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Chapter 9 : Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid
Authors:
Elizabeth Sykes1,
Yvonne Posey2
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Affiliations:
1: Beaumont Hospital – Royal Oak, Clinical Pathology, 3601 W. 13 Mile Road, Royal Oak, MI 48073;
2: Beaumont Hospital – Royal Oak, Clinical Pathology, 3601 W. 13 Mile Road, Royal Oak, MI 48073
Content Type: Reference
Publication Year:
2016
Category: Immunology
Book DOI:
10.1128/9781555818722
Chapter DOI:
10.1128/9781555818722.ch9
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Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid, Page 1 of 2
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Abstract:
The characterization of immunoglobulins spans a spectrum of methods, including molecular analysis of gene usage and rearrangement, quantitation of immunoglobulin heavy chains as well as intact and free light chains, qualitative assessment and characterization of clonality, and identification of abnormalities that may be clinically significant, such as hyperviscosity syndrome, cryoglobulinemia, and amyloidosis (AL). This chapter focuses on qualitative methods for the assessment and characterization of clonality. The methods include agarose gel electrophoresis (AGE) with immunofixation, capillary electrophoresis (CE) with immunosubtraction (ISUB), and isoelectric focusing with immunoblotting or immunofixation. 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 ISUB electrophoresis are diagnostic tools used for the identification of monoclonal gammopathies and, conversely, for the confirmation of polyclonal hypergammaglobulinemia. Isoelectric focusing with immunoblotting or immunofixation is a cerebrospinal fluid (CSF) diagnostic test for the identification of oligoclonal bands in multiple sclerosis (MS).
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
Figures
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid
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Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig1
FIGURE 1
(A) Normal serum electropherogram; (B) normal serum immunofixation gel.
10.1128/9781555818722/8722ch9fig1_thmb.gif
10.1128/9781555818722/8722ch9fig1.gif
FIGURE 1
(A) Normal serum electropherogram; (B) normal serum immunofixation gel.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig2
FIGURE 2
(A) Electropherogram with monoclonal protein in the gamma region; (B) immunofixation gel showing an IgG lambda monoclonal protein.
10.1128/9781555818722/8722ch9fig2_thmb.gif
10.1128/9781555818722/8722ch9fig2.gif
FIGURE 2
(A) Electropherogram with monoclonal protein in the gamma region; (B) immunofixation gel showing an IgG lambda monoclonal protein.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig3
FIGURE 3
Serum immunofixation gel with no staining in the lambda lane due to lambda antiserum not being applied.
10.1128/9781555818722/8722ch9fig3_thmb.gif
10.1128/9781555818722/8722ch9fig3.gif
FIGURE 3
Serum immunofixation gel with no staining in the lambda lane due to lambda antiserum not being applied.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig4
FIGURE 4
Normal serum immunosubtraction pattern.
10.1128/9781555818722/8722ch9fig4_thmb.gif
10.1128/9781555818722/8722ch9fig4.gif
FIGURE 4
Normal serum immunosubtraction pattern.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig5
FIGURE 5
Serum immunosubtraction pattern with IgG lambda monoclonal protein.
10.1128/9781555818722/8722ch9fig5_thmb.gif
10.1128/9781555818722/8722ch9fig5.gif
FIGURE 5
Serum immunosubtraction pattern with IgG lambda monoclonal protein.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig6
FIGURE 6
(A) Electropherogram with polyclonal increase in gamma globulins; (B) immunofixation gel with polyclonal increase in IgG (a “fuzzy” band is noted in the lambda lane).
10.1128/9781555818722/8722ch9fig6_thmb.gif
10.1128/9781555818722/8722ch9fig6.gif
FIGURE 6
(A) Electropherogram with polyclonal increase in gamma globulins; (B) immunofixation gel with polyclonal increase in IgG (a “fuzzy” band is noted in the lambda lane).
