R.M.A. Hoet

Generation and Application of Type-specific Anti-Heparan Sulfate Antibodies Using Phage Display Technology FURTHER EVIDENCE FOR HEPARAN SULFATE HETEROGENEITY IN THE KIDNEY

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A “single pot” semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene,DP-42), and YYHYKVN (VH1 gene,DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. K d values for HS are about 0.1 μm. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- andN-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.

Antiperinuclear factor, a marker autoantibody for rheumatoid arthritis: colocalisation of the perinuclear factor and profilaggrin.

The antiperinuclear factor, an autoantibody specific for rheumatoid arthritis, was found in 51/63 (81%) patients with rheumatoid arthritis by indirect immunofluorescence on human buccal mucosa cells. The sensitivity of the antiperinuclear factor test was increased by pretreating the buccal mucosa cells with 0.5% Triton-X100. The specificity of the test for rheumatoid arthritis as compared with control serum samples was maintained. The localisation of the perinuclear factor in the keratohyalin granules of the buccal mucosa cells was verified by immunoelectron microscopy. The perinuclear factor was found to be an insoluble protein whose antigenicity was sensitive to various fixation procedures. In serum samples from patients with rheumatoid arthritis there was a positive correlation between the presence of antiperinuclear factor and the presence of the so called antikeratin antibodies as detected by immunofluorescence on unfixed rat oesophagus cryostat sections. No relation was found between the presence of the perinuclear factor and either the rheumatoid factor, Epstein-Barr virus components, or any cytokeratin. By double immunofluorescence an exact colocalisation of the perinuclear factor and profilaggrin was found. Although the precise biochemical identity of the perinuclear factor remains unclear, our results suggest that it is a protein only present in the fully differentiated squamous epithelial cell layer.

Anti-(U1) small nuclear RNA antibodies in anti-small nuclear ribonucleoprotein sera from patients with connective tissue diseases.

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. Sera from patients with connective tissue diseases often contain antibodies against the proteins present in these snRNPs. Antibodies against the RNA components of snRNPs, the U snRNAs, are thought to be rare. We tested 118 anti-snRNP sera for the presence of anti-snRNA antibodies and found them in 45 sera (38%). In all sera the antibodies (IgG and F(ab)2 fragments thereof) were exclusively directed against U1 snRNA. The anti-(U1) RNA antibodies were always accompanied by anti-(U1)RNP antibodies but were not found in sera which contain antibodies of the Sm serotype directed against all nucleoplasmic U snRNP particles. Like anti-RNP antibodies, anti-U1 RNA activity is confined to sera from patients with SLE or SLE overlap syndromes and is rarely found in patients with other connective tissue diseases. By analyzing binding to subfragments of U1 snRNA made in vitro, it was demonstrated that anti-(U1)RNA antibodies recognize epitopes distributed throughout the U1 RNA molecule. In most sera, however, either the second or the fourth hairpin loop is the main target of the antibody. The possible mechanisms that could lead to the production of this new type of autoantibody are discussed.

The Antiperinuclear Factor (APF) and Antikeratin Antibodies (AKA) in Rheumatoid Arthritis

The diagnosis of rheumatoid arthritis (RA) is first of all based on clinical manifestations. Serological support for such a diagnosis is not very well established and it is based mainly on the presence of rheumatoid factors (RF). A positive RF test has a predictive value [1] and is related to disease with a more severe outcome [2]. However, RF is also present in other (autoimmune) diseases and in control sera from healthy persons [3]. Therefore, testing for a second RA-specific antibody would be very useful, and might even be necessary for the management of seronegative RA patients.

A new method for the analysis and production of monoclonal antibody fragments originating from single human B cells

The phage display approach has proven to be a major step forward in studies on the human autoimmune repertoire. However, it remains doubtful whether the heavy and light chains of the antibodies obtained from these libraries resemble original in vivo pairings. Here we describe a novel, simple method for the immortalization of the variable heavy and light chain regions originating from individual, nonboosted, autoantigen-specific human B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD40L) as well as soluble factors were shown to be essential. This B cell culture system combined with a selection on antigen (the U1A protein, a frequent autoantigenic target in patients with systemic lupus erythematosus) and single cell sorting resulted in the isolation of U1A-specific human B cells and the subsequent expression of an U1A-specific single chain variable fragment (scFv). Our method circumvents laborious plating and screening and has the advantage that original heavy/light chain pairings can be isolated. Due to the high growth efficiency of single cultured B cells (50-70% outgrowth) even rare B cell activities can be studied using this system.