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RayBio® Human IgG1 ELISA Kit

1. 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 lt 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 IgG1 ELISA Kit Protocol 11 RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over 500 list visit www raybiotech com for details RayBio Human IgG1 ELISA Kit Protocol 12 This product is for research use only 2004 RayBiotech Inc RayBio Human IgG1 ELISA Kit Protocol 13
2. 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 0 1 1 10 100 Human IgG1 concentration ng ml B SENSITIVITY The minimum detectable dose of IgG1 is typically less than 0 05 ng ml RayBio Human IgG1 ELISA Kit Protocol 8 C LINEARITY Sample Type 12 Average of Expected Range 1 4 Average of Expected Range D REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Serum 88 77 97 86 75 96 Plasma 82 72 94 81 71 92 Detect human IgG1 No cross reactivity was found againt IgG2 IgG3 IgG4 IgM IgA IgD and IgE X REFERENCES 1 Thorpe R C A R Mire sluis and M Wadhwa 2001 Immunoglobulin Standardization In Immunoglobulin Reference Volume 1 Ligands Oppenheim J J Feldmann M Durum S K Hirano T Vilcek J and Nicola N A eds Academic Press San Diego CA pp83 91 2 Biological Reference Materials 2000 National Institute for Biological Standards and Control RayBio Human IgG1 ELISA Kit Protocol 3 Harlow E and Lane D 1999 Using Antibodies A Laboratory Manual Cold Spring Harbor Laboratory 4 Barry R and Soloviev M 2004 Quantitative protein profiling using antibody arrays Proteomics 4 3717 3726 RayBio Human IgG1 ELISA Kit Protocol 10 XI TROUBLESHOOTING GUIDE Problem Cause Solution
3. DDITIONAL MATERIALS REQUIRED 1 Microplate reader capable of measuring absorbance at 450 nm 2 Precision pipettes to deliver 2 ul to I ml volumes 3 Adjustable 1 25 ml pipettes for reagent preparation 4 100 ml and I liter graduated cylinders RayBio Human IgG1 ELISA Kit Protocol 3 5 Absorbent paper Distilled or deionized water 7 Log log graph paper or computer and software for ELISA data analysis 8 Tubes to prepare standard or sample dilutions ON V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Sample dilution 1x Assay Diluent Item E should be used for dilution of serum plasma samples Suggested dilution for normal serum plasma 10 000 000 fold For example add I ul of serum plasma into a tube with 99 ul Ix Assay Diluent to prepare a 100 fold diluted sample Mix through and then pipette I ul of prepared 100 fold diluted sample into a tube with 99 ul Ix Assay Diluent to prepare a 10 000 fold diluted sample Mix through and then pipette I ul of prepared 10 000 fold diluted sample into a tube with 999 ul Ix Assay Diluent to prepare a final 10 000 000 fold diluted sample 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 should be diluted 5 fold with deionized or distilled water before use 4 Preparation of standard Briefl
4. RayBio Human IgG1 ELISA Kit User Manual Revised Mar 1 2012 RayBio Human IgG1 ELISA Kit Protocol Cat ELH IGG1 001 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 Ray Biotech Inc RayBio Human IgG1 ELISA Kit Protocol TABLE OF CONTENTS MR bel ea EE EE TL REASONS ccc ccsedavinaciwesssesdatacsseonwaseseneeanten Pre IV Additional Materials Required V Reagent Preparanom sc cchovasevsseccaamnetuaweensee VI Assay PROCedUte sc cncyuuscospraarisercanteuonendiene 6 VII Assay Procedure Summary 00eee eee VIII Calculation of Results A Veldre B S lve as C Linearity EE EEE D Repode bl ct ccestcasscoesedccsavies beis IX Je 9 Reverse XI Troubleshooting Guide aaannnnnnnnnnnnennerrnn 11 RayBio Human IgG1 ELISA Kit Protocol 1 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 dis
5. efore use HRP Streptavidin concentrate should be diluted 2 000 fold with 1x Assay Diluent For example Briefly spin the vial Item G and pipette up and down RayBio Human IgG1 ELISA Kit Protocol 5 to mix gently Add 6 ul of HRP Streptavidin concentrate into a tube with 12 ml Ix Assay Diluent to prepare a final 2 000 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 I hour at room temperature with gentle shaking Discard the solution Repeat the wash as in step 3 Add 100 ul of prepared Streptavidin solution see Rea
6. gent 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 RayBio Human IgG1 ELISA Kit Protocol 6 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 VIL ASSAY PROCEDURE SUMMARY l Oo P Nn Prepare all reagents samples and standards as instructed J Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C J Add 100 ul prepared biotin antibody to each well Incubate 1 hour at room temperature U Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature J Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature J Add 50 ul Stop Solution to each well Read at 450 nm immediately RayBio Human IgG1 ELISA Kit Protocol 7 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
7. n is added to the wells and color develops in proportion to the amount of IgG1 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Human IgG1 ELISA Kit Protocol 2 I REAGENTS 1 IgG1 Microplate Item A 96 wells 12 strips x 8 wells coated with anti human IgG1 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution 3 Standards Item C 2 vials of human IgG1 4 Assay Diluent Item E 2 bottles of 15 ml 5x concentrated buffer For Sample and reagent diluent 5 Detection Antibody IgG Item F 2 vial of biotinylated anti human IgG each vial is enough to assay half microplate 6 HRP Streptavidin Concentrate Item G 200 ul 2 000x concentrated HRP conjugated streptavidin 7 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffer solution 8 Stop Solution Item I 8 ml of 0 2 M sulfuric acid HI STORAGE May be stored for up to 6 months 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 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 A
8. ulfide bonds While the amino terminal portions 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 IgG4 were first demonstrated in the 1960 s by using polyclonal antisera prepared in animals immunized with human myeloma proteins The RayBio Human IgG1 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human IgG1 in serum and plasma This assay employs an antibody specific for human IgG1 coated on a 96 well plate Standards and samples are pipetted into the wells and IgG1 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 solutio
9. y spin the vial of Item C and then add 600 ul 1x Assay Diluent into Item C vial to prepare a 300 ng ml stock standard solution Dissolve the powder thoroughly by a gentle mix RayBio Human IgG1 ELISA Kit Protocol 4 Add 80 ul IgG1 standard 300 ng ml from the vial of Item C into a tube with 520 ul 1x Assay Diluent to prepare a 40 ng ml standard Pipette 400 ul Ix Assay Diluent into each tube Use the stock 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 80 ul standard 200 ul 200 ul 520 ul 200ul 200ul 200ul 200 ul m H Perr E 40 13 3 4 44 1 48 0 49 0 16 0 05 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 ul of 1x Assay Diluent 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 and used in 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 b

RayBio® Human IgG1 ELISA Kit

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RayBio&reg; Human IgG1 ELISA Kit

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RayBio® Human IgG1 ELISA Kit