Manual - RayBiotech, Inc.
Contents
1. or room temperature before making serial dilutions Improper dilution Check pipettes and ensure proper serial dilutions Poor standard curve Increase laser power so the highest standard Inadequate detection concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed Always use new cytokine standard vial for new cytokine standards set of experiment Discard any leftover Lower the PMT or sigmal gain eee remove wash buffer in each wash Insufficient wash Increase wash time and use more wash buffer Dust Work in clean environment a iS aOWed 1o cly Don t dry out slides during experiment 16 XIV Publications Citing This Product 1 Du Y Wei X He Y Wei G Hampel H et al P2 380 Identification and characterization of human autoantibodies that may be used for the treatment of prion diseases Alzheimer Dementia 2008 4 4 Suppl T484 Abstract P2 380 Species Human Sample Type Plasma 2 Wei X Roettger Y Tan B et al Human Anti prion Antibodies Block Prion Peptide Fibril Formation and Neurotoxicity The Journal of Biological Chemistry 2012 287 16 12858 12866 doi 10 1074 jbc M111 255836 Species Human Sample Type Conditioned Media More citations for this product may be available Contact techsupport raybiotech com XV Experiment Record Form Date File Name Laser Power PMT Sample Name Dilution factor CNTRL St2. Quantibody Human lg Isotype Array 1 Quantitative measurement of 8 human Immnunoglobulins Catalog QAH ISO 1 User Manual Last revised March 2015 Caution Extraordinarily useful information enclosed R RayBiotech ee 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 D O O O D Page 7 Additional Materials Required 3 3 5 TET VI VII General Considerations A Sample Preparation B Handling Glass Slides C Incubation VIII Protocol A Completely Air Dry The Glass Slide B Prepare Cytokine Standard Dilutions C Blocking amp Incubation D Incubation with Biotinylated Antibody Cocktail amp Wash E Incubation with Cy3 Equivalent Dye Streptavidin amp Wash F Fluorescence Detection G Data Analysis IX Array Map amp Standard Curves X Standard Concentrations X X V l FF X Experiment Record Form How To Choose A Quantibody 20 Please read the entire manual carefully before starting your experiment XV 4 4 ma N NNN o N O o1 gt ie N OO 2 l Overview Cytokines Detected 8 IgA IgD IgE IgG1 IlgG2 IgG3 IgG4 IgM See Section IX for Array Map One standard glass slide is spotted with 16 wells o
3. d with human myeloma proteins Quantitatively the relative abundance of the four subclasses in adult human serum follows IgG1 gt IgG2 gt IgG3 IgG4 which accounts for 6 98 3 8 0 56 and 0 56 mg ml respectively ll How It Works Yyy ee Antibody array support a Y Kg Y glass slide Incubation of sample amp standard protein cocktail T of 1 2 O Incubation with biotinylated antibody cocktail 7 3 1 2 hours ee aii Incubation with Cy3 equivalent dye labeled streptavidin 1 hour Scan and perform data extraction amp analysis IV Materials Provided Catalog Component Name 1 Slide Box pron ere Human lg Isotype Array 1 Lyophilized QAH ISO 1 STD Standard Mix 1 Vial 2 Vials jEr Human lg Isotype Array 1 Biotinylated A QAH ISO 1B Antibody Cocktail 1 25 ul 2 x 1 25 ul Cy3 equivalent dye conjugated QA CY3E Streptavidin 5 ul 2x5 ul QA SWD Slide Washer Dryer 1 x 30 ml Tube oonan pares Fim 4 slide kits are comprised of 2 separate 2 slide kits See Section X for detailed cytokine concentrations after reconstitution V Storage Upon receipt all components should be stored at 20 C The kit will retain activity for up to 6 months Once thawed the glass slide standard mix antibody cocktail and dye conjugated Streptavidin should be kept at 20 C All other components may be stored at 4 C The entire kit should be used within 6 months of purchase VI Additional Mat
4. d7 Std6 Std5 Std4 Std3 Std2 1 Well No Sample Name on om oe w p38 w os w ps w oe w oz w oe w S S a e e of o a a K sl g E _ JL LLL We dete XVI How to Choose a Quantibody Array Species based selection aman OAH Carne OAC Egune OAE Raon AL Function based selection Adhesion Molecule Angiogenesis Arrays Bone Metabolism Chemokine Arrays Arrays Arrays Custom Arrays Cytokine Arrays Growth Factor Arrays ae IL 1 Family Arrays ie RESPONS Inflammation Arrays Interleukin Arrays Isotyping Arrays MMP Arrays Obesity Arrays Ophthalmic Arrays ee Disease Receptor Arrays Thi Th2 Th17 Arrays Cytokine Number based selection Arrays are available in the Quantibody platform to detect 440 human 200 mouse or 67 rat proteins GLP Compliant testing services are also available To learn more about the Quantibody Antibody Array visit www RayBiotech com Quantibody Multiplex Elisa Array html Quantibody is the trademark of RayBiotech Inc This product is for research use only 2015 RayBiotech Inc
