Quantitative immunoassay of biotoxins on hydrogel-based protein microchips

Rubina, A. Yu.; Dyukova, V.I.; Dementieva, Ekaterina; Stomakhin, A. A.; Nesmeyanov, V. A.; Ev, Grishin; Заседателев, А. С.

doi:10.1016/j.ab.2005.01.042

Cited by 91 publications

(63 citation statements)

References 24 publications

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“…The signal measured directly after printing was considered as reference value (value set to 1). The immobilization efficiency measured for the 3D immobilization technique was 60 %, which is in good agreement with the literature (Rendl et al, 2011;Moschallski et al, 2010;Rubina et al, 2005;Rubina et al, 2003). In case of the coated arrays, a second loss in immobilized biomolecules was observed and the obtained signal was dropped to 30% of the initial value obtained (Figure 5.7).…”

Section: Study Of the Diffusion In The Hydrogelsupporting

confidence: 80%

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The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

Mahmoud¹

2015

JOSHA

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JOSHA is a service that helps scholars, researchers, and students descover, use, and build upon a wide range of content DeclarationI hereby confirm that this master thesis at hand is solely my own written work and that no other sources, than those indicated in the references, were used.All text passages and diagrams, quoted or paraphrased, from these sources have been properly acknowledged as such and fully cited.This thesis has not been submitted in the same, similar or even partial version to another examination board and was not published elsewhere. In the past few years, this technology has developed a lot with the implementation of novel surface chemistries, detection techniques, and different assay formats. 3D-hydrogel microarrays were developed using unique immobilization techniques based on hydrophilic polymer networks. The 3D polymer network attaches and immobilizes the capture molecules onto the surface in form of a spot with molecules immobilized both on the surface and inside the gel matrix. This retains the molecule's natural confirmation and structure and enables higher immobilization efficiency, which allows for higher sensitivity. However, the achieved sensitivities are still far from the theoretical limit and in many cases, long incubation times are required. These limitations are caused by both the resolution of the used detection techniques and the assay kinetics. This hinders the transfer of the technology from the laboratory to routine diagnostics.The kinetics of a biochemical assay in a microarray can be controlled either by the transport of molecules to the spot or by the binding interaction itself. This work focuses on studying the assay kinetics in 3D-hydrogel microarrays in order to understand and characterize the limiting step for signal development. To simulate conditions of high affinity binding partners and therefore reduce the influence of the reaction kinetics, the biotin-streptavidin system was selected as the biological model for this work.A microarray to study the kinetic processes involved in the signal development was designed and the optimum working concentrations for kinetic characterization were defined. To confirm the mass transport limited kinetics, the measured kinetics was compared to the ideal reaction kinetics depending on the affinity parameters for biotin-streptavidin interaction. The ideal reaction kinetics was three orders of magnitude faster than measured kinetics. The two-compartment model, which is widely used to describe the assay kinetics in 2D microarrays, was used to fit the observed kinetics. However, a deviation from the model after the initial phase of signal development was observed. A hypothesis was made that this deviation is due to an additional diffusion step in the hydrogel. Therefore, a microarray model to study this diffusion step was designed. In this model, the microarrays were dip coated with hydrogel layers of various thickness and mesh sizes. This model simulated conditions where the signal development should depend only on the diffusion t...

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Section: Study Of the Diffusion In The Hydrogelsupporting

confidence: 80%

“…One antibody is immobilized on the substrate called capture antibody and another labeled antibody is used for the detection namely, the detection antibody (Figure 1.2.c) (Rubina et al, 2005). First, the analyte is added to the capture antibody followed by a washing step to remove non-bound molecules.…”

Section: Sandwich Immunoassaymentioning

confidence: 99%

The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

Mahmoud¹

2015

JOSHA

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“…Methods were developed based principally on immunoassays [208] and mass spectrometry analysis [209]. An immuno-PCR set up for the determination of viscumin [210] could also be employed.…”

Section: Bioweaponsmentioning

confidence: 99%

Ribosome-inactivating proteins: progress and problems

Stirpe

Battelli

2006

Cell. Mol. Life Sci.

314

225

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Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apoptotic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies ('immunotoxins') or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.

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“…A rapid and unquestionable identification of corynebacteria that are tox-positive and express active DT is necessary for an appropriate diagnosis of a diphtheria infection. All techniques known to date rely on 3 basic principles: (a) the immunologic detection of DT such as the common Elek test (29 ) and a number of derivatives such as immunoblots, ELISAs (30,31 ), immunochromatographic strip assays (32 ), assays based on fluorescence nanocrystal quantum-dots (33 ), hydrogel-based protein microchips (34 ), and antibody-based microarrays (35 ); (b) the molecular biological detection of the tox-gene by PCR assays (36 ); and (c) the detection of the enzymatic activity of DT by infecting guinea pigs (4,31 ), in vitro translation assays, and ADP-ribosylation assays (22 ).…”

Section: Discussionmentioning

confidence: 99%

Quantification of Diphtheria Toxin–Mediated ADP-Ribosylation in a Solid-Phase Assay

et al. 2007

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Background: Because of reduced vaccination programs, the number of diphtheria infections has increased in the last decade. Diphtheria toxin (DT) is expressed by Corynebacterium diphtheriae and is responsible for the lethality of diphtheria. DT inhibits cellular protein synthesis by ADP-ribosylation of the eukaryotic elongation factor 2 (eEF2). No in vitro system for the quantification of DT enzymatic activity exists. We developed a solid-phase assay for the specific detection of ADPribosylation by DT. Methods: Solid phase-bound his-tag eEF2 is ADPribosylated by toxins using biotinylated NAD؉ as substrate, and the transferred biotinylated ADP-ribose is detected by streptavidin-peroxidase. DT enzymatic activity correlated with absorbance. We measured the amount of ADP-ribosylated eEF2 after precipitation with streptavidin-Sepharose. Quantification was done after Western blotting and detection with anti-his-tag antibody using an LAS-1000 System. Results: The assay detected enzymatically active DT at 30 ng/L, equivalent to 5 mU/L ADP-ribosylating activity. Pseudomonas exotoxin A (PE) activity was also detected at 100 ng/L. We verified the assay with chimeric toxins composed of the catalytic domain of DT or PE and a tumor-specific ligand. These chimeric toxins revealed increased signals at 1000 ng/L. Heat-inactivated DT and cholera toxin that ADP-ribosylates G-proteins did not show any signal increase. Conclusions: The assay may be the basis for the development of a routine diagnostic assay for the detection of DT activity and highly specific inhibitors of DT.

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Quantitative immunoassay of biotoxins on hydrogel-based protein microchips

Cited by 91 publications

References 24 publications

The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

Ribosome-inactivating proteins: progress and problems

Quantification of Diphtheria Toxin–Mediated ADP-Ribosylation in a Solid-Phase Assay

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