TB532 Rev. B 0510JN WS Human Hormone 1
Contents
1. 10 sec e Shake for 1 h 750 rpm room temperature in the dark Detection Antibody Incubation e Wash and vacuum plate 2X 100 ul Assay Buffer Type 2 e Add 40 ul Detection Antibodies mix to each well e Vortex mix plate 10 sec e Shake for 1 h 750 rpm room temperature in the dark e NOTE Do NOT wash or vacuum filter plate after incubation Streptavidin PE SA PE Incubation e Dilute 15X SA PE as needed For entire plate Add 144 ul 10X SA PE 2016 ul Assay Buffer Type 2 e Add 20 tl diluted 1X SA PE to each well e Shake for 30 min 750 rpm room temperature in the dark e Wash and vacuum plate 2X 100 ul Assay Buffer Type 2 e Resuspend beads in 100 ul Assay Buffer Type 2 Analysis e Shake approx 5 min 750 rpm room temperature in the dark Analyze on xMAP system Recommended e CAL2 Gain Setting default Luminex or RP1low BioPlex e DD Gate 7500 15500 Luminex or default BioPlex e Sample size 50 ul e Collect 50 100 events per bead region e Timeout 60 sec Luminex or default BioPlex USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 628 8470 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com bioscienceshelp techservice merckbiosciences de Ireland Toll Free 1800 409445 bioscienceshelp emdchemicals com customer service merckbiosciences co uk emdchemicals com2. filter plate with plate sealer included or lab tape for future use 2 Pre wet 96 well filter plate wells with 50 ul Assay Buffer Type 2 and incubate for a minimum of 5 min Immediately prior to Step 3 remove liquid from filter plate by vacuum filtration Do not exceed 5 in Hg or 127 mm Hg vacuum liquid should drain in 2 5 sec Tap filter plate on a paper towel to remove any buffer remaining on the underside Note It is critical to remove excess buffer from the underside of the filter plate before adding samples or reagents Otherwise samples may wick out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations If a well does not drain use the non tip end of a 200 ul pipet tip to flick the center of the plastic support on the underside of the well then reapply vacuum 3 Add 10 ul of Human Hormone Panel 1 Blocking Buffer to each filter plate well that will be used 4 Add 30 ul of each standard sample or control to appropriate well of the 96 well filter plate Note Human Hormone Panel 1 Control 1 and Control 2 do not need to be diluted 5 Vortex the plate by gently gliding the plate over the vortex mixer Note Gradually increase the vortex speed from low to medium Hold the plate with a loose grip Mix thoroughly for 10 sec Avoid splashing Alternatively mix using a plate shaker for 10 sec on high speed 1200 rpm 6 Sonicate 10 sec optional and vortex the
3. tube of Human Hormone Panel 1 Capture Beads for 10 sec Add 10 ul to each well Vortex or shake the plate 10 sec as described above in Step 5 Cover plate with aluminum foil to protect from light and incubate 1 hr at room temperature on a plate shaker 750 rpm 9 Remove liquid from filter plate by vacuum filtration 5 in Hg or 127 mm Hg maximum 10 Wash beads by adding 100 ul Assay Buffer Type 2 to each well and applying vacuum to remove buffer Repeat wash step for total of two washes in Assay Buffer Type 2 After the second wash and vacuum tap the filter plate on paper towels to remove any buffer remaining on the underside Note Do not resuspend beads in Assay Buffer Type 2 after second wash 11 Add 40 ul Human Hormone Panel 1 Detection Antibodies to each well Vortex or shake the plate as described in Step 5 12 Cover plate with aluminum foil to protect from light and incubate 1 h at room temperature on a plate shaker 750 rpm Note Do not wash beads after Detection Antibody incubation 13 Microcentrifuge 15X Streptavidin PE briefly 5 sec to ensure all material is in the bottom of the tube If using all 96 wells dilute 15X Streptavidin PE to 1X by adding 144 ul concentrated Streptavidin PE to 2016 ul Assay Buffer Type 2 Note Do not dilute the whole vial of Streptavidin PE if the entire kit will not be used Dilute only what is needed based on the number of wells Allow 10 extra for pipetting error If there is an insufficien
