step 2 Selecting the plate type The OptiMax microplate fits the 96 well SBS standard in all specifications Please use 96 well standard or similar selection when setting the plate type Step 3 Selecting the probe direction 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 readers is important for obtaining useful measurements A manual sensitivity gain setting is recommended for reading OptiMax microplates The procedure is described below 1 2 3 4 5 6 7 Dispense 50 uL of OptiGlow A 50 uL of OptiGlow B 1 uL of OptiGlow C and 1uL of horseradish peroxidase labeled streptavidin SAv HRP stock solution Siloam Biosciences in a clean plastic tube mix well and wait for 2 minutes The substrate will be fully developed and stable for hours Load 4 uL of the mixture into well A1 of an OptiMax microplate and wait until the well is empty Read well A1 using a range of gain settings Select the gain setting which gives an RFU reading for well A1 closest to 11 000 Using the same gain setting read one blank well of the OptiMax plate The RFU reading should be less than 50 Save or record this gain setting This defines the maximum reading RFUmax that OptiMax based assays can reach with this reader gain sensitivity setting an
16. nd signal observed in the OptiMax plate was about 30 higher than that seen in the Optimiser plate Siloam expects that the concentration of the LOD or LOQ will also be approximately 30 5096 higher when using the OptiMax plate under the conditions used here for example a 3 pg mL LOD with Optimiser and 4 4 5 pg mL LOD with OptiMax Oooo NEM HN Concentration Replicate Wells E Replicate Wells ei _Meanaru Replicate wels The increased background is attributed to the black sealing tape used in the manufacture of OptiMax plates Clear sealing tape is used in the manufacture of Optimiser plates Page 12 APPENDIX 4 DETAILED PROCEDURE FOR READER GAIN SETTING VERIFICATION Most fluorescence multi mode readers tested with Optimiser and OptiMax plates show excellent performance and ability to set reader gain as described in the Reader Setup section If users believe that the reader does not offer satisfactory sensitivity and or dynamic range the following procedure can be used for additional verification A list of readers tested with Optimiser plates is available on Siloam s website under the Technical Support Tab Chemifluorescence Solution Preparation NOTE MIX RATIO 1 In well A1 of a clean 96 well v bottom plate add 50 uL of OptiGlow A 50 uL of OptiGlow B 5 pL of OptiGlow C and 1 uL of supplied SAv HRP stock solution mix well and wait for 2 minutes The substrate will be fully dev
17. ng product Table of Contents hdieniedonwee 2 A tomatic Pipetting System Setup iine tee eve og ead le ex YER x LER Y de sd onde le Pax V EYE ERR cade ce Pax vex ERR ea vx DV RR EY ERE cate 3 Kerle EM Re 3 Reagent and Sample Preparation ccccccssssssscececsesssssensececeesessnssensececessesesaseesececeeseausaeseseceeeesensaasaesecesseseausensececessesssananes 3 Position of Tips While Dispensing esssseseseeee eene nnne nhnnnn eaS rannas EEEE assises assassin esse tia sans ass sese a nEn 3 Liguid Class Configurations snsc Em 4 Fluorescence Plate Reader Set p aseisiin eie nied dee sasasesanceedsespasuessnucee Paene NOE ss syaaueesdussddsesvassesausesdecsvnseseeaues 5 Sandwich Assay Procedure Using OptiMax Plates ccccsessssccececessesssaececeescesseeaaeeeeecsseesesaaaeeeeeesseeseaaeaeeeeeeessesauaeeeeeeeeeeees 6 KEEPING INCUBATION TIMES CONSISTENT FOR EACH WELL ssseeseeeeeeneeenneen nennen nennen tnnt nnn innt nnne nnne tnter ensis nnne 7 uei sieen cee mL 8 APPENDIX 1 CUSTOMER DRAWING OF OPTIMAX MICROPLATE essere nennen ennt nnnetnnr inner innen enses 10 APPENDIX 2 ALTERNATIVE ASSAY PROCEDURES ON OPTIMAX cscceseceseceeceseceseceeeeeeeseeeseaeeeaeesaeesaeecaaesaaeseaeseaeseaeeeeeeees 11 Rapid PA CCI ITSESIe dE d
