RayBio Human Allergen Protein Array G2

Transcription

1 RayBio Human Allergen Protein Array G2 Glass-chip Based Allergen Protein Array User Manual (Revised Jul 26, 2017) For simultaneous detection of human IgE and IgG associated with 22 common allergens in human serum samples, including foods, animals, mites, fungi (molds), insects, and pollen from trees, grass and weeds. RayBio Allergen Protein Array G2 (Cat. # PAH-AGN-G2) RayBio Custom Allergen Protein Array (Cat. # PAH-CUST-G) RayBio Allergen Protein Array Service (Cat. # PAH-AGN-G2-SERV) Please read manual carefully before starting experiment For research use only. Not for diagnostic or therapeutic use. Tel: , Fax: Website: info@raybiotech.com RayBiotech, Inc. We Provide You with Excellent Protein Array Systems and Service

2 Human Allergen Protein Array Manual RayBio Human Allergen Protein Array G1 TABLE OF CONTENTS I. Introduction II. Materials Provided 6 Additional Materials Required 7 III. Overview and General Considerations 8 1. Preparation of Samples Handling of Glass Arrays 8 3. Incubation Layout of Human Glass Arrays Incubation Chamber Assembly...10 IV. Protocol Blocking and Sample Incubation Biotin-conjugated Anti-human IgE/IgG Incubation Cy3-conjugated Streptavidin Incubation Fluorescence Detection Data Analysis.. 16 V. Array List VI. Array Map.. 19 VII. Troubleshooting Guide VIII. Reference List RayBio is the trademark of RayBiotech, Inc. 2

3 I. Introduction Human Allergen Protein Array Manual RayBio Allergen Protein Arrays are a series of allergen identification products developed by RayBiotech, Inc., The Protein Array Pioneer Company. Recombinant allergen proteins or extracted native allergen proteins are spotted onto a solid glass slide surface, and then probed for the presence of serum specific antibodies to these allergen targets. This detection kits can then be applied in screening specific allergens based on IgE and/or IgG levels present in patient serum samples. Custom allergen protein arrays are also available from RayBiotech, Inc. You can select your own proteins of interest or provide your own allergen proteins to RayBiotech, Inc. After we receive your custom panel, we produce this tailored protein arrays for you. How it works The microarray-based allergen detection is the foundation of high throughput allergen screening in human samples. In this assay (see the flow chart on the next page), the allergen proteins are arrayed on glass slide surface in triplicate to create the allergen array including the positive and negative controls. The serum samples are then incubated on the allergen array, allowing serum IgE and IgG against these allergens to specifically bind. After comprehensive washing steps, the arrays are incubated with biotin-conjugated anti-human IgE and/or IgG, followed by addition of the fluorescence dye Cy3-conjugated streptavidin. The target fluorescence is then captured using a laser scanner and the presence of serum IgE and IgG specific to target allergens can be evaluated. 3

4 Human Allergen Protein Array Manual How RayBio Allergen Protein Arrays Works Applications This array kit can be used to test the allergens reactivity to human IgE and IgG in serum samples and find out the potential allergens based on IgE and IgG levels. 4

5 Features of RayBio Protein Arrays Human Allergen Protein Array Manual Low sample consumption: as little as 25 µl of original sample required per array. Fully customizable: create a custom array from our list of targets. High sensitivity: both biotin-streptavidin pair and fluorescent detection enable the most sensitive assay available to measure serum IgE and IgG. High efficiency and accuracy: high throughput screening of multiple allergens in a single assay. Each slide can test up to 16 samples simultaneously, and contains internal positives to normalize between slides, thereby minimizing the variation from assay to assay. Additionally, the assay duration is less than 8 hours. Large dynamic range of detection (4 orders of magnitude) with highly accurate data that can be normalized between arrays. Affordable and simple to use. 5