4. CH USE ONLY NOT FOR HUMAN OR DIAGNOSTIC USE User Protocol TB532 Rev B 0510JN Page 2 of 9 About the Kit WideScreen Human Hormone Panel 1 72276 3 Overview Bead based flow cytometric assays enable sensitive precise quantitation of analytes within a sample When directed toward proteins or small molecules such assays are essentially ELISAs on a bead In the conventional sandwich format samples are combined with fluorescently labeled microparticles beads covalently conjugated to a capture antibody Analytes captured on the beads are identified with detection antibodies and a fluorescent label A major advantage of bead based assays over traditional protein quantitation methods such as ELISA is the capacity for multiplexing as bead based assays allow simultaneous quantitation of multiple analytes in a small sample volume WideScreen Human Hormone Panel 1 is a pre mixed multiplex bead kit of quantitative antibody based assays for simultaneous detection of five human reproductive hormones FSH follicle stimulating hormone hCG human chorionic gonadotropin beta subunit LH luteinizing hormone progesterone and testosterone The kit includes all the reagents and buffers needed to analyze the above molecules in serum plasma urine and tissue culture supernatants using the Luminex xMAP System WideScreen Human Hormone Panel 1 contains two types of immunoassays conventional and competitive FSH hCG B and LH assays are con
5. Multichannel pipet optional USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 628 8470 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com bioscienceshelp techservice merckbiosciences de Ireland Toll Free 1800 409445 bioscienceshelp emdchemicals com customer service merckbiosciences co uk emdchemicals com User Protocol TB532 Rev B 0510JN Page 4 of 9 Human Hormone Panel 1 Protocol Considerations Before You Begin e Guidelines when using filter plates and vacuum manifold e Excessive vacuum will cause the filter plate membrane to perforate Adjust the pressure using a non filter ELISA or tissue culture plate ensuring that vacuum does not exceed 5 in 127 mm Hg e After adjusting the vacuum with a non filter plate place filter plate on the manifold Use fingertips to apply pressure evenly across the plate The liquid should drain in 2 5 sec e To avoid drying out the beads vacuum only long enough to drain all wells Do not allow drained beads to sit for gt 5 min before rehydrating with buffer e It is critical to remove excess buffer from the underside of the filter plate by tapping it on a paper towel several times before adding samples or reagents This prevents samples from wicking out of the wells during incubation steps For the same reason avoid placing filter plate on an absorbent surface during incubations e To avoid perforating the filter plate membrane make certain that the probe
6. WideScreen User Protocol TB532 Rev B 0510JN Paqe 1 of 9 WideScreen Human Hormone Panel 1 Table of Contents About tnecKits favs asticct we actus dens Parsee A ces iti ren ieee ee eee Suction Overview Components and Storage Additional Materials Required But Not Supplied ww n do Human Hormone Panel 1 Protocol ccccccccccsceeeeeeeeeeeeeeeeeeseeeeeseseeeseseeeeeeeeeees Considerations Before You Begin Reagent Preparation Test Sample Preparation Standard Dilution Series Preparation Immunoassay Protocol ONAA A Collecting Data and Data Analysis cccccceeeeeeceeeeeeeeeeeaaeeeeeeeseaeeesaeeeeeeesenees Data Acquisition Generation of Standard Curves and Quantitation of Experimental Samples SNN Tro blesh o n rrsan ieia e a AA ENAS ATES EA 8 Appendix A Flowchart for Human Hormone Panel 1 Immunoassaj 5 9 2010 EMD Chemicals Inc an Affiliate of Merck KGaA Darmstadt Germany All rights reserved The WideScreen name and logo are registered trademarks of EMD Chemicals Inc in the United States and in certain other jurisdictions Bio Plex is a registered trademark and Bio Plex Manager is a trademark of Bio Rad Laboratories Inc Luminex and xMAP are registered trademarks and Luminex 100 IS is a trademark of Luminex Corporation ProClin is a registered trademark of Rohm and Haas Co Tween is a registered trademark of ICI Americas Inc Manufactured by Rules Ba
