User Manual - RayBiotech, Inc.
Contents
1. 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 ONOnNRWOND V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Assay Diluent Item E2 should be diluted 5 fold with deionized or distilled water before use 3 Sample dilution 1X Assay Diluent Item E2 should be used for dilution of serum and plasma samples The suggested dilution for normal serum plasma is 10 000 000 fold For example add 1 ul of serum plasma into a tube with 99 ul 1X Assay Diluent B to prepare a 100 fold diluted sample Mix thoroughly and then pipette 1 ul of prepared 100 fold diluted sample into a tube with 99 ul 1X Assay Diluent to prepare a 10 000 fold diluted sample Mix thoroughly and then pipette 1 ul of prepared 10 000 fold diluted sample into a tube with 999 ul 1X Assay Diluent to prepare a final 10 000 000 fold diluted sample Note Levels of IgG may vary between different samples Optimal dilution factors for each sample must be determined by the investigator 4 Preparation of standard Briefly spin a vial of Item C Add 4002. concentrate should be diluted 4 000 fold with 1X Assay Diluent Item E2 For example Briefly spin the vial Item G and pipette up and down to mix gently Add 3 ul of HRP Streptavidin concentrate into a tube with 12 ml 1X Assay Diluent to prepare a final 4 000 fold diluted 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 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 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 100 ul of prepared Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature
3. ul 1X Assay Diluent into Item C vial to prepare a 50 ng ml standard solution Dissolve the powder thoroughly by a gentle mix Add 180 ul IgG standard 50 ng ml from the vial of Item C into a tube with 420 ul 1X Assay Diluent to prepare a 15 ng ml standard solution Pipette 400 ul 1X Assay Diluent into each tube Use the 15 ng ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1X Assay Diluent serves as the zero standard 0 ng ml 180 ul standard 420 200l 200ul 200 ut 200 ul 200 ul 200 ul GS OS IS OWS Os Os 15 1 67 0 56 0 19 0 062 0 021 ng ml EP ng ml ng ml ng ml ng ml ng ml octal 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 Item E2 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 Item E2 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 precipitates may form during storage HRP Streptavidin
4. 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 curve must be run with each assay Assay Diluent OD 450 nm 0 1 4 0 01 r r 0 0 1 1 10 100 Human IgG co
5. RayBio Human IgG ELISA Kit Catalog ELH IGG User Manual Last revised December 7 2015 Caution Extraordinarily useful information enclosed The protein array pioneer Ke RayBiotech 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 IgG ELISA Kit Protocol Table of Contents Pose invoamion Cd sensed essere Cd V Additional Materials Required Reagent Preparation VI Assay Procedure VII Assay Procedure Summary Calculation of Results A Typical Data B Sensitivity EEE C Spiking amp Recovery D Linearity E Reproducibility Specificity Troubleshooting Guide 11 0 WMO N on A A TB ol o ae Please read the entire manual carefully before starting your experiment I INTRODUCTION The human immune system consists of two functional components classified as the innate system the physical biochemical and cellular barriers and the adaptive immune system including lymphocytes and immunoglobulins Immunoglobulins are the key elements of the humoral immune response in vertebrate against parasitic invasion The polypeptide chains of immunoglobulins composed of two identical heavy H chains and two identical light L chains linked together by inter chain disulfide bonds While the amino terminal p
6. 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 Storage Stay Stability oa micropiate tem A Microplate Item A Iu micropat tem ay wells 12 rac x 8 wells coated with anti imonha month at aor Human een Ween Suet ier 25 ml 25 mi of 20x concentrated soluton 20X concentrated solution 1 month at ft monthatas ier Item B Standard Protein temo Standard Protein temo Item C 2 vials of Human oo 1 vial is enough to run 1 jt wockat 30 at jt wockat 30 each standard in oo oe Antibody a Item 2 vials of biotinylated anti Human E T Each vial 5 5 Bdysase at 4 Bdysase is E T to assay half the microplate HRP 200 ee 4 000X concentrated HRP conjugated oe not store and Concentrate ee G ee 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 CET iaaa Assay Diluent Item E2 2 bottles of 15 ml 5X concentrated buffer 1 month at 4 C Return unused wells to the pouch containing desiccant pack reseal along entire edge
7. ncentration ng ml B SENSITIVITY The minimum detectable dose of Human IgG was determined to be 150 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 IgG into the sample types listed below Mean recoveries are as follows Sample Type Average Recovery Range Serum 116 1 105 123 Plasma 118 1 99 126 D LINEARITY Sample Type Serum Plasma 1 2 Average of Expected 111 3 123 Range 101 122 104 121 1 4 Average of Expected 113 2 114 4 103 122 105 120 Range E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY The sandwich ELISA antibody pair detects Human IgG 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 insufficiently 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 sufficien
8. ortions that exhibits highly variable amino acid composition are involved in antigen binding the C terminal constant parts are involved in complement binding placental passage and binding to cell membranes Based upon the variation of the constant region of the heavy chain nine immunoglobulin heavy chain isotypes are found in humans IgA with subclasses IgA1 and IgA2 IgD IgE IgM and IgG with subclasses IgG1 IgG2 IgG3 and IgG4 IgG is the predominant immunoglobulin in the serum which accounts for 75 of the total serum antibody of healthy individuals IgG has a molecular weight of about 150 kDa Four distinct subgroups of human IgG IgG1 IgG2 IgG3 and lgG4 were first demonstrated in the 1960s by using polyclonal antisera prepared in animals immunized with human myeloma proteins The RayBio Human IgG ELISA kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human IgG in serum and plasma This assay employs an antibody specific for human IgG coated on a 96 well plate Standards and samples are pipetted into the wells and IgG present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human IgG 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 IgG bound
9. t 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
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