6 II. Materials Provided Human Allergen Protein Array Manual Array Kit Components Item Component 4 sample kit 16 sample kit A B D E F G H I J K L M Allergen Protein Array, Glass Slides 1,000 X Biotin-conjugated Anti-Human IgG, 1.5 μl/vial 1,000 X Biotin-conjugated Anti-Human IgE, 1.5 μl/vial 1,000 x Cy3-Conjugated Streptavidin, 1.5 μl/vial Blocking Buffer 8 ml/bottle 20 X Wash Buffer I 30 ml/bottle 20 X Wash Buffer II 30 ml/bottle Adhesive Plastic Strips 30-mL Centrifuge Tube User Manual Target List Array Map 1 slide (4 sub-arrays) 1 slide (16 sub-arrays) 1 vial 2 vials 1 vial 2 vials 1 vial 2 vials 1 bottle 1 bottle 1 bottle 1 strip 1 tube Please download online ( ) Please download online ( ); Manual, Section V Please download online ( ); Manual, Section VI Notes: Items B, D & E: dilute with Blocking Buffer (Item F) prior to use. Items G & H: dilute with distilled water prior to use. Storage Upon receipt, all components of array kit should be stored at -20 ºC to -80 ºC until just before the experiment. At -20 ºC to -80 ºC 6

7 Human Allergen Protein Array Manual the kit will retain complete activity for up to 6 months. Please use within six months of purchase. Once thawed, protein array glass slide (Item A) and Blocking Buffer (Item F) should be kept at -20 ºC and all other components (Items B, D, E, G, & H) should be stored at 4 ºC. Use within three months after reagents have been thawed. Additional Materials Required Distilled water Aluminum foil Small plastic boxes or containers Pipettors, pipette tips and other common lab consumables Orbital shaker or oscillating rocker Laser scanner for fluorescence detection 7

8 III. Overview and General Considerations Human Allergen Protein Array Manual 1. Preparation of Samples Optimal sample dilutions and amounts will need to be determined empirically by each experimenter, but 50 to 200-fold dilution may be used as a starting point. Blocking Buffer (Item F) can be used to dilute samples if necessary, but PBS or other buffers may be used as diluters as well. Normalize samples by loading equal amounts of normal serum or equal dilutions onto the array(s). Optimization of experimental conditions: If you experience high background, you may need to further dilute your sample and/or to wash slides in Wash Buffer I (Item G) overnight at 4 C. If the signal is too weak, you may increase the amount of your sample and/or increase incubation times of one or more steps. 2. Handling Glass Arrays The microarray slides are delicate. Do not touch the array surface with pipette tips, forceps or your fingers. Hold the slides by the edges only. Handle the slides with powder-free gloves and in a clean environment. Remove reagents/sample by gently applying suction with a pipette to corners of each chamber (see picture, right). Do not touch the printed area of the array, only the sides of the chamber assembly. 8

9 3. Incubation Human Allergen Protein Array Manual Completely cover array area with sample or buffer during incubation steps. Cover the incubation chamber with adhesive strips (Item I) or plastic sheet protector during incubation to avoid drying, particularly when incubation lasts more than 2 hours or less than 70 l of sample or reagent is used. During incubation and wash steps, avoid foaming and remove any bubbles from the sub-array surface. Perform all incubation and wash steps under gentle rotation or rocking motion (~0.5 to 1 cycle/second). Avoid cross-contamination of samples to neighboring wells. To remove Wash Buffers and other reagents from chamber wells, you may invert the Glass Slide Assembly to decant and aspirate the remaining liquid as shown in picture above. Several incubation steps such as blocking, sample incubation, biotin-conjugated antibody incubation, or fluorescenceconjugated streptavidin incubation may be done at 4 ºC overnight. Before overnight incubations cover the incubation chamber tightly (Item I or similar) to prevent evaporation. Protect glass slides from direct, strong light and temperatures above room temperature. 9

