User Manual - RayBiotech, Inc.
Contents
1. 100 ul of prepared Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle shaking 8 Discard the solution Repeat the wash as in step 4 9 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with gentle shaking 10 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately V I ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C 3 Add 100 ul prepared biotin antibody to each well Incubate 1 hour at room temperature 4 Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately Vill CALCULATION OF RESULTS Calculate the mean absorbance for each set of duplicate standards controls and samples and subtract the average zero standard optical density Plot the standard curve on log log graph paper or using Sigma plot software with standard concentration on the x axis and absorbance on the y axis Draw the best fit straight line through the standard points A TYPICAL DATA These standard curves are for demonstration only A standard c2. RayBio Human PD 1 ELISA Kit Catalog ELH PD1 User Manual Last revised December 7 2015 Caution Extraordinarily useful information enclosed Ke RayBiotech The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com RayBiotech Inc RayBio Human PD 1 ELISA Kit Protocol Table of Contents Pose Invoanion E sure essen v roor maras Reged OOOO Tione OOOO Pere SS assay Pace sonnen OOO OO Calculation of Results A Typical Data B Sensitivity FEET V l VI C Spiking amp Recovery D Linearity E Reproducibility Specificity Troubleshooting Guide 11 F FF 0 WMO N on A A TB ol o Please read the entire manual carefully before starting your experiment Il INTRODUCTION The RayBio Human PD 1 Programmed Death 1 ELISA kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human PD 1 in serum plasma and cell culture supernatants This assay employs an antibody specific for human PD 1 coated on a 96 well plate Standards and samples are pipetted into the wells and PD 1 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human PD 1 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavi
3. cipitates may form during storage HRP Streptavidin concentrate should be diluted 200 fold with 1X Assay Diluent B Item E For example Briefly spin the vial Item G and pipette up and down to mix gently Add 50 ul of HRP Streptavidin concentrate into a tube with 10 ml 1X Assay Diluent B to prepare a 200 fold diluted HRP Streptavidin solution do not store the diluted solution for next day use Mix well VI ASSAY PROCEDURE 1 2 3 Bring all reagents and samples to room temperature 18 25 C before use It is recommended that all standards and samples be run at least in duplicate Label removable 8 well strips as appropriate for your experiment Add 100 ul of each standard see Reagent Preparation step 3 and sample into appropriate wells Cover well and incubate for 2 5 hours at room temperature or over night at 4 C with gentle shaking Discard the solution and wash 4 times with 1X Wash Solution Wash by filling each well with Wash Buffer 300 ul using a multi channel Pipette or autowasher Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels 5 Add 100 ul of 1X prepared biotinylated antibody Reagent Preparation step 6 to each well Incubate for 1 hour at room temperature with gentle shaking 6 Discard the solution Repeat the wash as in step 4 7 Add
4. din is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of PD 1 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm ll STORAGE The entire kit may be stored at 20 C for up to 1 year from the date of shipment Avoid repeated freeze thaw cycles The kit may be stored at 4 C for up to 6 months For extended storage it is recommended to store at 80 C For prepared reagent storage see table below lil REAGENTS Size Description aeea Stay Stability PD 1 PO 1 Microplate dema Item A Poi Microplate tema wells 12 roa x 8 wells coated with anti imonha month at aor Human a 1 Wesneuie ieee 25 ml 25 mi of 20x concentrated soluton 20X concentrated solution 1 month at ft monthatas ieee Item B Standar Protein em Standar Protein em Item C Avil or Human PE of VALS ENQuUgRIO rn 1 jt wockat 30 at jt wockat 30 each standard in duplicate Detection oe PD 1 2 vials of biotinylated anti Human PD 1 Each 5 sack 5 Bdysase at 4 Bdysase oe F vial is enough to assay half the microplate HRP ee 200 eo eee f 200X concentrated HRP conjugated oe not store and Concentrate ee G eo eee f oe TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i Reagent Item H buffer solution Stop Solution Item 1 8 ml of 0 2 M sulfuric acid iaaa Assay Dil
