Reproducible detection of relevant autoantibodies in some human autoimmune diseases including multiple sclerosis (MS) has proven to be a difficult task, in part because commonly used techniques only permit detection of antibodies directed against linear epitopes on denatured proteins (Western blot) or do not adequately discriminate between denatured and folded proteins (ELISA) (1,2). Solution-phase binding of antibodies to monomeric radiolabeled autoantigens is a powerful technique for identification of pre-diabetic individuals (3-5), but only enables detection of relatively high affinity autoantibodies. In animal models of antibody-mediated autoimmunity, such as hemolytic anemia, low affinity autoantibodies to membrane proteins can be highly pathogenic in vivo because antigen clustering enables bivalent antibody binding (6). Multimeric ligands have proven useful for detection of antigen-specific T and B cells (7,8), and we reasoned that increasing the valency of a target antigen could permit detection of rare and/or low affinity antibodies by enabling bivalent binding.
We have developed a method for the production of a series of multimeric antigens based on the ability of the streptavidin (SA) monomer to spontaneously assemble into a stable tetrameric structure. Multimerization of the antigen enables identification of antibodies that have a low binding affinity or are present at low concentrations. In vitro translation with ER microsomes (9) allows rapid expression of radiolabeled proteins that require disulfide bond formation, glycosylation and chaperone-assisted folding. The assay can be multiplexed by combining multiple tetramers that differ in molecular weight, such as the MOG-SA and CD2-SA tetramers described in the accompanying Nature Medicine article. The assembled SA tetramer is resistant to a variety of denaturing agents, permitting selective unfolding of the antigen domain for parallel assessment of conformation-sensitive and conformation-insensitive antibodies with the same technique. Tetrameric RIA offers a high level of specificity, as antibodies are rarely detected at low levels in control sera. Comparisons with different assays have demonstrated that tetramer RIA is more sensitive than monomer RIA, ELISA or FACS. Our method permits characterization of autoantibodies that are difficult to detect with classical techniques, and could be applied to the study of antibodies in a variety of autoimmune diseases.
The protocol outlined below details how to produce radiolabeled antigen-streptavidin tetramers for detection of antibodies by immunoprecipitation. Optionally, the antigen tetramers can be denatured to compare responses to folded and unfolded antigen in the same system. This technique can be applied to a large or small number of samples, and a given sample can be simultaneously assayed with multiple antigens.