User Manual - RayBiotech, Inc.
Contents
1. Cytokine Capture antibody Glass Slide Support Mouse Ig Isotyping Array Q1 1 TABLE OF CONTENTS Ie OEI EE ls 1 A A di 3 LOW LE WOKS ata aorta 6 IL Materials Provided cun 7 Additional Materials Required 7 IL General Considerations iii tt 8 A Preparation of Samples 8 B Handling Glass ChipS 22 8 A ERO E 8 EM i o meres alia 9 A Complete Air Dry the Glass Chip 9 B Prepare Immunoglobulin Standard Dilutions 9 C Blocking and Incubation 10 D Incubation with Detection Antibody Cocktail 11 E Fluorescence Detection ais 11 E Dat Analy SiG a ao zen 12 V Immunoglobulin Array Map amp Standard Curves 13 VI 8 PomEsStandards er ie te edi a 14 VI System EW Sci 15 VIII Quantibody Q Analyzer iii 16 IX Troubleshooting Guide 00000 17 X Select Quantibody Publications 18 XI Experimental Record Form 19 XII How to Choose Quantibody Products Lat 20 Mouse Ig Isotyping Array Q1 2 I Introduction Immunoglobulins are the key elements of the humoral immune response in vertebrate against parasitic invasion The polypeptide chains of immunoglobulins are composed of two identical heavy H chains and two identical light L chains linked together by inter chain dis2. QAH ANG 3 QAM INT 1000 QAR CYT 3 QAM CHE 1 e 20 cytokines QAH CYT 1 QAH CYT 2 QAM CYT 1 QAM CYT 2 QAM CYT 3 e 10 cytokines QAH TH 1 QAH INF 1 QAH INF 2 QAH ANG 1 QAH MMP 1 e less than 10 cytokines QAH ISO 1 QAH ADI 2 QAP CYT 1 QAM ISO G1 Purpose based selection Custom Arrays e Choose from over 1000 cytokine pool Any kind Any number e Order slide only or full service in house e Desired marker not in our pool No problem For certain developmental fee we may be able to add the marker to your panel if the paired antibodies are available on the market Mouse Ig Isotyping Array Q1 20 Note Quantibody is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for three months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price This product is for research use only 2015 RayBiotech Inc Mouse Ig Isotyping Arr
3. Rapid Mouse Ig Isotyping Array Cat AAM ISO 1 This semi quantitative kit has following features e One step mouse monoclonal antibody isotyping e Use only 1 2 ul sample e The whole experiment can be done within 1 hour e Sandwich based technology for high specificity and sensitivity e Low system CV with high reproducibility e High throughput sample processing e Qualitative visual inspection or semi quantitative result e Processed slides can be stored for years without signal decay Mouse Ig Isotyping Array Q1 5 How It Works Array support YY Y YY o md Incubation of Sample 88681 VVith arrayed antibody i Samples 9 Supports Dye labeled i KKK x Incubation with YY Cy3 equivalent 1 hr dye labeled Ab Detection of signals ES Data analysis and graph Mouse Ig Isotyping Array Q1 I Materials Provided Upon receipt all components of the Quantibody Array kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 6 months Once thawed the glass chip Immunoglobulin standard mix and detection antibody cocktail should be kept at 20 C and all other components may be stored at 4 C The entire kit should be used within 6 months of purchase Components Item Description 1 Slide kit 2 Slide kit 1 Quantibody Array Glass Chip 1 2 2 Sample Diluent 1 1 3 20X Wash Buffer I 2 3 4 20X Wash Buffer II 1 1 5 Lyophiliz
4. Diluent to each of the tubes 4 Pipette 100ul Stdl into tube Std2 and mix gently Perform 5 more serial dilutions by adding 100ul Std2 to tube Std3 and so on Mouse Ig Isotyping Array Q1 9 5 Add 100u1 Sample Diluent to another tube labeled as CNTRL Do not add standard Immunoglobulin or samples to the CNTRL tube which will be used as negative control For best results include a set of standards in each slide Note Since the starting concentration of each Immunoglobulin is different their serial concentrations from Stdl to Std7 are varied which can be found in section VI C Blocking and Incubation 6 Add 100ul Sample Diluent into each well and incubate at room temperature for 30 min to block slides 7 Decant buffer from each well Add 100ul standard Immunoglobulins or samples to each well Incubate arrays at room temperature for 1 hour Longer incubation time is preferable for higher signals Note The sample volume can be 50 100 ul If sample volume is less than 70 ul cover the gasket with adhesive sealer to prevent evaporation during incubation This step may be done overnight at 4 C for best results Note See Section II for recommendations of sample dilutions 8 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of 1x Wash Buffer I at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer I with H O e Optional Put the gl
