Comparative characteristic of the methods of protein antigens epitope mapping

OIu, Halkin

doi:10.15407/ubj86.04.164

Cited by 4 publications

(2 citation statements)

References 43 publications

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“…Epitope mapping enables one tos obtain genetic engineering peptides (proteins) and their subsequent study as potential antigenic determinants. At present, many variants of this methodological approach have been developed, in particular: phage and bacterial display, site-directed mutagenesis, mapping of recombinant proteins with tag (Galkin, 2014b). The methodological principles of use of MPs in epitope mapping technologies were formed at the end of the 20th century (Kala et al, 1997;McConnell et al, 1999).…”

Section: Methods For Detecting Of Systems Based On Mpsmentioning

confidence: 99%

Modern magnetic immunoassay: Biophysical and biochemical aspects

Galkin

Besarab

Pysmenna

et al. 2017

Regul. Mech. Biosyst.

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In this review article an analysis of the biochemical and biophysical aspects of modern magnetic immunoassay (MIA) is conducted and additionally the problems and perspectives of its application in biology, biotechnology and medicine are defined. Magnetic immunoassay should be considered as an evolutionary extension of the classical immunoassay. MIA can have many variants of modifications, similar to the classic immunoenzymatic assay. The key distinctive element of the MIA is the use of magnetic particles (MPs), which are usually nanoparticles. MPs in the MIA can act as a marker for detection, or the solid phase at which the immunochemical reaction takes place. MIA possesses basic advantages over classical immunoassay methods: thanks to the unique magnetic properties of the MPs and the ability to manipulate it in the external magnetic field, it is possible to increase the informative value of the analysis (first of all, sensitivity and specificity), as well as the rigid requirements for “purity” of tested samples. For the purposes of immunoassay, magnetic particles of size from 10 to 200 nm are important, since such particles are in a superparamagnetic state, in the absence of strong magnetic fields; they are not agglomerated in a liquid medium. The size of the spherical particle determines the rate of sedimentation and mobility in the solution. The outer polymeric membrane serves as a matrix in which the surface functional groups are added, and also protects the core of the metal from the external environment. The outer shell may also consist of agarose, cellulose, porous glass, silicon dioxide etc. There are several strategies for the synthesis of nanoparticles: mechanical (dispersion), physical (gas phase deposition), wet chemical methods (chemical comprecipitation, thermal decomposition, methods of micro emulsion, hydrothermal reactions) and physico-chemical methods. Also used are magnesite nanoparticles of biogenic origin. Magnetic particles may function, and this is important for immunoassay. Surface functional groups include carboxylic, amino, epoxy, hydroxyl, tosyl, and N-hydroxysuccinate-activated groups. Magnetic spherical particles usually interact with surface molecules such as streptovidine, biotin, protein A, protein G, and immunoglobulin etc. Directions and prospects of the development of methods of magnetic immunoassay are determined, mainly, by the development of methods for detecting or influencing magnetic particles. In this case, the classical methods of detection are electrochemical methods, electrochemiluminescence, fluorescence. More modern ones include giant magnetoresistance, superconducting quantum interference devices, surface-enhanced Raman spectroscopy, biosensors based on nonlinear magnetization, magneto-PCR immunoassay. The current trend is to combine or integrate the application of various biochemical, physical, molecular and genetic, physico-chemical detection methods. In fact, all of these benefits undoubtedly open up broad prospects for the practical application of MIA in biology, biotechnology and medicine.

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Section: Methods For Detecting Of Systems Based On Mpsmentioning

confidence: 99%

Modern magnetic immunoassay: Biophysical and biochemical aspects

Galkin

Besarab

Pysmenna

et al. 2017

Regul. Mech. Biosyst.

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“…The first bond connects the chain N-end and helix С Cys 1-4 (Cys-2-Cys-92); the second one is formed between the loop AB and the beginning of helix D Cys 3-6 (Cys 47-Cys 141). The third disulfide bond is between the loop AB and the lower part of the helix D Cys 2-5 (Сys 34-Сys 129) The molecule also has 3 sites of N-glycosylation in the positions Asn 95, Asn 116 and Asn 141 [6,7].…”

Section: Introductionmentioning

confidence: 99%

Development of the method of isolation and purification of the recombinant human interleukin-7

Lutsenko

Motronenko

2022

Biopolym. Cell

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To develop a method for isolation and purification of recombinant human interleukin-7 from bacterial inclusion bodies. Methods. Protein synthesis via fermentation, densitometry, refolding method of gel filtration, two-stage chromatographic purification of the protein.Results. The inclusion bodies obtained via fermentation in E.coli cells contained recombinant human interleukin-7, which was solubilized at 22ºС for 1 hour in the following buffer solution: 7M of guanidine hydrochloride, 100mM of Tris-НСl, 0.1 % Tween-20 and 50mM of dithiothreitol. Renaturation was conducted by replacing the buffer on column XK26 / 40 (GE Healthcare) with Sephadex G-25 fine equilibrated Clark-Labs solution containing L-arginine hydrochloride and tween-20. Subsequent purification of rIL-7 on the gel filtration column was performed in two stages: at the first stage, purification of rIL-7 was performed on a column packed with 20 ml of Q sepharose, at the second stage, the rIL-7 fraction purified on Q sepharose was purified on SP sepharose. Elution of rIL-7 was performed using double-stage gradient NaCL (0.2 and 0.7M) in a buffer 0.05 M of KH 2 PO 4 /NaOH with pH 6.0; 0.1 М of L-arginine; 0.1 % Tween-80. Conclusions. The amount of the target protein after all the proposed stages of purification was 10 % with the purity ratio about 95 %, which is sufficient for further development of the various pharmaceutical forms.

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Specificity of Manifacturing Process Validation for Diagnostic Serological Devices

Galkin¹

2018

Biotechnol. acta

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directly applied to medical devices for in vitro diagnostics. It was shown that the specifics of application and raw materials require individual validation parameters and process validations for serological diagnostics devices. Critical parameters to consider in validation plans were provided for every typical stage of production of in vitro diagnostics devices on the example of immunoassay kits, such as obtaining protein antigens, including recombinant ones, preparations of mono-and polyclonal antibodies, immunoenzyme conjugates and immunosorbents, chemical reagents etc. The bottlenecks of technologies for in vitro diagnostics devices were analyzed from the bioethical and biosafety points of view.

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Comparative characteristic of the methods of protein antigens epitope mapping

Cited by 4 publications

References 43 publications

Modern magnetic immunoassay: Biophysical and biochemical aspects

Modern magnetic immunoassay: Biophysical and biochemical aspects

Development of the method of isolation and purification of the recombinant human interleukin-7

Specificity of Manifacturing Process Validation for Diagnostic Serological Devices

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