10 4. Layout of Glass Arrays Human Allergen Protein Array Manual RayBio Allergen Protein Arrays are available in 4 and 16 subarray/sample kit sizes per glass slide. Depending on sample kit sizes, each glass slide contains different numbers of identical sub-arrays (see picture, below). Only the sub-arrays printed with allergen proteins should be loaded with reagents or samples, as the other wells are blank. Don t touch the printed surface of the glass slide, which is on the same side as the barcode. 16-subarray glass slide has no place to print bar code and other marks indicate the printed side (below). 4 sample kit 16 sample kit * * 5. Incubation Chamber Assembly After finishing your experiment, if you need to repeat any of the incubation or wash steps, you must first re-assemble the glass slide into the incubation chamber by following the steps as shown 10

11 Human Allergen Protein Array Manual in the figures below. To avoid breaking the printed glass slide, it is recommended that you first practice assembling the device with a regular blank standard histology glass slide. Apply slide to incubation chamber, barcode facing upward (Image A). Gently snap one edge of a snap-on side (Image B). Gently press other of side against lab bench and push in lengthwise direction (Image C). Repeat with the other side (Image D) A B C D 11

12 IV. Protocols Human Allergen Protein Array Manual 1. Blocking and Sample Incubation 1.1 Take the package containing the Assembled Glass Slide (Item A) from the freezer. Place unopened package on the bench top for approx. 30 minutes, and allow the Assembled Glass Slide to equilibrate to room temperature. 1.2 Open package, and take the Assembled Glass Slide out of the sleeve (Do not disassemble the Glass Slide from the chamber assembly). Peel off the cover film and let Glass Slide Assembly air-dry in clean environment for 1 hour at room temperature. Note: Protect the slide from dust or others contaminants. Incomplete drying of slides before use may cause the formation of comet tails. 1.3 Block sub-arrays by adding 100 μl of Blocking Buffer (Item F) into each well of Assembled Glass Slide (Item A) and incubating at room temperature for 1 hour. Ensure there are no bubbles on the array surfaces. Note: Only add reagents to wells printed with allergens. Be careful not to add reagents forcefully or directly to the glass slide. Always add reagents slowly to the sides of the plastic well assembly. 1.4 Decant Blocking Buffer from each well. Dilute serum samples using Blocking Buffer (Item F) and load 100 μl of diluted serum samples to each well. Remove any bubbles from the array surfaces. Incubate arrays with gentle rocking or shaking 12

13 Human Allergen Protein Array Manual at room temperature for 1 to 2 hours, or other condition as appropriate. Notes: The serum dilution fold needs to be optimized in array pretests. Normally, we applied for 100 μl of 200-fold diluted serum samples per array. Cover the incubation chamber with adhesive film during incubation if less than 70 ul of sample is used. Blocking Buffer (Item F) can be used to dilute samples if necessary. This step may be done overnight at 4 0 C for higher optical signals. 1.5 Dilute 20 Wash Buffer I (Item G) with distilled water to 1. Decant the samples from each well, and wash the wells 5 times with 150 μl of 1 Wash Buffer I. Wash at room temperature with gentle shaking for 5 minutes per wash. Completely remove 1 Wash Buffer I in each wash step. Note: Avoid solution flowing into neighboring wells. 1.6 Dilute 20 Wash Buffer II (Item H) with distilled water to 1. Wash 2 times with 150 μl of 1 Wash Buffer II. Wash at room temperature with shaking for 5 minutes per wash. Completely remove 1 Wash Buffer II in each wash step. Note: Incomplete removal of the wash buffer in each wash step may cause dark spots (Background signals higher than that of the spot). 13