5. IgM IgG1 IgG2a IgG2b IgG3 lambda kappa QAM ISO 1 Standard Curves 105 104 4 Signal Intensity 108 102 10 100 10 102 108 104 105 108 Concentration pg ml Mouse Ig Isotyping Array Q1 13 V Point Standards After reconstitution of the lyophilized Immunoglobulin standard mix the 8 point Immunoglobulin concentration used for generating the standard curve of a given antigen is listed below The detection sensitivity of each protein in one experiment is user dependent Try our array specific Quantibody Q Analyzer to see your Limit of Detection LOD Section VIII Serial standard concentration pg ml pg ml Control Std7 Std6 Std5 Std4 Std3 Std2 Std1 IgA 0 137 412 1 235 3 704 11 111 33 333 100 000 IgE 0 27 82 247 741 2 222 6 667 20 000 IgM 0 1 12 37 111 333 1 000 IgG1 0 3 25 74 222 667 2 000 IgG2a 0 14 41 123 370 1 111 3 333 10 000 IgG2b 0 3 8 25 74 222 667 2 000 IgG3 0 137 412 1 235 3 704 11 111 33 333 100 000 Mouse Ig Isotyping Array Q1 14 VL System Flow Experiments y Image scan laser scanner y Data extraction GenePix etc y Data computation Q Analyzer y Final Result pg ml Mouse Ig Isotyping Array Q1 15 y 455 433 443 442 121 122 132 119 2 1 3 2 89 88 90 91 21 22 21 23 222 223 232 213 55 54 57 56 188 178 189 190 E VII Quantibod
6. Quantibody Mouse Ig Isotyping Array 1 Quantitative measurement of 7 Mouse Immnunoglobulins Patent Pending Technology User Manual Version 1 1 2015 Cat QAM ISO 1 RayBiotech Inc We Provide You With Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info Oraybiotech com Immunoglobulin Detected 7 3 Quantitative IgA IgE IgM IgG 1 IgG2a IgG2b and IgG3 Semi Quantitative IgD and two light chains x and A One standard glass slide is spotted with 16 wells of Format identical Immunoglobulin antibody arrays Each antibody is arrayed in quadruplicate Detection Method Fluorescence with laser scanner Cy3 equivalent dye Sample Volume 50 100 ul per array Sample Dilution Hybridoma Supernatant 1 10 1 1 000 Purified mouse monoclonal antibody 10 1000 ng ml Ascitic fluids Serum Plasma 1 1 000 1 100 000 Reproducibility CV lt 20 Assay duration 6 hrs 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 00000000 S See Section V For Array Map so gt Fluor dye cy3 equivalent Biotin Streptavidin complex Detect antibody
7. Wash Buffer I 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 with gentle and gently shake at room temperature for 5 minutes Mouse Ig Isotyping Array Q1 11 14 Remove water droplets completely by gently applying suction with a pipette to remove water droplets Do not touch the array only the sides Note You may dry the glass chip by a compressed N stream 15 Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength such as Axon GenePix Make sure that the signal from the well containing the highest standard concentration Std1 receives the highest possible reading yet remains unsaturated Note In case the signal intensity for different Immunoglobulin varies greatly in the same array we recommend using multiple scans with a higher PMT for low signal Immunoglobulins and a low PMT for high signal Immunoglobulins F Data Analysis 16 Data extraction can be done with most of the microarray analysis software GenePix ScanArray Express ArrayVision or MicroVigene For quantitative data analysis our Quantibody Q Analyzer software is available It gives visual output as well as digital values More information can be found in section VIII Mouse Ig Isotyping Array Q1 12 IV Immunoglobulin Array Map Standard Curves POS1 POS2 IgA IgD IgE
