Off the Shelf Kits and Reagents for Luminex-Based Proteomic Assays
While the Luminex platform can be used for the development of genomic and proteomic assays, most of Luminex’s partners have developed a vast selection of proteomic assays for the analysis of cytokines and other protein or small molecule analytes. These assays require specific combinations of capture and/or detection antibodies or other proteins to work effectively in a multiplex mix.
To find validated kits for different sets of analytes offered by Luminex partners, users can search with the Kit Finder search engine on the Luminex web website. Some partners supply antibodies separately or will help users build a custom assay. For example, R&D systems has a Luminex Assay Online Configuration Tool that allows users to select from specific lists of antibodies to create a custom multiples kit on the Luminex platform. Origene also has lists of proteins and matched capture/detection antibody sets for Luminex based assays that users can choose from for analysis of certain analytes.
Listings of Commercial and Public Sources of Proteomic Reagents
For users developing their own Luminex-based proteomic assays and lacking the resources to make their own reagents, finding the right combination of commercial or publicly available antibodies or proteins can seem challenging.
Whether the assay requires specific capture and detection antibodies for a sandwich capture method or specific detection antibodies for an indirect ELISA type chemistry, selection of the right combination of antibodies is critical for optimum assay performance. For the successful use of an antibody on any platform, the method of antibody generation against native or denatured proteins, the antibody purification method, the stability of certain preparations, buffer conditions of the detection assay along with a number of other factors can have a serious impact on any assay’s reliability and data quality.1
With so many variables impacting the performance in the hands of a user, knowing how an antibody performs for a given application is useful starting information, however, the reagents still need to be tested by the user for their specific application.
While many companies provide assay platform compatibility information for their antibodies, only a few have information about the Luminex platform. For example, Atlas Antibodies, eBioscience, Jackson Immunoresearch Laboratories, Origene, R&D Systems, KPL, Cell Signaling Technologies, IMGENEX, Proteintech, Fitzgerald, and others offer a wide range of potentially useful antibodies and some proteins. However, not all of these websites provide notations about their utility for different applications. Several government sponsored websites are also available for searching for useful antibodies or proteins. Some, such as the Nonhuman Primate Reagent Resource, NeuroMab, The Developmental Studies Hybridoma Bank, BEI Resources, and AbMiner, can be useful for finding general use reagents or reagents focused on specific disease topics or species. Of all these websites, only a few, such as eBioscience, Origene, and R&D Systems, currently provide some type of information about the compatibility of certain antibodies with the Luminex platform.
With this wide selection of potentially useful antibodies from different sources, development of an effective multiplex custom assay may require testing the compatibility of antibodies from several different sources. For example, when Lawson et al developed an 8-plex porcine cytokine assay for IL-1β, IL-8, IFN-α, TNF-α, IL-12, IL-10, IFN-γ, and IL-4, they described specific capture and detection antibodies from six different commercial sources.2 While these authors reported the final set of 15 antibodies used, they had to test over 30 different antibodies from nine different sources before finding the right combination to use in the final multiplex assay (J. Lunney, personal communication).
Higher plex assays require screening even more antibodies. For example, Skogstrand et al. developed several inflammatory marker assays from 24 to 26 plex that required screening over 100 antibodies from several manufacturers before finding the best capture/detection pairs.3,4 While this process required testing a large number of reagents, it allowed the author to generate single plex data to evaluate the best antibody pairs that were further tested for their compatibility in multiplex assays (K. Skogstrand, personal communication). The author was then able to develop several multiplex assays suitable for the analysis of multiple inflammatory markers from several different sample types such as plasma/serum, dried blood spot samples, amniotic fluid, cervical fluid, nasal lavage, spinal fluid, and cell culture.
When a user needs to find antibodies (or proteins) from multiple sources, websites like the Antibody Directory, Antibodies-online, and Antibody Resource are a good resource. These websites provide information about antibodies from commercial suppliers that pay to be listed. As a result, any platform specific information about a company’s antibodies are provided by the antibody supplier, not the website. The website determines if and how these details about the antibodies are displayed.
