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Biomedical subjects

M Fránek

Publications and source records attributed to M Fránek.

15 recordsLinked to original sources
PubMed2003

A highly sensitive flow-through amperometric immunosensor based on the Peroxidase chip and enzyme-channeling principle.

A concept based on the Peroxidase-chip (P-chip), antibody co-immobilization, competitive and enzyme-channeling principle was exploited to develop an integrated flow-through amperometric biosensor for detection of environmental pollutants such as s-triazine herbicides. In this concept, recombinant peroxidase is immobilized on the gold electrode (P-chip) in such a way that direct electron transfer is achieved. The recognition and quantitation the target analyte is realized through the competition between the simazine-glucose oxidase (GOD) conjugate and free simazine for the binding sites of the monoclonal antibody co-immobilized with peroxidase on the gold electrode. The arrangement allows to generate a specific signal in the presence of glucose through the channeling of H2O2 produced by GOD conjugate bound to the antibody. The immunosensor exhibited 50% signal decrease (IC50 value) at approximately 0.02 microg l(-1). A concentration of 0.1 ng l(-1) gave a signal clearly distinguishable from the blank whereas the ELISA using the same antibody had a typical detection limit of about 1 microg l(-1), which is four orders of magnitude higher compared to the presented biosensor system. The results demonstrated that gene engineering biomolecules, in this case recombinant peroxidase, might be attractive reagents for the development of electrochemical immunosensors.

Biosensing Techniques↗
PubMed2001

Antibody-based methods for surfactant screening.

This brief overview summarises the immunoassay-based results obtained in the course of two years of the European INCO-Copernicus project BIOTOOLS. The project is aimed at simplifying the procedures for detection of surface active compounds (SAC) using, among others, antibody-based methods, i.e., microtiter plate-based enzyme-linked immunosorbent assays (ELISA), polarisation fluoro immunoassays (PFIA), and enzyme flow injection immunoassays (FIIA). Thirty-three rabbits were immunised with five different sulphophenyl moieties and three p-hydroxyphenyl moieties conjugated to protein immunogens to produce analytical antibodies against linear alkylbenzene sulphonates (LAS) and nonylphenol (NP). Although most of the antibodies exhibited binding reaction in indirect ELISA, only a few showed the required assay sensitivity. The best antibodies for LAS exhibited a 50% binding inhibition at IC50 19.8 microg L(-1) in indirect ELISA. Similar inhibition was observed for direct ELISA using peroxidase tracers. Antibodies against NP allowed the establishment of an indirect assay operating in the mg L(-1) range. A rapid and simple protocol for the screening of NP and LAS using homogeneous PFIA is described. The assay time for 10 samples was 7 minutes, thus allowing fast detection of the selected SAC at the mg L(-1) level. A generic competitive FIIA system, using a protein G column for separation of free and antibody-bound beta-galactosidase (beta-Gal) tracer, was developed for the screening of LAS, NP, and nonylphenol decaethoxylate (NPEO10). The FIIA had a sample throughput (STP) of 5-10 samples per hour, with limits of detection (LOD) for LAS, NP, and NPEO10 of 19.5, 52, and 2.4 microg L(-1), respectively. The developed FIIAs were applied to spiked rain and surface water.

Animals↗
PubMed1994

Characterization of monoclonal antibodies to 2,4-dichlorophenoxyacetic acid using a piezoelectric quartz crystal microbalance in solution.

Piezoelectric quartz crystals (resonance frequency 10 MHz) were used for an investigation of the immunochemical reaction between 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide and several monoclonal antibodies prepared against 2,4-D. The herbicide was immobilized on the gold electrodes of the crystals silanized with 3-aminopropyltriethoxysilane. The activated carboxylic group of 2,4-D was linked either directly to the silanized surface or through hexamethylenediamine or albumin as a macromolecule. The interaction of the immobilized antigen with monoclonal antibody in solution was followed as a change in the resonant frequency of the crystals. The best results were achieved using 2,4-D attached to albumin. The affinity binding of five monoclonal antibodies was characterized by association (ka) and dissociation (kd) kinetic rate constants and by equilibrium association constants (KA) which were obtained from the experimental frequency vs. time curves.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1987

Structural aspects of steroid-antibody specificity.

The paper gives a review of the immunogens most frequently used for preparing antibodies against steroid haptens. The structural concepts of immunological specificity and complementarity are applied to a description of the formation of antibodies in immunized animals and the binding mechanisms of steroid-antibody interactions in systems in vitro. Recent experimental findings show that Ehrlich's lock and key principle is too static for many steroid-antibody complexes and that a more important factor in the binding events is the overall flexibility of these complexes. In this connection a discussion is undertaken of the properties of unsaturated steroid skeletons, the multispecificity of binding sites and further critical factors involved in the formation of the final specificity. Special attention is paid to considerations of the size of steroid determinants in connection with the question of the so-called bridge effect. The relevant expositions of this phenomenon are based in this article more on conformation changes of the steroid ligands than on direct binding interaction of the bridge substituent with the antibody. This interpretation makes it possible to interpret more broadly the experimental facts and gives new stimuli for an improvement of the present strategies of steroid immunoanalysis.

Antibodies↗
PubMed1985

Specificity of the immune response to the oestrone-azo-hapten structure.