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig7
FIGURE 7
Polyclonal increase in an IgG4 subclass. (A) Electropherogram with a rounded peak in the beta-gamma region; (B) immunofixation gel showing an increase in staining in the beta-gamma region in the IgG and both kappa and lambda lanes.
10.1128/9781555818722/8722ch9fig7_thmb.gif
10.1128/9781555818722/8722ch9fig7.gif
FIGURE 7
Polyclonal increase in an IgG4 subclass. (A) Electropherogram with a rounded peak in the beta-gamma region; (B) immunofixation gel showing an increase in staining in the beta-gamma region in the IgG and both kappa and lambda lanes.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig8
FIGURE 8
Serum immunofixation gel with a biclonal gammopathy (IgG lambda and IgA kappa). Antigen excess is also present in the IgA and kappa lanes.
10.1128/9781555818722/8722ch9fig8_thmb.gif
10.1128/9781555818722/8722ch9fig8.gif
FIGURE 8
Serum immunofixation gel with a biclonal gammopathy (IgG lambda and IgA kappa). Antigen excess is also present in the IgA and kappa lanes.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig9
FIGURE 9
Serum immunofixation gel with a free lambda monoclonal protein.
10.1128/9781555818722/8722ch9fig9_thmb.gif
10.1128/9781555818722/8722ch9fig9.gif
FIGURE 9
Serum immunofixation gel with a free lambda monoclonal protein.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig10
FIGURE 10
IgG heavy-chain disease. (A) Immunosubtraction showing removal of only IgG from the small gamma peak (no change is seen with kappa and lambda antisera); (B) immunofixation gel with only an IgG band.
10.1128/9781555818722/8722ch9fig10_thmb.gif
10.1128/9781555818722/8722ch9fig10.gif
FIGURE 10
IgG heavy-chain disease. (A) Immunosubtraction showing removal of only IgG from the small gamma peak (no change is seen with kappa and lambda antisera); (B) immunofixation gel with only an IgG band.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig11
FIGURE 11
Prominent oligoclonal banding. (A) Immunosubtraction; (B) immunofixation gel.
10.1128/9781555818722/8722ch9fig11_thmb.gif
10.1128/9781555818722/8722ch9fig11.gif
FIGURE 11
Prominent oligoclonal banding. (A) Immunosubtraction; (B) immunofixation gel.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig12
FIGURE 12
Contrast media artifact. (A) Electropherogram from capillary electrophoresis showing a peak (arrow) in the alpha-2/beta region; (B) immunofixation gel with no band corresponding to the contrast medium peak.
10.1128/9781555818722/8722ch9fig12_thmb.gif
10.1128/9781555818722/8722ch9fig12.gif
FIGURE 12
Contrast media artifact. (A) Electropherogram from capillary electrophoresis showing a peak (arrow) in the alpha-2/beta region; (B) immunofixation gel with no band corresponding to the contrast medium peak.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig13
FIGURE 13
“Shadow” artifact on an immunofixation gel caused by incomplete washing of monoclonal protein from the gel (lanes IgA, IgM, and lambda).
10.1128/9781555818722/8722ch9fig13_thmb.gif
10.1128/9781555818722/8722ch9fig13.gif
FIGURE 13
“Shadow” artifact on an immunofixation gel caused by incomplete washing of monoclonal protein from the gel (lanes IgA, IgM, and lambda).
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig14
FIGURE 14
(A) Densitometric scan from a concentrated urine specimen showing a monoclonal protein (1,000 mg/24 h) in the gamma region on urine PE. The patient had 0.7 g/dl of IgG lambda monoclonal protein (including C3 complement) in the beta globulin region on serum PE/IFE. (B) Urine IFE performed on the specimen used in panel A demonstrates a free lambda monoclonal protein.
