Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo
- 3.3k Downloads
Functional characterization of antibodies that inhibit soluble cytokines or chemokines requires robust, sensitive in vitro and in vivo bioassays. Testing an antibody in vitro requires consideration of antigen source, integrity, and concentration, as well as the magnitude of the biologic response and assay interference by components in the antibody test sample. This chapter describes several exemplary in vitro bioassays, including an assay to determine species cross-reactivity of an anti–tumor necrosis factor antibody and a whole blood assay for interleukin–18 (IL–18) to determine neutralization of native antigen. Testing an antibody in vivo requires consideration of species cross-reactivity, selection of the dose range and the route of administration, analyses of the pharmacokinetics and tissue distribution of the antibody, and evaluation of host antibody responses. An example of an in vivo bioassay, in which human peripheral blood mononuclear cells are injected into severe combined immunodeficient mice and activated in vivo to produce human IL–18, is provided.
KeywordsAntigen Concentration L929 Culture Medium Human Tumor Necrosis Factor Native Antigen Antigen Source
Editorial support was provided by Robin L. Stromberg, PhD, of Arbor Communications, Inc. (Ann Arbor, MI, USA) and funded by Abbott Laboratories.
- Fernandez-Botran R, Vètvička V (2001) Assays of cytokines. In: Methods in cellular immunology, 2nd edn. CRC Press, Boca Raton, FL. pp 87–158Google Scholar
- Foster B, Prussin C, Liu F, Whitmire JK, Whitton JL (2007) Detection of intracellular cytokines by flow cytometry. Curr Protoc Immunol Chap. 6 :Unit 6.24
- Guenat S, Rouleau N, Bielmann C, Bedard J, Maurer F, Allaman-Pillet N, Nicod P, Bielefeld-Sévigny M, Beckmann JS, Bonny C, Bossé R, Roduit R (2006) Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates. J Biomol Screen 11:1015–1026PubMedCrossRefGoogle Scholar
- Haringman JJ, Gerlag DM, Smeets TJ, Baeten D, van den Bosch F, Bresnihan B, Breedveld FC, Dinant HJ, Legay F, Gram H, Loetscher P, Schmouder R, Woodworth T, Tak PP (2006) A randomized controlled trial with an anti-CCL2 (anti-monocyte chemotactic protein 1) monoclonal antibody in patients with rheumatoid arthritis. Arthritis Rheum 54:2387–2392PubMedCrossRefGoogle Scholar
- Jeffes EW 3rd, Schmitz K, Yamamoto R, Tomich JM, Beckman M, Nep R, Knauer M (1991) A simple nonisotopic in vitro bioassay for LT and TNF employing sodium fluoride-treated L-929 target cells that detects picogram quantities of LT and TNF and is as sensitive as TNF assays done with ELISA methodology. Lymphokine Cytokine Res 10:147–151PubMedGoogle Scholar
- Kasaian MT, Tan XY, Jin M, Fitz L, Marquette K, Wood N, Cook TA, Lee J, Widom A, Agostinelli R, Bree A, Schlerman FJ, Olland S, Wadanoli M, Sypek J, Gill D, Goldman SJ, Tchistiakova L (2008) Interleukin-13 neutralization by two distinct receptor blocking mechanisms reduces immunoglobulin E responses and lung inflammation in cynomolgus monkeys. J Pharmacol Exp Ther 325:882–892PubMedCrossRefGoogle Scholar
- Li J, Tomkinson KN, Tan XY, Wu P, Yan G, Spaulding V, Deng B, Annis-Freeman B, Heveron K, Zollner R, De Zutter G, Wright JF, Crawford TK, Liu W, Jacobs KA, Wolfman NM, Ling V, Pittman DD, Veldman GM, Fouser LA (2004) Temporal associations between interleukin 22 and the extracellular domains of IL-22R and IL-10R2. Int Immunopharmacol 4:693–708PubMedCrossRefGoogle Scholar
- Llopis J, Westin S, Ricote M, Wang Z, Cho CY, Kurokawa R, Mullen TM, Rose DW, Rosenfeld MG, Tsien RY, Glass CK (2000) Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. Proc Natl Acad Sci USA 97:4363–4368PubMedCrossRefGoogle Scholar
- Pappu BP, Dong C (2007) Measurement of interleukin–17. Curr Protoc Immunol Chap. 6:Unit 6.25Google Scholar
- Wu C, Ying H, Grinnell C, Bryant S, Miller R, Clabbers A, Bose S, McCarthy D, Zhu RR, Santora L, Davis-Taber R, Kunes Y, Fung E, Schwartz A, Sakorafas P, Gu J, Tarcsa E, Murtaza A, Ghayur T (2007) Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 25:1290–1297PubMedCrossRefGoogle Scholar
- Zeng R, Spolski R, Leonard WJ (2007) Measurement of interleukin–21. Curr Protoc Immunol Chap. 6:Unit 6.30Google Scholar