5. e signal from the well containing the highest standard concentration Std1 receives the highest possible reading yet remains unsaturated In case the signal intensity for different cytokine varies greatly in the same array we recommend using multiple scans with a higher PMT for low signal cytokines and alow PMT for high signal cytokines G Data Analysis 19 Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software GenePix ScanArray Express ArrayVision MicroVigene etc GAL files can be found here www RayBiotech com Gal Files html Need help analyzing all that data Copy and paste your data into the Q Analyzer Tool specific for this array catalog number QAH ISO 1 SW More information can be found in Section XIl Experiment 4 Image Scan A list of compatible laser scanners can be found here RayBiotech com Scanners 4 Data Extraction GenePix etc 4 Data Computation Q Analyzer software sold separately see section VIII 4 Analyze Results pg ml concentrations IX Array Map amp Standard Curves Each antibody is printed in quadruplicate horizontally Me SE SE Ae IE BE Ae aE Se A POSI T POS2 S E m C WE QM Di Gt G E WgGS IgG4 ig Concentration pg ml Signal IU Ig Concentration pg m I lig Concentration pg ml ig Concentration pg ml X Standard Concentrations After reco
6. erials Required e Benchtop rocker or orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water 1 5 ml Polypropylene microcentrifuge tubes VII General Considerations A Preparation of Samples e Blood samples should be collected by venipuncture Allow to clot naturally Undiluted samples may be stored at 2 8 C for up to 72 hours or in 20 C for longer periods Avoid repeated freezing and thawing e Sample dilution The suggested dilution for the patient sample is 1 40 000 However user may decide to use the optimum range for his own sample Dilute 1ul serum sample in 199ul sample diluent Gently mix well and then proceed with another 1 200 dilution by adding 1ul of the diluted sample to 199ul sample diluent The net dilution is 1 40 000 B Handling Glass Slides e Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only e Handle all buffers and slides with powder free gloves e Handle glass slide s in clean environment e Because there is no barcode on the slide transcribe the slide serial number from the slide bag to the back of the slide with a permanent marker before discarding the slide bag Once the slide is disassembled you might not have enough info to distinguish one slide from the other C Incubation e Completely cover array area with sample or buffer during incubation e Avoid foaming during incubation steps e Perform a
7. f identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Method list of compatible laser scanners Il 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 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 about 12 mg ml 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 lgG1 IgG2 IgG3 and IlgG4 were first demonstrated in the 1960 s by using polyclonal antisera prepared in animals immunize
8. l of sample or reagent is used 8 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of 1X Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer with H2O e Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with 1X Wash Buffer cover the whole glass slide and frame with Wash Buffer and wash at room temperature with gentle shaking for 20 min e Decant the 1x Wash Buffer from each well wash 2 times 5 min each with 150 ul of 1X Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20X Wash Buffer II with H2O Incomplete removal of the wash buffer in each wash step may cause dark spots the background signals higher than the spots D Incubation with Biotinylated Antibody Cocktail amp Wash 9 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly 10 Add 80 ul of the detection antibody cocktail to each well Incubate at room temperature for 1 2 hour Longer incubation time is preferable for higher signals and backgrounds 11 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer and then 2 times with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step E Incubation with Cy3 E
9. ll incubation and wash steps under gentle rocking or rotation e Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or lt 70 ul of sample or reagent is used e Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation VIII Protocol A Completely Air Dry The Glass Slide 1 Take out the glass slide from the box and let it equilibrate to room temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry for another 1 2 hours Incomplete drying of slides before use may cause the formation of comet tails thin directional smearing of antibody spots B Prepare Cytokine Standard Dilutions There is only one vial of standard provided in the two slide kit which is enough for making two standard curves Reconstitute the lyophilized standard within one hour of usage If you must use the standard for two different days store only the Std1 dilution at 80 TC 100ul 100ul 100ul 100u 100ul 100ul GS TSOS TSO YD Add 500u1 Sample Diluent 2001 200u 2001 200ul 200l 200n1 100ul Vial Labels Stdl Std2 Std3 Std4 Std5 Std6 Std7 CNTRL 2 Reconstitute the Cytokine Standard Mi