7. e container is not full the sheath fluid is not empty and the SD fluidics module is turned on Check system calibration using approved calibration beads Verify correct system pressure Confirm that the system is free of air or particulate buildup Follow maintenance steps in the instrument user manual Insufficient volume of an immunoassay reagent Solutions were not prepared or used as described in protocol Confirm correct buffer dilutions and use If additional Assay Buffer Type 2 is needed PBS can be used for the final bead resuspension step If additional 96 well filter plates are required we recommend Millipore Cat No MSBVN1210 If there is insufficient volume of 15X Streptavidin PE for your final experiment making a slightly more dilute working stock e g 20 fold instead of 15 fold will not adversely affect results Sample measurements not within the standard curve Dilution of sample is too low or too high If values are higher than the standard curve dilute samples further in Sample Dilution Buffer Type 1 and repeat assay Target concentration is below detection Verify that curve fitting at the lower end of the standard curve is accurate Not all serum plasma samples contain detectable levels of all analytes USA and Canada Tel 800 628 8470 bioscienceshelp emdchemicals com Germany Tel 0800 100 3496 techservice merckbiosciences de All Other Countries www novagen com biosciencesh
8. ed with the concentration standards are plotted using Median Fluorescent Intensity MFI as the signal readout Y axis against concentration of standard dilutions X axis e Five parameter logistic SPL curve fitting is recommended for modeling data obtained from bead based immunoassays Most ranges of standard concentrations are too wide for accurate linear regression analysis Four parameter logistic 4PL equations will often give a good fit but are not ideal because they assume the standard curve is symmetrical e If the signal from an unknown sample exceeds the highest point of the standard curve the concentration of the unknown should not be calculated by extrapolation because the non linear shape of the standard curve cannot be accurately modeled past the last measured point In this case dilute the samples and test again e When concentrations of unknowns have been determined by reading off of the standard curve remember to multiply this value by the dilution factor to obtain the concentration of the target in the original sample USA and Canada Tel 800 628 8470 bioscienceshelp emdchemicals com Germany Tel 0800 100 3496 techservice merckbiosciences de United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk All Other Countries www novagen com bioscienceshelp emdchemicals com User Protocol TB532 Rev B 0510JN Page 8 of 9 Troubleshooting Pr
9. eland Toll Free 1800 409445 bioscienceshelp emdchemicals com customer service merckbiosciences co uk emdchemicals com User Protocol TB532 Rev B 0510JN Page 3 of 9 Components and Storage Additional Materials Required But Not Supplied The kit includes all the reagents and buffers needed to assay the above proteins in serum plasma urine and tissue culture supernatants using the Luminex xMAP System Whole blood or grossly hemolyzed samples cannot be used with this kit The kit contains sufficient components to assay one 96 well plate WideScreen Human Hormone Panel 1 72276 3 1 1 ml Human Hormone Panel 1 Capture Beads PBS with BSA Tween 20 and 0 009 ProClin 300 1 vial Human Hormone Panel Detection Antibodies Lyophilized biotinylated detection antibody premix 1 vial Human Hormone Panel 1 Standards Mix Lyophilized purified protein FSH hCG B and LH and synthesized progesterone and testosterone standards 1 vial Human Hormone Panel 1 Control 1 Lyophilized low levels of FSH hCG f LH progesterone and testosterone in human serum 1 vial Human Hormone Panel 1 Control 2 Store all components at Lyophilized high levels of FSH hCG B LH progesterone and testosterone in human serum 4 C 60 ml Assay Buffer Type 2 1X proprietary mix of domestic animal proteins in PBS with 0 025 ProClin 300 1 vial Human Hormone Panel 1 Blocking Buffer Lyophilized proprietary mix of antibodies biotinyla
