Chemiluminescent Alkaline Phosphatase ELISA Systems

Transcription

1 Chemiluminescent Alkaline Phosphatase ELISA Systems Catalog nos. C10552, C10553, C10554, C10555, C10556 Table 1. Contents and storage information. Material Composition Amount C10552 C10553 C10554 C10555 C10556 Storage 10X assay buffer (Component A) 200 mm Tris (ph 9.8), 10 mm MgCl ml 100 ml 100 ml 100 ml 100 ml Blocking reagent (Component B) Highly purified casein, dry powder 7.5 g 7.5 g 7.5 g 7.5 g 7.5 g CSPD substrate/ Sapphire II enhancer (Component C) CSPD substrate/ Emerald-II enhancer (Component C) CDP-Star substrate/ Sapphire II enhancer (Component C) 0.4 mm ready to use CSPD or CDP Star substrate containing 10% (v/v)sapphire II or Emerald II enhancer 100 ml 25 ml 100 ml 25 ml 100 ml 25 ml 2 6 C Dessicate Protect from light CDP-Star substrate/ Emerald-II enhancer (Component C) 100 ml 25 ml These storage conditions are appropriate when storing the entire kit upon receipt. For optimal storage conditions for each component, see labels on the individual vials. When stored as directed, this kit is stable for at least 1 year. C10556 is a sampler kit that contains 25 ml of each of the four substrate/enhancer combinations (Components C F). Number of assays: Sufficient material is supplied for 1,000 assays based on the protocol below. Introduction Description of the System The Chemiluminescent Alkaline Phosphatase ELISA detection system incorporates CSPD or CDP-Star 1,2-dioxetane substrates for alkaline phosphatase with Sapphire-II or Emerald II enhancer in a system designed for rapid and ultrasensitive analyte detection in enzyme-linked immunoassays. 1 7 Light emission from alkaline phosphatase-activated CSPD or CDP-Star substrate is in the form of a glow. Maximum light emission is reached in 5 to 60 minutes, depending on the temperature and the substrate chosen. Enzymatic dephosphorylation of substrate occurs at a constant rate proportional to enzyme concentration; the resulting anion decomposes with a finite half-life. Light emission can be quantitated with a variety of luminometers without the need for solution injection. Enzyme-linked immunosorbent assays (ELISAs) can be formatted in several configurations on a variety of solid supports including Revised: 12 June 2009 MP 10552

2 microplate wells, tubes, polystyrene beads, or ferrite particles. The high sensitivity obtained with 1,2-dioxetane substrates is demonstrated in a sandwich immunoassay format ELISA that employs a biotinylated detector antibody and streptavidinalkaline phosphatase conjugate for quantitating recombinant human IL-6 (rhil-6). The results obtained with CSPD substrate/sapphire-ii enhancer (Figure 1) show a significant improvement in signal-to-noise performance at all concentrations of rhil-6 and a much wider assay dynamic range compared to those obtained with the fluorescent substrate 4-methylumbelliferyl phosphate (4-MUP), and the colorimetric substrate, p-nitrophenyl phosphate (pnpp). This benefit can be expected when any colorimetric ELISA is converted to 1,2-dioxetane/enhancer chemiluminescence. 10,000 1,000 S/N 100 CSPD/Sapphire II pnpp 4-MUP ,000 10,000 rhil-6 Conc (pg/ml) Figure 1. Comparison of Chemiluminescent, Fluorescent, and Colorimetric Detection for ELISA Quantitation of rhil-6.. Sandwich Immunoassay A direct sandwich ELISA is used for the detection of large molecules with multiple antigenic sites, usually proteins. In this format, a solid support is coated with a capture antibody that immunoadsorbs the antigen from the sample; a detector antibody conjugated to alkaline phosphatase, specific for a second site on the antigen, is then added. Alternatively, an unlabeled or hapten-labeled detector antibody (not from the same species as the capture antibody) can be used, followed by a secondary antibody, anti-hapten antibody, or streptavidin-alkaline phosphatase conjugate. The enzyme-generated signal is proportional to the amount of antigen. Sandwich immunoassay formats with 1,2-dioxetane substrates have been used for the quantitation of a variety of animal and human proteins from plasma and tissue extracts. 2,3,6 13 In a related assay format, antigen-coated solid support has been used with 1,2-dioxetanes for detection of HTLV-I antibodies 14 and calculation of antibody binding constants. 15 CDP Star substrate with Sapphire-II enhancer or Emerald-II enhancer has become widely used for immunoassay protein detection applications such as detection of plasma proteins 16 and viral antigens in both clinical serum samples 17 and in cell culture media as a viral infection assay. 18,19 Competitive Immunoassay Competitive ELISA formats, in which a competition for available antibody binding sites occurs between a labeled and an unlabeled antigen, are typically used for detection of small molecules. 1,2-Dioxetanes have been utilized in these assays for detection of peptides and hormones Competitive assays generate an inverse standard curve; for increasing concentrations of antigen, a decrease in signal is observed. Because the standard curve in a competitive ELISA exhibits maximum signal at the lowest analyte concentration, it may be necessary to adjust reagent concentrations to optimize detection of low analyte concentrations. The sensitivity of chemiluminescent detection enables the use of lower concentrations of capture antibody and competing antigen to generate a standard curve which spans a much lower concentration range compared to colorimetric methods. Chemiluminescent Alkaline Phosphatase ELISA Systems 2

