Publications by authors named "Pavel Damborsky"

6 Publications

  • Page 1 of 1

Full-length antibodies versus single-chain antibody fragments for a selective impedimetric lectin-based glycoprofiling of prostate specific antigen.

Electrochim Acta 2017 08;246:399-405

Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 845 38 Bratislava, Slovakia.

The main aim of the research was to design a functional impedimetric biosensor able to glycoprofile prostate specific antigen (PSA), a biomarker for prostate cancer (PCa), with high specificity using lectins as glycan recognising proteins. Traditionally, full-length antibody is immobilised on the biosensor interface for specific capture of PSA with subsequent glycoprofiling of PSA by addition of lectins. Since full-length antibodies contain glycans in the Fc domain, particular attention has to be paid to suppress direct binding of lectins to immobilised full-length antibodies, which would compromise accurate glycoprofiling. This issue is addressed here using a recombinant single-chain antibody fragments (scAb), which do not contain any carbohydrate moiety. Surface plasmon resonance was applied to prove negligible interaction of lectins with immobilised scAb fragments, while substantial binding of lectins to full length antibodies was observed. Eight different biosensor designs were tested for their ability to detect PSA. The biosensor device based on scAb fragments covalently immobilised on the gold electrode surface, patterned by a mixed SAM using standard amine coupling chemistry, proved to be the most sensitive. The scAb fragment-based biosensor exhibited sensitivity of 15.9 ± 0.8% decade (R = 0.991 with an average RSD of 4.9%), while the full antibody-based biosensor offered sensitivity towards PSA of 4.2 ± 0.1% decade (R = 0.999 with an average RSD of 4.8%). Moreover, the selectivity of the scAb-based biosensor was tested using a kallikrein 2 protein, a protein structurally similar to PSA, and the results indicated high selectivity for PSA detection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.electacta.2017.06.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667744PMC
August 2017

Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody: Application to the Construction of an Ultrasensitive Glycan Biosensor.

Langmuir 2017 03 8;33(11):2709-2716. Epub 2017 Mar 8.

Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences , Dubravska cesta 9, 845 38 Bratislava, Slovakia.

The main aim of the study was to optimize the interfacial presentation of a small antigen-a Tn antigen (N-acetylgalactosamine)-for binding to its analyte anti-Tn antibody. Three different methods for the interfacial display of a small glycan are compared here, including two methods based on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding with better kinetics of binding (t = 137 s, t = the time needed to attain 50% of a steady-state signal) compared to the 2D biosensor configuration with t = 354 s. The 3D glycan biosensor was finally applied for the analysis of a human serum sample spiked with an analyte.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.langmuir.6b04021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659382PMC
March 2017

Determining the binding affinities of prostate-specific antigen to lectins: SPR and microarray approaches.

Proteomics 2016 12;16(24):3096-3104

Department of Glycobiotechnology, Center for Glycomics, Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia.

Prostate cancer (PCa) is one of the most common newly diagnosed cancers among men and we focused on its traditional biomarker, prostate-specific antigen (PSA), using targeted glycomics-based strategies. The aberrant glycosylation pattern of PSA may serve as a valuable tool for improving PCa diagnosis including its early-stage. In this study, we evaluated the usability of two techniques, surface plasmon resonance and protein microarray assay, for the study and characterization of interactions of PSA (both free and complexed) with six lectins (SNA, ConA, RCA, AAL, WGA and MAA II). The information on the character of such interactions is important for the application of lectins as prospective bioreceptors for biomarker glycoprofiling in a follow-up biosensing assays. SPR as well as established bioanalytical techniques allowed determination of K values of PSA-lectin interactions in a more reliable way than protein microarray. The protein microarray method did not allow accurate quantification of K values. However, the features of a microarray approach, such as speed and costs, enabled the screening and estimation of the nature of lectin-glycan biomarker interaction in an effective and time-saving way. All of the tested lectins interacted with commercial PSA standard isolated from healthy persons, except MAA II which reacted only very weakly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201500466DOI Listing
December 2016

Lectin-based lateral flow assay: proof-of-concept.

Analyst 2016 Nov;141(23):6444-6448

Department of Glycobiotechnology, Center for Glycomics, Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 84105, Slovakia.

Lateral flow assays (LFAs) enable the simple and rapid detection and quantification of analytes and is popular for point-of-care (PoC), point-of-use and outdoor testing applications. LFAs typically depend on antibody or nucleic acid based recognition. We present the innovative concept of a LFA using lectins in the role of the biorecognition element. Lectins are a special kind of glycan-binding protein and the lectin-based LFA herein described was developed for the determination of the glycosylation of free prostate specific antigen (PSA). PSA is routinely used as a biomarker of prostate cancer (PCa) and the glycosylation status of PSA is a more specific marker of disease progress than only the PSA level. Using the lectin-based LFA we were able to detect α-2,6 sialic acid present in fPSA using Sambucus nigra (SNA) lectin. As a negative control, we employed Maackia amurensis lectin II (MAA II) which specifically binds α-2,3 sialic acid. The novel approach presented here can be applied to a wide range of biomarkers that have a significant impact on clinical diagnosis and prognosis, providing an alternative to standard lectin-based assays. The assay uses commercial components and is easily performed by applying a sample to the sampling pad on the lectin-based LFA strip, with results obtained within 10 minutes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c6an01746kDOI Listing
November 2016

Optical biosensors.

Essays Biochem 2016 06;60(1):91-100

Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 845 38 Bratislava, Slovakia

Optical biosensors represent the most common type of biosensor. Here we provide a brief classification, a description of underlying principles of operation and their bioanalytical applications. The main focus is placed on the most widely used optical biosensors which are surface plasmon resonance (SPR)-based biosensors including SPR imaging and localized SPR. In addition, other optical biosensor systems are described, such as evanescent wave fluorescence and bioluminescent optical fibre biosensors, as well as interferometric, ellipsometric and reflectometric interference spectroscopy and surface-enhanced Raman scattering biosensors. The optical biosensors discussed here allow the sensitive and selective detection of a wide range of analytes including viruses, toxins, drugs, antibodies, tumour biomarkers and tumour cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/EBC20150010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986466PMC
June 2016

DNA aptamer-based sandwich microfluidic assays for dual quantification and multi-glycan profiling of cancer biomarkers.

Biosens Bioelectron 2016 May 19;79:313-9. Epub 2015 Dec 19.

Department of Electronic & Electrical Engineering, University of Bath, Bath BA2 7AY, United Kingdom. Electronic address:

Two novel sandwich-based immunoassays for prostate cancer (PCa) diagnosis are reported, in which the primary antibody for capture is replaced by a DNA aptamer. The assays, which can be performed in parallel, were developed in a microfluidic device and tested for the detection of free Prostate Specific Antigen (fPSA). A secondary antibody (Aptamer-Antibody Assay) or a lectin (Aptamer-Lectin Assay) is used to quantify, by chemiluminescence, both the amount of fPSA and its glycosylation levels. The use of aptamers enables a more reliable, selective and controlled sensing of the analyte. The dual approach provides sensitive detection of fPSA along with selective fPSA glycoprofiling, which is of significant importance in the diagnosis and prognosis of PCa, as tumor progression is associated with changes in fPSA glycosylation. With these approaches, we can potentially detect 0.5 ng/mL of fPSA and 3 ng/mL of glycosylated fPSA using Sambucus nigra (SNA) lectin, both within the relevant clinical range. The approach can be applied to a wide range of biomarkers, thus providing a good alternative to standard antibody-based immunoassays with significant impact in medical diagnosis and prognosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bios.2015.12.058DOI Listing
May 2016
-->