Development of a borreliosis assay: Mannan coated polyethylene sinter bodies as a new platform technology.

Rapid diagnosis of Lyme borreliosis has been carried out on chemically modified porous polyethylene sinter bodies. Photografting of 2-propenol on sinter body's surface was performed as a first step, introducing active hydroxyl groups as a result of polyalcohol formation. The hydroxyl groups were used for further immobilization and could be linked via 3-aminopropyltriethoxysilane (APTES) to polysaccharides like mannan. Prone to coupling, mannan was activated using N, N'-disuccinimidyl carbonate (DSC) to allow smooth reaction with the primary amine groups of the silane layer. In a final preparation step, a recombinant fusion protein consisting of the mannan-binding domain of the lectin Concanavalin A (ConA) and a specific Borrelia surface antigen was immobilized by self-organization on the mannan surface. The fusion protein was used as biological interface structure. This strategy is highly efficient and resulted in a defined orientation of the antigen part of the fusion protein. Rapid and convenient differentiation could be then established between Borrelia-negative and a -positive serum even in 1000-fold diluted samples and detection of Lyme borreliosis in a rather early stage is likely. Furthermore, this generic strategy can be easily transferred to other bacterial or viral antigen structures.

Insights into the Value of Lyso-Gb1 as a Predictive Biomarker in Treatment-Naïve Patients with Gaucher Disease Type 1 in the LYSO-PROOF Study.

Gaucher disease (GD) is a rare autosomal recessive disorder arising from bi-allelic variants in the GBA1 gene, encoding glucocerebrosidase. Deficiency of this enzyme leads to progressive accumulation of the sphingolipid glucosylsphingosine (lyso-Gb1). The international, multicenter, observational "Lyso-Gb1 as a Long-term Prognostic Biomarker in Gaucher Disease"-LYSO-PROOF study succeeded in enrolling a cohort of 160 treatment-naïve GD patients from diverse geographic regions and evaluated the potential of lyso-Gb1 as a specific biomarker for GD. Using genotypes based on established classifications for clinical presentation, patients were stratified into type 1 GD (n = 114) and further subdivided into mild (n = 66) and severe type 1 GD (n = 48). Due to having previously unreported genotypes, 46 patients could not be classified. Though lyso-Gb1 values at enrollment were widely distributed, they displayed a moderate and statistically highly significant correlation with disease severity measured by the GD-DS3 scoring system in all GD patients (r = 0.602, p < 0.0001). These findings support the utility of lyso-Gb1 as a sensitive biomarker for GD and indicate that it could help to predict the clinical course of patients with undescribed genotypes to improve personalized care in the future.

An integrated multiomic approach as an excellent tool for the diagnosis of metabolic diseases: our first 3720 patients.

To present our experience using a multiomic approach, which integrates genetic and biochemical testing as a first-line diagnostic tool for patients with inherited metabolic disorders (IMDs). A cohort of 3720 patients from 62 countries was tested using a panel including 206 genes with single nucleotide and copy number variant (SNV/CNV) detection, followed by semi-automatic variant filtering and reflex biochemical testing (25 assays). In 1389 patients (37%), a genetic diagnosis was achieved. Within this cohort, the highest diagnostic yield was obtained for patients from Asia (57.5%, mainly from Pakistan). Overall, 701 pathogenic/likely pathogenic unique SNVs and 40 CNVs were identified. In 620 patients, the result of the biochemical tests guided variant classification and reporting. Top five diagnosed diseases were: Gaucher disease, Niemann-Pick disease type A/B, phenylketonuria, mucopolysaccharidosis type I, and Wilson disease. We show that integrated genetic and biochemical testing facilitated the decision on clinical relevance of the variants and led to a high diagnostic yield (37%), which is comparable to exome/genome sequencing. More importantly, up to 43% of these patients (n = 610) could benefit from medical treatments (e.g., enzyme replacement therapy). This multiomic approach constitutes a unique and highly effective tool for the genetic diagnosis of IMDs.

