PATH Protein Microarray Slide (Product Numbers and ) Sandwich Immunoassay Protocol

Transcription

1 L002 Rev of 6 Table of Contents 1.0 Introduction Contents Materials Required, But Not Included Storage and Stability Safety and Handling Assay Considerations Methods for PATH Sandwich Immunoassay 7.1 Primary Antibody Printing Blocking Antigen Addition Wash Secondary Antibody Addition Wash Detection Reagent Addition Wash Scanning Sample Data Ordering Information Protein Microarray Products Introduction The PATH protein microarray slide features an ultra-thin nitrocellulose coating specifically designed for multiplex immunoassays. The PATH slide is the first protein microarray slide to provide the many benefits of nitrocellulose in a low-fluorescence background thin film. As a result, PATH technology delivers the highest signal-tonoise and sensitivity for antibody microarray applications. A variety of assays can be performed using the PATH slide, including sandwich immunoassays, capture immunoassays, protein profiling, and protein characterization. Guidelines are provided for a typical fluorescence-based sandwich immunoassay. It may be necessary to modify some of your steps to fit specific requirements. The TOP surface of the PATH slide can be easily identified by the etched barcode and accompanying numbers. The PATH slide is ready to use right out of the package. 2.0 Contents Product Description Quantity Prod. No PATH Protein Microarray Slides Ultra-Thin Nitrocellulose Film, Bar-coded Materials Required, But Not Included Component Recommended Description Prod. No. Print Buffer PATH print Protein Microarray Print Buffer (5X), 10 ml Block Buffer PATH block Protein Microarray Block Buffer (5X), 30 ml Wash Buffer PATH wash Protein Microarray Wash Buffer (10X), 250 ml Rinse Buffer PATH rinse Protein Microarray Rinse Buffer (10X), 250 ml Separator SIMplex 16 Multi-Array System 2 x 8 well format Microarray Spotter Compatible with standard microarray spotters. NA Microarray Scanner Compatible with standard microarray scanners. NA

2 L002 Rev of Storage and Stability PATH slides should be stored at room temperature (20-30 C) in the original packaging until used. Slides are stable for 1 year from the date of purchase if stored and handled properly. 5.0 Safety and Handling Normal precautions exercised in handling laboratory materials should be followed. The material is not considered hazardous according to 29 CFR The chemical, physical, and toxicological properties of this product may not, as yet, have been thoroughly investigated. We recommend the use of gloves, lab coats, and eye protection when working with any material. 6.0 Assay Considerations 6.1 Incubation Chambers or Gaskets are often used to keep multiple assays separate during incubation and wash steps. To best maintain the integrity of the ultra-thin nitrocellulose film, we recommend using the SIMplex 16 Multi-Array System (Prod. No ). We do not recommend the use of gaskets and chambers with adhesive backing. 6.2 We highly recommend blocking the PATH surface before applying a gasket or chamber to the slide, as described in sections Methods for PATH Sandwich Immunoassay 7.1 Primary Antibody Printing Note 1: It has been shown that the optimal primary antibody concentration for microarray spotting on the PATH slide is lower than that for conventional (porous) nitrocellulose protein microarray slides. Users should optimize primary antibody concentration to achieve the best possible spot morphology (e.g. elimination of comet tails, feathering, etc.) and signal-to-noise for their application starting at around 0.5 mg/ml, within a range of mg/ml Proteins should be printed onto the PATH slide at room temperature (20-30 C) The slide can be blocked immediately after printing; however, some protein types will benefit from postprinting incubation prior to blocking. The length of the post-printing incubation period varies from protein to protein. For example, some proteins may require overnight incubation at room temperature to achieve maximum immobilization efficiency and spot morphology. For best results, determine the postprinting incubation period for your protein. 7.2 Blocking Note 2: As described in sections , we highly recommend blocking the PATH surface before applying a gasket or chamber to the slide. To best maintain the integrity of the ultra-thin nitrocellulose film, we recommend using the SIMplex 16 Multi-Array System (Prod. No ). Gaskets or chambers with adhesive backing are not recommended To minimize comet tails, fuzzy spots or streaking, we recommend blocking by rapid perpendicular immersion ( plunging ) of slides into a bath or slide mailer filled with 1X PATH block reagent. We recommend the following:

