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Biomedical subjects

S E Kakabakos

Publications and source records attributed to S E Kakabakos.

At least 19 recordsLinked to original sources
PubMed2002

Multi-analyte capillary immunosensor for the determination of hormones in human serum samples.

In this work we present the development of a multi-analyte immunosensor for the determination of follitropin, human chorionic gonadotropin and prolactin in human serum. The immunosensor is based on plastic capillaries. According to the methodology, discrete areas of the internal capillary surface are coated with different antibodies, which are highly specific for each one of the analytes to be determined. The sample that will be analyzed along with a mixture of analyte-specific biotinylated antibodies is introduced into the capillary. The coated and the detection antibodies react with different epitopes of the analytes in the sample to form a 'sandwich'. The detection is based on reaction of the immobilized biotinylated antibody with streptavidin labeled with R-phycoerythrin. The fluorescent areas formed were quantified by scanning the capillary with a light beam of appropriate wavelength. A light sensor placed at the end of the capillary detects the emitted photons, that are trapped and waveguided into the capillary walls. The multi-analyte immunosensor assays were characterized by high specificity and short analysis time. In addition, the results obtained by the multi-analyte optical capillary immunosensor were comparable to those obtained by immunofluorimetric assays performed in microtitration wells. Potential applications of the proposed immunosensor include determination of several analyte panels in a broad spectrum of disciplines such as endocrinology, hematology, and oncology.

Animals↗
PubMed2001

Improved antibody coating protocol using a second antibody antiserum. Application to total thyroxin immunoassay.

A complete antibody coating protocol for the preparation of dry antibody coated tubes is presented. This protocol is based on a recently described antibody immobilization principle. We modify this immobilization principle in order to improve and simplify the coating procedure. In addition, we propose a drying procedure that provides long-term storage stability of the antibody coated tubes. According to the modified protocol, polystyrene plastic tubes are first coated with rabbit gamma-globulins. The tubes are incubated with a sheep anti-rabbitIgG antiserum dilution. After incubation, antigen-specific antibody antiserum raised in rabbits is added directly into the tubes containing the sheep anti-rabbit IgG antiserum solution (difference from the original protocol). Finally, the tubes are washed, blocked, and dried following the drying procedure developed. The suitability of the modified protocol for the development of immunoassays requiring high loading of antibody was exemplified through the development of a RIA for total thyroxin. The estimated assay characteristics (detection limit 4 microg/L, dynamic range up to 210 microg/L, within-run CV 2.7-5.7%, between-run CV 5.1-7.3%, recovery 84.4-112%, cross-reactivity for T3 1.9%) were comparable with those provided by commercially available RIA kits for the determination of thyroxin.

Animals↗
PubMed1999

Heterogeneous fluoroimmunoassays using fluorescein as label with measurement of the fluorescence signal directly onto the solid-phase.

A heterogeneous fluoroimmunoassay approach with measurement of the fluorescence signal directly onto the solid support is described. Fluorescein was employed as fluorescent label whereas plastic microtitration wells were used as solid carriers. The influence of the type of the plastic wells on the proposed approach was investigated. Different types of commercially available plastic microtitration wells (white-opaque, black, and transparent) were tested. The solid supports were judged on the grounds of the maximum fluorescence signal, the precision, and the sensitivity they provided in a model non-competitive assay for mouse IgG. Among the solid supports tested, the white-opaque wells provided the most promising results with respect to the parameters in question. This support was also used for the development of a competitive fluoroimmunoassay for the determination of total thyroxin in human serum samples in order to assess the validity of the proposed approach under real immunoassay conditions. The assay developed had appropriate analytical characteristics for the determination of thyroxin and the serum sample values obtained were well correlated with those determined by a commercial solid-phase radioimmunoassay.

Animals↗
PubMed1998

A multi-band capillary immunosensor.

In the present work we propose a new optical immunosensor based on capillary geometry and capable of multianalyte determinations. The device is made of a polystyrene capillary tube. The inner walls of the capillary are segmented into distinct bands which are coated with appropriate binding molecules. Following excitation, some of the fluorescent photons emitted by the label are trapped and waveguided into the capillary walls provided they are launched towards the walls and within the critical angle. Here, Europium-labeled streptavidin reacted with different amounts of biotinylated bovine serum albumin immobilized onto each one of the bands. Due to the small inner volume of the capillary and the multianalyte feature we expect that the proposed device can be used for fast and inexpensive assays.