8. ass chip with frame into a box with 1x Wash Buffer I cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min e Decant the 1x Wash Buffer I from each well wash 2 times 5 min each with 150 ul of 1x Wash Buffer II at room temperature with Mouse Ig Isotyping Array Q1 10 gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer II with H O Note Incomplete removal of the wash buffer in each wash step may cause dark spots Background signal is higher than that of the spot D Incubation with dye labeled detection antibody and 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 dye labeled detection antibody to each well Incubate at room temperature for 1 hour Longer incubation time is preferable for higher signals and backgrounds 11 Decant the samples from each well and wash 5 times with 150 ul of 1x Wash Buffer I 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 Fluorescence Detection 12 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 13 Place the slide in the slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x
9. ay Q1 21
10. ed Immunoglobulin standard mix 1 1 6 Dye labeled detection antibody 1 2 7 Slide Washer Dryer 1 1 8 Adhesive device sealer 5 10 9 Manual 1 1 See Section VI for detailed Immunoglobulin concentrations after reconstitution Additional Materials Required e Orbital shaker e Laser scanner for fluorescence detection e Aluminum foil e Distilled water e 1 5ml Polypropylene microcentrifuge tubes Mouse Ig Isotyping Array Q1 7 II General Considerations A Preparation of Samples Sample types hybridoma supernatant purified antibodies serum plasma and ascitic fluids etc Suggested sample dilution a Hybridoma supernatant 1 10 1 1 000 b Purified mouse monoclonal antibody 10 1000 ng ml c Serum plasma and ascitic fluids 1 1 000 1 100 000 Handling glass chips Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only Handle all buffers and slides with latex free gloves Handle glass chip in clean environment 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 Completely cover array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash
11. h k wash step High E Insufficient wash Increase wash time and use more wash background buffer Dust Work in clean environment Slide is allowed to dry out Don t dry out slides during experiment Mouse Ig Isotyping Array Q1 17 IX Select Quantibody Publications Stechova et al Influence of Maternal Hyperglycaemia on Cord Blood Mononuclear Cells in Response to Diabetes associated Autoantigens Scandinavian Journal of Immunology 2009 70 2 149 158 Willingham SB et al NLRP3 NALP3 Cryopyrin facilitates in vivo caspase 1 activation necrosis and HMGB 1 release via inflammasome dependent and independent pathways J Immunol 2009 183 3 2008 15 El Karim et al Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts Journal of Endodontics 2009 35 6 829 833 Souqui re S et al T Cell tropism of simian T cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills Mandrillus sphinx J Med Primatol 2009 38 4 279 89 Sharma et al Induction of multiple pro inflammatory cytokines by respiratory viruses and reversal by standardized Echinacea a potent antiviral herbal extract Antiviral Research 2009 83 2 165 170 Altamirano Dimas et al Echinacea and anti inflammatory cytokine responses Results of a gene and protein arra
12. o Sample Name Dilution factor 1 CNTRL 2 Std7 3 Std6 El el 4 Std5 al al 5 Std4 6 Std3 sl 6 1 Std2 8 Std Ld JE 11 E d JE 14 15 16 Mouse Ig Isotyping Array Q1 19 XI How to Choose Quantibody Products Species based selection e Human QAH e Mouse QAM Rat QAR e Porcine QAP CYT 1 e Non Human Primates NHP QAN CYT 1 e Canine QAC CYT 1 e Feline QAF CYT 1 e Equine QAE CYT 1 e Bovine QAB CYT 1 e Rabbit QAL CYT 1 Function based selection e THI TH2 TH17 Array QAH TH 1 QAH TH17 QAM TH17 e Inflammation Arrays QAH INF 1 QAH INF 2 QAH INF 3 QAM INF 1 QAR INF 1 e Angiogenesis Arrays QAH ANG 1 QAH ANG 2 QAH ANG 3 QAH ANG 1000 e Chemokine Arrays QAH CHE 1 QAM CHE 1 e MMPArray QAH MMP 1 Immunoglobin Isotype Array QAH ISO 1 AAM ISO G1 e Dried Eye Disease QAH DED 1 e Periodontal Disease QAH PDD 1 e Sepsis Biomarker QAH SEP 1 Cytokine Number based selection e 440 cytokines QAH CAA 440 e 400 cytokines QAH CAA 9000 e 360 cytokines QAH CAA 8000 e 320 cytokines QAH CAA 7000 e 280 cytokines QAH CAA 6000 e 240 cytokines QAH CAA 5000 e 200 cytokines QAH CAA 4000 QAM CAA 4000 e 160 cytokines QAH CAA 3000 QAM CAA 3000 e 120 cytokines QAH CAA 2000 QAM CAA 2000 e 80 cytokines QAH CAA 1000 QAM CAA 1000 e 60 cytokines QAH ANG 1000 QAM CYT Q2000 e 40 cytokines QAH INF 3 QAH CHE 1 QAH GF 1 QAH REC 1 QAH CYT 4 e 30 cytokines QAH ANG 2