10. elp emdchemicals com United Kingdom and Ireland UK Freephone 0800 622935 Ireland Toll Free 1800 409445 customer service merckbiosciences co uk User Protocol TB532 Rev B 0510JN Page 9 of 9 Appendix A Flowchart for Human Hormone Panel Immunoassay Pre wet Filter Plate e Add 50 ul Assay Buffer Type 2 to each well being used Prepare Reagents e Reconstitute all lyophilized reagents Standards Mix 150 ul dH2O Controls 1 and 2 100 ul dH20 each Blocking Buffer 1 5 ml dH20 Standard Curve Diluent 1 0 ml dH20 Detection Antibodies 4 4 ml dH20 Prepare Diluted Test Samples e Dilute serum plasma urine or tissue culture supernatants 5 fold in Sample Dilution Buffer Type 1 e If further dilutions are desired perform in Sample Dilution Buffer Type 1 Prepare 8 point Standard Dilution Series Duplicates 80 ul Standard Curve Diluent Type 1 in tubes S7 S1 e 150 ul Standards Mix in tube S8 e 3 fold serial dilutions mix thoroughly 40 ul from tube S8 to tube S7 etc Blocker Analyte Capture Bead Incubation e Remove liquid from pre wet filter plate by vacuum e Add 10 ul Human Hormone Panel 1 Blocking Buffer per well being used e Add 30 ul of the following and mix Test sample diluted or Controls 1 or 2 undiluted or Standard Dilution Series e Vortex sonicate Capture Beads Premix e Add 10 ul Capture Beads Premix to each well e Vortex mix plate
11. f 5 min not to exceed 30 min and repeat vortexing step Human Hormone Panel Detection Antibodies can remain at room temperature for up to 2 hours Note Following reconstitution store any unused reagents at 70 C Unused reagents can be stored at 70 C for up to one month Avoid multiple freeze thaw cycles USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 628 8470 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com bioscienceshelp techservice merckbiosciences de Ireland Toll Free 1800 409445 bioscienceshelp emdchemicals com customer service merckbiosciences co uk emdchemicals com User Protocol TB532 Rev B 0510JN Page 5 of 9 Test Sample Preparation 1 Thaw and dilute samples within 1 h of use Remove any particulates by centrifugation or filtration Avoid multiple freeze thaw cycles 2 Dilute serum plasma urine or tissue culture supernatant samples 1 5 in Sample Dilution Buffer Type 1 Duplicate Samples 15 ul sample 60 ul Sample Dilution Buffer Type 1 Assaying duplicate samples is recommended Mix well and store on ice If desired further dilutions of samples can also be performed in Sample Dilution Buffer Type 1 to ensure reading within the range of the assay standards Note Depending on the sample type empirical testing may be needed to determine the optimal dilution factor For example dilutions and representative data for serum plasma and urine samples please refer to the Cer
12. height on the xMAP system is adjusted correctly Do not touch the membrane with pipet tips For accurate pipetting touch tips to the sides of the filter plate wells Change tips as necessary to prevent cross contamination e Capture Beads and Streptavidin PE are light sensitive To avoid photobleaching keep beads and Streptavidin PE in microcentrifuge tubes covered Cover filter plates with aluminum foil during incubation steps To prevent fluorescent dye loss do not use organic solvents with capture beads Beads are incompatible with DMSO concentrations gt 1 e Many of the washing and reagent dispensing steps may be done with an 8 channel or 12 channel pipet manual or automatic For accurate results use calibrated single channel pipets for manipulation of standards and test samples Test samples serum plasma urine tissue culture supernatant should be stored at 70 C prior to use Reagent Preparation 1 Resuspend each of the following lyophilized reagents in deionized water immediately prior to performing the assay Reagent dH 0 Volume Human Hormone Panel 1 Standards Mix 150 pl Human Hormone Panel 1 Control 1 100 pl Human Hormone Panel 1 Control 2 100 pl Human Hormone Panel Blocking Buffer 1 5 ml Standard Curve Diluent Type 1 1 0 ml Human Hormone Panel Detection Antibodies 4 4 ml 2 Mix each vial by vortexing at medium speed for 15 sec Incubate at room temperature for a minimum o