Animals↗
PubMed1998

Antibody coating approach involving gamma globulins from non-immunized animal and second antibody antiserum.

An alternative protocol for immobilization of antibodies onto plastic solid supports is presented. According to the proposed protocol, tubes are first coated with gamma-globulins from non-immunized animal of the same species as that from which the antigen-specific antibody has been developed. Then, an excess of second antibody is added to the tubes and the anti-species specific antibodies present in the antiserum are immunoadsorbed on the immobilized gamma-globulins. Finally, the antigen specific antibody is immunoadsorbed on the immobilized second antibody. We found that the coating protocol developed allows the use of antigen-specific and second antibody antisera dilutions, thus avoiding the need for affinity purification of antibodies. Additionally, it provides solid-phase second antibody with increased binding capacity compared to the directly adsorbed onto the solid second antibody. The advantages of the proposed coating protocol were demonstrated through the development of a solid-phase radioimmunoassay for the determination of total triiodothyronine in human serum samples.

Animals↗
PubMed1994

High-performance liquid chromatographic separation of biotinylamide analogues used as substrates in biotinidase radioassays.

A simple, one-step protocol for synthesizing biotinylmono[125I]iodotyramine and biotinyldi[125I]iodotyramine, which are used as tracer substrates in biotinidase radioassays, is presented. This synthetic protocol uses reversed-phase HPLC for the isolation of the biotinylamide analogues from the reaction mixture. The HPLC method developed can potentially be applied to a scaled-up synthetic protocol for non-radioactive biotinylmono- and diiodotyramine. In addition, it can be used for the identification of the above-mentioned compounds.

Amidohydrolases↗
PubMed1994

Direct colorimetric determination of solid-supported functional groups and ligands using bicinchoninic acid.

We describe new colorimetric methods for the direct determination of total solid-supported sulfydryl, aldehydo, hydrazido, and N-hydroxysuccinimido carboxylate groups as well as of immobilized cysteine, tyrosine, and thyroxin using only the commercially available bicinchoninic acid/copper protein assay reagent. The method is based on the ability of these groups to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic acid absorbing at 562 nm. Each assay requires only one incubation step of the solids with the reagent for 1 h at 60 degrees C. The quantitation of the different groups is finally carried out through standard curves of appropriate substances. Using the assays developed we determined the amount of the above-mentioned functional groups and ligands onto several commercially available solid supports. The values obtained were in agreement with those provided by relative literature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, allowing the determination of nmol quantities of functional groups per assay tube.

Aldehydes↗
PubMed1994

Colorimetric determination of reactive solid-supported primary and secondary amino groups.

A simple and sensitive method for the quantitative determination of solid-supported primary and/or secondary amino groups using commercially available reagents is described. The solid supports are treated in an aqueous environment with either 2-iminothiolane (ITL) or sulpho-succinimidyl-3-(4-hydroxyphenyl)propionate (sulpho-SHPP), which introduce one sulphydryl or one hydroxyphenyl group per amino group reacted, respectively. These groups are capable of reducing Cu2+ to Cu+ in alkaline medium. Thus, after removal of the excess reagents through washing, subsequent incubation of the solids with 2,2'-bicinchoninic acid (BCA) copper protein reagent results in production of Cu+ in the solution, which forms a chelate complex with BCA absorbing at 562 nm. The quantitation of the groups introduced on the surfaces, and therefore of the reacted amino groups, is carried out through standard curves of cysteine solutions for ITL, or tyrosine solutions for sulpho-SHPP-treated solids. Using ITL, only the primary amino groups are determined, whereas sulpho-SHPP provided the primary and secondary reactive amino groups. The method is versatile and can be used for the estimation of amino groups onto several biomedical solid matrices, and should provide useful information for the covalent immobilization of ligands (e.g. drugs, antibodies).

