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1. 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 curve must be run with each assay RayBio Human CEACAM 1 ELISA Kit Protocol 7 Assay Diluent A Assay Diluent B 10 10 E 1 E 1 C c e eo LO wo H Q 0 11 Q 0 14 O O 0 01 4 0 01 4 10 100 1 000 10 000 100 000 10 100 1 000 10 000 100 000 Human CEACAM 1 concentration pg ml Human CEACAM 1 concentration pg ml B SENSITIVITY The minimum detectable dose of CEACAM 1 is typically less than 20 pg ml C RECOVERY Recovery was determined by spiking various levels human CEACAM 1 into human serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 96 92 82 112 Plasma 86 50 70 105 Cell culture media 83 49 75 90 RayBio Human CEACAM 1 ELISA Kit Protocol 8 D LINEARITY Sample Type 1 2 Average of Expected Range 1 4 Average of Expected Range E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Serum 101 3 90 118 119 3 108 128 Plasma Cell Culture Media 108 8 124 5 93 119 117 129 85 14 134 3 78 91 127 141 Cross Reactivity This ELISA kit shows no cross reactivity with the following cytokines tested human Angiogenin BDNF BLC ENA 78 FGF 4 IL 1la IL 1 IL 2 IL 3 IL2. 4 IL 5 IL 6 IL 7 IL 8 IL 9 IL 11 IL 12 p70 IL 12 p40 IL 13 IL 15 IL 309 IP 10 G CSF GM CSF IEN Leptin OB MCP 1 MCP 2 MCP 3 MDC MIP 1a MIP 1 D MIP 16 PARC PDGF RANTES SCF TARC TGF B TIMP 1 TIMP 2 TNF a TNF p TPO VEGF RayBio Human CEACAM 1 ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Poor standard curve 1 Inaccurate pipetting 2 Improper standard dilution 1 Check pipettes 2 Ensure a brief spin of Item C and dissolve the powder thoroughly by a gentle mix 2 Low signal 1 Too brief incubation 1 Ensure sufficient times incubation time assay procedure step 2 may change to over night 2 Inadequate reagent 2 Check pipettes and volumes or improper ensure correct dilution preparation 3 Large CV 1 Inaccurate pipetting 1 Check pipettes 4 High background 1 Plate is insufficiently washed 2 Contaminated wash buffer 1 Review the manual for proper wash If using a plate washer check that all ports are unobstructed 2 Make fresh wash buffer 5 Low sensitivity 1 Improper storage of the ELISA kit 2 Stop solution 1 Store your standard at 20 C after reconstitution others at 4 C Keep substrate solution protected from light 2 Stop solution should be added to each well before measure RayBio Human CEACAM 1 ELISA Kit Protocol 10 RayBio ELISA kits Over 200 ELISA kits custom ELIS
3. A kit choose from over 500 list visit www raybiotech com for details RayBio Human CEACAM 1 ELISA Kit Protocol 11 RayBio Human CEACAM 1 ELISA Kit Protocol 12 This product is for research use only i 2004 RayBiotech Inc RayBio Human CEACAM 1 ELISA Kit Protocol 13
4. RayBio Human CEACAM 1 ELISA Kit User Manual RayBio Human CEACAM 1 ELISA Kit Protocol Cat ELH CEACAMI 001 RayBiotech Inc We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Human CEACAM 1 ELISA Kit Protocol TABLE OF CONTENTS L Jir HC O a gt 445425555 500 44n8 4 y n bAn aa kr 4 n nka 2 U rw EE 2 Wi RR Suc N DD E E E IV Additional Materials Required 3 V Reagent Preparation iiec kk 3 VI Assay Procedure sse 5 VII Assay Procedure Summary 6 VIII Calculation of Results LEER RE 7 B EE RTT v i v MEN ISO 9 E Reprod cibility iicet tret er rer Een IX edi vibn an c PS X Troubleshooting Guide sss RayBio Human CEACAM 1 ELISA Kit Protocol 1 I INTRODUCTION The RayBio Human CEACAM 1 Carcinoembryonic Antigen related Cell Adhesion Molecule 1 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human CEACAM 1 in serum plasma cell culture supernatants and urine This assay employs an antibody specific for human CEACAM 1 coated on a 96 well plate Standards and samples are pipetted into the wells and CEACAM 1 present in a sample is bound to the wells by t
5. he immobilized antibody The wells are washed and biotinylated anti human CEACAM 1 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin 1s 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 CEACAM 1 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm II REAGENTS I CEACAM 1 Microplate Item A 96 wells 12 strips x 8 wells coated with anti human CEACAM 1 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials of recombinant human CEACAM 1 Assay Diluent A Item D 30 ml diluent buffer 0 09 sodium azide as preservative For Standard Sample serum plasma diluent Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture medium urine diluent Detection Antibody CEACAM 1 Item F 2 vial of biotinylated anti human CEACAM 1 each vial is enough to assay half microplate HRP Streptavidin Concentrate Item G 8 ul 20 000x concentrated HRP conjugated streptavidin TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffer solution RayBio Human CEACAM 1 ELISA Kit Protocol 2 9 Stop Solution Item I 8 ml of 2 M sulfuric acid HI STORAGE May be stored for up to 6 m
