Chapter 88 : Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays

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Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays, Page 1 of 2

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Autoantibodies directed against intracellular antigens are characteristic features of a number of human autoimmune diseases and certain malignancies (1–3). Studies of systemic autoimmune rheumatic diseases have provided strong evidence that autoantibodies are maintained by antigen-driven responses (4, 5) and that autoantibodies can be reporters from the immune system, revealing the identities of antigens involved in disease pathogenesis. Historically, autoantibody detection and analysis have relied on a number of different technologies, such as hemagglutination and particle aggregation, immunodiffusion, indirect immunofluorescence (IIF), complement fixation, counterimmunoelectrophoresis (CIE), Western and dot blotting, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA), and on functional assays that demonstrate inhibition of the catalytic or other functional activity of the antigen of interest. These technologies have limitations because they tend to be labor-intensive and time-consuming, are limited in throughput, are semiquantitative, and are not adaptable to leading-edge research. Immunodiffusion has been used for over 50 years, and it is still used in some clinical laboratories because it is inexpensive and has high specificity, but it lacks sensitivity and can take up to 48 h before precipitin lines are interpretable. Western blotting is more costly and time-consuming, and not all autoantibodies are detected by this technique. For example, in the SS-A/Ro system, it has been observed that IP techniques are required to identify some sera that contain antibodies reacting only with the “native” SS-A/Ro particle (6). IP protocols that use extracts from [S]methionine-labeled cells are not suitable for the detection of all autoantibodies, such as antibodies to Ro52/TRIM21 protein (7). ELISA techniques have rapidly advanced, but highly specific, sensitive, and reliable assays that use highly purified or recombinant proteins are limited by intermanufacturer and interlaboratory variation of results (8). Immunodiffusion and CIE generally favor high-titer sera and often cannot discriminate multiple autoantibody responses that are characteristic of systemic autoimmune rheumatic disease sera.

Citation: Chan E, Burlingame R, Fritzler M. 2016. Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays, p 859-867. 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.ch88
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Enhanced expression of recombinant proteins is illustrated by the effect on the bacterial host. A construct, pET-p115, was transformed into either JM109(DE3) or Rosetta(DE3), and recombinant proteins were produced with the transformed bacteria using the same culture medium (2YT broth) and conditions. To the cultured bacteria was added 1 mM isopropyl-b--thiogalactopyranoside at time zero, and cells were harvested as pellets after 1 or 2 h, solubilized in gel sample buffer, and analyzed with SDS-PAGE using a 5% gel. Proteins were detected with Coomassie brilliant blue G-250 stain. Note that the recombinant JM109 culture generated major products of ~60 kDa (arrowhead) plus other lower-molecular-mass products, whereas the Rosetta culture yielded major products (arrow) corresponding to the native protein. Lane M, molecular mass markers (numbers at the left are masses in kilodaltons).

Citation: Chan E, Burlingame R, Fritzler M. 2016. Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays, p 859-867. 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.ch88
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Comparison of results of ELISA and ALBIA detection of anti-ribosomal P autoantibodies in 30 sera

Citation: Chan E, Burlingame R, Fritzler M. 2016. Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays, p 859-867. 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.ch88
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Frequency of autoantibodies detected by the INOVA Quanta Plex assay in cohorts of patients with systemic rheumatic diseases and multiple sclerosis and in controls

Citation: Chan E, Burlingame R, Fritzler M. 2016. Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays, p 859-867. 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.ch88
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Advantages, challenges, and opportunities relating to new array technologies

Citation: Chan E, Burlingame R, Fritzler M. 2016. Detection of Autoantibodies by Enzyme-Linked Immunosorbent Assay and Bead Assays, p 859-867. 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.ch88

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