3 Whole-Cell ELISA Immunoassay detection of a surface antigen on whole cells has been demonstrated with 1,2-dioxetane chemiluminescent detection. 23 β-galactosidase enzyme conjugates can also be used with Galacton-Star substrate with Sapphire-II enhancer for chemiluminescent immunoassay detection, particularly for whole cell ELISA applications that may exhibit high levels of cellular alkaline phosphatase. 24 Protein Detection Applications Anti-phosphopeptide immunoassays with CSPD or CDP-Star substrates and Sapphire II or Emerald-II enhancers have been developed for quantitating several protein kinase activities, including PKA, PKC, CAM-KII, receptor interacting protein and src kinases, 25 WaaP protein tyrosine kinase and sugar kinase, 26 and p38 kinase. 27 In addition, a receptor binding assay of a neurotrophic factor to a tyrosine kinase receptor, 28 as well as viral foci imaging with immunodetection, 29 has been demonstrated. Quantitation of protein-protein interactions with an ELISA assay 30 and quantitation of sirna-mediated protein knockdown 31 have been demonstrated using the CDP-Star substrate with Sapphire II enhancer. Nucleic Acid and Nucleic Acid- Protein Interaction Detection Applications DNA probe hybridization assays, DNA-protein interaction assays, and DNA aptamer binding assays are often formatted in microplate wells or on other solid phases. Alkaline phosphatase (AP)-labeled probes, hapten-labeled probes, or antibodies to DNA-DNA or DNA-RNA duplexes 32 can be used to detect hybridization or binding with AP-conjugated detection reagents and 1,2-dioxetane substrates. Chemiluminescent enzyme-linked oligonucleotide assay (ELONA) has been used to quantitate DNA aptamer binding to protein. 33 CDP-Star substrate is used in microplate-based assay systems for detection of viral RNA or DNA by immunodetection, 34,35 quantitative detection of labeled PCR products, 36 and ELISA-PCR for mrna quantitation. 37 In addition, detection of chemical-dna adducts in mammalian tissues has been demonstrated with CDP Star substrate with Emerald-II enhancer. 38 Before Starting Materials Required but Not Provided Appropriate antibodies, andtigens, antibody- or antigen-alkaline phosphatase conjugates, or biotinylated antigens Phosphate buffered saline (PBS) Tween -20 detergent Deionized water 96-well white (opaque) luminometer microplates Microplate luminometer Caution The 10X assay buffer (Component A) and the CSPD or CDP-Star substrates with Sapphire II or Emerald-II enhancers (Components C F) are irritating to eyes and skin. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing and eye/face protection. Chemiluminescent Alkaline Phosphatase ELISA Systems 3

