Cortisol Kit Manual - Seattle Sensor Systems Corporation
Contents
1. a ate Calibri Ju jaa ag Ei wrap Text General El B a oy W a SSN Pid wa Paste J Format Painter B u gt A E E E H Merge amp Centers 150 38 conan oe ae ___ Insert Delete Format Rye Pate Joa Clipboard g Font g Alignment g Number g Styles Cells Editing 12 j x A B c D E F G H oN Pn a R s 1 Concentration computed based on assumption 234 are averaged Results of scan for time Run 1 3 Standard Curve coeffecient are Coef1 0 00 and Coef2 0 00 0 1 Sensor Slope R 5 Run 5234 Avg S1C1 0 006 5 1 0 009 sic2 0 016 2 0 121 oan s1c3 0 021 3 0 091 ps E Si 0 014 4 0138 yy Add Level X Delete Level Za Copy Level options 7 My data has headers a Column Sort On Order ee LU e oe Sortby sensor Values Smallest to Largest Saz ous 2 __ 8 0897 Then by channel values i Smalest to Largest 520 2 002 8 0 135 2 0 001 9 0 096 10 0 127 S3C1 0 005 11 0 087 3C2 0 003 S303 0 007 3 0 002 sacl 0 005 1 2 S4C2 0 071 1 2 0 002 0 005 s4c3 0 1 2 0 002 0 011 S4 0 025 N 1 3 0 021 0 404 1 3 0 027 0 58 1 3 0 01 0 151 Results of scan for time 27 1 3 0 007 0 057 Run 2 M 4 gt M Alldata Sensor 2 channel 1 Sensor 2 channel 2 Sensor 2 channel 3 Sensor 3 channel 1 Sensor 3 channel 2 Sensor 3 channel 3 Sensor 4 channel 1 Sensor 4 d Ready Average 2 249368741 Count 665 Sum 1482334 Tao Ve 4 sal hi2. 7 Following the data sorting the runs will be in increasing order see column I above as well as the sensors channels and slopes columns J K L respectively This arrangement facilitates the selection of all of the slopes from one sensor s channel for all of the runs which is the method we utilize for quantifying the cortisol concentration 8 We usually sort the individual channel slopes to separate worksheets where they can be charted and analyzed separately see tab labels at bottom of screen shot 9 Once all of a particular channel s slopes have been grouped into a new worksheet for example sensor 2 channel 1 the percent decrease in slope which equals the amount of free cortisol competing for antibody binding to the sensor surface as a function of the preceding no cortisol sample is calculated for each cortisol containing sample The correlation of this value with a particular cortisol concentration will define a point in the reference curve For example if the slope of a no cortisol sample was 0 2 and the following sample contained 1 ng ml cortisol with a slope of 0 1 the percent decrease in slope mediated by 1 ng ml cortisol would be 50 0 1 0 2 X 100 Thus saliva or other samples that similarly mediated a 50 decrease in slope compared to ano cortisol control would be quantified to contain cortisol at a concentration of 1 ng ml based on this example 10 Using this approach the percent decrease in
3. 1 0 003 0 011 2013 02 14 12 39 00 1 1 2 0 003 0 018 1 1 3 0 019 0 325 Analysis of file 2013 02 04 16 10 22spr txt 1 2 1 0 020 0 374 1 2 2 0 018 0 437 Number of data rows 6392 1 2 3 0 010 0 157 Duration of post sample injection flow 100 1 3 1 0 008 0 161 Number of Injections 24 1 3 2 0 011 0 136 Injections windowed from 50 through 100 t 3 3 0 014 0 315 1 4 1 0 029 0 460 1 4 2 0 028 0 517 Results of scan for time of start of Injection block 1893 r 4 3 0 030 0 426 Run 1 v 2 1 1 1 457 0 716 v Sanana siana naa 3 1 2 nonan ncen On some Windows system this procedure doesn t work If you have trouble an alternate approach is to double click on the file SpiritAnalysisSystem bat This will initiate the software application in a slightly different way that we have found works on most computers Please contact Seattle Sensors if you have any trouble installing or running your system Note On some Windows based computers the graphic as shown in Figure 1 does not appear For reasons that are unclear the work around is to grab a corner of the display window and resize it slightly as this corrects the display problem Seattle Sensor Systems Free Cortisol Assay CT 1317 15 3 Analysis Operation The procedure for analyzing a data set from the Spirit instrument is as follows 1 Set Experiment Run Parameters These values should be set to be compatible with the cortisol assay parameters The default assay collects targe
4. instrument 13 5 Cortisol Dilution Vials 0 5 1 0 Keep refrigerated Instrument 1 5 2 0 3 0 ng ml in antibody until use familiarization buffer with 0 05 azide 10ml performance monitoring 14 1 Shutdown Solution with 0 05 Store at or below Used prior to long azide 50ml room temperature term storage 15 2 Syringe Needles sample amp Store at room Sample and initialization temperature initialization solution injection 16 lt Reserved for future use gt Seattle Sensor Systems Free Cortisol Assay CT 1317 3 Preparing the SPIRIT Instrument for Use 1 Setup Unpack all items in the provided assay kit be sure to identify each item correctly using the table above and labels on items Immediately prior to use the sensors will need to be prepared by rinsing with Buffer Solution 1 in the provided Petri Dish 11 Holding the sensors with the gold surface down and the connector pointed up immerse the gold surface in the solution and agitate manually for at least one minute Take care not to touch or scratch the gold surface or the electrical connectors on the sensors This step will remove the protective shipping coating on the cortisol and prepare the sensors for first use Follow instructions in SPIRIT Operations amp Appendix document for inserting sensors into their respective compartments in the instrument Two cortisol biosensors are provided in the kit They are to be used in parallel and should be
5. set to 500 ul volume then attach tip in preparation for use Mix beads very well making sure they are all in suspension Rapidly open tube containing the mixed beads and pipet out 500 ul of bead suspension 5 mg of beads with the pipette Aliquot entire volume to first labeled 1 5 ml tube Close tube with cap 4 Re close tube containing beads mix well and repeat bead allocation procedure described in step 3 for all labeled tubes The same pipette tip can be used for all samples unless it has been contaminated by contact with improper surfaces hands etc The tip should be replaced with a new one if any unwanted contact is suspected 5 Obtain magnetic separation rack MSR Insert tubes containing allocated beads into MSR with tops oriented to open away from the rack 6 Set p1000 pipette to 600 ul volume carefully open all of the tubes on the MSR and starting with the left most tube and going left to right pipette out the entire bead buffer from each tube into a waste container Be careful to not pipette out any beads which are lining the tube adjacent to the MSR by observing the aspirated buffer in the pipet tip This and additional steps which result in the beads being exposed to air should be performed as rapidly but accurately as possible as extensive drying of the beads can reduce the conjugated antibody s ability to bind cortisol 7 Once bead buffer has been removed from the last tube set the p1000 pipette to 1 ml and begin
