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1. III General Considerations ueuuuuuue A Preparation of Samples B Handling Glass Chips ueeeeee o Dic UD IO To ose vates bod een PONI E areaneontroeee es IVs POCO once ento mte uda EE RDUM A Complete Air Dry the Glass Chip Be Sample IncubattOn acis u a vip dUR Pese AMA C Inor sce nce Detection aester FREU PREVEDE ECVPE IB BEISPIEL V Specificity amp Accuracy sssssesssesse eese WIL Assay SOUSIUVIDV cocos arara EEEE tom M RE MIEL Typical RESU S sex sconti tap noo ERES VIII Raybio Rapid Rat Ig Isotyping Analyzer IX Troubleshooting Guide Luueeeeeusee X Experimental Record Form Xl Laser Scanners for Glass Slide Arrays Raybio Rapid Rat Ig Isotyping Array 2 Un ut U O ND o o oco NQ N Oc p pi pb pb Lm NO MO Uu N e e O Introduction Immunoglobulins are the key elements of the vertebrate humoral immune response against pathogen invasion and infection The polypeptide chains of immunoglobulins are 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 exhibit highly variable amino acid composition are involved in antigen binding the C terminal constant regions are involved in complement binding plac
2. Detection Output 16 bit TIFF Recommended Scanners e GenePix 4000A Molecular Devices Corporation GenePix 4000B Molecular Devices Corporation GenePix 4100A Molecular Devices Corporation GenePix Professional 4200A Molecular Devices Corporation ScanArray 9 Lite PerkinElmer Inc ScanArray Express PerkinElmer Inc ScanArray Express HT PerkinElmer Inc LS Series Laser Scanner Tecan Group AG AlphaScan Microarray Scanner Alpha Innotech The DNAscope LM Biomedical Photometrics Inc The DNAscope IV amp V Biomedical Photometrics Inc Open Frame DNAscope Biomedical Photometrics Inc Revolution 4200 Microarray Scanner VIDAR Systems Co aQuire 110V Genetix aQuire 240V Genetix VersArray ChipReader 5um System Bio Rad VersArray ChipReader 3um System Bio Rad InnoScan 710 Microarray Scanner Innopsys InnoScan 900 Microarray Scanner Innopsys Compatible Scanners e NovaRay Detection Platform Alpha Innotech e arrayWoRx Applied Precision LLC e GSD 501 System Calibration Kit Invitrogen Please note that this is not an exhaustive list In general most gene microarray scanners should be compatible as long as they have a Cy3 green channel pixel resolution of 10um and able to scan a standard histology slide Raybio Rapid Rat Ig Isotyping Array 16 This product 1s for research use only 2014 RayBiotech Inc 3607 Parkway Lane Suite 200 Norcross GA 30092 Tel 770 729 2992 1 888 494 8555
3. Fax 770 206 2393 Web www raybiotech com Raybio Rapid Rat Ig Isotyping Array 17
4. 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 1s 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 there might not be enough information to distinguish one slide from another C Incubation Completely cover array area with sample or buffer during incubation Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Incubation can be done at room temperature for hr or 30 min at BTC Raybio Rapid Rat Ig Isotyping Array 8 III Protocol e 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 e Sample Incubation 2 Add 1 7 ml Sample Diluent into the detection antibody cocktail tube Spin briefly 3 Based upon the sample numbers aliquot 100 ul of the detection antibody to corresponding number of wells in the 96 well plate 4 Add 1 ul of each sample to each well mix well through pipetting Note This dilution
5. 1 IgG2a IgG2b and IgG2c will generally have much stronger signals than the low abundant group IgA and IgM In general the light chain is generally more dominant than chain Hepresentative Images IgA K IgM IgG2a X lgG1 x POS lgG1 X Lambda IgG2b IgG2c IgG2a Raybio Rapid Rat Ig Isotyping Array 12 VII Raybio Rapid Rat Ig Isotyping Analyzer Raybio Rapid Rat Ig Isotyping Analyzer is an Excel based program specifically designed for this product It facilitates the semi quantitative data analysis as well as output the final sample isotypes With a simple copy and paste process the sample Ig 1sotype 1s determined Semi quantitative Data Output lA E6066 782 336 396 315 369 320 326 218 313 Hd pe es pes pen ps pne Ds a 88 3 8 Err ss e12 Emme es a 872 Ties p sn pem p zs de poss e ce Teac em p ee ie p 7e ions T 487 uses eoo o0 am arr Sample lg Isotypes Raybio Rapid Rat Ig Isotyping Array 13 VIII Troubleshooting guide Inadequate detection Increase laser power and PMT parameters improper dilution preparation Short incubation time Ensure sufficient incubation time or increase Weak Signal sample incubation step to overnight sample freeze thaw the slide Bubble formed during incubation Avoid bubble formation during incubation Arrays are not completed covered by Completely cover arrays with solution reagent Uneven