14 Human Allergen Protein Array Manual 2. Biotin-conjugated Anti-human IgE/IgG Incubation 2.1 Add 100 μl of 1,000-fold diluted Biotin-conjugated Antihuman IgE (Item D) and/or IgG (Item B) to each well. To prepare 1,000-fold diluted Biotin-labeled Anti-human IgE or IgG, spin down the vials (Items B & D) briefly, reconstitute the contents with 1.5 ml of Blocking Buffer (Item F) and mix well. Note: If Biotin-conjugated Anti-human IgE and IgG need to be combined for use, dilute one vial first with 1.5 ml of Blocking Buffer (Item F). After spin down briefly, then transfer the mixture to the other vial and mix well. 2.2 Incubate at room temperature for 1 hour. 2.3 Wash with 1 Wash Buffer I as described in Step 1.5, then wash with 1 Wash Buffer II as described in Step 1.6, above. 3. Cy3-conjugated Streptavidin Incubation 3.1 Brief spinning the vial containing 1,000 Fluorescenceconjugated Streptavidin (Item E) prior to use, add 1.5 ml of Blocking Buffer (Item F) and mix well. Add 100 μl of diluted Fluorescence-conjugated Streptavidin to each sub-array. 3.2 Cover the incubation chamber with aluminum foil to avoid exposure to light or incubate in dark room. 3.3 Incubate at room temperature for 1 hour with gentle rocking or shaking. 3.4 Wash with 1 Wash Buffer I as described in Step 1.5, then with 1 Wash Buffer II as described in Step 1.6, above. 14

15 4. Fluorescence Detection Human Allergen Protein Array Manual 4.1 Decant excess 1 Wash Buffer II from wells. 4.2 Carefully disassemble the glass slide from the incubation frame and chamber by pushing clips outward from the sides, as shown below. Carefully remove the glass slide from the gasket. Note: Be careful not to touch the printed surface of the glass slide, which is on the same side as the barcode or other marks. 4.3 Place the whole slide in the included 30-mL Centrifuge Tube (Item J) or a glass slide holder with the cap. Add enough 1 Wash Buffer I (about 30 ml) to cover the whole slide and gently shake or rock at room temperature for 10 minutes. Decant 1 Wash Buffer I. 4.4 Wash with 1 Wash Buffer II (about 30 ml) with gentle shaking at room temperature for 10 minutes. Decant 1x Wash Buffer II. 4.5 Rinse the glass slide with 30 ml of distilled water for 5 minutes. 4.6 Take glass slide out of the wash container, and gently apply suction with a pipette to remove any water droplets. Do not touch the array area, only the unprinted area. Air-dry for 10 minutes, protected from light. 15

16 Human Allergen Protein Array Manual Note: Make sure the slides are absolutely dry before starting the scanning procedure or storage. High background can result from incomplete drying of the slide. 4.7 Imaging: signals can be visualized through use of a laser scanner equipped with a Cy3/green channel capable wavelength (excitation of 532 nm), such as an Axon GenePix. For additional scanners and slide scanning information, please visit web: Support/Laser_Scanners_for_Glass_Slide_Arrays.pdf Notes: In case the signal intensity for different allergens varies greatly in the same array, we recommend performing multiple scans, starting with a low PMT for high signal allergens, then a higher PMT for low signal allergens. Although we recommend scanning slides right after the experiment, you also can store the slides at room temperature or C in dark for several days. The Cy3 fluor dye used in this kit is very stable at room temperature and resistant to photo bleaching on completed glass slides. Don t have a laser scanner? Just send your slides to us and we can scan them for you. For details, please visit Array Scanning and Analysis Service at our website: 5. Data Analysis 5.1 Data extraction: the captured array signal can be extracted with most of the microarray analysis software (GenePix, 16

17 Human Allergen Protein Array Manual ScanArray Express, ArrayVision, etc.) associated with the laser scanner. 5.2 Data normalization: The raw data normalization is used to compare data between arrays (i.e., different samples) by accounting for the differences in signal intensities of the positive control spots on those arrays. The positive control is a controlled amount of biotinylated protein printed on the arrays in triplicate. The amount of signal from each of those spots is dependent on the amount of the reporter (Cy3- streptavidin) bound to biotinylated protein. Since the reporter amount proportionally affects the signal intensity of every spot on the array, the differences in the positive control signals between arrays will accurately reflect the differences between other spots on those arrays. To normalize the data, one array must be defined as the Reference Array (r) to which the signals of other Sample Arrays (s) are normalized. It is up to the customers to define which array should be the reference. The normalized values are calculated as follows: Pr: the average signal density of the positive control spots on the reference array (r) Ps: the average signal density of the positive control spots on the sample array (s) Xs: the signal density for a particular spot (X) on sample array (s) nxs: the normalized Xs value 17

