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Chapter 125 : Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis

Authors: A. Bernard Collins, Robert B. Colvin
Content Type: Reference
Publication Year: 2006

Category: Immunology

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Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis, Page 1 of 2

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Abstract:

Several diseases of the kidney with overlapping histological and clinical features are characterized by a rapid loss of renal function, crescentic glomerulonephritis, and sometimes lung hemorrhage. Serological assays for the detection of anti-neutrophil cytoplasmic antibodies (ANCA) and anti-glomerular basement membrane (anti-GBM) autoantibodies are important diagnostic tools in the evaluation of patients with rapidly progressive renal failure and/or pulmonary hemorrhage, providing a rapid noninvasive method for establishing a diagnosis. These tests are sensitive and highly specific and can provide definitive diagnostic information for the effective clinical management of patients with a rapidly progressive course of disease. This chapter describes the detection of circulating anti-GBM antibodies by serological techniques, including Western blot analysis and enzyme-linked immunosorbent assay (ELISA).

Citation: Collins A, Colvin R. 2006. Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis, p 1110-1115. 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.ch125

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Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis
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Citation: Collins A, Colvin R. 2006. Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis, p 1110-1115. 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.ch125
/content/10.1128/9781555815905.ch125.ch125fig01
FIGURE 1
Western blot analysis of a serum sample from an Alport syndrome patient who developed posttransplant anti-GBM nephritis (lane 1). The reactivity of the antibody is to the α3(IV) chain, the Goodpasture antigen, at 28 and 48 to 50 kDa. The reactivity is identical to that seen with a monoclonal antibody to the α3(IV) collagen chain (lane 2) and with that of a serum from a patient with renal-biopsy-proven Goodpasture syndrome (lane 3).
10.1128/9781555815905/f1111-01_thmb.gif
10.1128/9781555815905/f1111-01.gif
Image of FIGURE 1
FIGURE 1

Western blot analysis of a serum sample from an Alport syndrome patient who developed posttransplant anti-GBM nephritis (lane 1). The reactivity of the antibody is to the α3(IV) chain, the Goodpasture antigen, at 28 and 48 to 50 kDa. The reactivity is identical to that seen with a monoclonal antibody to the α3(IV) collagen chain (lane 2) and with that of a serum from a patient with renal-biopsy-proven Goodpasture syndrome (lane 3).

Citation: Collins A, Colvin R. 2006. Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis, p 1110-1115. 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.ch125
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/content/10.1128/9781555815905.ch125.ch125fig02
FIGURE 2
Western blot analysis of another serum sample from an Alport syndrome patient who also developed post-transplant anti-GBM nephritis. However, when immunoblotted on the collagenase digest of the human GBM it shows reactivity to a lower-molecular-mass antigen in the monomer region of the NC1 domain, at approximately 26 kDa instead of 28 kDa (lane 3). It shows the same reactivity to a higher-molecular-mass antigen in the dimer region, at approximately 48 to 50 kDa. The reactivity is to α5(IV) chain collagen and is identical to reactivity of a monoclonal antibody to the α5(IV) chain collagen (lane 2). Serum from a patient with renal-biopsy-proven Goodpasture syndrome is in lane 1.
10.1128/9781555815905/f1111-02_thmb.gif
10.1128/9781555815905/f1111-02.gif
Image of FIGURE 2
FIGURE 2

Western blot analysis of another serum sample from an Alport syndrome patient who also developed post-transplant anti-GBM nephritis. However, when immunoblotted on the collagenase digest of the human GBM it shows reactivity to a lower-molecular-mass antigen in the monomer region of the NC1 domain, at approximately 26 kDa instead of 28 kDa (lane 3). It shows the same reactivity to a higher-molecular-mass antigen in the dimer region, at approximately 48 to 50 kDa. The reactivity is to α5(IV) chain collagen and is identical to reactivity of a monoclonal antibody to the α5(IV) chain collagen (lane 2). Serum from a patient with renal-biopsy-proven Goodpasture syndrome is in lane 1.

Citation: Collins A, Colvin R. 2006. Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis, p 1110-1115. 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.ch125
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/content/10.1128/9781555815905.ch125.ch125fig03
FIGURE 3
Western blot analysis of sera from seven patients with biopsy-proven anti-GBM nephritis (lanes 3 to 8). Note the reduced intensity of the bands in lane 7 compared to lane 8. The serum sample in lane 7 was from the same patient as that in lane 8, but after 11 plasmapheresis exchanges. Positive control serum and normal control serum are in lanes 1 and 2, respectively. The antigen preparation is a collagenase digest of human GBM, which is separated by PAGE and immunoblotted onto nitrocellulose paper. The reactivity seen in the positive sera is to the same two molecular mass regions at 28 and 50 kDa, which represent the a3(IV) NC1 (Goodpasture antigen) in its monomeric and dimeric forms.
10.1128/9781555815905/f1113-01_thmb.gif
10.1128/9781555815905/f1113-01.gif
Image of FIGURE 3
FIGURE 3

Western blot analysis of sera from seven patients with biopsy-proven anti-GBM nephritis (lanes 3 to 8). Note the reduced intensity of the bands in lane 7 compared to lane 8. The serum sample in lane 7 was from the same patient as that in lane 8, but after 11 plasmapheresis exchanges. Positive control serum and normal control serum are in lanes 1 and 2, respectively. The antigen preparation is a collagenase digest of human GBM, which is separated by PAGE and immunoblotted onto nitrocellulose paper. The reactivity seen in the positive sera is to the same two molecular mass regions at 28 and 50 kDa, which represent the a3(IV) NC1 (Goodpasture antigen) in its monomeric and dimeric forms.

Citation: Collins A, Colvin R. 2006. Kidney and Lung Disease Mediated by Anti-Glomerular Basement Membrane Antibodies: Detection by Western Blot Analysis, p 1110-1115. 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.ch125
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References

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