Human sICAM-1ELISA Kit（KT20353） User Manual
Contents
1. Human sICAM 1ELISA Kit KT20353 gt User Manual For research use only Not intended for diagnostic testing gt warinontec A ABGENT IL TIL IV VI VII VIII xe MOO Pp TABLE OF CONTENTS Introduction nn 2 Reagents nasse 2 A ER REEN HERR IRERUEEBEREREREREREIR 3 Additional Materials Required 3 Reagent Preparation 222scseseeeeeeeee een 4 Assay Procedure rad 6 Assay Procedure Summary 7 Calculation of Results Typical Data on tree 8 Sensilivity 2 an 2 ita 8 RECO A HIRTEN 8 Linearity nr ans Uae 9 Reproducibility ooooooooooconconcononconocnncnoss 9 Specificity i 9 Troubleshooting Guide oooooccocconconononnonoso 11 I INTRODUCTION sICAM 1 soluble Intercellular Adhesion Molecular 1 has been reported in serum cerebrospinal fluid and bronchoalveolar lavage ICAM 1 expression is weak on leukocytes epithelial and resting endothelial cells ICAM 1 is a ligand for LFA 1 CD11a CD18 and Mac 1 CD11b CD18 Its expression is up regulated upon stimulation by inflammatory mediators such as cytokines and LPS The Human sICAM 1 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human soluble ICAM 1 in serum plasma cell culture supernatants and urine This assay employs an antibody specific for human sICAM 1 coated on a 96 well plate Standards and samples are pipetted into the wells a2. Problem Cause Solution 1 Poor standard curve 1 Inaccurate pipetting 2 Improper standard dilution Check pipettes 2 Ensure a brief spin of Item C and dissolve the powder thoroughly bya gentle mix 2 Low signal 1 Too brief incubation times Ensure sufficient 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 1 Review the manual washed for proper wash If using a plate washer check that all ports are unobstructed 2 Contaminated wash 2 Make fresh wash buffer buffer 5 Low sensitivity 1 Improper storage of the 1 Store your standard ELISA kit at lt 20 C after reconstitution others at 4 C Keep substrate solution protected from light 2 Stop solution 2 Stop solution should be added to each well before measure Note This product is for research use only aR 18 USA Abgent Inc Toll Free 888 735 7227 Or 858 875 1900 info_us abgent wuxiapptec com CHINA Abgent Suzhou 86 512 69369088 sales abgent wuxiapptec com EUROPE Abgent Europe 44 0 1235 854042 eurosales abgent wuxiapptec com For other countries www abgent com
3. 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 store 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 OAANNABRWN 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 V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Sample dilution If your samples need to be diluted Assay Diluent A Item D should be used for dilution of serum plasma samples Assay Diluent B Item E should be used for dilution of culture supernatants and urine Suggested dilution for normal serum plasma 5 200 fold Please note that levels of the target protein may vary between different specimens Optimal dilution factors for each sample must be determined by the investigator 3 Assay Diluent B should be diluted 5 f
4. DATA These standard curves are for demonstration only A standard curve must be run with each assay Assay Diluent A Assay Diluent B 10 10 14 1 o 2 8 Y Q Q O o1 O 01 0 01 r r 0 01 0 1 1 10 100 0 1 1 10 100 Human sICAM 1 concentration ng ml Human sICAM 1 concentration ng ml B SENSITIVITY The minimum detectable dose of sICAM 1 is typically less than 150 pg ml C RECOVERY Recovery was determined by spiking various levels of human sICAM 1 into human serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 97 48 88 106 Plasma 98 67 90 107 Cell culture media 97 37 89 107 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 98 97 96 Range 91 106 89 105 89 106 1 4 Average of Expected 97 95 96 Range 89 106 88 105 90 107 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Cross Reactivity This ELISA kit shows no cross reactivity with any of the cytokines tested e g human Angiogenin BDNF BLC ENA 78 FGF 4 IL lo IL 1 IL 2 IL 3 IL 4 IL 5 IL 7 IL 8 IL 9 IL 10 IL 11 IL 12 p70 IL 12 p40 IL 13 IL 15 IL 309 IP 10 G CSF GM CSF IFN y Leptin MCP 1 MCP 2 MCP 3 MDC MIP la MIP 1 B MIP 16 PARC 9 PDGF RANTES SCF TARC TGF TIMP 1 TIMP 2 TNF a TNF TPO VEGF X TROUBLE SHOOTING GUIDE
5. epeat the wash as in step 3 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 Discard the solution Repeat the wash as in step 3 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 VI ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed y 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C y 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 y 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 standard concentration on the x axis and absorbance on the y axis Draw the best fit straight line through the standard points A TYPICAL
6. n step 4 of Part VI Assay Procedure 7 Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use HRP Streptavidin concentrate should be diluted 340 fold with 1x Assay Diluent B For example Briefly spin the vial Item G and pipette up and down to mix gently Add 25 ul of HRP Streptavidin concentrate into a tube with 8 5 ml 1x Assay Diluent B to prepare a 340 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well 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 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 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 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 Discard the solution R
7. nd sICAM 1 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human sICAM 1 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin 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 sICAM 1 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm Il REAGENTS 1 sICAM 1 Microplate Item A 96 wells 12 strips x 8 wells coated with anti human sICAM 1 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials recombinant human sICAM 1 4 Assay Diluent A Item D 30 ml 0 09 sodium azide as preservative For Standard Sample serum plasma diluent 5 Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture medium urine diluent W Detection Antibody sICAM 1 Item F 2 vial of biotinylated anti human sICAM 1 each vial is enough to assay half microplate HRP Streptavidin Concentrate Item G 200 ul of 340x concentrated HRP conjugated streptavidin TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution Stop Solution Item I 8 ml of 0 2 M sulfuric acid IH STORAGE May be stored for up to 6 months at 2
8. old with deionized or distilled water 4 Preparation of standard Briefly spin the vial of Item C and then add 750 ul Assay Diluent A for serum plasma samples or 1x Assay Diluent B for cell culture medium and urine into Item C vial to prepare a 20 ng ml standard Dissolve the powder thoroughly by a gentle mix Pipette 250 ul Assay Diluent A or 1x Assay Diluent B into each tube Use the 20 ng 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 The 20 ng ml standard in Assay Diluent B may be saturated we recommend 10 ng ml serves as starting point the highest standard point for Assay Diluent B Standard Item C 750 ul 250u1 2501 25041 2504 250ml 2504 20 10 5 2 5 1 25 0 625 0 313 0 ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng 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 pl of 1x 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 4 C for 5 days The detection antibody concentrate should be diluted 80 fold with 1x Assay Diluent B and used i
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