18 Human Allergen Protein Array Manual The signal intensities obtained from laser scanner can simply be imported into our analysis tool (Cat. #. S02-PAH-AGN-G22). This analysis tool is very simple and affordable, which will not only assist in compiling and organizing your data, but also reduces your calculations to a copy and paste step. The analysis tool will help you: Locate your signal intensities to array map Protein list sorting Average signal intensities Subtract background Normalize the data from different samples Obtain protein level comparison charts among different samples 18

19 V. Array List Human Allergen Protein Array Manual The RayBio Allergen Protein Array kit (Cat #. PAH-AGN-G22) includes 22 common allergens from foods, animals, mites, fungi (molds), insects, and pollen from trees, grass and weeds (see Table 1). If you want to add your allergens of interest or remove any allergens from this list, please contact us so we can customize your specific array as needed. For your convenience, or any editing, please visit our website to download this allergen list in the Excel format. VI. Array Map The RayBio Allergen Protein Array (Cat #. PAH-ANG-G22) is printed in 12 spot 8 spot format (see table below). 22 allergens are printed in triplicate, including the positive control for data normalization and orientation determination, and negative controls. For your convenience, or any editing, please visit our website to download this allergen map in the Excel format Biotin-BSA (1) #7, Che a 3 #15, Fus c 1 Human IgG (1) 1 2 Biotin-BSA (2) #8, Cla h 5 #16, Gly d 2 Human IgG (2) 2 3 #1, Alt a 1 #9, Cla h 8 #17, Lep d 2 0.1% BSA-PBS 3 4 #2, Asp f 1 #10, Cte f 1 #18, Mus m 1 PBS 4 5 #3, Bla g 5 #11, Der f 2 #19, Pen c 3 Human IgE (1) 5 6 #4, Bla g 6 #12, Der p 1 #20, Pen n 13 Human IgE (2) 6 7 #5, Can f 1 #13, Der p 2 #21, Per a 1 Blank 7 8 #6, Che a 2 #14, Fel d 1 #22, Rat n 1 Biotin-BSA (1)

20 Human Allergen Protein Array Manual For your convenience, or any editing, please visit our website to download follwoing allergen list in the Excel format. Table 1. Target List of RayBio Allergen Protein Array UniProt Allergen Species - Common # Allergen Type Complete Protein Name Species - Scientific Name Accession Name Name #. #1 Alt a 1 Fungi (moulds) Major allergen Alt a 1 Alternaria alternata Alternaria rot fungus Q6Q128 #2 Asp f 1 Fungi (moulds) Ribonuclease mitogillin Aspergillus fumigatus P67875 #3 Bla g 5 Insects Glutathione transferase Blattella germanica German cockroach O18598 #4 Bla g 6 Insects Troponin C Blattella germanica German cockroach Q1A7B3 #5 Can f 1 Animals Dog 1 allergen Canis lupus familiaris Dog O18873 #6 Che a 2 Weed Pollen Profilin Chenopodium album Lamb's-quarters, pigweed Q84V37 #7 Che a 3 Weed Pollen Polcalcin Chenopodium album Lamb's-quarters, pigweed Q84V36 #8 Cla h 5 Fungi (moulds) Heat shock 70 k Cladosporium herbarum mold P42039 #9 Cla h 8 Fungi (moulds) Mannitol dehydrogenase Cladosporium herbarum mold P0C0Y5 #10 Cte f 1 Insects Salivary antigen 1; FS-I Ctenocephalides felis Cat flea Q94424 #11 Der f 2 Mites Cysteine protease Dermatophagoides farinae American house dust mite Q00855 #12 Der p 1 Mites Cysteine protease Dermatophagoides pteronyssinuseuropean house dust mite P08176 #13 Der p 2 Mites NPC2 family Dermatophagoides pteronyssinuseuropean house dust mite Q1H8P8 #14 Fel d 1 Animals Cat-1; AG4 Felis catus Cat (saliva) P30440 #15 Fus c 1 Fungi (moulds) Ribosomal protein P2 Fusarium culmorum Q8TFM9 #16 Gly d 2 Mites Glycyphagus domesticus Storage mite Q9NFQ4 #17 Lep d 2 Mites Lepidoglyphus destructor Storage mite P80384 #18 Mus m 1 Animals Major urinary protein II Mus musculus Mouse P02762 #19 Pen c 3 Fungi (moulds) Peroxisomal membrane proteinpenicillium citrinum Q9Y8B8 #20 Pen n 13 Fungi (moulds) Serine protease Penicillium chrysogenum Q9URR2 #21 Per a 1 Insects Cr-PII Periplaneta americana American cockroach O18528 #22 Rat n 1 Animals Major urinary protein (MUP) Rattus norvegicus Rat (urine) P