5. les into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 60 ul PD 1 standard into a tube with 440 ul Assay Diluent A or 1X Assay Diluent B to prepare a 6 000 pg ml standard solution Pipette 300 ul of Assay Diluent A or 1X Assay Diluent B into each tube Use the 6 000 pg ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer Assay Diluent A or 1X Assay Diluent B serves as the zero standard 0 ng ml 60 ul standard 2 2 2 2 200 ul 2 440 pl OO 200 ul 00 ul 00 ul 200 ul OOS I OO OY A 35558856 ae 2 6 000 2 400 960 384 153 6 61 44 24 58 0 pg ml pe ml pg ml pg ml pg ml peml pgml pg ml 5 If the Wash Concentrate 20X Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer Briefly spin the Detection Antibody vial Item F before use Add 100 ul of 1X Assay Diluent B Item E into the vial to prepare a detection antibody concentrate Pipette up and down to mix gently the concentrate can be stored at 4 C for 5 days The detection antibody concentrate should be diluted 80 fold with 1X Assay Diluent B Item E and used in step 5 of Part VI Assay Procedure Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use as pre
6. ntly washed Contaminated wash buffer e Improper storage of the ELISA kit e Stop solution Low sensitivity e Check pipettes Briefly centrifuge Item C and dissolve the powder thoroughly by gently mixing Briefly spin down vials before opening Dissolve the powder thoroughly Ensure sufficient incubation time assay procedure step 2 may be done overnight Check pipettes and ensure correct preparation Check pipettes Remove bubbles in wells Review the manual for proper wash If using a plate washer ensure that all ports are unobstructed Make fresh wash buffer Store your standard at lt 70 C after reconstitution others at 4 C Keep substrate solution protected from light e Add stop solution to each well before reading plate RayBio ELISA Kits Over 2 000 ELISA kits available visit www RayBiotech com ELISA Kits html for details This product is for research use only 2015 RayBiotech Inc
7. uent A Item D 30 ml of diluent buffer 0 09 sodium azide as preservative Assay Diluent B Item E 15 ml of 5x concentrated buffer 1 month at 4 C Return unused wells to the pouch containing desiccant pack reseal along entire edge ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbent paper Distilled or deionized water Log log graph paper or computer and software for ELISA data analysis Tubes to prepare standard or sample dilutions lt ONOnNROND V REAGENT PREPARATION 1 2 Bring all reagents and samples to room temperature 18 25 C before use Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water before use Sample dilution Assay Diluent A Item D should be used for dilution of serum plasma samples 1X Assay Diluent B Item E should be used for dilution of culture supernatants The suggested dilution for normal serum plasma is 2 fold Note Levels of PD 1 may vary between different samples Optimal dilution factors for each sample must be determined by the investigator Preparation of standard Briefly spin a vial of Item C Add 400 ul of Assay Diluent A for serum and plasma samples or 1X Assay Diluent B for cell culture supernatant samp
8. urve must be run with each assay Assay Diluent A Assay Diluent B 10 10 Dt 0 1 a 0 01 0 01 10 100 1000 10000 10 100 1000 10000 OD 450 nm OD 450 nm Human PD 1 concentration pg ml Human PD 1 concentration pg ml B SENSITIVITY The minimum detectable dose of Human PD 1 was determined to be 20 pg ml Minimum detectable dose is defined as the analyte concentration resulting in an absorbance that is 2 standard deviations higher than that of the blank diluent buffer C SPIKING amp RECOVERY Recovery was determined by spiking various levels of Human php echo PD 1 into the sample types listed below Mean recoveries are as follows Sample Type Average Recovery Range Serum 92 05 86 96 Plasma 84 41 78 88 Cell culture media 97 77 92 102 D LINEARITY Sample Type Serum Plasma Cell Culture Media ee Average of Expected 107 5 107 6 104 1 Range 100 116 100 116 96 112 1 4 Average of Expected 96 16 101 3 92 29 Range 88 104 93 109 84 100 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY This ELISA antibody pair detects human PD 1 Other species not determined yet X TROUBLESHOOTING GUIDE Inaccurate pipetting Improper standard dilution Improper preparation of standard and or biotinylated antibody Too brief incubation times Inadequate reagent volumes or improper dilution Low signal e Inaccurate pipetting Air bubbles in wells Plate is insufficie
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