6. to add 1 ml of saliva to each tube starting with the left most tube which had buffer removed first and moving from left to right Be sure to add the correct saliva sample to the correctly labeled tube and switch pipet tips between different samples 8 Following addition of saliva to the last tube close all of the beads saliva containing tubes and remove all tubes from the MSR Briefly mix each tube to re suspend the beads then place onto rotating apparatus and rotate for 5 minutes Free cortisol will be bound by the beads during this time 9 During the bead saliva incubation label equivalent sets of tubes as either supernatant elution 1 or elution 2 10 Following completion of the 5 minute incubation place the tubes into the MSR as in step 6 Obtain PBST buffer for next steps 11 Noting each saliva tube s label set the p1000 pipette to 1 ml and begin to transfer the supernatant from each tube to the corresponding supernatant tube being careful to avoid removing any beads Change pipette tips after removing each supernatant Supernatant Seattle Sensor Systems Free Cortisol Assay CT 1317 26 12 13 14 15 16 17 18 19 20 21 removal should occur in a left to right order as described above The saved supernatants can be re tested if there are any issues with the subsequent bead analysis Close and freeze the supernatant tubes can be stored on ice temporarily Moving f
7. reliable for a minimum of 40 detection cycles Place the first cortisol sensor cortisol 1 into sensor position 4 the right most slot as you face the instrument and the second cortisol sensor cortisol 2 in position 3 Place the BSA background sensor in slot 3 You can use one of the sensors shipped with the instrument as a dummy sensor in position 1 Data read from this slot is not used in the detection algorithm Insert the buffer solution 1 regeneration solution 2 and waste container 3 into the instrument see p16 of the operators manual Follow the instructions in the SPIRIT Operations and Appendix document for connecting the instrument to the laptop and starting the software Note that you will select a location for storing the instrument data file Make note of this because you will need it when you run the SPIRIT Analysis Software If the unit is dry after a period of storage or shipment you will need to prime the fluidics circuits to remove all the air before using the instrument Inject 1 0ml of Buffer Solution 1 into the injection port Using the software interface select Flow and run buffer solution through the instrument for at least 5 minutes or until no more bubbles are seen in the lines and liquid is flowing into the waste container 2 Initialization Seattle Sensor Systems Free Cortisol Assay CT 1317 6 Since this is a new assay for your instrument you must program in the protocol seq
8. slope for each cortisol standard 0 5 1 1 5 2 3 ng ml cortisol is charted using the Scatter Chart with only Markers charting function in Excel Percent competition values from unknown cortisol samples obtained by comparison to no cortisol controls can then be used in an equation derived from the curve assigned to the cortisol standard points to determine cortisol concentrations of the unknown samples We have found that the most accurate cortisol concentrations can be obtained by plotting the 0 5 1 1 5 Seattle Sensor Systems Free Cortisol Assay CT 1317 22 and 2 ng ml cortisol samples then using an equation derived from a linear fit trendline An example of a cortisol competition standard curve is shown below Cortisol Competition Standard Curve j N oO o oO fon Oo Series1 D O N oO oO 0 5 1 1 5 2 Q igo N n D pa e QO e 2 Ge e c cD i gt pes co a Cortisol Concentration ng ml Seattle Sensor Systems Free Cortisol Assay CT 1317 23 Appendix 4 Saliva Depletion and Spiking for Saliva Reference Curve Generation Detailed Protocol 1 Obtain 6 mls of saliva from the species of interest pooled or from a single animal 2 Retrieve 15ml tube containing antibody coated beads the magnetic separation rack MSR and the p1000 pipettor 3 Remove the plastic tube rack surrounding the magnet of the MSR by pulling it up and off the ma
9. system has been shut down for a period of time 4 hours or more or needs to be cleaned following a sample run described below Enter and save the following program as program 2 Seattle Sensor Systems Command Time Air Pump Flow Speed Flow 3 seconds On 0 Inject 20 seconds Off 0 Flow 100 seconds Off 20 Stop z p This will be the Experimental Analysis program for sample analysis Aspirate 1 ml of PBST buffer into a syringe in preparation for injection into the instrument Large air bubbles in the syringe should be removed by holding the syringe vertically needle side up flicking the syringe to promote movement of large air bubbles to the top of the syringe and then expelling the air bubbles by slowly compressing the plunger while holding the needle into a clean Kimwipe or paper towel When only fluid is expelled the sample is ready for injection and the needle can be inserted into the injection port but do not try to inject the sample at this time Confirm or set program 1 as the selected program on the Main Screen then click on the green Run Experiment button above the program selection box You will hear the air pump as the program initiates the first command When you hear the Inject function valves click and the inject step appears in the Messages box press the syringe plunger to inject the entire sample There are 20 seconds allotted for the injection step and the e
