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

Abstract:

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 [35S]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.