6. o each well Incubate for 1 hour at room temperature with gentle shaking 5 Discard the solution Repeat the wash as in step 3 6 Add 100 ul of prepared Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle shaking 7 Discard the solution Repeat the wash as in step 3 8 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 9 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately VII 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 RayBio Human CEACAM 1 ELISA Kit Protocol 6 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature J 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately VIII 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
7. onths at 2 to 8 C from the date of shipment Standard recombinant protein should be stored at 20 C or 80 C recommended at 80 C after reconstitution Opened Microplate Wells or reagents may be stored for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant pack reseal along entire edge Note the kit can be used within one year if the whole kit is stored at 20 C Avoid repeated freeze thaw cycles IV 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 8 Tubes to prepare standard or sample dilutions NAD BWN r V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Sample dilution Assay Diluent A Item D is used for dilution of serum plasma samples 1x Assay Diluent B Item E can be used for dilution of cell culture supernates urine RayBio Human CEACAM 1 ELISA Kit Protocol 3 3 Assay Diluent B should be diluted 5 fold with deionized or distilled water before use 4 Preparation of standard Briefly spin the vial of Item C and then add 400 ul Assay Diluent A for serum plasma samples or 1x Assay Diluent B for cell cult
8. tte up and down to mix gently before use HRP Streptavidin concentrate should be diluted 20 000 fold with 1x Assay Diluent B For example Briefly spin the vial Item G and pipette up and down to mix gently Add 2 ul of HRP Streptavidin concentrate into a tube with 198 ul Ix Assay Diluent B to prepare a 100 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix through and then pipette 50 ul of prepared 100 fold diluted solution into a tube with 10 ml Ix Assay Diluent B to prepare a final 20 000 fold diluted HRP Streptavidin solution VI ASSAY PROCEDURE 1 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 2 Add 100 ul of each standard see Reagent Preparation step 2 and sample into appropriate wells Cover well and incubate for 2 5 hours at room temperature or over night at 49C with gentle shaking 3 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 RayBio Human CEACAM 1 ELISA Kit Protocol 5 4 Add 100 ul of 1x prepared biotinylated antibody Reagent Preparation step 6 t
9. ure supernates urine into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 150 ul standard 50 ng ml from the vial of Item C into a tube with 350 ul Ix Assay Diluent A or 1x Assay Diluent B to prepare a 15 000 pg ml standard solution Pipette 400 ul Assay Diluent A or 1x Assay Diluent B into each tube Use the 15 000 pg ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer Gently vortex to mix Assay Diluent A or 1x Assay Diluent B serves as the zero standard 0 pg ml 150 Standard 350 ul 200u 200g 200g 200g 200 ul 200 ul 15 000 5 000 1 667 555 6 185 2 61 73 20 58 0 pg ml pg ml pg ml pg ml pg ml pg ml pg ml 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 6 Briefly spin the Detection Antibody vial Item F before use Add 100 ul of Ix Assay Diluent B into the vial to prepare a detection antibody concentrate Pipette up and down to mix gently the concentrate can be stored at 4C for 5 days The detection antibody concentrate should be RayBio Human CEACAM 1 ELISA Kit Protocol 4 diluted 80 fold with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure 7 Briefly spin the HRP Streptavidin concentrate vial Item G and pipe
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