10. tips so carefully observe the tip as you transfer the 10 ul to the 990 ul of buffer as rapidly as possible Once transferred to the 10 ug tube which now contains 10 ug of cortisol mix the tube s contents well and then transfer 10 ul from the 10 ug tube to the 100 ng labeled tube and mix well The 100 ng ml cortisol dilution tube will be used for spiking the depleted saliva samples as described below Using the p20 pipettor pipette 5 ul of the 100 ng ml cortisol dilution to the saliva containing microfuge tube labeled 0 5 and mix This tube now contains 0 5 ng ml cortisol Add the appropriate volume from the 100 ng ml tube to the 4 remaining labeled saliva tubes being sure to change tips following each addition of cortisol to the saliva The volumes are 10 ul to the 1 tube 15 ul to the 1 5 tube 20 ul to the 2 tube and 30 ul to the 3 tube These 5 cortisol spiked saliva samples can now be used for generation of a saliva reference curve Seattle Sensor Systems Free Cortisol Assay CT 1317 25 Appendix 5 Assay Procedure for Reference Curve Generation and Endogenous Free Cortisol Analysis Detailed Protocol 1 Obtain saliva samples 5 samples is the maximum number of samples which can be assayed at one time 2 Label 1 5 ml microfuge tubes to match the number of samples to be assayed and leave in rack without closing caps Obtain tube containing cortisol antibody conjugated beads 3 Obtain p1000 pipette
11. very good slope estimate even if the R 2 value is small For any slope that has a low R 2 value it is good to zoom in and view the dataset in the user interface plot screen to see if the R 2 value is small because of noise or if in fact the binding rate was non linear To analyze another dataset exit the software and restart selecting the new dataset as described above in step 1 When running a series of injections with the Spirit instrument the data file is continuously appended with new data so it is possible to copy and Load the current version of the dataset into the Analysis Software and do analysis of binding rates while additional injections are still underway This is one way of getting faster results during a field test The Plot Window of the user interface has a few interactive commands that can be used for quick inspection of the loaded dataset Seattle Sensor Systems Free Cortisol Assay CT 1317 16 e Left click redraw plot potentially with new sensor activation settings changed e Left button pushed dragged and released Zoom in on highlighted windowed section of plot e Right button click zoom back to full size and redraw the plot Note that whenever the plot window is redrawn the start and end times are updated and for all the sensors currently active the average slope is computed and displayed under the plot as well This gives you a way to quickly check the slope of the binding curve from a
12. 1 as described in this section Switching to Program 2 inject and run 2 consecutive 0 cortisol samples prepared in step 23 followed by the first cortisol sample then another 0 cortisol sample etc The first of the 2 consecutive O cortisol samples is a blocking sample which will not be used to determine cortisol levels in subsequent samples while the 2 control sample will be used for this purpose Once the sample slopes have been obtained as described in the Data Analysis section a standard curve can be generated from the spiked saliva samples see step 10 in the Data Analysis section Saliva samples containing unknown levels of cortisol can now be assayed by starting at step 1 in this Appendix 5 The standard curve produced in step 25 is then used to calculate cortisol levels in the samples Thus the equation of a standard curve for a particular sensor or sensor channel which is solved for y slope in Excel must be solved for x concentration then the obtained slope for a cortisol containing sample is used in the equation substituted for y in the equation to obtain the sample s cortisol concentration Seattle Sensor Systems Free Cortisol Assay CT 1317 28
13. before each sample 1 pre sample block run so an equal volume of diluted antibody one extra control will be required for the controls To compensate for any loss during pipet steps a small excess of antibody dilution should always be prepared Thus adding 2 ul of the antibody stock to 148 ul of PBST Seattle Sensor Systems Free Cortisol Assay CT 1317 27 22 23 24 25 26 would be sufficient for 5 samples controls For the final dilution aliquot 12 mls of PBST toa 15 ml conical tube using the p1000 pipette The 1 75 diluted antibody should then be mixed by carefully flicking the tube with your finger Using the p200 pipette transfer 120 ul of the 1 75 diluted antibody to the 12 mls of PBST and mix well Using the p200 pipette transfer 50 ul of glycine to 6 1 5 ml tubes for no cortisol controls Using the p1000 pipette transfer 950 ul of the 1 7500 diluted antibody to each of the 5 tubes containing the 50 ul of glycine eluate from step 20 the 6 controls prepared in step 21 assuming 11 samples are being run so this number will vary depending on the number of samples Mix then rotate for 5 minutes The samples are now ready for injection into the instrument During the 5 min sample rotation the instrument should be prepared for sample injection according to the Instrument Preparation and Sample Analysis section First inject buffer and run the System Preparation and Cleaning Program Program
14. ccssscccscsscsccesssssscsscsessensvssssassesesaennsssssccosesancveessscdesssaneessesssccsassseaasessssseaass 24 Appendix 5 Assay Procedure for Reference Curve Generation and Endogenous Free Cortisol PUNY SES E E A E E E T E E A E 26 Detailed Protocol wiisssscccissssdeccissssccscesdeccasaccssccscasdecedicsescscecsddedecsceesssecssedeccabevssteadessdsaseverssisccasdedeseres 26 Seattle Sensor Systems Free Cortisol Assay CT 1317 2 1 Limitations and Cautionary Notices NOT FOR USE IN DIAGNOSTIC PROCEDURES FOR RESEARCH USE ONLY e The kit should not be used beyond six months of the date of manufacture e Do not substitute reagents or mix with those from other kits or sources e Before using this kit read the user manual for the SPIRIT instrument and familiarize yourself with the use and operation of the device Any variation in techniques used kit age or reagents may cause variation in molecular binding rates and impact results achieved from this kit e Differences in results may be introduced by variations in sample preparation and treatment including storage e While this assay has been designed to eliminate interference through non specific binding by other contaminants and factors that may be present in samples used in this assay until all such potential contaminants and factors have been tested the possibility of interference cannot be excluded Seattle Sensor Systems Free Cortisol Assay CT 1317 3 2 Materials Pr