4 General Guidelines Ultrasensitive immunoassay detection is often limited by non-specific binding of enzyme conjugates to the plates; high quality, purified conjugates are critical. 25 Detailed protocols for immunoassay formats and microplate coating are available in published literature. 26 The light produced from the alkaline phosphatase catalyzed decomposition of CSPD or CDP-Star substrate is a glow-type emission, and the incubation time required to achieve maximum light emission is a function of alkaline phosphatase activity and temperature. The chemiluminescent assay for alkaline phosphatase activity is subject to interference by hemoglobin and by high concentrations of protein (greater than 11.6 g/l). Bilirubin (up to 173 μmol/l) and lipid (2 100 g/l) do not interfere with the assay. 1 Determine the optimal blocking reagents and conditions for the individual assay. Avoid any materials which may contain contaminating alkaline phosphatase. Perform chemiluminescent ELISAs, which are read in a microplate luminometer, in opaque white plates. Perform all steps at room temperature, unless otherwise indicated. Recommended volumes and terminology used in the protocols below are based upon use of microplates. Luminometers We recommend using a single-mode luminometer or a multi-mode detection instrument set for luminescence measurement to measure light emission from 96- or 384-well microplates. Measure light emission for 1 second per well, or as appropriate for the instrument. Preparing Working Solutions Prepare all solutions with deionized water. Keep 10X PBS sterile at room temperature 1X Assay Buffer 1.1 Dilute 10X assay buffer (Component A) 1:10 in deionized water before use. Store at 4 C. Blocking Buffer 1.2 Dilute 3 ml of 10X PBS 1:10 in 27 ml of deionized water, and microwave for 40 seconds. Do not boil. 1.3 Add 0.06 g of blocking reagent (Component B) and allow the solution to cool. 1.4 After the solution has cooled, add 15 μl of Tween -20 detergent. Cool to room temperature before use. The solution will remain opaque, but particles will be dissolved. Wash Buffer 1.5 Add 50 μl of Tween -20 detergent to 10 ml of 10X PBS, and mix well. 1.6 Add deionized water to 100 ml. Coating Microplates Empirically determine the optimal coating buffer and protein concentration (usually 0.2 to 10 μg/ml). 2.1 Add μl coating solution per well, seal plate, and incubate for 2 hours at 37 C or overnight at room temperature or 4 C. Chemiluminescent Alkaline Phosphatase ELISA Systems 4

5 Experimental Protocols Direct Sandwich ELISA Direct sandwich immunoassays are the most sensitive format for detecting soluble antigen. We recommend this format for antigens for which two distinct antibodies are available. Many variations on this format are possible, including monoclonal antibody screening of hybridoma culture supernatants. 3.1 Coat plate with capture antibody (see step 2.1), then wash the coated plate 3 times with wash buffer (steps ) by filling wells with wash buffer from a squirt bottle. Shake buffer into a sink after each wash. After the last wash, remove residual liquid by tapping plate face down on clean paper towels. Note: If you are using automated 96-well microplate washer instrumentation, fill the wells to slightly below well capacity. 3.2 Incubate wells with blocking buffer (steps ) for 1 hour at room temperature or overnight at 4 C. 3.3 Wash blocked plate 3 times with wash buffer following the same wash procedure as in step Dilute antigen samples in blocking buffer, and add 100 μl of the diluted antigen into each well. Incubate for 1 hour with shaking at room temperature. 3.5 Wash wells 3 times with wash buffer following the same wash procedure as in step Dilute detector antibody-alkaline phosphatase (AP) conjugate in blocking buffer, and add 100 μl of the dilution into each well. Incubate for at least 1 hour with shaking at room temperature. Note: Determine the optimal dilution of antigen samples empirically. 3.7 Wash wells 4 times with wash buffer, then twice with 1X assay buffer (prepared in step 1.1). 3.8 Add 100 μl of substrate/enhancer solution (Component C) into each well and incubate for 10 minutes at room temperature. 3.9 Measure chemiluminescence in a luminometer at 10 minute intervals until light emission has reached plateau. Competitive ELISA using Antigen-Coated Plates This assay format is useful for detection of small antigen molecules, such as therapeutic drugs, for which a specific antibody and milligram quantities of antigen are available. 4.1 Coat plate with antigen (see step 2.1), then wash the coated plate 3 times with wash buffer (steps ) by filling wells with wash buffer from a squirt bottle. Shake buffer into a sink after each wash. After the last wash, remove residual liquid by tapping plate face down on clean paper towels. Note: If you are using automated 96-well microplate washer instrumentation, fill the wells to slightly below well capacity. 4.2 Incubate wells with blocking buffer (steps ) for 1 hour at room temperature or overnight at 4 C. 4.3 Wash blocked plate 3 times with wash buffer following the same wash procedure as in step 4.1. Chemiluminescent Alkaline Phosphatase ELISA Systems 5