13. l sanitize alcohol flush probe sonication etc Make sure that the probe height is set correctly Make sure that beads are in suspension by incubating plate for 3 5 min on plate shaker 750 rpm immediately before analysis Microbial growth in buffers can cause beads to stick to the filter plate membrane Do not use contaminated reagents Timeout limit is set too low 50 100 events per bead region should be acquired within the 60 sec timeout limit If necessary the timeout limit can be set higher e g 75 sec Beads are not falling into the gates properly Beads were not resuspended in Assay Buffer Type 2 before analysis The setting of the Doublet Discriminator DD gate is buffer specific This gate can be adjusted but Assay Buffer Type 2 is the recommended buffer for running samples Other buffers may also cause bead aggregation DD gate setting not optimal Use the DD gate setting recommended in the Data Acquisition Section If necessary raw data results can be reanalyzed with different DD gate settings see software user manual Beads were exposed to organic solvents Do not use organic solvents in the immunoassay as they will damage beads irreversibly Beads are falling outside the bead region gates due to photobleaching Do not use expired beads Do not expose the beads to ambient light for gt 10 min Avoid intense light Fluidics system is not running properly Confirm that the wast
14. oblem Probable Cause Solution Leaking wells in filter plate Wicking due to adherent drops Tap filter plate several times on paper towel before adding samples or reagents Do not place filter plate on an absorbent surface during incubations If wells leaked during data acquisition it is possible to reacquire these wells Blot underside of wells and add 100 ul well Assay Buffer Type 2 Perforation of filter plate membranes Adjust the vacuum setting to lt 5 in 127 mm Hg Do not touch membranes with pipet tips Filter plate wells not draining under vacuum Vacuum is too low Adjust vacuum setting to 3 5 in 76 127 mm Hg Clean rubber seals Apply fingertip pressure to filter plate to ensure formation of a good seal Use the plate sealer to cover wells not in use Clogged membranes Clarify samples by centrifugation or filtration If samples are viscous dilute further before assaying Use the non tip end of a 200 ul pipette to flick the center support on the underside of the well then reapply vacuum Low bead counts during data acquisition No beads in the wells See Leaking wells in filter plate solutions above Verify that beads were added at the correct concentration and that correct bead regions and wells were selected during acquisition setup xMAP fluidics system is clogged Clear system of clogs or air using maintenance steps described in the instrument user manua
15. ogesterone to maintain the pregnancy A chromatographic hCG B immunoassay is the basis for standard home pregnancy tests hCG f is also secreted by some types of tumors and can be used as a tumor marker in non pregnant individuals e Luteinizing hormone LH is secreted by the anterior pituitary gland Elevated LH levels stimulate ovulation in females and ultimately act to maintain elevated progesterone levels necessary for implantation of a fertilized embryo In males LH stimulates testosterone production by Leydig cells which is required for spermatogenesis e Progesterone is a steroid hormone secreted by the female ovaries the brain and by the placenta during pregnancy Progesterone levels are consistently low in males and vary in females during the menstrual cycle In the case of pregnancy progesterone levels remain elevated which is required for maintenance e Testosterone is a steroid hormone produced by the male testes female ovaries and in small amounts from the adrenal gland Testosterone is the primary male sex hormone found on average in higher levels in males compared to females and is involved in the development of primary and secondary male sexual characteristics In both sexes testosterone is important for various anabolic effects USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 628 8470 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com bioscienceshelp techservice merckbiosciences de Ir