15. dcesseceateodsssscvacsecesesosssssesescccessonseddeecacdenes 7 Mu Assay Procedures sc das testesdenddndeniasdicidelsstavtiacuncasastuucins ennasaundencindevstndesstacessiasaaseameiasaseteias 8 1 Instrument Familiarization Cortisol Measurement in Buffer cccccccccssssssesceeeceecesssssssceeees 8 2 Free Cortisol Assay and Analysis Protocol s ssssssscceeccecsssssseeceeeccecssssseseeceeeseeeasssseeeeeeeeeooas 9 Appendix 1 Loading Parameters for the Cortisol Assay ccccssssssscccccssssssceeceeenessseeeeeeees 10 Appendix 2 Data Analysis via the Spirit Analysis Software sssscccccssssssceecceeneesseeeeeeees 14 Le INthOGUCHION iis sesciscccsicedsensasveccenassessvacecccsnnsescsdeasescscieuncisevsbanddecsenscesvesbedecessasscocendssceassxevecsdesss 14 2 MnstallatiOnsss cccccenc ccsssusicvsseddecscssnscccocvostccoseesscoossencccecsnscececussscceshesscscensssteces esscocendesseassesesesess s 14 3 Analysis Operations sacscccccscecceccaassasccecsectetieasseses ct ceccceasasedsiacecetecs esadsdeetestececsssscasentetesccuadeseseseee 16 Appendix 3 Instrument Familiarization Cortisol Measurement in Buffer sceessseees 18 Detailed Protocol iss scsiswescscsnsisccccnsveccensseced csavccsdonsuscdisavncceceaunsssecebandcccsuvececeunbsdecessavsccdensesccacassecesdesed 18 Appendix 4 Saliva Depletion and Spiking for Saliva Reference Curve Generation 24 Detailed Protocol iiisscicsseccc
16. e Seattle Sensor Systems CORPORATION February 2013 The Measurement of Free Cortisol from Saliva Samples A fast and sensitive quantitative assay for use in the monitoring of animal stress Free Cortisol Measurement Assay CT 1317 Associated Consumable Kit KCT 1317 Seattle Sensor Systems Free cortisol assay CT 1317 1 Free cortisol Measurement Assay Kit CT 1317 CONTENTS 1 Limitations and Cautionary Notices sssssccccccsssssssceecccecssscsecescecesssesescccecsssssecesoeesssens 3 2 Materials Provided and Storage INStructiOns cccccccssssssececccsssssssceccccnessscescescessssssceeees 4 1 Hardware and Core Equipment specialized items cccccccsssssssssceeeccecassscssssceeeceecaaessseeeeees 4 2 Consumables standard lab items ccccccccssssssssssssceeeccecsssssseeceeeccecssssseeeeceeeceeeasseseeeeeseooees 4 3 Specific Assay Components Can be ordered separately as a pre configured kit KCT 1317 4 3 Preparing the SPIRIT Instrument for USC ssssscccceccssssssececcccesssescececcneesscescesceessssecesees 6 Dy SOUU Pisedidinssccdescsecad dusesnesccaenddancavanececeusescnscansndscduessacecdunsecdeastseccccnesecauessencsaassevscsssensdcanaevawsescassscs 6 P MPMIRT LIZ ARNO Missi 55k oes cd stows teceeccecescesevedectendecsceceeses teesdeeeccodausescelssasccoaksesscoeesseseesuasesssusvdssssbesssoces 6 3 Shut GOWN AN StOlage c ccicssccdecccescsdesssccsdcecseceeteosddes
17. e Cortisol Assay CT 1317 12 Now create a second program Program 2 with the following sequence steps This will be used as the Experimental Analysis program for measuring injected samples Step Pump Air Number Fluidics Step Duration Speed Pump 1 Flow 3 0 ON 2 Inject 20 0 OFF 3 Flow 100 20 OFF 4 Stop Seattle Sensor Systems Free Cortisol Assay CT 1317 13 Appendix 2 Data Analysis via the Spirit Analysis Software 1 Introduction The main purpose of this software is to compute the rate of binding slope of the injected sample to the biosensor surface The output of the software is the slope in units of uRIU sec during the post injection bind flow for every injection in the selected dataset This software package was developed as a working tool during the cortisol assay development activity so it reflects our experience in what to monitor for both the assay performance and in making the final call on target detected or not Please feel free to provide us feedback on changes to the Analysis Software that you would find valuable in supporting your project goals Currently this analysis package runs offline from the live data collection 2 Installation The analysis system is designed to run as a companion piece of software on the same computer that is running the Spirit instrument operation systems IOS software Assuming you have successfully installed the IOS onto your computer you now need to ins
18. for the cortisol measurement go to the View Set menu in the SPR Operating Software and select Fluidics Program Select Options for This Step n Program 1 Fluidics Step Flow Program 2 j Program 3 Duration 1 600 s Program 4 Pump Speed 0 100 Air Vacuum Settings Air Pump Auto Vacuum Air amp Vacuum No AirVac Use the Program menu to select which program you will use for cortisol detection This is stored on the instrument and the instrument is limited to four different program sequences For the cortisol assay Programs 1 and 2 will be set up Seattle Sensor Systems Free Cortisol Assay CT 1317 10 Select Options for This Step Fluidics Step Duration 1 600 s Pump Speed 0 100 Air Vacuum Settings Air Pump Auto Vacuum Air amp Vacuum No AirVac Use the list on the right to select the item in the sequence to program This will run through each step in the list until it reaches a Stop step without any user input Select Options for This Step Fluidics Step Duration 1 600 s Pump Speed 0 100 Air Vacuum Settings Regenerate Air Pump Stop Vacuum Air amp Vacuum No Air Vac Select the step type in the dropdown menu These steps will equilibrate the sensors prior to injection zero the sensor signal set the valves for injection flow the sample over t
19. gnet 4 With the magnet on a flat surface hold the tube containing the antibody coated beads directly against the magnet so the beads are lining the tube surface adjacent to the magnet 5 Carefully remove the bead buffer using the p1000 pipettor set to 1 ml Try to avoid removing any beads Repeat until the entire buffer volume has been removed The buffer can be saved for re addition to the beads at the end of the procedure note that it contains sodium azide 6 Using the p1000 pipettor pipette the 6 mls of saliva into the tube containing the magnetic beads The bead tube does not need to be adjacent to the magnet at this point and can be put into any tube rack 7 Once all of the saliva had been added to the beads use the p1000 pipette to resuspend the beads in the saliva by pipetting up and down multiple times Mix the saliva with the beads for 5 minutes using a rotator or other mixing device 9 Take the 15 ml tube containing the saliva and beads and put it next to the magnet as in step 4 10 Using the p1000 pipettor and a new tip remove all of the saliva and pipette into a new 15 ml tube being very careful to not transport any beads to the new tube This saliva has now been depleted of cortisol which is now bound to the antibody conjugated beads 11 Following removal of saliva from the beads use the p1000 pipettor to pipette 3 ml of PBST buffer to the beads and resuspend by pipetting up and down Use the magnet to remove the buffe
