CONICET | Buscador de Institutos y Recursos Humanos

Antibodies that bind to DNA are a hallmark of the autoimmune disease in systemic lupus erythematosus (SLE), but these are not specific to particular sequences of single- or double stranded DNA. In the clinic, this fact leads to important limitations in diagnostic assays, since their precisions mostly relies on the wide range of antibody affinities found in the patients´ anti-DNA serum antibodies. In camelids, a subset of the immunoglobulins consists of heavy-chain homodimers devoid of light chains, called heavy-chain IgGs. Their antigen-binding site consists of one single domain, referred as VHH or nanobodies (Nb). These Nb are highly soluble, stable and bear long CDR3 loops than can reach into and fill out molecular cavities on proteins that are usually inaccessible to conventional antibodies, constituting an interesting alternative for molecular mimicry and diagnostic assays.ED84 is a high-affinity and specific anti-DNA monoclonal antibody generated against a double-stranded 18 bp oligonucleotide. Llama immunization with ED84 led to a strong anti-idiotypic (anti-Id) response, allowing us to generate several anti-Id Nb against ED84. In particular, Nb79 was shown to compete with DNA for ED84 binding, demonstrating its functional mimicry with the anti-DNA ED84 immunogen. In this work, we will present our latest results in Nb79:ED84 biophysical characterization by size exclusion chromatography, crystal growth and optimization screenings. Over 500 different crystallization conditions from Hampton Research and Jena Bioscience were tested with our Honeybee963 robot, yielding preliminary plate-shaped crystals in a single condition (PEG Rx 1 screen, condition No. 5, with 25% PEG 300 + 0.1 M Bis-Tris, pH 6.5). These crystallographic and biophysical studies will shed light on the underlying bases of molecular mimicry and will also contribute to the development of useful diagnostic tools for clinical management of SLE patients.