10. nstitution the lyophilized cytokine standard mix contains the following concentrations for each antigen included Serial standard concentration pg ml Kassab eed bl inn tne fal bad Bi al ad kail beaa bla hes Ec biel al lal aa Seal eel nl fel linc ea IgG2 549 4 938 14 815 44 444 133 333 400 000 Notes xII Quantibody Q Analyzer The Q Analyzer is an array specific Excel based program It is much more than a simple calculation macro it performs sophisticated data analysis see below for description The Q Analyzer Tool specific for this array is catalog number QAH ISO 1 SW Key features e Simplicity Easy to operate and requires no professional training With a simple copy and paste process the cytokine concentration is determined e Outlier Marking amp Removing The software can automatically mark and remove the outlier spots for more accurate data analysis e Normalization The program allows for intra and inter slide normalization for large numbers of samples e Two Positive Controls The program utilizes the two positive controls in each array for normalization e Two Analytical Algorithms Users can choose either linear regression or log log algorithms to meet their analytical needs e Two Data Outputs standard curves and digital concentration e User Intervention The program allows for user manual handling of outliers and other analytical data e Lower and Upper Limits Determination The prog
11. quivalent Dye Streptavidin amp Wash 12 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix gently 13 Add 80 ul of Cy3 equivalent dye conjugated streptavidin to each well Cover the device with aluminum foil to avoid exposure to light or incubate in dark room Incubate at room temperature for 1 hour 14 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step F Fluorescence Detection 15 16 17 18 Disassemble the device by pushing clips outward from the slide side Carefully remove the slide from the gasket Be careful not to touch the surface of the array side Place the slide in the Slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x Wash Buffer about 30 ml to cover the whole slide and then gently shake at room temperature for 15 minutes Decant Wash Buffer I Wash with 1x Wash Buffer II about 30 ml and gently shake at room temperature for 5 minutes Remove water droplets completely by gently applying suction with a pipette to remove water droplets Do not touch the array only the sides You may dry the glass slide by a compressed N2 stream Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength green channel such as Axon GenePix Make sure that th
12. ram automatically marks out the values below or above the detection range e Standard Deviation The program outputs the standard deviations of the quadruplicate spots for data accuracy e Analytical Tips Q Analyzer analysis tips are included in the program XIII Troubleshooting Guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent volumes or improper Check pipettes and ensure correct preparation dilution l EET Increase incubation time or change sample Weak Signal Short incubation time fj Ip o wangi Too low protein Lessen dilution or do not dilute sample concentration in sample Concentrate sample if necessary morasestordearki Store kit as suggested temperature Don t prap g freeze thaw the slide Bubble formed during Decrease amount of rocking shaking during incubations check for bubble formation and incubation remove bubbles Arrays are not Uneven signal completed covered by Completely cover arrays with solution for all required steps reagent Cover the incubation chamber with adhesive film Reagent evaporation poo during incubation Cross contamination Avoid overflowing wash buffer and other from neighboring wells _ solutions into neighboring wells Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard Reconstitute the lyophilized standard well at the reconstitution
13. x lyophilized by adding 500 ul Sample Diluent to the tube For best recovery always quick spin vial prior to opening Dissolve the powder thoroughly by a gentle mix Labeled the tube as Std1 3 Label 6 clean microcentrifuge tubes as Std2 to Std7 Add 200 ul Sample Diluent to each of the tubes 4 Pipette 100 ul Std1 into tube Std2 and mix gently Perform 5 more serial dilutions by adding 100 ul Std2 to tube Std3 and so on 5 Add 100 ul Sample Diluent to another tube labeled as CNTRL Do not add standard cytokines or samples to the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Since the starting concentration of each cytokine is different the serial concentrations from Std1 to Std7 for each cytokine are varied which can be found in Section X C Blocking amp Incubation 6 Add 100 ul Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides 7 Decant buffer from each well Add 100 ul standard cytokines or samples to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals This step may be done overnight at 4 We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation especially if less than 70 u
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