User Reviewed or Ranked Listings of Commercial and Public Reagent Sources
Another website that collects information from commercial sources as well as the scientific community is CiteAb, which is a citation-ranked antibody search engine hosted by the University of Bath. In addition to these sources, it also uses information collected from peer reviewed journals to rank a reagent’s use by the scientific community. CiteAb indicates what platforms the antibodies have been used on, including Luminex, when this information is provided by the supplier or mentioned in published papers. When antibodies are available from multiple suppliers, the website ranks them by the number of situations they have appeared in. While this ranking is useful to determine how often the antibody is used by the scientific community, the website does not provide details about how to use the antibodies for different applications. Details on how to use the reagent for different applications may be obtained from the antibody supplier or in the scientific literature.
A few blogs combine information about antibodies from different sources for different applications with reviews by other users such as Antibodies That Work and pAbmAbs. Antibodies That Work is aimed mostly toward posting reviews about antibody related issues and doesn’t provide users with options to search for antibodies for different applications with user reviews about reagent use or performance.
The pAbmAbs website is designed to let users search for antibodies against different proteins or small molecules and post reviews about their utility for a broad range of applications, including Western blotting, Immuno staining, Immuno precipitation, ELISA, Flow cytometry, and the Luminex platform. Users can post reviews about the performance of antibodies they have used and can rate an antibody’s utility on these different platforms. Posted information can contain recommendations for reagent concentrations and other protocol details for using the reagent for different applications. If an antibody is not listed, a user can submit a request to the system administrator at info@pabmabs.com to have antibodies added to the list.
Whether using these websites or other resources, once candidate antibodies are identified for a specific application, the next step in developing an assay is to test the antibodies on the Luminex platform to find the right combinations that give optimum results in a multiplex assay. Instructions for coupling and testing different combinations of antibodies can be found in the xMAP® Cookbook and in Dunbar and Hoffmeyer 2013.5 Additional information and tips can also be searched for on the Luminex blog or by contacting one of Luminex’s Field Application Scientists.
So, when designing a Luminex based study, if you can’t find an off the shelf kit, a number of resources are available to help you quickly find the best reagents for your application needs. If you are moving from a single plex application to developing a Luminex based multiplex assay, these resources can help find the best reagent sources to start developing a Luminex-based multiplex assay. For studies analyzing multiple analytes in many samples, moving to a multiplex Luminex-based assay will be more time and cost effective. Ask your sales manager to provide you with a cost comparison, if needed. For additional assistance with developing custom assays, you can also contact Luminex technical support or one of Luminex’s Field Application Scientists.
References
- Marx, V. Finding the right antibody for the job. Nat Methods 2013;10(8):703-707.
- Lawson, S, Lunney J, Zuckermann F, Osorio F, Nelson E, Welbon C, Clement T, Fang T, Wong S, Kulas K, Christopher-Hennings J. Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: Assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination. Vaccine 2010;28(32): 5356-5364.
- Skogstrand K. Multiplex assays of inflammatory markers, a description of methods and discussion of precautions – Our experience through the last ten years. Methods 2012;56(2):204-212.
- Skogstrand K, Thorsen P, Norgaard-Pedersen B, Schendel D, Sorensen L, Hougaard D. Simultaneous measurement of 25 inflammatory markers and neurotrophinsin neonatal dried blood spots by immunoassay with xMAP technology. Clinical Chemistry 2005;51(10):1854-1866.
- Dunbar SA, Hoffmeyer MR. Microsphere-Based Multiplex Immunoassays-Development and Applications Using Luminex xMAP Technology. In: Wild D, editor. The Immunoassay Handbook (Fourth Edition): Theory and Applications of Ligand Binding, ELISA and Related Techniques. Elsevier Ltd.:157–174. Doi: 10.1016/B978-0-08-097037-0.00012-9.