Biomarker quantitation assay guide Quantitate protein: ELISA l Antibody pairs l ProQuantum HS immunoassays l ProcartaPlex immunoassays Quantitate mRNA: QuantiGene multiplexed gene expression assays Our Invitrogen™ portfolio offers a variety of assays for quantitation of single proteins and mRNA, as well as mix-and-match and ready-to-use panels for correlated multiplexing using the Luminex® platform. Our extensive portfolio of assays includes: • ELISA and antibody pair kits • ProQuantum™ high-sensitivity immunoassay kits • ProcartaPlex™ multiplex immunoassay kits • QuantiGene™ Plex gene expression assays Additionally, Thermo Fisher Scientific supports your quantitation assay needs with accessory reagents and instruments for a comprehensive offering. Use this guide to help identify your needs, and then contact your sales representative if you have questions or go to thermofisher.com/quantitatebiomarkers Contents Biomarker background 4 Biomarker quantitation assay platforms 7 Single-analyte quantitation ELISA kits 9 Antibody pair kits 11 ProQuantum high-sensitivity immunoassay kits 12 Accessory reagents and equipment 14 Multianalyte quantitation Luminex xMAP technology 17 ProcartaPlex multiplex immunoassay kits 19 ProcartaPlex high-sensitivity immunoassay kits 27 ProcartaPlex Platinum immunoassay kits 28 Custom assay development service 30 Multiplexed gene expression assays 32 QuantiGene Plex assays 32 Additional gene expression solutions 38 Comprehensive immunoassay product listing 40 Biomarker background Highly referenced kits you can trust We proudly offer a selection of quantitation assays for Assays within these areas of research assess the measurement of protein and RNA as biomarkers in cellular mechanisms from basic drug discovery key disease research areas. From single-analyte analysis to development through multiple ELISA formats to multiplex quantitation • Adaptive immunity using Invitrogen™ ProcartaPlex™ and QuantiGene™ Plex • Adhesion and migration assays for the Luminex platform, you can study the mechanisms of disease pathways from expression of the • Angiogenesis gene to the protein. • Apoptosis This guide describes all the options available for single- • Bone biology analyte and multianalyte assays, as well as showing the correlation between these assays. With high correlative • Cancer biology values between these various assays, you have the flexibility to use the assay you need to continue your • Cell cycle and proliferation research and publish with confidence. • Cell signaling and cell structure • Cytokines, chemokines, and growth factors • Innate immunity • Metabolism • Stem cell biology • Transcription factors The importance of biomarker quantitation assays As part of the immune response, inflammation plays an Acute versus chronic inflammation important role in defending the body against pathogens, Acute inflammation is a short-lived response that is such as viruses, bacteria, fungi, and other parasites. characterized by extravasation of leukocytes, erythrocytes, However, inappropriate activation of inflammatory and plasma components into the injured tissue. If left processes is an underlying contributor to many common unchecked, the acute inflammatory process can lead to pathological conditions. For example, autoimmune chronic inflammation. conditions arise when our immune system mistakes our cells or tissues for pathogens and attacks them. Unlike acute inflammation, chronic inflammation is In addition, studies show that tumor proliferation and characterized primarily by tissue infiltration by lymphocytes metastasis may occur when inflammatory cytokines create and macrophages. Chronic inflammation is closely a microenvironment conducive to cancer progression. associated with allergy, atherosclerosis, cancer, arthritis, Figure 1 shows a simplified representation of cytokine and Alzheimer’s disease, as well as autoimmune interactions during inflammation. diseases. The process of acute inflammation is well defined, but the causes of chronic inflammation and its associated molecular and cellular pathways are still not well understood [1]. 4 Inflammation research PECAM-1, VCAM-1, Bacterial products ICAM-1, P-selectin, (LPS, peptidoglycans, etc.) E-selectin Fibroblast liver TNF-α Endothelial I L-1 Acute phase β cell proteins TGF-β T N F, I L-1β PDGF I L- 6 Cardiovascular M-CSF, TNF-α, GM- CSF, pathology G - C S F, I L-1, I L- 6 , I L-11, Histamine I L-12 Fever I L- 8 , Macrophage TNF-α Hematopoiesis I L-1, I L- 6 , I L- 8 , I L-10 , IL-12, IL-15, IFN-α, IFN-β, TNF-α, MCP-1, MIP-1α Neutrophil elastase Neutrophil Respiratory burst I L-18 Reactive oxygen species I L- 3 , I L- 6 , I L- 3 , I L- 4, I L-10 , IL-7, GM-CSF IL-13, IFN-γ, TNF-β Resting T cell I L- 3 , I L- 4, Mast cell Allergy anaphylaxis Resting B cell I L-10 , I L-14, I L-10 I L-12, I L-15 I L- 4, I L- 6 Innate immunity I L- 3 , I L- 4, I L- 5 Activated T cell LFA-1, VLA-4, Mac-1 Eotaxin IFN-γ Eosinophil I L-2 I L- 6 Activated B cell Lung I L-2 epithelium I L-2 3 Viral infection, tumor surveillance NK cell TGF-β, IL-2, I L- 4, I L- 5 , I L-13 , Th17/22 IFN-γ CTL Plasma cell Th1/Th2 Th9 IgE IL-2, IFN-γ, I L- 4 Fibrosis IgM/G/A Allergy anaphylaxis Autoimmunity Viral infection, Humoral immunity tumor surveillance I L-17A , I L-17 F, Tissue repair, I L-2 2, I L- 6 , protozoan immunity GM-CSF Figure 1. Interaction of cytokines during inflammation. 