3 L002 Rev of 6 Blocking directions using a slide mailer: Place about 24 ml of 1X PATH block reagent into a slide mailer Position the slide so it lines up with one of the end slots in the mailer. Do not allow the slide to contact the reagent at this time. Be sure the arrayed side is facing inward (towards the center of the mailer), Figure 1(a) Rapidly plunge the slide into the mailer, Figure 1(b) Immediately cap the mailer, Figure 1(c) Immediately mix by inverting the slide mailer back and forth for approximately 20 seconds, Figure 1(d). It is not necessary to shake Incubate one hour. Occasionally mix the mailer over the course of incubation by brief inversion. Mailers are not always liquid-tight and will leak if left inverted. Figure 1. Rapid immersion of PATH slide in PATH block immediately followed by several repeated inversions. Note 3: Optimal results can be achieved with as many as two slides in one mailer. To do this, place the slides on opposite sides of the mailer with the arrayed surface facing inward. The blocking solution in the mailer can be used multiple times. A 50 ml conical can also be used instead of a slide mailer; additional blocking buffer is required. Similar results can be attained by rapid immersion of slides in blocking buffer contained in a glass staining dish w/removable rack (Wheaton catalog # ). If your protocol requires direct application of the block buffer to the surface (rather than plunging), apply it quickly, but not directly, onto the array so that there is a slight washing action as the blocker is added. Immediately agitate the solution after addition to each well by hand shaking or tapping with a finger Following incubation in the blocking buffer, remove the slide and allow excess liquid to run off the surface by gently flicking or pipeting. Allow the surface to dry by leaning the slide vertically with its edge on a piece of absorbent paper to allow excess blocking buffer to wick off Place the slide in a SIMplex 16 multi-array device and continue to section Antigen Addition Apply the antigen to the appropriate wells and seal with SIMplex 16 Well Seal (Prod. No ) Incubate the slide for 1 hour at room temperature. We recommend gentle agitation by rocking during incubation. Note that orbital shakers can cause inconsistent protein binding within a chamber. Note 4: Do not allow the surface to completely dry at any time after antigen addition until you are ready to scan the slide.

4 L002 Rev of Wash 1 Note 5: For the following wash and reagent incubation steps: sections 7.4, 7.5, 7.6, 7.7 and 7.8, we recommend one of the following methods to wash and incubate your prepared slide. If sufficient reagents are available, we recommend Method 1. Method 1 Remove the gasket and submerge the entire PATH slide into the wash or incubation bath. Method 2 With the gasket adhered to the PATH slide, wash or incubate within each well or partition. ONCE THE GASKET HAS BEEN REMOVED IT CAN NO LONGER BE RETURNED TO THE SLIDE Following incubation in section 7.3.2, allow the excess liquid to run off the slide by gently flicking or pipeting, but do not allow the surface to completely dry Incubate the slide in wash buffer for 2-3 minutes at room temperature with gentle agitation using the selected method from Note 5 above Repeat the wash two more times using the selected method from Note 5 above. 7.5 Secondary Antibody Addition Following the wash in section 7.4, allow the excess liquid to run off the slide by gently flicking or pipeting, but do not allow the surface to completely dry Incubate with the secondary antibody for 1 hour at room temperature with gentle agitation using the selected method from Note 5 above. 7.6 Wash Following incubation with the secondary antibody, allow excess liquid to run off the slide by gently flicking or pipeting, but do not allow the slide to completely dry Incubate with 1X PATH wash reagent for 2-3 minutes at room temperature with gentle agitation using the selected method from Note 5 above Repeat the wash two more times using the selected method from Note 5 above. 7.7 Detection Reagent Addition Note 6: Typically, the optimal concentration of the labeled detection reagent should be between µg/ml. However, some optimization should be performed to determine the best possible concentration of the labeled detection reagent before running an assay. Additionally, the incubation period should be optimized for each system; typical incubation times range from 30 minutes to 3 hours Incubate the samples in appropriate detection reagent for your optimal time period at room temperature with gentle agitation. The recommended starting point for optimization of the detection reagent concentration is 0.5 µg/ml. The recommended starting point for optimization of the incubation time is one hour If multiple probing steps are required, follow the wash procedure outlined in Note 5.

