21st European Workshop for Rheumatology Research
- Meeting abstract
Phage display as a tool to study human autoantibodies and autoantigens in systemic autoimmune disease. Selection of recombinant (auto)-antibodies specific for human autoantigens in rheumatic disease (RA, SLE, SSc) from human autoimmune-patient and immunized chicken derived phage display libraries
Arthritis Research & Therapy volume 3, Article number: P116 (2001)
One important property of the immune system is its ability to discriminate between self and non-self antigens. The unresponsiveness of the immune system to self-antigens is called self-tolerance, loss of this property results in immune reactions against own or autologous antigens. Such reactions often are associated with an autoimmune disorder and eventually may contribute to clinical manifestations. The humoral immune response plays a crucial role in the onset of autoimmunity, and it is a general observation that autoimmune diseases are associated with distinct profiles of autoantibodies. During the last five years there has been a growing interest in finding possible connections between apoptosis and autoimmunity. It has been hypothesized that the recognition, uptake, processing, or presentation of modified self-antigens may promote autoantibody production. At present, many autoantigens have been found that are modified (i.e. cleaved, phosphorylated or dephosphorylated) during apoptosis. Using autoimmune patient sera in immunoprecipitation and Western blotting assays with cell extracts derived from non-apoptotic and apotptic cells, we identified sera reactivities specific for (novel) modifications of autoantigens.
In this paper we give an overview of our studies on human recombinant autoantibodies derived from patients suffering from rheumatic diseases. Furthermore, we describe the generation of recombinant chicken antibodies specific for human autoantigens that will also be used to study these antigens and (apoptotic) modifications thereof in more detail.
We selected human recombinant antibodies from patient phage display scFv combinatorial antibody libraries (complexity of 107 or higher) derived from peripheral blood or bone marrow lymphocytes of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and scleroderma (SSc). Next to the human patient libraries we also used chicken libraries made from spleens of animals immunized with 7 human (auto)antigens simultaneously. Screening of both patient and animal derived libraries with recombinant or purified autoantigens resulted in several recombinant monoclonal human and chicken (auto)-antibodies. From our SLE patient libraries, autoantibodies against U1snRNP components U1-A, U1-C, U1-70k and U1-RNA were selected. Moreover, from our SLE libraries we selected anti Ro52 and anti ribosomal P protein antibodies, and from both our SLE and scleroderma libraries we selected anti-La and anti a-fodrine recombinant antibodies. From our RA patient libraries antibodies specific for RA related peptides were obtained. All human scFv clones obtained were characterized by ELISA, immunoprecipitation assays, western blotting, epitope mapping and of some clones the affinities were measured and competition experiments with patient sera were performed. Sequence analysis was performed to study the germline usage. All chicken clones were sequenced and analyzed by LIA, Western blot and ELISA.
We will discuss characteristics of some of the selected scFv's i.e. epitope mapping, germline gene usage, and competition experiments with patient sera.
The phage autoantibodies selected from autoimmune patient libraries were also analyzed for their specificity for (apoptotically) modified forms of their target autoantigens by Western blotting and immunprecipitation assays using apoptotic cell-extracts. Some anti-La, anti-70K and anti-RA peptide scFv's recognized (apoptotically)-modified forms of their target antigens.
The use of antibody phage display proves to be an extremely helpful technique in studying autoantibodies and autoantigens. Modifications of autoantigens (by apoptosis and/or necrosis) seem to play a major role in the ontogeny of autoimmune diseases. Currently, by using antibody phage display libraries in combination with patient sera we continue our search for possible modifications of autoantigens involved in the ontogeny of autoimmune disease.
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Raats, J., Degen, W., Litjens, S. et al. Phage display as a tool to study human autoantibodies and autoantigens in systemic autoimmune disease. Selection of recombinant (auto)-antibodies specific for human autoantigens in rheumatic disease (RA, SLE, SSc) from human autoimmune-patient and immunized chicken derived phage display libraries. Arthritis Res Ther 3 (Suppl 2), P116 (2001). https://doi.org/10.1186/ar285
- Systemic Lupus Erythematosus
- Systemic Lupus Erythematosus Patient
- Phage Display
- Patient Seron
- Recombinant Antibody