13. reagent volumes or Check pipettes and ensure correct improper dilution preparation Short incubation time Ensure sufficient incubation time and Weak Signal change sample incubation step to overnight Too low protein concentration in sample Don t make too low dilution or concentrate sample Improper storage of kit Store kit as suggested temperature Don t freeze thaw the slide Uneven signal Bubble formed during incubation Avoid bubble formation during incubation Arrays are not completed covered by reagent Completely cover arrays with solution Reagent evaporation Cover the incubation chamber with adhesive film during incubation Poor standard Cross contamination from neighboring wells Avoid overflowing wash buffer Comet tail formation Air dry the slide for at least 1 hour before usage Inadequate standard reconstitution or Improper dilution Reconstitute the lyophilized standard well at the room temperature before making serial dilutions Check pipettes and ensure proper curve serial dilutions Inadequate detection Increase laser power that the highest standard concentration for each cytokine receives the highest possible reading yet remains unsaturated Use freeze thawed cytokine standards Always use new cytokine standard vial for new set of experiment Discard any leftover Overexposure Lower the laser power Dark spots Completely remove wash buffer in eac
14. ruplicate together with two positive controls with 16 identical sub arrays in one standard glass slide The kit also provides a myeloma derived standard mixture of these 7 immunoglobulins whose concentration has been predetermined In the experiment standard immunoglobulins and samples are assayed in each well simultaneously through a sandwich like ELISA procedure The signals will be detected using fluorescence based detection method for consistency and reliability By comparing signals from unknown samples to the standard curve generated for each of the 7 immunoglobulins the unknown immunoglobulin concentration in the samples will be determined The kit provides a highly sensitive approach within nano gram range to simultaneously detect 7 immunoglobulin subclasses expression levels The experimental procedure is simple and can be performed in any laboratory This kit can be used for many applications such as Antibody producing hybridoma screening and selection Mouse monoclonal antibody heavy and light chain identification Selection and isolation of immunoglobulin isotype switch variants Mouse model immune disease research autoimmune disease allergies Ig deficiency disorders malignancies GI disorders or repeated bacterial infections etc Mouse Ig Isotyping Array Q1 4 For researchers who don t need quantitative data but only want to determine the mouse immunoglobulin isotypes a semi quantitative version 1s available Raybio
15. steps under gentle rotation 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 Several incubation steps such as step 6 blocking step 7 sample incubation or step 10 detection antibody incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation Mouse Ig Isotyping Array Q1 8 III Protocol A Completely air dry the glass chip 1 Take out the glass chip 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 at room temperature for another 1 2 hours Note Incomplete drying of slides before use may cause the formation of comet tails B Prepare Immunoglobulin Standard Dilutions Prepare serial dilution of Immunoglobulin standards 100u1 100u1 100ul 100u1 100ul 100ul IS SOs CS CS TY TY Add 500ul Sample Diluent 20011 200u1 20041 200u1 20041 20041 100ul Vial Labels Stdl Std2 Std3 Std4 Std5 Std6 Std7 CNTRL 2 Reconstitute the Immunoglobulin Standard Mix lyophilized by adding 500ul 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 200u1 Sample