Comparison of the cross-reactions between 14 related steroids with eight antibodies showed that each antibody had its individual molecular specificity. During the 183-day period covering eight immunizations, cross-reaction between oestrone and 1-methyloestrone showed only very little change with five antibodies, while the great differences in cross-reactivity values for the individual antibodies were retained. With the same five antibodies, the course of cross-reaction between oestrone and 3-sulphooestrone as well as 6-oxooestrone was also fairly constant, the differences in cross-reactivity in relation to the individual antibodies being considerably large. The blood plasma antibody concentrations measured at the same intervals showed considerable fluctuation, whereas the affinity constants of the respective antibodies, except one of them, showed a moderate upward trend. The suggested molecular parameters of binding sites of the antibodies to the oestrone-4-azo-hapten structure were in surprisingly good agreement with those reported in the literature for mouse myeloma immunoglobulin A proteins possessing dinitrophenyl-binding activity. The individual antibodies exhibited highly sensitive radioimmunoassay curves for both oestrone and 3-sulphooestrone.

Animals↗
PubMed1983

Characterization of antibodies against oestrone-azo-protein conjugates using 3H and 125I radioligands.

This report describes the synthesis of six oestrone-azo-protein immunogens differing in the position of the azo group in the steroid skeleton and in the nature of carrier protein. By nitration of oestrone, 2-nitro- and 4-nitro-isomers were prepared and converted to amino-derivatives by reduction of the nitro group. The diazotized amino-oestrones were coupled to albumin, thyreoglobulin and haemocyanin. The immunogens thus prepared were administered to rabbits to assess the effect of position of the bridge, carrier protein and adjuvant on antibody production and specificity. Very weak immune responses were obtained with both types of albumin immunogen. The presence of the azo group at C-2 position led to a lower antibody production as compared with C-4 derivatives. The relatively high antibody production was achieved by immunization with oestrone-4-azo-thyreoglobulin or oestrone-4-azo-haemocyanin, with the oil component of adjuvant omitted. The antibodies combined with tritiated and iodinated radioligands yielded typical curves showing the sensitivity of 10-20 pg for 3H labelled oestrone and that of 3-10 pg for 125I labelled oestrone. The specificity of the 6 antibodies was tested using 25 related structures. In all these cases there was low cross-reaction with oestradiol, oestriol and their derivatives. On the other hand, the discriminating ability of the antibodies with regard to the opposite end of the molecule, i.e. with regard to the A-ring and partly the B-ring was very low. This makes it possible to use these antibodies for the radioimmunoassay of oestrone alone or some of its metabolites modified in the A-ring. The results of cross reactivity are generalized to the effect that in the oestrone-azo-hapten structure the antigenic determinant area can be ascribed to the size not exceeding 2-3 steroid rings.

Animals↗
PubMed1983

Characteristics of antibodies against 17 beta-oestradiol in homologous and heterologous systems of radioimmunoassay.

Directly iodinated oestradiol-2(4)-iodo-[125I] and oestradiol-6-O-(CMO)-[125I]iodohistamine were prepared and used in conjunction with anti-oestradiol-2(4)-azo and anti-oestradiol-6-O-(CMO) sera for the development of various radioimmunoassay (RIA) systems which showed marked differences in sensitivity and relatively small differences in specificity. Whereas the heterologous combination of oestradiol-6-O-(CMO)-[125I]iodohistamine and anti-oestradiol-2(4)-azo-serum showed a sensitivity expressed in femtograms, the homologous combination using oestradiol-6-O-(CMO)-[125I]iodohistamine radioligand exhibited a sensitivity two orders lower. The specificity of both the heterologous and homologous system did not differ significantly from the RIA system using tritiated radioligand. The combination of directly iodinated oestradiol and anti-oestradiol-2(4)-azo-serum showed a higher sensitivity and a lower specificity as compared with tritiated radioligand.

Antibodies↗
PubMed1983

Radioimmunoassay of progesterone in cow milk.

Fertilization checks in dairy cattle performed by estimation of progesterone in milk require a simple technique, the use of a sample taken independently of milking procedure and a simple management of the results. The laboratory equipped with a sample processor and pipetting device, high throughput counter for 125iodine and a tabletop computer can analyse about 1000 samples of fat-free milk in one day. The results are used for computer management of reproduction and veterinary service in farms with 600 to 2000 cows.

Animals↗
PubMed1978

[Variation of the serum testosterone level in boars during 24 hours and during a period of several days].

Seven sexually mature boars were studied in two-hour intervals for 8 to 32 hours, as to the variation of testosterone levels in the blood plasma. Blood samples were obtained by means of a cannula inserted in the v. cava cranialis. The hormone level under study showed marked fluctuation. The coefficient of variation of testosterone concentration ranged from 12.7% to 35.2% in the individual animals and for the whole part of the study it was 33.0%. It appeared impossible to derive seriously any regular periodicity of testosterone concentration in boar blood in the 24-hour period from the analysis of the variation of the levels of the hormone in individual animals during the period under study. Further, the study revealed a marked fluctuation of testosterone levels in the blood plasma of twelve sexually mature boars in the course of several days' study. The coefficient of variation of testosterone concentration in individual animals ranged from 18.1% to 106.5%, reaching 77.0% for the whole part of study. The analysis of variance showed statistically significant differences of hormonal levels in individual boars in which the fluctuation of testosterone was studied in the 8- to 32-hour period as well as in several days' period. This proved the role of the animals' individuality in determining the concentration of testosterone in their blood. The discussion concerns the importance of these findings for the evaluation of the role of basal testosterone levels in the blood in classifying the incretion function of the gonads and in the diagnosis of incretion hypogonadism of boars.

Age Factors↗
PubMed1976

Separation of free and protein-bound ligands in the radioimmunoassay by gel filtration-centrifugation.

The application of gel filtration-centrifugation to the separation of free and protein-bound ligands in radioimmunoassay is described. The method is simple and rapid. A large number of samples can be handled simultaneously and the bount radioactivity is directly transferred to the scintillation vial. Using a 9 X 36 mm column of Sephadex G-50 (coarse), 400 mul of the reaction mixture can be separated during 2 min centrifugation.

Animals↗