18. osable Page4 Fluorescence Plate Reader Setup OptiMax based assays are compatible with standard fluorescence plate readers and multi mode plate readers with fluorescence read capability Below is the general guidance for setting up the readers The Technical Support section on Siloam s website offers detailed guidance on the set up of several major brands of instruments as illustrative examples Step 1 Selecting the wavelength for excitation and emission light 450 Figure 3 Normalized absorption left and emission right spectra of Assays performed on the OptiMax platform use the OptiGlow substrate system which can be detected using the appropriate excitation and emission settings Figure 3 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 can be set at 530 575 nm for excitation and at 585 630 nm for emission Below are examples for different types of readers 570 4 585 T T Be nA Filter based readers Install 528 20 nm or similar filter for excitation Wavelength nm and 590 35 nm or similar filter for emission e Monochromator based readers Set excitation wavelength at 544 nm and emission wavelength at 590 nm OptiGlow chemifluorescent substrate e Readers with pre configured optical set Select the wavelength setting for Rhoda
19. shows that the reaction adsorption in the microchannel will reach equilibrium in 10 minutes in most assays To achieve optimal assay precision the incubation times must be consistent for each well in each REACTION STEP The rapid incubation times of OptiMax based assays require particular attention to this detail Some examples are listed below 1 Use 8 channel pipettor with 200 uL disposable tips for all steps During the assay procedure the pipettor will perform multiple dispensing over 12 columns with same solution for most steps example adding capture antibody solution followed by buffer For incubation following these steps the timing will be consistent for each well since the pipettor follows the same pattern for each load i e Column 1 will have first load of capture at t 00 00 min and buffer at t 10 00 min Corresponding duration for Column 12 may be 00 20 and 10 20 min respectively The 2 exceptions to this will occur when a antigen is added since pipettor tips needs to replaced washed and b 30 ul wash steps after HRP addition since pipettor cannot accommodate volume for 12 columns Suggested workarounds include e To keep consistent time for each well in antigen incubation the pipettor needs to dispense the wash buffer following the same pattern as antigen addition Time delay may be applied between each dispensing in this washing step The incubation duration for wash buffer following antigen step will be set at 10 min
20. t drain within 10 minutes A bubble is in the well e Follow recommended liquid class configurations Sample contains particulates e Centrifuge sample for 10 min at 13 000 RPM or e Filter the sample using a 0 2 um filter OptiBind A E coating buffer step followed by a OptiBlock or b OptiWash e Use Protein free blocking buffer Pierce Cat 37572 following the capture antibody load step for assays using OptiBind A E No signal or unexpectedly low signal Standard has degraded e Use standard on the day of its reconstitution Incorrect reader filters e Confirm filters meet requirements for substrate Antibodies or SAv HRP are degraded e Use within specified expiration period e Store according to recommended storage temperature Substrate was prepared e 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 e Confirm filters meet requirements for substrate Dust in the well e Minimize the dust in working environment Reagent contamination e Avoid cross contamination in reagents Always change the tips when handling different buffers reagents Poor precision Inconsistent incubation time between wells e Program to
21. utes starting after wash buffer is added in ast column The slightly longer duration for prior columns will have no effect on assay performance e To keep consistent time for each well in SAv HRP incubation add an additional 5 uL wash step before load 30 uL washing buffer see workflow above The 5 ul wash buffer addition step can be completed using a single pass with a 200 ul tip and flushes out the HRP solution Additional washes 30 ul can follow different timing patterns without affecting assay performance 2 Use 8 channel pipettor with 200 uL disposable tips for most reagent amp buffer addition 96 channel pipettor for antigen addition Using 96 channel pipettor can reduce the pipetting time especially in sample addition It may also lead to inconsistent incubation times if 8 channel pipettor is used to dispense the wash buffer after antigen step Hence the wash buffer after antigen addition should also be dispensed with 96 channel pipettor Page 7 TROUBLESHOOTING The OptiMax microplate and 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 OptiMax well or does no
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