16. 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 eight immunoglobulin heavy chain isotypes are found in mice IgA IgD IgE IgM and IgG with subclasses IgG1 IgG2a IgG2b and IgG3 Identification of class and subclass of immunoglobulins is essential for determination of immunochemical and functional properties Detection of specific Ig isotype is a powerful tool in the study of immunoglobulin deficiency disorders allergies autoimmune diseases malignancies GI disorders or repeated bacterial infections Meanwhile the growth and widespread use of mouse monoclonal antibody technology have created a need for a fast accurate and simple means of determining immunoglobulin class and sub class Identification is essential since chemical and biological properties of the various classes are unique They differ in their solubility and electrophoretic properties in their susceptibility to cleavage enzymes and in their reactivity with protein A Determining the class and subclass of a monoclonal antibody is thus useful in planning the best immunoglobulin purification method For example mouse IgA and IgM are best purified by size 1 e gel exclusion or using immunoaffinity separation col
17. umns Mouse IgG2a and IgG2b are purified with immobilized Protein A at pH 7 8 while Mouse IgG1 binds best to Protein A at pH 8 9 Immunoglobulin that contains kappa light chains can be purified using immobilized Protein L Quantitative measurement of the immunoglobulin subclasses can be done with Radial Immunodiffusion assay RID Nephelometry and turbidimetry assay Radio Immuno Assay RIA Immuno affnity chromatography Direct Antiglobulin Test DAT or Enzyme linked Immunosorbent Assay ELISA While most assays can detect only one subclass of the immunoglobulin a time taking advantage of the array technology and the availability of the Mouse Ig Isotyping Array Q1 3 isotype specific monoclonal antibodies Raybiotech Inc is proudly offering the research community with the Quantibody Mouse Ig Isotype kit which can simultaneously and quantitatively detect multiple immunoglobulin subclasses in one experiment Quantibody Mouse Ig Isotype Array uses sandwich ELISA based technology for quantitative measurement of the seven mouse isotype immunoglobulins IgG1 IgG2a IgG2b IgG3 IgA IgE and IgM and semi quantitative measurement of IgD and the two light chain types Similar technology has been successfully used in our other quantibody products for quantitative measurement of up to 40 cytokines in human mouse rat and porcine samples See Section XI Briefly the 7 mouse immunoglobulin subclass specific antibodies are arrayed in quad
18. y Q Analyzer Quantibody Q Analyzer is an array specific Excel based program However it is not a simple calculation macro as it contains sophisticated data analysis 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 number of samples e Two Positive Controls The program takes 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 those outliers and other analytical data e Lower and Upper Limits Determination The program 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 Mouse Ig Isotyping Array Q1 16 VII Troubleshooting guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate
19. y analysis Pharmacuetical Biology 2009 47 6 500 508 Cheung et al Cordysinocan a polysaccharide isolated from cultured Cordyceps activates immune responses in cultured T lymphocytes and macrophages Signaling cascade and induction of cytokines Journal of Ethonopharmacology 2009 124 1 61 68 Du et al P2 380 Identification and characterization of human autoantibodies that may be used for the treatment of prion diseases Alzheimer s and Dementia 2009 4 4 T484 T484 Van Rossum et al Granulocytosis and thrombocytosis in renal cell carcinoma a pro inflammatory cytokine response originating in the tumour Neth J Med 2009 67 5 191 4 10 Zhai et al Coordinated Changes in mRNA Turnover Translation and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation Molecular and Cellular Biology 2008 28 24 7414 7426 11 Gao et al A Chinese herbal decoction Danggui Buxue Tang activates extracellular signal regulated kinase in cultured T lymphocytes FEBS Letters 2007 581 26 5087 5093 This reference validates mulitplex ELISA results for several analytes with standard ELISA test results 12 Piganelli et al Autoreactive T cell responses new technology in pursuit of an old nemesis Editorial Review Pediatric Diabetes 2007 8 249 251 Mouse Ig Isotyping Array Q1 18 X Experiment Record Form IN Well N
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