20. have found that copying the output to an Excel file is the easiest method for data analysis First select the data in each of the 2 windows separately and past into an Excel worksheet Use control C to copy the selected data The 2 data types would be 1 the labeled individual channel and sensor data The beginning of this data type will indicate the date SPR file ID number of data rows etc 2 the data from individual channels grouped together as a table without extra labels and designed for Excel analysis This is the easiest file to manipulate in Excel By copying all of the data into the Excel file it is centralized for easy access and future analysis if required 5 Select the tabled data data type 2 mentioned in step 4 and use the Custom Sort command under the Sort amp Filter button to group the data as needed You can also analyze the data by a different approach depending on your preference 6 Inthe Custom Sort box select My data has headers then first sort by sensor number smallest to largest then add a level and sort by channel smallest to largest The screen shot below shows an Excel worksheet containing the 2 types of data output mentioned in step 2 as well as the Sort box under Custom Sort Seattle Sensor Systems Free Cortisol Assay CT 1317 21 Da Tr z 77 home Insert Pagelayout Formulas Data Review View 7 x
21. he sensor surface flush the Seattle Sensor Systems Free Cortisol Assay CT 1317 11 injection loop with buffer and then regenerate or elute bound analyte from the sensor surface preparing the sensor for the next detection Select Options for This Step Program Fluidics Step Flow Flow j Baseline Inject Duration 1 600 s Flow Flush Flow Regenerate Air Vacuum Settings er Air Pump Auto Flow Stop Stop Stop i Stop D No Air Vac Stop Pump Speed 0 100 Vacuum Air amp Vacuum Load Programs Use the Duration and Pump Speed boxes to select the appropriate values for these Use the Air Vacuum Settings to select whether the air pump is turned on or off This pressurizes the buffer chamber to prevent air bubbles from forming This will be used for System Preparation and Cleaning Step Fluidics Pump Air Number Step Duration Speed Pump 1 Flow 3 0 ON 2 Baseline 2 0 OFF 3 Inject 20 0 OFF 4 Flow 100 20 OFF 5 Flush 20 0 ON 6 Regenerate 300 100 OFF 7 Flow 300 20 OFF 8 Stop After selecting the correct values hit the Save Programs button As soon as you make a change it gets loaded onto the instrument provided you aren t currently running an experiment However if you power the system off that currently loaded program won t get saved unless you ve used Save Programs Seattle Sensor Systems Fre
22. ibed in step 17 Following the last sample wait 3 minutes to permit completion of the cortisol elution Return the tubes to the MSR then use the p20 pipette set to 19 ul to carefully remove the glycine from the bottom of the tubes avoiding the beads along the side of the tube A few aspiration steps should be performed to confirm removal of all of the glycine Note the tube label and pipet the glycine into the appropriately labeled elution 1 tube Repeat for each tube using a different pipet tip for each sample After removing glycine from the last sample set the p1000 pipette to 1 ml and pipet 1 ml of PBST into each bead containing tube on the MSR Remove tubes from the MSR and place on ice for later washing and regeneration Take the elution 1 tubes containing glycine and return to the MSR then again remove the glycine from the bottom of the tubes and transfer to the appropriately labeled elution 2 tube This step will ensure that there are no beads in the final glycine eluate Prepare an appropriate volume of diluted anti cortisol antibody according to the number of samples being run An initial 1 75 antibody dilution will be subsequently diluted 1 100 10 ul per 1 ml in a larger volume of PBST for addition to the samples 1 7500 final dilution For example if 5 samples are being processed then 50 ul of 1 75 diluted antibody will be required for the samples alone In addition 0 cortisol antibody controls will be run
23. mmended that the unit be depressurized by selecting Stop on the user interface and then manually releasing any residual pressure from the buffer tube by remove the tube momentarily and then reattaching For storage beyond 72 hours it is recommended that the unit be cleaned with DI water see section viii Maintenance in the operator s manual After storage be sure to follow the instructions for Setup section 3 1 above Any active sensors should be replaced or returned to Seattle Sensors after this step and new functionalized sensors installed next time the unit is used Seattle Sensor Systems Free Cortisol Assay CT 1317 7 4 Assay Procedures Running the assay to measure cortisol levels in the 0 5 to 3 0 ng ml range occurs in four steps 1 extraction of cortisol from saliva samples 2 running the assay to create the dataset 3 calculation of binding rates slope in uRIU sec using the provided data analysis software and 4 computation of cortisol concentration typically in Excel or with a calculator Note In order to familiarize yourselves with the instrument it is recommended that you first run the steps in Section 1 below to familiarize yourself with the measurement of cortisol in buffer The final assay will extract cortisol from saliva and then re elute the cortisol into buffer for measurement In order to get the best reference curve for converting binding rate to cortisol concentration the creation of the act