16. sed Medicine Inc RBM By opening the packaging containing this product which contains fluorescently labeled microsphere beads authorized by Luminex Corporation or using this product in any manner you are consenting and agreeing to be bound by the following terms and conditions You are also agreeing that the following terms and conditions constitute a legally valid and binding contract that is enforceable against you If you do not agree to all of the terms and conditions set forth below you must promptly return this product for a full refund prior to using it in any manner You the buyer acquire the right under Luminex Corporation s patent rights if any to use the product or any portion of this product including without limitation the microsphere beads contained herein only with Luminex Corporation s laser based fluorescent analytical test instrumentation marketed under the name Luminex Instrument The terms and conditions governing EMD Chemicals sale of this product are as indicated on our website www emdbiosciences com USA and Canada Europe All Other Countries Tel 800 628 8470 France Germany Ireland United Kingdom All other Contact Your Local Distributor bioscienceshelp Freephone Freecall Toll Free Freephone European Countries www novagen com emdchemicals com 0800 126 461 0800 100 3496 1800 409 445 0800 622 935 44 115 943 0840 bioscienceshelp emdchemicals com techservice merckbio eu www novagen com FOR RESEAR
17. t volume of 15X Streptavidin PE for your final experiment making a slightly more dilute working stock will not adversely affect results 14 Add 20 pl 1X Streptavidin PE to each well 15 Cover plate with aluminum foil to protect from light and incubate 30 min at room temperature on a plate shaker 750 rpm 16 Remove liquid from filter plate by vacuum filtration 17 Wash beads by adding 100 ul Assay Buffer Type 2 to each well and applying vacuum to remove buffer Repeat wash step for total of two washes in Assay Buffer Type 2 After second wash and vacuum tap filter plate on paper towels to remove any buffer remaining on the underside 18 Add 100 ul Assay Buffer Type 2 to each well 19 Cover plate to protect from light Incubate 3 5 min at room temperature on a plate shaker 750 rpm 20 Analyze using a Luminex instrument USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 628 8470 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com bioscienceshelp techservice merckbiosciences de Ireland Toll Free 1800 409445 bioscienceshelp emdchemicals com customer service merckbiosciences co uk emdchemicals com User Protocol TB532 Rev B 0510JN Page 7 of 9 Collecting Data and Data Analysis Data Acquisition For detailed instructions on the operation of Luminex systems refer to the user guide for your specific instrument and software General recommendations are given below 1 Using your Luminex s
18. ted antigen for competitive assays and buffered domestic animal proteins to minimize non specific interactions 2x 3 6 ml Sample Dilution Buffer Type 1 1X proprietary mix of buffered domestic animal proteins in PBS with 0 025 ProClin 300 1 vial Standard Curve Diluent Type 1 Lyophilized proprietary mix of domestic animal proteins 250 ul 15X Streptavidin Phycoerythrin PBS with 2 mM NaN 1 96 well Filter Plate and Sealer Following reconstitution of lyophilized reagents store any unused reagent at 70 C See Reagent Preparation section p 4 Note WideScreen Human Hormone Panel 1 is not compatible with other bead kits and buffers sold by EMD or other vendors Caution Human Hormone Panel 1 Standards Mix Control 1 and Control 2 contain components derived from human sources All human source materials have been tested negative for HIV 1 HIV 2 HCV antibodies HIV Ag and HBsAg However all materials derived from human fluids or tissues should be considered biohazardous and handled accordingly Refer to MSDS for additional information Additional Materials Required But Not Supplied e Luminex xMAP System or equivalent e Vacuum manifold for filter plates Pall 5017 or Millipore MSVMHTSO0 e 96 well plate platform shaker such as IKA MTS4 e Polypropylene microcentrifuge tubes e 15 ml polypropylene centrifuge tubes e Vortex mixer e Ultrasonic bath such as Cole Parmer EW 08849 optional e