10.1128/9781555818722/8722ch9fig14_thmb.gif
10.1128/9781555818722/8722ch9fig14.gif
FIGURE 14
(A) Densitometric scan from a concentrated urine specimen showing a monoclonal protein (1,000 mg/24 h) in the gamma region on urine PE. The patient had 0.7 g/dl of IgG lambda monoclonal protein (including C3 complement) in the beta globulin region on serum PE/IFE. (B) Urine IFE performed on the specimen used in panel A demonstrates a free lambda monoclonal protein.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig15
FIGURE 15
IFE of a concentrated urine specimen demonstrating a dominant free kappa and a smaller amount of IgG kappa monoclonal protein.
10.1128/9781555818722/8722ch9fig15_thmb.gif
10.1128/9781555818722/8722ch9fig15.gif
FIGURE 15
IFE of a concentrated urine specimen demonstrating a dominant free kappa and a smaller amount of IgG kappa monoclonal protein.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig16
FIGURE 16
IFE of a concentrated urine specimen demonstrating a free kappa and a free lambda monoclonal protein in a patient with IgA kappa and IgA lambda monoclonal proteins detected on serum IFE.
10.1128/9781555818722/8722ch9fig16_thmb.gif
10.1128/9781555818722/8722ch9fig16.gif
FIGURE 16
IFE of a concentrated urine specimen demonstrating a free kappa and a free lambda monoclonal protein in a patient with IgA kappa and IgA lambda monoclonal proteins detected on serum IFE.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9fig17
FIGURE 17
Seven paired CSF (lanes 1 to 4 and 6 to 8) and serum (lanes 1′ to 4′ and 6′ to 8′) specimens along with two positive CSF controls (lanes 5 and 9) and two negative CSF controls (lanes 5′ and 9′) were subjected to IEF for the detection of oligoclonal bands. The patient specimens in lanes 1 and 1′ and 7 and 7′ reveal oligoclonal bands consistent with MS. The pattern for the patient in lanes 2 and 2′ are consistent with the presence of a monoclonal protein (posttranslational changes account for the multiple monoclonal protein bands seen with IEF). The patterns in lanes 3 and 3′ have faint bands appearing in both the CSF and serum specimens; this is nondiagnostic. The patterns in lanes 4 and 4′ and 8 and 8′ are normal.
10.1128/9781555818722/8722ch9fig17_thmb.gif
10.1128/9781555818722/8722ch9fig17.gif
FIGURE 17
Seven paired CSF (lanes 1 to 4 and 6 to 8) and serum (lanes 1′ to 4′ and 6′ to 8′) specimens along with two positive CSF controls (lanes 5 and 9) and two negative CSF controls (lanes 5′ and 9′) were subjected to IEF for the detection of oligoclonal bands. The patient specimens in lanes 1 and 1′ and 7 and 7′ reveal oligoclonal bands consistent with MS. The pattern for the patient in lanes 2 and 2′ are consistent with the presence of a monoclonal protein (posttranslational changes account for the multiple monoclonal protein bands seen with IEF). The patterns in lanes 3 and 3′ have faint bands appearing in both the CSF and serum specimens; this is nondiagnostic. The patterns in lanes 4 and 4′ and 8 and 8′ are normal.
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
References
/content/book/10.1128/9781555818722.ch9
Tables
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid
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Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9tab1
TABLE 1
Distribution of plasma cell proliferative disorders at the Mayo Clinic from 1960 to 2003 a
TABLE 1
Distribution of plasma cell proliferative disorders at the Mayo Clinic from 1960 to 2003 a
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9
/content/10.1128/9781555818722.ch9.ch9tab2
TABLE 2
Protocol for urine concentration used at Beaumont Laboratory
TABLE 2
Protocol for urine concentration used at Beaumont Laboratory
Citation:
Sykes E, Posey Y.
2016.
Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid,
p 89-100.
In
Detrick B, Schmitz J, Hamilton R (ed),
Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition.
ASM Press, Washington, DC.
doi: 10.1128/9781555818722.ch9