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Chapter 113 : Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation

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Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, Page 1 of 2

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Abstract:

Antinuclear antibodies (ANA) are markers for systemic autoimmune disease but also can be seen at low titers in healthy individuals. Thus, there has been considerable interest in identifying ANA subsets with greater diagnostic specificity for particular diseases. Autoantibodies specific for systemic lupus erythematosus (SLE), systemic sclerosis (SSc), polymyositis or dermatomyositis (PM/DM), and other systemic autoimmune disorders have been identified and are of considerable importance as diagnostic markers. The prevalence of some of the more useful autoantibody markers for various diseases is summarized. Double immunodiffusion (DID) (Ouchterlony method) is a classic immunoassay to detect interactions of antigens and antibodies. Antigenic proteins or protein complexes in the cell extract and antibodies in the serum diffuse toward one another in the gel and form insoluble antigen-antibody complexes when the antigen-to-antibody ratio is near equivalence. Gel diffusion and enzyme-linked immunosorbent assay (ELISA), unlike Western blot assays, allow the detection of autoantibodies to native anti-genic determinants, but the composition of the antigenic particles and fine specificities of the autoantibodies cannot readily be determined. Immunoprecipitation is a highly sensitive and specific technique for detecting a variety of autoantibodies. Immunoprecipitation is the gold standard for detecting autoantibodies against Ku/DNA-dependent protein kinase (DNA-PK). Autoantibodies against aminoacyl tRNA synthetases are diagnostic markers for PM/DM and the antisynthetase autoantibody syndrome.

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113

Key Concept Ranking

Small Nuclear RNA
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Small Nucleolar RNA
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Small Nuclear RNA
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Small Nucleolar RNA
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Small Nuclear RNA
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Small Nucleolar RNA
0.5113636
Small Nuclear RNA
0.5113636
Small Nucleolar RNA
0.5113636
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Figures

Image of FIGURE 1
FIGURE 1

Homemade gel puncher for DID. A design for assembling a simple “cookie cutter” punch for agarose gels out of drinking straws is shown. (A) Arrangement of the straws. Seven straws (black) are used to punch holes in the gel, and six additional straws (white) are used as spacers and recessed so that they do not contact the agarose gel. (B) Assembled hole puncher. Straws are assembled as shown in the diagram, using Super Glue. Note the recessed spacer straws that maintain the proper distance between holes.

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
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Image of FIGURE 2
FIGURE 2

DID in agarose gels. A photograph of the assay is shown on the left, and an interpretation is diagrammed on the right. (A) Appearance of precipitin lines when properly diluted reference sera with the following specificities are used: anti-nRNP anti-Sm (note line of partial identity with anti-nRNP), anti-Ro, anti-Jo-1, anti-ribosomal P (r-P), and anti-La. (B) Determining the optimal serum dilution. Test serum was diluted 1:1, 1:4, 1:16, 1:64, 1:256, and 1:1,024 with PBS. The optimal dilution for precipitin line 1 is 1:4; optimal dilution for line 2 is 1:64. (C and D) DID using dilutions of 1:4 (C) and 1:64 (D) of the serum shown in panel B. Precipitin line 1 is identified as anti-La (C). Precipitin line 2 is identified as anti-nRNP (D).

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
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Image of FIGURE 3
FIGURE 3

Typical arrangements of sera and antigens for DID. (A and B) Arrangements for testing an unknown precipitin line for several specificities. Panel A illustrates the use of a single antigen; Panel B illustrates use of two different test antigens (e.g., RTE and human lymphocyte extract). (C and D) Arrangements for testing several sera for a single autoantibody specificity when reference serum is precious. Ag1 and Ag2, antigens; u1 to u4, sera with unknown specificities; a to d, reference sera.

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
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Image of FIGURE 4
FIGURE 4

Protein immunoprecipitation for analyzing SLE sera. Sera were tested according to the protocol in the text, and immunoprecipitated proteins were resolved on a 12.5% gel. Sera containing the following autoantibodies were tested: anti-nRNP (RNP), anti-Sm, anti-ribosomal P (r-P), anti-PCNA, anti-Ki, anti-Ro60, anti-La (SS-B), anti-Ku, anti-Ku plus anti-DNA-PKcs (Ku-DNAPK) anti-Su, anti-RNAP II, anti-RNA helicase A (RHA), and normal human serum (NHS). Positions of molecular weight standards are shown on the left (in kilodaltons). Protein components of the antigens are indicated (dots).

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
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Image of FIGURE 5
FIGURE 5

Protein immunoprecipitation for analyzing myositis (PM/DM) sera. Sera were tested according to the protocol in the text, and immunoprecipitated proteins were resolved on an SDS-8% polyacrylamide gel. Sera containing the following autoantibodies were tested: anti-histidyl tRNA synthetase (Jo-1, 50 kDa), anti-alanyl tRNA synthetase (PL-12, 110 kDa), anti-glycyl tRNA synthetase (EJ, 75 kDa), anti-threonyl tRNA synthetase (PL-7, 80 kDa), anti-isoleucyl tRNA synthetase (OJ, 150 kDa and multiprotein complex), and anti-signal recognition particle (SRP, 54-kDa protein and multiprotein complex). Protein components of the antigens are indicated (dots).

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
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Image of FIGURE 6
FIGURE 6

Protein immunoprecipitation for analyzing scleroderma sera. Sera were tested according to the protocol in the text, and immunoprecipitated proteins were resolved on an SDS-8% polyacrylamide gel. Sera containing the following autoantibodies were tested: anti-RNAP I, II, and III (RNAP I/II/III); anti-RNAP I and III (RNAP I/III); anti-RNAP II, unphosphorylated and phosphorylated forms (RNAP IIA/O); anti-RNAP II, phosphorylated form (RNAP IIO); anti-topo I (100 kDa); antifibrillarin (34 kDa); and anti-NOR90 (doublet--95 kDa). Protein components of the antigens are indicated (dots).

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
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References

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1. Arnett, F. C.,, J. D. Reveille,, H. M. Moutsopoulos,, L. Georgescu, and , K. B. Elkon. 1996. Ribosomal P autoantibodies in systemic lupus erythematosus. Arthritis Rheum. 39:18331839.
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19. Satoh, M.,, H. B. Richards,, K. J. Hamilton, and , W H. Reeves. 1997. Human anti-nRNP autoimmune sera contain a novel subset of autoantibodies that stabilizes the molecular interaction of U1RNP-C protein with the Sm core proteins. J. Immunol. 158:50175025.
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Tables

Generic image for table
TABLE 1

Some non-DNA/chromatin autoantibodies useful in the diagnosis of systemic autoimmune disease

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
Generic image for table
TABLE 2

Prevalence of some non-DNA/chromatin autoantibodies by disease

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113
Generic image for table
TABLE 3

Gold standards for autoantibody testing (non-DNA/chromatin antigens)

Citation: Reeves W, Satoh M, Lyons R, Nichols C, Narain S. 2006. Detection of Autoantibodies against Proteins and Ribonucleoproteins by Double Immunodiffusion and Immunoprecipitation, p 1007-1018. 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.ch113

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