6 4.4 Dilute standard or test antigen samples separately in blocking buffer to 2X final concentration. 4.5 Dilute antibody-alkaline phosphatase (AP) conjugate in blocking buffer to 2X final concentration. 4.6 Add equal volumes of antigen dilution (from step 4.4) and antibody-(ap) conjugate dilution (from step 4.5) to wells (50 75 μl/well). Incubate for 2 hours with shaking at room temperature. 4.7 Wash wells 4 times with wash buffer, then once with 1X assay buffer (prepared in step 1.1). 4.8 Add 100 μl of substrate/enhancer solution (Component C) into each well and incubate for 10 minutes at room temperature. 4.9 Measure chemiluminescence in a luminometer at 10 minute intervals until light emission has reached plateau. Competitive ELISA using Antibody-Coated Plates This assay format, which is recommended for small antigens that can be modified without affecting antibody affinity, requires a biotinylated or alkaline phosphatase-conjugated antigen. 5.1 Coat plate with capture antibody (see step 2.1), then wash the coated plate 3 times with wash buffer (steps ) by filling wells with wash buffer from a squirt bottle. Shake buffer into a sink after each wash. After the last wash, remove residual liquid by tapping plate face down on clean paper towels. Note: If you are using automated 96-well microplate washer instrumentation, fill the wells to slightly below well capacity. 5.2 Incubate wells with blocking buffer (steps ) for 1 hour at room temperature or overnight at 4 C. 5.3 Wash blocked plate 3 times with wash buffer following the same wash procedure as in step Dilute standard or test antigen samples separately in blocking buffer, and 100 μl of the dilution per well. Incubate for 10 minutes with shaking at room temperature. 5.5 Dilute biotinylated antigen or antigen-alkaline phosphatase (AP) conjugate in blocking buffer, and add 50 μl per well containing the diluted standard or test antigen samples (step 5.4). 5.6 Wash wells 4 times with wash buffer, then once with 1X assay buffer (prepared in step 1.1). 5.7 Add 100 μl of substrate/enhancer solution (Component C) into each well and incubate for 10 minutes at room temperature Measure chemiluminescence in a luminometer at 10 minute intervals until light emission has reached plateau. Chemiluminescent Alkaline Phosphatase ELISA Systems 6

