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Chapter 10 : Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid

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Abstract:

This chapter focuses on qualitative methods for the assessment and characterization of clonality. The methods include nondenaturing agarose gel electrophoresis (AGE) with immunofixation, capillary zone electrophoresis (CZE) with immunosubtraction, and isoelectric focusing with immunoblotting. All three methods can be used to identify monoclonal, oligoclonal, and polyclonal immunoglobulin populations and to identify the heavy and/or light chains contained in the population. Immunofixation electrophoresis (IFE) and immunosubtraction electrophoresis (ISE) are diagnostic tools used for the identification of monoclonal gammopathies and, conversely, for the confirmation of polyclonal hypergammaglobulinemia. Isoelectric focusing with immunoblotting is a cerebrospinal fluid (CSF) diagnostic test for the identification of oligoclonal bands (OCB) in multiple sclerosis (MS). The monoclonal gammopathies include a spectrum of diseases, from ones that may have little clinical significance to ones that may be rapidly fatal. These include multiple myeloma (MM), Waldenström’s macroglobulinemia, smoldering MM, monoclonal gammopathy of undetermined significance (MGUS), primary systemic amyloidosis (AL), lymphoproliferative diseases, and plasmacytomas. High-resolution agarose gel protein electrophoresis (PEL) and CZE fulfill the requirements for a screening procedure for detection of monoclonal proteins. Monoclonal free light chains are uncommon in MGUS and are associated predominantly with MM or AL. The assessment of urine samples by IFE is similar to serum IFE. The major difference is the need to concentrate urine samples to achieve an appropriate protein concentration. The concentration of immunoglobulins is increased in the CSF of patients with inflammatory diseases of the central nervous system, such as MS, neurosyphilis, and acute inflammatory polyradiculoneuropathy.

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10

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Figures

Image of FIGURE 1
FIGURE 1

PEL and IFE of normal serum. (Top) PEL gel scan and electrophoretogram with the albumin (Alb), α, α, β, and γ fractions. (Bottom) IFE gel scan. Lanes were immunofixed with antisera to γ (G), α (A), μ (M), κ (K), and λ (L). The distribution of normal serum immunoglobulins is illustrated.

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 2
FIGURE 2

PEL and IFE of a serum containing a monoclonal protein. The PEL indicates that the M-spike equals 7.2 g/liter, and the IFE indicates that it is a monoclonal IgG λ protein. The γ and λ bands are sharp and comigrate. There is very little polyclonal immunoglobulin in this serum sample. Alb, albumin; lanes G to L, as defined in the legend to Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 3
FIGURE 3

PEL gel scan and IFE of a serum containing a biclonal gammopathy. The PEL gel contains two discrete bands in the γ fraction. The IFE indicates that there is a γ band with a corresponding κ band plus a μ band with a corresponding κ band. The biclonal gammopathy contains a monoclonal IgG κ and a monoclonal IgM κ protein. Lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 4
FIGURE 4

PEL and IFE of a serum containing a small monoclonal IgG κ protein. The protein is too small to be fractionated and quantitated as an M-spike. The γ fraction contains mostly polyclonal immunoglobulin, and the PEL electrophoretogram shows a small bump on top of the γ fraction. The PEL gel scan contains a small, fuzzy band corresponding to the asymmetry in the electrophoretogram, and the IFE shows some corresponding increased, restricted reactivity in the γ and κ lanes. Lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 5
FIGURE 5

PEL and IFE of a serum specimen from a patient with LCMM. Although this patient has myeloma, there is no obvious PEL abnormality. The IFE, however, clearly shows a discrete λ band with no corresponding γ, α, or μ reactivity. Lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 6
FIGURE 6

PEL and IFE of a serum from a patient with an IgD myeloma. The PEL contains a small M-spike that has a corresponding λ band as determined by IFE. The IFE was repeated with antisera to κ, λ, δ (D), and ɛ (E) and indicated that the monoclonal protein is an IgD λ immunoglobulin. Lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 7
FIGURE 7

PEL and IFE of a sample containing fibrinogen. The PEL contains a fast-γ peak that is indistinguishable from a small monoclonal protein. The IFE shows no corresponding heavy- or light-chain bands. Lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 8
FIGURE 8

PEL of a hemolyzed serum sample. The gel scan appears to contain a small monoclonal protein in the α-β region. Alb, albumin.

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 9
FIGURE 9

PEL, IFE, and quantitative immunoglobulins of a serum sample that contains a large IgG κ M-spike. The large, narrow IgG band has saturated the amount of stain in the PEL agarose gel. The M-spike is 44.9 g/liter, whereas the IgG nephelometric quantitation is 50% higher at 64.9 g/liter. Alb, albumin; lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 10
FIGURE 10

PEL and IFE of urine from a patient with LCMM. The PEL shows a large M-spike in the β fraction. The IFE shows a dense λ band corresponding to the M-spike. In addition, there are small amounts of additional λ reactivity in the γ fraction that most likely represent multimers of the monoclonal λ protein. Alb, albumin; lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 11
FIGURE 11

PEL and IFE of urine from a patient with primary systemic AL. The urine contains 5 g of protein per 24-h collection. The PEL shows mostly albumin and no apparent monoclonal protein. The IFE shows a faint λ band in the fast-γ region. Alb, albumin; lanes G to L, as defined for Fig. 1 .

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 12
FIGURE 12

ISE of a normal serum sample. The CZE pattern is shown in the upper left panel (SPE, serum protein electrophoresis). The upper right panel shows the CZE pattern after subtraction of IgG from the sample. Except for the remaining IgM and IgA near the β fraction, almost the entire γ fraction is removed. The IgA panel shows the CZE after subtraction of IgA, and the troughs surrounding the β fraction now appear closer to the baseline. The κ and λ panels show the reduction in the γ fraction after removal of the immunoglobulins that contain κ or λ light chains.

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 13
FIGURE 13

Immunosubtraction of a serum sample from a patient with an IgG κ monoclonal protein. The M-spike is apparent by AGE and CZE. After subtraction, the M-spike is removed with antiserum to IgG and κ. Note that the polyclonal portion of the γ fraction is also removed by the IgG reagent and is reduced by the κ and λ reagents. CE/IS, capillary electrophoresis/immunosubtraction; SPE, serum protein electrophoresis.

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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Image of FIGURE 14
FIGURE 14

IEF with IgG immunoblotting of paired CSF-serum samples. (A) Positive oligoclonal banding result for an MS patient. The CSF contains more than four IgG OCB that are not seen in the serum. (B) Negative result with no IgG bands detected in the CSF or serum. (C) Negative result with no IgG bands that are CSF specific. (D) Banding pattern obtained with a sample that contains a monoclonal protein.

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
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References

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Tables

Generic image for table
TABLE 1

Distribution of plasma cell proliferative disorders at Mayo Clinic, 1960 to 2003

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
Generic image for table
TABLE 2

Monoclonal serum proteins detected at Mayo Clinic, 1960 to 2003

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10
Generic image for table
TABLE 3

Monoclonal serum proteins: newly diagnosed MM

Citation: Katzmann J, Kyle R. 2006. Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, p 88-100. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch10

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