6. Raybio Rapid Rat Ig Isotyping Array One step determination of 6 Rat Immunoglobulin sub classes and 2 light chain types in one experiment Patent Pending Technology User Manual Version Sept 2014 Cat AAR ISO G1 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 raybiotech com I Overview Immunoglobulin Heavy Chains 6 IgA IgM IgG1 IgG2a IgG2b IgG2c Detected Light Chains 2 Kappa i Lambda A One standard glass slide 1s spotted with 16 wells of Format identical Immunoglobulin antibody arrays Each Ig specific antibody 1s arrayed in quadruplicate Detection Method Fluorescence with laser scanner Cy3 equivalent dye sample Volume 1 2 ul Hybridoma Supernatant 1 10 1 1 000 Sample Dilutions Purified rat monoclonal antibody 10 1000 ng ml Ascitic fluids Serum Plasma 1 10 000 1 1 000 000 Reproducibility CV 1090 Rat Ig Array Map POSI POS2 EN IgGl IgG2a IgG2b IgG2c Raybio Rapid Rat Ig Isotyping Array I TABLE OF CONTENTS ee WON CIVIC rc Tritt OCC D ODD a aec dut tot IN EEUEH PEU NO ORLEANS Research Applications ccccee cece eee eeaees IT Deque S iret aceite caeenaw aaah ENS ERU Sew Asean ENDS HOw IO WW OUKS seiretan Ea Ih Materials Provided ia err roten E TER EER hei terna Additional Materials Required
7. ayBio Rapid Rat Ig Isotyping Array uses sandwich ELISA based technology for determination of the six rat immunoglobulin sub classes IgA IgM IgGl IgG2a IgG2b and IgG2c and the two light chain types Briefly the 6 rat immunoglobulin subclass and 2 light chain specific antibodies are arrayed in quadruplicate together with two positive controls with 16 Raybio Rapid Rat Ig Isotyping Array 3 identical sub arrays in one standard glass slide Sixteen samples can be assayed in one slide simultaneously through a sandwich ELISA procedure While the traditional sandwich based assay is time consuming and contains multiple steps such as blocking sample incubation addition of detection antibody and signal generation through fluorescence or chemiluminescence detection methods our one step rat Ig isotyping kit uses an innovative one step strategy which greatly simplifies the whole procedure while retaining similar detection specificity and sensitivity In this system samples are first mixed with the Cy3 equivalent dye labeled detection antibody and then applied to the array After washing away the non specific signals the slides can then be visualized with a laser scanner Results can be evaluated qualitatively by visual inspection or semi quantitatively through data extraction The kit provides a highly sensitive approach within nanogram range for simultaneous detection of 6 immunoglobulin subclasses and the 2 light chain types in cell cultur
8. e supernatants or from purified rat monoclonal antibodies Because rat serum plasma and ascites fluids contain all the six Ig isotypes this assay may be used for disease associated isotype profiling of these sample types The abundance of each isotype in each sample can be determined semi quantitatively The experimental procedure is simple and can be performed in any laboratory For researchers who want to quantify each Ig isotype in the rat serum or plasma a quantitative version of this kit is available Cat QAR ISO 1 Raybio Rapid Rat Ig Isotyping Array 4 Research Applications e Antibody producing hybridoma screening and selection e Rat monoclonal antibody heavy and light chain identification e Selection and isolation of immunoglobulin isotype switch variants e Rat model immune disease research autoimmune disease allergies Ig deficiency disorders malignancies GI disorders or repeated bacterial infections etc Kit Features e One step rat monoclonal antibody isotyping e Requires only 1 2 ul sample Entire experiment can be done within 1 hour e Sandwich based technology allows for high specificity and sensitivity e Low system CV with high reproducibility e High throughput sample processing e Processed slides can be stored for years without signal decay e Qualitative visual inspection or semi quantitative result A quantitative version of this kit is available with the Cat QAR ISO 1 Raybio Rapid Rat Ig Isotypi
9. ental and intestinal passage and binding to cell membranes Based upon the variation of the constant region of the heavy chain six immunoglobulin heavy chain isotypes are found in rats IgA IgM IgG1 IgG2a IgG2b and IgG2c 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 rat 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 rat IgA and IgM are best purified by size 1 e gel exclusion or using immunoaffinity separation columns Rat IgG2a and IgG2b are purified with immobilized Protein A at pH 7 8 while rat IgG1 binds best to Protein A at pH 8 9 Immunoglobulin that contains kappa light chains can be purified using immobilized Protein L The R