24. nd Usage Handling 1 3 Buffer Solution with 0 05 azide Store at or below General buffer 50ml room temperature 2 1 15 mM NaOH Regeneration Store at or below Regeneration of Seattle Sensor Systems Free Cortisol Assay CT 1317 Solution 15ml room temperature sensor surface 3 2 Waste Repository Tube Dispose following Waste collection regulations 4 10 Sample Injection Syringe 1ml Dispose following Sample injection regulations 5 10 Medium syringes for washing 3ml Dispose following Fluidics rinse regulations following initialization 6 2 Cortisol Biosensor Keep refrigerated Used in and sealed until use instrument 7 1 BSA Biosensor Keep refrigerated Used in and sealed until use instrument 8 1 Cortisol stock 1 mg ml diluted in Keep refrigerated Used to create methanol 2ml tube and sealed until use positive control samples 9 2 Antibody buffer for no cortisol Keep refrigerated Used to create controls with 0 05 azide 15ml and sealed until use negative control samples 10 250ug Anti cortisol antibody 2 mg ml Keep refrigerated Used in sample needs diluting before use with until use assay and in 0 05 azide controls 11 1 Glycine pH 2 2 with 0 05 azide Keep refrigerated Elution of cortisol 10ml until use from beads 12 1 Initialization Solution with 0 05 Keep refrigerated Preparing cortisol azide 15ml until use sensor following installation in
25. ntire sample must be injected during this time frame The syringe can be left in the injection port following injection Free Cortisol Assay CT 1317 19 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 There will be no significant slope present during the flow step as this procedure is just performed before each run to make sure the sensors and flow path are cleaned of any non specifically bound material and the instrument is operating correctly All of the program steps presented in step 2 should occur and be listed in the Messages box as the program is run At the end of the program change the speed setting on the main screen to 20 and manually activate the flow command using the button on the main screen This will keep buffer flowing through the system while preparing for sample injections Obtain the 11 Standard Curve samples prepared in the first section Set program 2 as the selected program on the Main Screen Aliquot 30 ml of PBST into a 50 ml conical tube This will be used to clean the syringe between injections as described below Have a waste container available for expelling buffer used to wash the syringe Remove the syringe needle from the injection port and then move the syringe plunger back and forth 5 10 times while aiming the needle into a waste container to empty the syringe Wipe the syringe needle on a clean paper
26. ny sensor for any time sub window of the SPR sensor data Seattle Sensor Systems Free Cortisol Assay CT 1317 17 Appendix 3 Instrument Familiarization Cortisol Measurement in Buffer Detailed Protocol 1 2 Remove 5 cortisol standards from kit 0 5 1 1 5 2 3 ng ml cortisol Aliquot 1 ml of each cortisol concentration to a 1 5 ml microfuge tube 5 tubes total Label each tube first to identify the correct tube for each aliquot Obtain anti cortisol antibody from kit and prepare diluted antibody stock 1 75 in PBST buffer First label a 1 5 ml microfuge tube as the antibody dilution then add 148 ul of PBST to the labeled microfuge tube Add 2 ul using the p20 pipettor of the antibody stock to the 148 ul of PBST and mix To each of the 5 one ml cortisol standard aliquots add 10 ul of the diluted anti cortisol antibody for a final antibody dilution of 1 7500 Mix each tube for 5 minutes Do not throw away the remaining antibody dilution as it will be used in the next step During the antibody cortisol incubation time prepare the no cortisol antibody controls First aliquot 6 ml of the antibody buffer reagent to a 15 ml conical tube Then add 60 ul of the diluted antibody stock prepared in step 3 to the 6 ml of antibody buffer 10 ul per ml of reagent and mix well Following mixing aliquot 1 ml of the antibody buffer antibody solution to six labeled 1 5 ml microfuge tubes These no cortisol controls will be
27. ovided and Storage Instructions 1 Hardware and Core Equipment specialized items Item Quantity Description Storage and Usage Handling 1 1 1ml pipettor p1000 Store at room Sample temperature preparation 2 1 200ul pipettor p200 Store at room Bead and antibody temperature 3 1 20ul pipettor p20 Store at room Bead and antibody temperature 4 25mg Anti cortisol antibody coated Keep refrigerated Extraction of immunomagnetic beads in PBST and sealed until use analyte from with 0 05 azide 2 5ml Handle with care saliva or plasma reusable 5 1 Magnetic separation rack MSR for Store at room Separation of analyte separation temperature beads from matrix 2 Consumables standard lab items Item Quantity Description Storage and Usage Handling 1 1 box 1ml pipettor tips Store at room Sample and temperature reference 2 1 box 200ul pipettor tips Store at room Antibody and temperature bead 3 1 box 20ul pipettor tips Store at room Antibody and temperature bead 4 1 Small petri dish Keep with kit Rinsing sensors 5 1 bag 1 5ml microfuge tubes Store at or below Preparing samples room temperature and controls 6 1 box 15 ml conical tubes Store at room Preparing controls temperature in bulk 3 Specific Assay Components Can be ordered separately as a pre configured kit KCT 1317 Item Quantity Description Storage a
28. r wash and repeat one more time 12 Obtain glycine solution from the kit Following removal of the 2 PBST wash add 1 ml of glycine to the beads resuspend the beads in glycine with the p1000 pipettor and let incubate for 5 minutes This will remove the bound cortisol from the beads 13 Following completion of the bead glycine incubation use the magnet to collect the beads and remove the glycine which can be discarded note it contains sodium azide 14 Wash the beads 2X with PBST as in step 11 and then add 2 5 ml of PBST The beads have now been stripped of bound cortisol and are ready to be used in the assay of spiked or endogenous cortisol containing saliva samples Important Note The bead stripping procedure steps 11 14 must be performed following any bead based cortisol extraction to ensure consistent results in the next assay the beads can be stripped at any time as long as it precedes the next assay 15 Obtain cortisol stock solution 7 1 5 ml microfuge tubes p1000 and p20 pipettors Seattle Sensor Systems Free Cortisol Assay CT 1317 24 16 17 18 19 20 Label 5 of the 1 5 ml tubes as 0 5 1 1 5 2 3 and add 1 ml of depleted saliva from step 10 to each of the 5 tubes Label the 2 remaining 1 5 ml tubes as 10 ug or 100 ng and add 990 ul of PBST to each tube Then using the p20 pipettor add 10 ul of the cortisol stock to the 10 ug tube The cortisol is in methanol and quickly runs out of pipette
