Measurement of Human Serum IgD Levels

Catherine L. Overed‐Sayer1, David E. Mosedale2, Margaret Goodall3, David J. Grainger1

1 Department of Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom, 2 Pronostics Ltd., Babraham, Cambridge, United Kingdom, 3 Division of Immunity and Infection, The Medical School, University of Birmingham, Birmingham, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 2.9B
DOI:  10.1002/0471142735.im0209bs85
Online Posting Date:  April, 2009
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This unit describes an ELISA for the quantitative measurement of IgD levels in human serum. The ELISA is highly specific and sensitive, with a minimum detectable concentration of 30 pg/ml and more than 10,000‐fold specificity for IgD over all other human immunoglobulins. Linear dilution characteristics enable measurement of IgD concentrations ranging over 5 orders of magnitude. These factors are vital for the IgD assay, since IgD makes up only a small proportion of the total immunoglobulins present in normal sera, and IgD serum concentrations are known to vary widely between individuals. Curr. Protoc. Immunol. 85:2.9B.1‐2.9B.7. © 2009 by John Wiley & Sons, Inc.

Keywords: immunoglobulin D; serum; ELISA

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 50 mM Na 2CO 3, pH 9.6 (store up to 1 month at 4°C; allow to warm to room temperature before use, and ensure no crystals are present)
  • Mouse anti‐IgD antibody (clone JA11, Skybio, cat. no. M01015;
  • Wash buffer (see recipe)
  • Blocking buffer (see recipe)
  • Serum samples to be assayed
  • IgD purified from normal human serum, used as the standard (Athens Research and Technology, cat. no. 16‐16‐090704;
  • Rabbit anti‐IgD antibody (Dako, cat. no. A0093)
  • Donkey anti–rabbit IgG horseradish peroxidase (Jackson ImmunoResearch, cat. no. 711‐035‐152)
  • K‐Blue TMB substrate solution (Skybio, cat no. KB‐176)
  • 2 M H 2SO 4
  • Nunc Maxisorp ELISA plate wells (available in various formats)
  • Orbital microtiter plate shaker
  • Microtiter plate reader with 450‐nm filter
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Literature Cited

   Bahna, S.L., Heiner, D.C., and Myhre, B.A. 1983. Changes in serum IgD in cigarette smokers. Clin. Exp. Immunol. 51:624‐630.
   Drenth, J.P.H., Goertz, J., Daha, M.R., and van der Meer, J.W.M. 1996. Immunoglobulin D enhances the release of tumour necrosis factor‐α, and interleukin‐1β as well as interleukin‐1 receptor antagonist from human mononuclear cells. Immunology 88:355‐362.
   Dunnette, S.L., Gleich, G.J., Miller, R.D., and Kyle, R.A. 1977. Measurement of IgD by a double antibody radioimmunoassay: Demonstration of an apparent trimodal distribution of IgD levels in normal human sera. J. Immunol. 119:1727‐1731.
   Fraser, P.A. and Schur, P.H. 1981. Hypoimmunoglobulinaemia D: Frequency, family studies and association with HLA. Clin. Immunol. Immunopath. 19:67‐74.
   Haraldsson, A., Weemaes, C.M., Jonasdottir, S., Olafsson, O., Van Der Wiel, G., Goertz, J., and Klasen, I. 2000. Serum immunoglobulin D in infants and children. Scand. J. Immunol. 51:415‐418.
   Leslie, G.A., Lopez Correa, R.H., and Holmes, J.N. 1975. Structure and biological functions of human IgD. IV. Ontogeny of human serum immunoglobulin D (IgD) as related to IgG, IgA and IgM. Int. Arch. Allergy Appl. Immunol. 49:350‐357.
   Levan‐Petit, I., Cardonna, J., Garcia, M., Migeon, J., Corbi, C., Preud'homme, J., and Lecron, J. 2000. Sensitive ELISA for human immunoglobulin D measurement in neonate, infant and adult sera. Clin. Chem. 46:876‐878.
   Litwin, S.D. and Zehr, B.D. 1987. Membrane IgD‐positive B cells of “low‐IgD serum phenotype” individuals fail to secrete IgD and fail to shift to preferential lambda light‐chain expression in vitro. J. Clin. Immunol. 7:114‐120.
   Litzman, J., Ward, A.M., Wild, G., Znojil, V., and Morgan, G. 1997. Serum IgD levels in children under investigation for and with defined immunodeficiency. Int. Arch. Allergy Immunol. 114:54‐58.
   Mosedale, D.E., Sandhu, M.S., Luan, J., Goodall, M., and Grainger, D.J. 2006. A new sensitive and specific enzyme‐linked immunosorbent assay for IgD. J. Immunol. Meth. 313:74‐80.
   Peng, Z., Fisher, R. and Adkinson, N.F. Jr. 1991. Total serum IgD is increased in atopic subjects. Allergy 46:436‐444.
   Plebani, A., Avanzini, M.A., Massa, M., and Ugazio, A.G. 1984. An avidin‐biotin ELISA for the measurement of serum and secretory IgD. J. Immunol. Meth. 71:133‐140.
   Preud'homme, J.L., Petit, I., Barra, A., Morel, F., Lecron, J., and Lelievre, E. 2000. Structural and functional properties of membrane and secreted IgD. Mol. Immunol. 37:871‐887.
   Rowe, D. and Fahey, J.L. 1965. A new class of human immunoglobulins. I. A unique myeloma protein. J. Exp. Med. 121:171‐178.
   Rowe, D.S., Hug, K., Forni, L., and Pernis, B. 1973. Immunoglobulin D as a lymphocyte receptor. J. Exp. Med. 138:965‐972.
   Stoica, G., Macarie, E., Michiu, V., and Stoica, R.C. 1980. Biologic variation of human immunoglobulin concentration. I. Sex‐age specific effects on serum levels of IgG, IgA, IgM and IgD. Med. Interne 18:323‐332.
   van Boxel, J.A., Paul, W.E., Terry, W.D., and Green, I. 1972. IgD‐bearing human lymphocytes. J. Immunol. 109:648‐651.
   Walzer, P.D. and Kunkel, H.G. 1974. The correlation of serum IgD concentration and Gm allotype. J. Immunol. 113:274‐278.
Key References
   Mosedale et al., 2006. See above.
  This reference is the basis for this protocol.
   Vladutiu, A.O., 2000. Immunoglobulin D: Properties, measurement and clinical relevance. Clin. Diag. Lab. Immunol. 7:131‐140.
  This provides a useful summary of the measurement of IgD in human serum.
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