5 The critical balance between pro- and anti-inflammatory cytokine and chemokine networks has anti-inflammatory mediators made it crucial to examine them in relevant functional The overall effect of an inflammatory response is dictated groups rather than individually (Figure 1, page 5). by the balance between pro- and anti-inflammatory mediators (Table 1). To facilitate the investigation of inflammatory and other biologically relevant biological markers, the Invitrogen Pro-inflammatory cytokines such as IL-1β, IL-6, and portfolio has a broad menu of immunoassays to easily TNF-α, are responsible for early responses and amplify detect and quantify proteins, and to facilitate tracking inflammatory reactions, whereas anti-inflammatory gene expression of RNAs with the QuantiGene Plex cytokines, which include IL-4, IL-10, and IL-13, have assays. Products are available for important research the opposite effect in that they limit the inflammatory areas, including immunology, Alzheimer’s disease, cancer, reactions (Table 1). The increasing complexity of pro- and cardiovascular diseases, metabolism, and more. Table 1. Pro- and anti-inflammatory mediators [2]. Pro-inflammatory mediators • Cell-surface CD14 • Leukemia inhibitory factor • Prostacyclins • Free radical generation • Monocyte chemoattractant protein-1 • Prostaglandins • I L-1β • Monocyte chemoattractant protein-2 • Protein kinase • I L-2 • Neopterin • Soluble adhesion molecules • I L- 6 • Neutrophil elastase • Thromboxane • I L- 8 • Phospholipase A2 • TNF-α • I L-15 • Plasminogen activator inhibitor-1 • Tyrosine kinase • IFN-γ • Platelet activating factor • Vasoactive neuropeptides Anti-inflammatory mediators • Epinephrine • Leukotriene B4 receptor antagonists • Soluble TNF-α receptors • I L- 4 • LPS binding protein • Transforming growth factor-β • I L-10 • Soluble recombinant CD-14 • Type II IL-1R • I L-13 • I L-1R A Plate- and bead-based solutions for your research needs across many species Human, mouse, rat, nonhuman primate, canine, porcine, bovine, equine, and feline The Invitrogen portfolio of immunoassay products • Keep your research relevant by incorporating analytes supports the evolution of research projects by offering pertinent to your area of work at any point within multiple platforms that, in most cases, use the same your study antibody content. This provides freedom to move from one • Conserve resources by using existing data from platform to another, or from a multiplex assay to an ELISA, previous experiments producing truly comparable data. Adding QuantiGene Plex assays to the workflow can provide an accurate • Verify at your discretion with identical antibody content and precise method for multiplexed gene expression and correlated measurement across multiple platforms quantitation, using Luminex® xMAP® technology. 6 Biomarker quantitation assay platforms Tailored to meet your needs A variety of ELISA kit formats are available that include ELISA, including ELISA-optimized matched antibody complete, ready-to-use kits as well as preoptimized pairs, standards, detection reagents, coating buffers, reagents to make your own. A variety of singleplex sample diluent, and tetramethylbenzidine (TMB) substrate and multiplex panels for the Luminex platform are also solution. Plates are optional. available. See Table 2 for a performance comparison of our immunoassay platforms and Table 3 for a component list. • ProQuantum high-sensitivity (HS) assay kits— Take advantage of a platform innovation that provides • Invitrogen™ coated ELISA kits—Quantitate with researchers with an easy-to-run, high-performance confidence. Highly verified ELISA kits with precoated assay, utilizing proximity ligation assay (PLA™) technology plates provide lower inter- and intra-assay variability with to combine the analyte specificity of high-affinity ready-to-use reagents that help ensure consistent data. antibody–antigen binding with the signal detection and amplification capabilities of real-time PCR to achieve a – Invitrogen™ Instant ELISA kits—Reduce steps and highly sensitive protein quantitation assay, using very hands-on time to increase productivity. The one-wash small