5 L002 Rev of Wash Following incubation with the detection reagent, allow the excess liquid to run off the slide by gently flicking or pipeting, and then incubate the samples in wash buffer for 2-3 minutes at room temperature with gentle agitation times using the selected method from Note 5 above Repeat the wash two more times using the selected method from Note 5 above. If using Method 2, remove the slide from the SIMplex 16 device Briefly rinse the slide with 1X PATH rinse reagent Immediately dry the slide using an air or nitrogen stream to remove salt residue that is potentially visible on the array image. If cloudy or spotty background remains evident on the dried slide, briefly rinse again with 0.1X PATH rinse reagent or deionized water to remove dried salts. Immediately repeat the drying step by applying an air or nitrogen stream. 7.9 Scanning The dried slide can be read with any standard fluorescence-based microarray scanner. 8.0 Sample Data Figure 1. Multiplexed Sandwich Immunoassays of Purified Recombinant Human Cytokines on a PATH Slide. 0.5 mg/ml cytokine-specific mouse monoclonal antibodies were spotted onto a PATH slide. The PATH slide was blocked by rapid immersion into 1X PATH block and assembled in the SIMplex 16 multi-array device. A cocktail of purified recombinant antigens was spiked into human serum at various concentrations in individual wells and incubated for one hour. A blank control (serum only) was added to a separate well. Following antigen incubation, the PATH slide was removed from the SIMplex 16 device. After rinsing, a cocktail of biotin-labeled, cytokine-specific detector antibodies (1 µg/ml) was applied and incubated for one hour. Finally, after a second wash, the wells were exposed to 75 ng/ml streptavidin-labelled, Cy3 detection reagent. The slide was rinsed and scanned on a ScanArray Data was calculated by subtracting local background and the blank control from all samples. The limit of detection (LOD) is determined as two times the standard deviation of the blank.

6 L002 Rev of Ordering Information Telephone: Toll Free in North America: order@gentelbio.com Fax: Website: PATH Protein Microarray Products PRODUCT QTY/PKG SIZE PRODUCT NUMBER Slides PATH Protein Microarray Slide 62 x 21 mm usable surface area PATH plus Protein Microarray Slide PATH HTS Protein Microarray Slide x 21 mm usable surface area x 71 mm usable surface area PATH Protein Microarray Starter Kit 2 Slides and Buffers x 21 mm usable surface area PATH Protein Microarray Kit 10 Slides and Buffers PATH plus Protein Microarray Starter Kit 2 Slides and Buffers x 21 mm usable surface area PATH plus Protein Microarray Kit 10 Slides and Buffers PATH HTS Protein Microarray Kit 2 Slides and Buffers 108 x 71 mm usable surface area Kits Separator System SIMplex 16 Multi-Array System x 8 well format SIMplex 16 Gasket SIMplex 16 Well Seal x 25 mm Buffers PATH print Buffer (5X) 1 10 ml PATH block Buffer (5X) 1 30 ml PATH wash Buffer (10X) ml PATH rinse Buffer (10X) ml United States and International Patents Pending on PATH Slides. GenTel is a registered trademark of GenTel BioSurfaces, Inc. PATH is licensed from and is a trademark of Decision Biomarkers, Inc. SIMplex is a trademark of GenTel BioSurfaces, Inc. Cy3 is a trademark of Amersham/GE. ScanArray is a trademark of PerkinElmer, Inc. 2005, GenTel BioSurfaces, Inc. All rights reserved. For Research Use Only PURCHASER SHALL USE THE PRODUCTS PURCHASED HEREUNDER (the Products ) SOLELY FOR THE PURPOSE OF CONDUCTING INTERNAL RESEARCH AT ITS ORGANIZATION ( RESEARCH ). Purchaser will not sell, transfer, disclose or otherwise provide access to the Products to any third party. Purchaser agrees that Purchaser shall not have the right to authorize any third party to use or sell any Products or derivatives thereof. Unless a license has been executed between Purchaser and GenTel BioSurfaces, Inc. explicitly providing otherwise, Purchaser will not: (i) reformulate or create derivatives of the Products; (ii) use the Products for providing services to a third party (e.g., screening or profiling); (iii) use the Products in a diagnostic process; (iv) use the Products in a quality control or quality assurance process for the manufacture of a product for sale; (v) use the Products as a component of a kit.