19. tificate of Analysis Standard Dilution Series Preparation This preparation provides sufficient volume to run duplicate standard dilution curves Label 8 polypropylene tubes S8 through S1 Alternatively prepare standard dilutions in a 96 well plate Pipet Standard Curve Diluent Type 1 into labeled tubes as described below Transfer the reconstituted Human Hormone Panel 1 Standards Mix to the S8 labeled tube Prepare 3 fold serial dilutions of S8 following the table below Ensure that each new standard is mixed well by vortexing before proceeding to the next dilution Change tips between each dilution Standard Volume of Standard Curve Diluent Volume of Standards Mix Type 1 S8 O ul 150 ul from vial S7 80 ul 40 ul of S8 S6 80 ul 40 ul of S7 S5 80 ul 40 ul of S6 S4 80 ul 40 ul of S5 S3 80 ul 40 ul of S4 S2 80 ul 40 ul of S3 S1 80 ul 40 ul of S2 Note Standard concentrations are lot specific Refer to Certificate of Analysis of appropriate lot for specific standard concentrations USA and Canada Germany United Kingdom and Ireland All Other Countries Tel 800 628 8470 Tel 0800 100 3496 UK Freephone 0800 622935 www novagen com bioscienceshelp techservice merckbiosciences de Ireland Toll Free 1800 409445 bioscienceshelp emdchemicals com customer service merckbiosciences co uk emdchemicals com User Protocol TB532 Rev B 0510JN Page 6 of 9 Immunoassay Protocol 1 Seal any unused wells of the 96 well
20. ventional non competitive sandwich based immunoassays The progesterone and testosterone assays are competitive assays in which a biotinylated antigen in the blocking buffer is competed off the beads by analyte resulting in a decrease in fluorescence signal Analyte Full name FSH Follicle stimulating hormone hCG B Human chorionic gonadotropin beta subunit LH Luteinizing hormone Progesterone Progesterone Testosterone Testosterone Hormones regulate many of the central functions of the body A host of hormones secreted into the bloodstream act in concert to control reproductive processes such as the menstrual cycle development of secondary sex characteristics and maintenance and growth of pregnancy Measurement of these biomarkers in biological samples is used in the clinical setting for detecting disease monitoring treatment and studying normal reproductive processes The WideScreen Human Hormone Panel 1 is a non diagnostic kit for use in the pre clinical and primary research settings e Follicle stimulating hormone FSH is secreted by the anterior pituitary gland to regulate reproductive development and processes In females FSH leads to ovulation by stimulating the maturation of Graafian follicles In males FSH induces production of androgen binding protein and is required for spermatogenesis e Human chorionic gonadotropin beta subunit hCG B is secreted after conception by the embryo and placenta and stimulates the production of pr
21. ystem software prepare a protocol for the assay you will run Enter information for each bead target and for the standards samples and controls 2 Select the following bead regions Analyte Bead Region Analyte Bead Region FSH 12 Progesterone 55 hCG B 0l Testosterone 02 LH 08 3 If desired enter the standard concentration values in the assay panel template Standard concentrations are lot specific Please refer to the Certificate of Analysis of appropriate lot for specific standard concentrations 4 Acquire data using the system settings shown below Software Sample Events per Timeout Doublet CAL2 Gain Size Bead Region Discriminator Setting Luminex IS or 50 ul 50 100 60 sec 7500 15500 default equivalent Bio Plex Manager default 50 100 60 sec default RP1 low 50 ul 4335 10000 If the time spent acquiring samples needs to be reduced collect as few as 50 events per bead region Generation of Standard Curves and Quantitation of Experimental Samples e Protein and synthetic standards are supplied in the Human Hormone Panel kit allowing for accurate quantitation using a titrated standard curve Representative standard curves and assay performance information can be found in the Certificate of Analysis for the specific lot e Refer to the Certificate of Analysis for standard dilution series concentrations and expected control ranges e The eight data points obtain
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