7 References 1. J Biolumin Chemilumin 4, 99 (1989); 2. Clin Chem 35, 1441 (1989); 3. Clin Chem 37, 1526 (1991); 4. Immunochemical Assays and Biosensor Technology for the 1990s (Nakamura, RM, Kasahara, Y and Rechnitz, GA, eds), American Society for Microbiology, Washington, DC (1992), p ; 5. Clin Chem 40, 347 (1994); 6. Clin Chem 35, 2319 (1989); 7. Methods in Molecular Biology, Vol. 63: Recombinant Proteins: Detection and Isolation Protocols (Tuan, R, ed), Humana Press, Inc, Totowa, NJ (1997), p ; 8. Clin Chem 37, 1639 (1991); 9. Bioluminescence and Chemiluminescence: Current Status (Stanley, PE and Kricka, LJ, eds), John Wiley, Chichester, England (2001), p ; 10. J immunol Methods 168, 111 (1994); 11. J Neurosci Res 99, 622 (2002); 12. J Molecular Endocrinol 32, 145 (2004); 13. Respiratory Research 8, 3 (2007); 14. Clin Chem 36, 1090 (1990); 15. J Biol Chem 270, (1995); 16. Endocrinol 143, 1166 (2002); 17. J Clin Microbiol 41, 1901 (2003); 18. J Virol 74, 6893 (2000); 19. J Virol 81, 7048 (2007); 20. Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. AK Campbell, LJ Kricka and PE Stanley, editors. John Wiley & Sons, Chichester, England (1994), p ; 21. Steroids 60, 686 (1995); 22. J Pharm Biom Anal 14, 1653 (1996); 23. J immunolog Methods 138, 129 (1991); 24. Biochemistry 38, 1866 (1999); 25. Anal Biochem 244, 340 (1997); 26. J Biol Chem 277, 4722 (2002); 27. Nature Biotech 21, 302 (2003); 28. Porc Natl Acad Sci USA 94, 6238 (1997); 29. J Virolog Methods 66, 311 (1997); 30. J Mol Biol 300, 1323 (2000); 31. Mol Neurodegen 2, 4 (2007); 32. Clin Chem 39, 1934 (1993); 33. Nature Biotech 14, 1021 (1996); 34. J Clin Microbiol 38, 2150 (2000); 35. Nuc Acids Res 26, 5230 (1998); 36. BMC Genetics ( (2004); 37. J Immunol 166, 3749 (2001); 38. Carcinogenesis 23, 2043 (2002). Product List Current prices may be obtained from our website or from our Customer Service Department. Cat. no. Product Name Unit Size C10552 Chemiluminescent Alkaline Phosphatase ELISA Kit #1 *with CSPD Substrate/Sapphire-II Enhancer* *1000 assays*... 1 kit C10553 Chemiluminescent Alkaline Phosphatase ELISA Kit #2 *with CSPD Substrate/Emerald-II Enhancer* *1000 assays*... 1 kit C10554 Chemiluminescent Alkaline Phosphatase ELISA Kit #3 *with CDP-Star Substrate/Sapphire-II Enhancer* *1000 assays*... 1 kit C10555 Chemiluminescent Alkaline Phosphatase ELISA Kit #4 *with CDP-Star Substrate/Emerald-II Enhancer* *1000 assays*... 1 kit C10556 Chemiluminescent Alkaline Phosphatase ELISA Sampler Kit *1000 assays*... 1 kit Chemiluminescent Alkaline Phosphatase ELISA Systems 7

8 Contact Information Corporate Headquarters 5791 Van Allen Way Carlsbad, CA Phone: (760) Fax: (760) European Headquarters Inchinnan Business Park. 3 Fountain Drive Paisley PA4 9RF, UK Phone: +44 (0) Fax: +44 (0) euroinfo@invitrogen.com Technical Services: eurotech@invitrogen.com Japanese Headquarters LOOP-X Bldg. 6F , Kaigan Minato-ku, Tokyo Phone: Fax: jpinfo@invitrogen.com For country-specific contact information, visit Limited Use Label License No. 223: Labeling and Detection Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, Willow Creek Road, Eugene, OR 97402, Tel: (541) Fax: (541) Limited Warranty Invitrogen (a part of Life Technologies Corporation) is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, contact our Technical Support Representatives. All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis. The Company will replace, free of charge, any product that does not meet those specifications. This warranty limits the Company s liability to only the price of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. The Company reserves the right to select the method(s) used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Support Representatives. Life Technologies Corporation shall have no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Copyright 2009, Life Technologies Corporation. All rights reserved. For research use only. Not intended for any animal or human therapeutic or diagnostic use. Chemiluminescent Alkaline Phosphatase ELISA Systems 8