10. is optimized for hybridoma supernatant and purified rat monoclonal antibody For serum plasma ascites add lul sample to 999ul Sample Diluent to make a 1000x diluted sample first then use I ul for sample testing 5 Transfer 100 ul of mixed samples to the appropriative wells on the glass slide Incubate at room temperature for 1 hour or 30 min in 37 C 6 Wash Decant the samples from each well and wash 5 times with 1x Wash Buffer I at room temperature Rinse briefly and then completely remove wash buffer in each wash step Dilute 20x Wash Buffer I with distilled water Raybio Rapid Rat Ig Isotyping Array 9 e Fluorescence Detection 7 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 Optional Place the slide in the slide Washer Dryer add enough 1x 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 8 Rinse briefly with distilled water and completely remove water droplets through gentle suction with a pipette Do not touch the array only the sides 9 Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength such as Axon GenePix List of specifications and compatible scanners can be found in Section XI e Data Analysis 10 Results can be evaluated qualitatively by visual i
11. ng Array 5 How it works Dye labeled detection Ab KKKK N Mix detection Ab and Array support AAN samples then incubate with arrayed antibody for 1hr Y Adi MM UA Detection of signals hr Data analysis and graph Samples Raybio Rapid Rat Ig Isotyping Array 6 I Materials Provided Upon receipt all components of the One Step Rat Ig Isotyping kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 12 months Components Item Description 1 Slide kit 2 Slide kit Rat Ig Isotyping Glass Chip sample Diluent 20X Wash Buffer I Detection Antibody Cocktail slide Washer Dryer 96 well Plate Manual Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Distilled water Microcentrifuge Raybio Rapid Rat Ig Isotyping Array 7 II General Considerations A Sample Preparation Sample types This is an Ig isotyping assay for hybridoma supernatant and other purified antibodies Since serum plasma and ascitic fluid contain all six Ig isotypes this kit can be used for monitoring the relative Ig isotype abundance through semi quantitative data analysis Sample dilution a Hybridoma supernatant 1 10 1 1 000 b Purified rat monoclonal antibody 10 1000 ng ml c Serum plasma and ascitic fluids 1 10 000 1 1 000 000 B Handling glass chips Do not touch the surface of the slides as the microarray slides are
12. nspection or semi quantitatively through data extraction with most microarray analysis software GenePix ScanArray Express ArrayVision or MicroVigene Our array specific Raybio Rapid Rat Ig Isotyping Analyzer software is available for one step data computation It outputs digital values as well as isotype names Raybio Rapid Rat Ig Isotyping Array 10 IV Specificity amp Accuracy The rat isotype specific capture antibodies in the kit have been tested to react only with their own isotype and do not react with other rat Ig isotypes up to 1 ug ml The accuracy of the kit is further confirmed by isotype predetermined rat monoclonal antibody controls pgs umpa amide fanis Koppe Kappe kappa m e lambda kappa kappa 28 IgG2c 449 484 380 660 2318 1347 V Assay Sensitivity The detection sensitivity for each rat Ig heavy or light chain has been determined with purified rat immunoglobulin antigens to be in the nano gram range see following Ig Isotype Sensitivity ng ml Raybio Rapid Rat Ig Isotyping Array 11 VI Typical Results In a typical set of results for hybridoma supernatants or purified monoclonal antibodies except for the positive controls only one of the six heavy chains and one of the light chains will show positive signals in each array see following examples For rat serum plasma or ascitic fluids since it contains all the six isotypes the most abundant isotypes IgG
13. signal Reagent evaporation Cover the incubation chamber with adhesive film during incubation Air dry the slide for at least 1 hour before usage Impure sample Use serum plasma or ascites free samples Use fresh culture supernatant or purified antibody Hybrodoma sample contains two or Reclone hybridoma cells by limited dilution more cell lines polyclonal antibodies Sample too concentrated Increase dilution of supernatant samples For purified antibodies the final concentration should be lower than 1 ug ml w ae is secreting antibody Overexposure Decrease Decrease the laser power laser Decrease the laser power Dark spots Completely remove wash buffer after each Hich wash step 18 Insufficient wash Increase wash time and use more wash background buffer Work in clean environment Slide is allowed to dry out Don t dry out slides during experiment Multiple heavy chains are detected Raybio Rapid Rat Ig Isotyping Array 14 IX Experiment Record Form Date File Name Laser Power PMI Raybio Rapid Rat Ig Isotyping Array 15 X Laser Scanners for Glass Slide Arrays Specifications Standard Glass Slide 1 x 3 25 mm x 75 mm microscope slides Thickness 1 mm Light and Detector Orientation Facing array Scanned Area 22 mm x 73 mm Focus Auto focus or adjustable 200 um Excitation Cy3 Green Channel 532 nm Resolution 10 um Dynamic Range gt 3 orders of magnitude

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