29. rom left to right begin adding 1 ml of PBST to each tube on the MSR the same pipet tip can be used for each tube unless contaminated to wash the beads Close tube caps and then remove each tube and mix vigorously until the beads are re suspended in the buffer Return tubes to MSR then remove and discard 1 wash using a separate tip for each tube Add PBST buffer again and repeat steps 13 to 14 for 2 bead wash Obtain elution buffer and p200 pipette set to 50 ul Return tubes to MSR remove and discard 2 wash solution from each tube Make sure that there are no large pools of buffer at the bottom of the tubes which can interfere with the subsequent elution step then add 50 ul of glycine elution solution to the bottom of each tube being careful to avoid the beads Use a different tip for each addition to eliminate any possibility of cross contamination Remove all tubes from MSR then starting with the tube which was left most on the MSR use the p200 pipette set to 50 ul and a new tip for each sample to aspirate the glycine from the bottom of the tube and pipet it down the side of the tube where most of the beads are located Repeat washing the beads down the side of the tube with glycine in this manner until all of the beads have collected at the bottom of the tube This is important for assuring exposure of all of the beads to the glycine for optimal cortisol elution Repeat washing all of the bead samples with glycine as descr
30. rt into the injection port in preparation for the next injection When the previous sample is complete and the program stops wait 5 10 seconds then click the green Run experiment button on the Main Screen and inject the sample as described in step 18 Follow steps 17 26 for each sample until all of the samples have been run Seattle Sensor Systems Free Cortisol Assay CT 1317 20 28 Following the end of the last sample run aspirate 1 ml of PBST into the cleaned syringe insert the syringe into the injection port set program 1 as the selected program and click the green Run Experiment button Inject the buffer sample at the appropriate time and let the program run until complete This will clean the instrument which can now either be manually set to flow until required for further experiments or disconnected from the software then shut off if no longer needed Data Analysis 1 Set the Experimental Run Parameters to 50 100 seconds out of a 100 second run The latter part of the 100 second flow step is where the most accurate slope values are typically obtained 2 Load the file as described in Appendix 2 above 3 The software output will include slope calculations for each channel in each sensor there are 3 channels per sensor The highest quality analysis is obtained by examining individual channels We will QC the sensors before shipment to identify the most accurate channels for cortisol analysis 4 We
31. run before each cortisol containing sample and one will serve as a pre sample block before the first control At the end of the 5 minute incubation arrange the cortisol samples and no cortisol controls in order for injection into the instrument A no cortisol control must always precede a cortisol containing sample Therefore the first sample will be the block followed by one of the no cortisol controls followed by the 0 5 ng ml cortisol sample then another no cortisol control followed by the 1 ng ml cortisol sample etc The order should be block 0 no cortisol 0 5 0 1 0 1 5 0 2 0 3 ng ml cortisol with 5 cortisol and 6 no cortisol samples for a total of 11 samples for analysis Instrument Preparation and Sample Analysis 1 Refer to the Spirit Operations Manual for a general description of instrument use and programming procedures Following initialization of the new sensors as described in the manual enter and save the following program as program 1 Command Time Air Pump Flow Speed Flow 3 seconds On 0 Baseline 2 seconds Off 0 Seattle Sensor Systems Free Cortisol Assay CT 1317 18 Inject 20 seconds Off 0 Flow 100 seconds Off 20 Flush 20 seconds On 0 Regeneration 300 seconds Off 100 Flow 300 seconds Off 20 Stop This will be the System Preparation and Cleaning program which should be used before each sample run if the
32. t binding data for 100 seconds after the sample is injected into the instrument The window parameters define a subset of the 100 seconds of data The default is to use the data between 50 seconds and 100 seconds The goal here is to select the segment of the curve that demonstrates a linear binding rate of anti cortisol antibody to the biosensor surface The greater the slope the lower the concentration of cortisol in the injected sample 2 Load SPIRIT Data file After setting the Experiment Run Parameters click this button and a file system navigation and file selection window will open Navigate to the directory that you selected as the data storage location in the Instrument Operating Software In that directory you will see one or more text files that are automatically created and named by concatenating date time spr txt yyyy mm dd hh mm ssspr txt Select and Open the file you want to analyze Note that the data file is a txt file and can also be easily loaded into Excel and then converted into data columns with the Convert text to columns command enabling additional custom data analysis 3 The analysis system will calculate the slope of the line and the goodness of fit R 2 of that line and display these numerical values along with text indicating no detect potential detect or definite detection of cortisol Note that the R 2 value is sensitive to the amount of noise in the data so it is possible to get a
33. tall the analysis software This code is written in Java so that it will run on both Windows based and Mac OS X based computers Your computer must have the Java Runtime Environment already loaded Since most modern web browsers use Java technology you most likely already have this installed on your machine The Spirit Analysis Software files were delivered to you either by a thumb drive included in the free cortisol kit or by email to ensure you received the most up to date copy The file will be a zip file that must first be uncompressed Typically if you right click on the zip file you will see an option for uncompressing Once uncompressed you will see a dist for distribution directory Move into the dist directory and then double click on SpiritAnalysisSystem jar file In most cases this will immediately open a new window on your screen and the software will begin to run Seattle Sensor Systems Free Cortisol Assay CT 1317 14 File Edit Help Set Experiment Run Parameters Select Active Sensors MJ 1 Me Wes Wea Enter Injection Duration 100 a n suet wedowsat Tl Seattle Sensor Systems CORPORATION Enter Window End 100 Load SPIRIT Data File Number of Runs Found X zoom 0 16 Y zoom 0 08 X offset 0 Y offset 76 Plot Start Time Avg Slope 0 Plot End Time Slope Calculations a Alternate Data Matrix a SPIRIT Analysis System Version 2 2 Run Sensor Channel Slope Fit RA2 for Binding Rate Based Assays i