21 VII. Troubleshooting Guide Human Allergen Protein Array Manual Problem Cause Recommendation Weak Signal High Background Uneven Signal Inadequate detection Inadequate reagent volumes or improper dilution Short incubation times Protein or antibody concentrations in sample are too low Improper storage of kit Excess of protein or antibody Excess of streptavidin Overexposure Dust Slide is allowed to dry out Dark Spots Insufficient wash Bubbles formed during incubation Reagent evaporation Arrays are not completely covered by reagent Increase laser power and PMT parameters Check pipettes and ensure correct preparation Ensure sufficient incubation time or change sample incubation to an overnight step Dilute starting sample less or concentrate sample Store kit as suggested temperature; Don t freeze/thaw the slide Further dilute protein or antibody Further dilute streptavidin Lower the laser power Minimize dust in work environment before starting experiment Take additional precautions to prevent slides from dying out during experiment Completely remove wash buffer in each wash step Increase wash time and use more wash buffer Handle and pipette solutions more gently; Degas solutions prior to use Cover the incubation chamber with adhesive film during incubation Prepare more reagent and completely cover arrays with solution 21

22 VIII. Reference List Human Allergen Protein Array Manual Palacín A, et al. (2012) The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray. PLoS One. 7 (9): e Cretich M, et al. (2010) Allergen microarrays on high-sensitivity silicon slides. Anal Bioanal Chem. 398 (4): Harwanegg C, Hiller R. (2005) Protein microarrays for the diagnosis of allergic diseases: state-of-the-art and future development. Clin Chem Lab Med. 43 (12): Chen,G., et al (2007). Auto-antibody profiles reveal ubiquilin 1 as a humoral immune response target in lung adenocarcinoma. Cancer Res. 67, Huang, R.P. (2003a). Cytokine antibody arrays: a promising tool to identify molecular targets for drug discovery. Comb. Chem. High Throughput. Screen. 6, Huang, R.P. (2003b). Protein arrays, an excellent tool in biomedical research. Front Biosci. 8, D559-D576. Zhu, H., et al. (2001). Global analysis of protein activities using proteome chips. Science 293,

23 Human Allergen Protein Array Manual Additional Custom Protein Array Services We also offer the option to fully customize any protein arrays, and we have many potential array options. For more information, please contact us at or at Toll Free at Experiment design: our protein array experts can assist you in your experiment design based on your project goals or experimental hypothesis. 2. Customized arrays: Select your targets from our target list. Send your protein targets to us, or other synthesized targets, DNA, and other molecules, and we can print them on an array of your choice. If your preferred targets are not available on the market, we can even produce your recombinant proteins using our rapid bacterial gene expression or state-of-the-art mammalian cell gene expression platforms. Please visit our Custom Protein Service website ( for details. 3. Full Array Testing Services: We also offer complete testing services for all of our arrays. Let our array experts run your samples on your array, and provide you expert analysis of your data. 23

24 Note: Human Allergen Protein Array Manual RayBio is the trademark of RayBiotech, Inc. 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 RayBiotech, Inc. 24