Promising alternatives for one-tier testing of Lyme borreliosis.

A main focus of human health studies is the early detection of infectious diseases to enable more rapid treatment and prevent disease transmission. Diagnosis of Lyme borreliosis has been always challenging because of the lack of specific, but simple assay formats. Two-tiered testing has been recommended by US Centers for Disease Control and Prevention to provide more specific results for diagnosis of Lyme disease. However, such a technique is time consuming and is not well suited for early stage detection. Therefore, many tests were proposed as alternatives to overcome these drawbacks. Simple assays, which are mainly performed in one-tier manner, could be conducted with better performance than the two-tiered testing. Proposed assays utilize both newly identified antigens and new platforms to improve detection performance. These assays can be classified into those based on employing a single antigen and assays based on using multiple antigens. In addition to assays to this type of assays, immunoassays on borreliosis-related biomarkers are available. We report here the most recent assays developed over the last 10 years, for detection of Lyme borreliosis in body fluids.

A new platform for serological analysis based on porous 3-dimensional polyethylene sinter bodies.

A new sensitive and selective platform, three-dimensional immunosensor, has been developed for a rapid serological diagnosis; detection of a Borrelia infection was considered as a model assay. The immunosensor is based on a 3-dimensional (3D) porous solid surface (sinter body) with dimensions of 2×2.5mm where a recombinant variable lipoprotein surface-exposed protein (VlsE; Borrelia-antigen) is immobilized by different techniques. The sinter body served as a robust and inexpensive carrier, which facilitated a successful hydrophobic adsorption as well as covalent immobilization of the antigen with sufficient amounts of on the surface. Because of sinter body's porosity, the detection could be performed in an immune affinity flow system based on a little disposable plastic column. The flow of reagents through the column is advantageous in terms of reducing the non-specific interaction and shortening the test time. Furthermore, three labels were tested for a colorimetric detection: i) a horseradish peroxidase (HRP) labeled secondary antibody, ii) nanoparticles based on Sudan IV, and iii) gold nanoparticles modified with protein A. HRP secondary labeled antibody provides the most sensitive test, 1000 fold dilution of serum sample can be clearly detected in only 20min. Gold nanoparticles modified with protein A were used as a direct label or as a catalyst for reduction of silver ions. Direct detection with gold nanoparticles provides short time of analysis (5min) while detection of metallic silver required longer time (12min) but with improved sensitivity. Nanoparticles based on Sudan IV showed high background and were less favorable. The assay is distinctive because of the rapid analysis time with all used labels, longest 20min. Compared to classical serological methods for Borrelia diagnosis, the developed method offers a simple, rapid and reliable tool of analysis with minimal cost and can be easily transferred to other infectious diseases.

Surface plasmon resonance for real-time study of lectin-carbohydrate interactions for the differentiation and identification of glycoproteins.

A study of specific interactions between lectins and glycoproteins has been carried out using surface plasmon resonance (SPR) in a flow-injection mode. Lectins were covalently immobilised on the surfaces of the microfluidic sensor chip via amine coupling and serum glycoproteins were injected into the flow channels. Specific lectin-glycoprotein interactions caused the shift of refractive index proportional to the mass concentration accumulated on the channel surface. Lectins showed different affinity to the tested glycoproteins and each glycoprotein displayed its own lectin-binding pattern. It is possible to distinguish and identify even glycoproteins with similar sugar structures by simple and quick screening. The working conditions of the assay were optimised. The lectin-based SPR made it possible to carry out the label-free detection of glycoproteins within a broad concentration range with a good linearity. Regeneration conditions for the surface of the sensor chip were found and optimised. Combination of 10mM HCl and 10mM glycine-HCl (pH 2.5) removes the bound glycoproteins from the lectin surface without damaging it. The kinetic and affinity parameters of lectin-glycoprotein binding were evaluated. The proposed method was tested on human glycosylated serum. Combination of the lectin panel with SPR is suitable both for specific screening and for sensitive assay of serum glycoproteins.