Biomedical subjects

Jos Raats

Publications and source records attributed to Jos Raats.

5 recordsLinked to original sources

PubMed2006

Experimental autoimmune encephalomyelitis induction in peptidylarginine deiminase 2 knockout mice.

During the development of multiple sclerosis the destruction of the myelin sheath surrounding the neurites is accompanied by citrullination of several central nervous system (CNS) proteins, including myelin basic protein and glial fibrillary acidic protein. In experimental autoimmune encephalomyelitis (EAE), a disease induced in animals by immunization with proteins or peptides from the CNS, the animals develop symptoms similar to multiple sclerosis (MS). The increased levels of citrullinated CNS proteins associated with MS are also observed during the development of EAE. To study the role of CNS protein citrullination in EAE development, we induced EAE with a peptide derived from myelin oligodendrocyte glycoprotein (MOG(35-55)) in mice lacking the peptidylarginine deiminase 2 (PAD2) protein, because this enzyme was the most likely candidate to be involved in catalyzing CNS protein citrullination in the diseased state. Even though the PAD2 knockout mice displayed a dramatic reduction in the amount of citrullination present in the CNS, indicating that PAD2 is indeed responsible for the majority of detectable citrullination observed in EAE, the development of EAE was not impaired by genetic deletion of PAD2, suggesting that PAD2 catalyzed citrullination is not essential to the development of EAE.

Animals↗

PubMed2005

Plexin D1 expression is induced on tumor vasculature and tumor cells: a novel target for diagnosis and therapy?

We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections.

Amino Acid Sequence↗

PubMed2004

Cloning and characterization of two human Ro52-specific monoclonal autoantibodies directed towards a domain associated with congenital heart block.

Autoantibodies against amino acid 200-239 (p200) in the predicted leucine zipper region of the Ro52 protein are associated with congenital heart block, a potentially fatal condition that may affect fetuses of women with Ro52 autoantibodies. To allow detailed studies of the antibodies associated with congenital heart block, B-cell derived combinatorial antibody libraries from patients were screened for Ro52 and p200 specific antibody clones. Two human monoclonal anti-p200 antibody fragments, S3A8 and M4H1, were isolated and analysed with regard to VHand VL gene utilization, somatic mutations and binding properties. Both identified clones recognized recombinant and native intact Ro52, and reacted only with p200 in a set of related Ro52 peptides. The specificity and affinity was confirmed by biosensor measurements. Structural analysis of overlapping peptides revealed increased helicity in the p200 peptide compared to non-recognized peptides, indicating epitope conformation as essential for antibody binding. Both monoclonals produced punctate nuclear and diffuse cytoplasmic staining in human and mouse cell lines. The identified antibodies, which react specifically with the leucine zipper structure of Ro52, will be valuable in further exploration of the mechanisms operating during development of Ro52 antibody-associated congenital heart block.

Antibodies, Monoclonal↗

PubMed2003

Generating recombinant anti-idiotypic antibodies for the detection of haptens in solution.

A new method is described for generating recombinant human and chicken antibody fragments for accurate quantification of haptens in solution. The chemistry of labelling small molecules has always been a problem in the development of immunoassays. Here, we describe a specific panning procedure that enables the selection of recombinant anti-idiotypic phage antibodies that bind to hapten binding molecules (e.g., antibodies) in the absence of the hapten, but are displaced in a highly specific and concentration dependent manner, in the presence of the hapten. The major advantage of such a detection system is that there is no need to label the hapten or to covalently attach it to a solid phase. In this study we demonstrate, using cortisol and aldosterone as model haptens, that the recombinant antibody phage display technology offers great possibilities to generate recombinant anti-idiotypic antibodies. Furthermore, we show that such antibodies can be used successfully to design highly sensitive immunoassays for the quantification of small molecules.

Aldosterone↗

PubMedDate not supplied

Phage display antibody-based proteomic device using resonance-enhanced detection.

The combination of phage display antibody arrays with a novel nanotransducer technique based on resonant nanoparticles in a nanosandwiched film enables the sensitive parallel screening of proteins. Using the resonance of nanoparticles with their induced mirror dipoles in a thin-film structure, limitations of fluorophores, such as unspecific background and nonvisibility to the eye, can be overcome, thereby leading to an optical signal significantly more sensitive than that of standard colloid techniques. The signal can be both directly observed as a color change of a microdot at the sensor surface and tuned throughout the visible range of the spectrum. Here we report the application of an optical chip using scFv-antibody-antigen interactions. Artificial scFv-antibodies against a variety of proteins, including yeast enzymes and bovine serum albumin (as a standard), were constructed via Phage Display. These scFv-antibodies were then coated onto metal nanoclusters and bound to their antigens that were arrayed as nanodroplets at the resonance layer of the chip. ScFv-Antibody-antigen interaction resulted in a visible array of microdots. Using resonance-enhanced absorption, the absorption signal of the spots was amplified by one to two orders of magnitude (compared to colloid-based techniques). For quantitative analysis, either an 8-micron scanner or a CCD camera (resolution 4 microns) was employed to gain direct-reflection spectra rather than unspecific scatter data (prone to dust and unspecific interaction). Our results demonstrate that this device enables high-throughput proteomics to overcome some limitations of fluorescence, enzyme labels, and colloid techniques.

Antibodies↗