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Measurement of Human Serum IgD Levels

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

¹Department of Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom
²Pronostics Ltd., Babraham, Cambridge, United Kingdom
³Division 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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Abstract

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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Basic Protocol
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol

Materials

50 mM Na₂CO₃, 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; http://home.btconnect.com/derek.price/skybio/)
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; http://www.athensresearch.com/)
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 H₂SO₄

Nunc Maxisorp ELISA plate wells (available in various formats)
Orbital microtiter plate shaker
Microtiter plate reader with 450-nm filter

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Figures

Figure 2.9B.1

Specificity of IgD ELISA. Purified immunoglobulins were assayed at 2 µg/ml using the IgD assay (black bars). Values are shown as percentage cross-reactivity of the IgD assay with each of the purified immunoglobulins, by calculation from an IgD standard curve. Purified immunoglobulins (2 µg/ml) were also spiked into samples containing 2.5 ng/ml purified IgD, and the data shown are percentage recovery versus 2.5 ng/ml IgD alone (gray bars). Adapted from Mosedale et al. (2006), with permission.

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Figure 2.9B.2

Linearity of dilution. Each serum sample was diluted in wash buffer by a range of dilution factors. The IgD concentration was determined from the standard curve by multiplying by the appropriate dilution factor. Only data from samples that fell within the range of the standard curve following dilution are included in the figure. Adapted from Mosedale et al. (2006), with permission.

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Figure 2.9B.3

Standard curve of purified IgD. The standard curve was run on eight separate occasions, and the values of the lower standards normalized to the optical density of the top standard. Values shown are means ± 95% confidence intervals. Adapted from Mosedale et al. (2006), with permission.

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Literature Cited

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-, and interleukin-1 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.