34. towel or Kimwipe to remove any excess fluid Aspirate the first sample block see step 6 Instrument Familiarization section above into the syringe and expel large air bubbles as described above Insert the needle into the injection port but do not attempt to inject the sample at this time Click the Zero Sensors button in the upper right corner of the screen to bring all of the sensor traces to zero Click the green Run Experiment button above the program selection box When the Inject function valves click as described above inject the entire sample during the 20 second time frame for injection The flow step will now start and sensor traces possessing particular slopes will begin to appear depending on the amount of free cortisol antibody present in the sample Once the flow step begins it is important to prepare for the next sample injection as described below Remove the syringe needle from the injection port and expel residual fluid from the needle as described above Wipe the needle tip on a clean paper towel or Kimwipe as described above and then aspirate 1 ml of PBST from the aliquot prepared in step 11 Expel the PBST into the waste container then move the plunger back and forth as described above to expel any remaining fluid Repeat steps 21 to 23 Wipe needle tip on a clean paper towel or Kimwipe then aspirate the next sample O cortisol into the syringe expel large air bubbles and inse
35. tration See Appendix 3 for the detailed protocol Seattle Sensor Systems Free Cortisol Assay CT 1317 8 2 Free Cortisol Assay and Analysis Protocol The purpose of this protocol is to start with pig or sheep saliva samples and end up witha digital dataset that can be converted to sample cortisol concentration First a reference curve using cortisol depleted saliva from the species to be assayed pig sheep etc spiked with cortisol must be constructed This reference curve will serve as the basis for all of the cortisol concentration calculations for this species See Appendix 4 for the detailed protocol Antibody coupled beads are used to extract the cortisol from saliva and then the Spirit s SPR measurement capability is utilized to measure binding rates of anti cortisol antibodies to cortisol on the biosensor surface The binding data is captured as a time sequence of uRIUs refractive index units uRIUs are the basic measurement value provided by SPR based instruments The measured uRIU vs Time dataset is then read into the provided Analysis System Software which converts the data into slope measurements These slopes when compared to standards curves provide a quantitative measure of the cortisol in the original saliva sample See Appendix 5 for the detailed protocol Seattle Sensor Systems Free Cortisol Assay CT 1317 9 Appendix 1 Loading Parameters for the Cortisol Assay To set up the programming
36. ual Standard Curve is mapped out in Section 2 below The protocol steps provided in the appendices may appear complex We have provided as much detail as possible to ensure the successful running of the assay As you become familiar with the assay many of these steps will become automatic 1 Instrument Familiarization Cortisol Measurement in Buffer There are two purposes to this protocol 1 for a new user to become familiar with the operation of the instrument and data analysis and 2 to create a standard curve that relates spiked concentration levels to measure changes in slope between control and spiked samples It should be noted that the standard curve generated from this protocol will not be used for concentration calculations from saliva samples as magnetic beads are not utilized during the procedure and the curve is buffer based not saliva based Note that this procedure can be helpful in monitoring assay performance The procedure will be to 1 aliquot a set of 5 standard samples of precise free cortisol concentration 2 assay cortisol in the samples by adding anti cortisol antibody 3 inject the antibody cortisol samples into the Spirit instrument while running a specific processing program 4 transfer the collected dataset to the Analysis Software System to compute the binding rate slopes for all the injected samples and 5 use Excel or a calculator to compute and plot the measured changes in slopes vs the spiked concen
37. uence and store it before use Follow the instructions at the back of this assay document in the Appendix 1 for loading the parameters for the cortisol detection assay into the instrument and operating software Follow the instructions on pages 21 and 22 of the SPIRIT Operations amp Appendix document for initializing the instrument and calibrating the sensors for first use Use the provided syringe 5 and the initialization solution 12 Please attach the metal syringe needle to the plastic body before use and use dedicated syringes and needles labeled I initialization or S sample injection for the appropriate applications Note that these are dull tipped needles Also take the time to install the Analysis Systems software that will be used to analyze the datasets created by the Spirit Instrument Please follow the instructions in Appendix 2 Initialization only has to be performed when any new sensor is mounted into one of the sensor Slots in the instrument So when you change sensors for any reason please repeat this initialization Note that when changing sensors you must always de pressurize the fluidics so that buffer solution does not leak 3 Shut down and storage If no more experiments are to be done within a 72 hour period make sure the injection port injection loop and flow cell are flushed of any proteins To do this inject 5 10 ml of shutdown solution 14 and flow for at least 5 minutes It is reco
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