Colorimetry↗
PubMed1993

Determination of serum biotinidase activity with biotinyl derivatives of lodotyramines as substrates.

We synthesized biotinylated mono- and di-iodotyramine and their radioactive counterparts and used these substances as substrates to estimate serum biotinidase activity in a radioassay system. The Km values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 microM, respectively, whereas, the maximum velocities of the enzymatic reaction were 27.0 and 8.7 nmol.min-1.mL-1, respectively. Both substrates competed with biocytin for the same active site of the enzyme and the Ki values were 7.30 and 9.56 microM for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [125I]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the well-established colorimetric method (r = 0.9377, n = 31). However, for routine use, the assay may be accomplished by diluting the radiotracer with biocytin instead of its "cold" counterpart, because it is a commercially available reagent. The values obtained in this case were very well correlated with those determined by the colorimetric assay as well (r = 0.9289, n = 31).

Amidohydrolases↗
PubMed1992

Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solid-phase time-resolved fluorometry.

We describe a new multianalyte immunoassay principle and apply it to the simultaneous immunoassay of lutropin, follitropin, choriogonadotropin, and prolactin in serum. The method is based on the coating of distinct areas of polystyrene with analyte-specific antibodies. These antibodies react with the analyte and immobilize it in a specific area while another biotinylated antibody also reacts with the analyte to form a sandwich. After addition of streptavidin labeled with the fluorescent europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid, fluorescent areas are formed, the intensity of which is related to the amount of each analyte present in the sample. The fluorescent areas are quantified on the dry solid phase with laser-excited time-resolved fluorometric measurements. The assays developed are highly sensitive, precise, and accurate. We believe that this system shows potential for multianalyte immunoassay of diverse groups of compounds in disciplines such as endocrinology, infectious disease, hematology, and oncology.

Chorionic Gonadotropin↗
PubMed1992

Direct time-resolved fluorescence immunoassay of progesterone in serum involving the biotin-streptavidin system and the immobilized-antibody approach.

We developed a direct competitive-type immunoassay for progesterone in serum that combines the advantages of the biotin-streptavidin system with the antibody-immunobilization approach. We synthesized biotinylated progesterone derivatives of five different proteins and, after initial evaluation of the conjugates, selected biotinylated bovine IgG-progesterone as the most suitable tracer. Progesterone released from binding proteins with danazol competes with the biotinylated tracer conjugate for binding to a limited amount of a mouse anti-progesterone monoclonal antibody in microtitration wells coated with a goat anti-mouse IgG antibody. The binding of the biotinylated tracer is then monitored by reaction with a streptavidin-based universal detection reagent developed for time-resolved fluorometry. The assay demonstrated typical performance characteristics with respect to the dynamic range, detection limit, and precision. Recovery averaged 99.3% (SD 8.3%) and dilution experiments showed good linearity. Measurements correlated well with those from three commercially available direct immunoassays for progesterone.

Animals↗
PubMed1991

Biotinidase radioassay using an 125I-biotin derivative, avidin, and polyethylene glycol reagents.

A radioassay for determining biotinidase activity in human serum was developed, using N-[beta-(4-OH-3-125I-phenyl)ethyl]-biotinamide in combination with biocytin as the substrate, avidin as a binding protein, and polyethylene glycol as a separation reagent. The gamma-emitting 125I-biotinamide (= tracer) was synthesized by coupling (pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to 125I-tyramine. Using polyethylene glycol as a separation reagent, it was possible to eliminate several problems that were encountered when other separation reagents were used. Biotinidase activity was evaluated following the cleavage of the 125I-biotinamide and expressed in fmol of tracer cleaved.min-1.ml-1 in the presence of 9 nmol of biocytin. Under the conditions used, the time response of the assay was linear up to 3 h. The method is simple to perform, more sensitive than the previously described methods, and reproducible (intra- and interassay CVs of 4.9 and 10.2%, respectively) and allows the simultaneous handling of more than 100 samples in less than 3 h.

Amidohydrolases↗
PubMed1991

Isolation of mono- and di-iodine 125 tyramines for conjugation labelling.

Here we describe a method for isolating mono- and di-iodine 125 tyramines from a crude tyramine radio-iodination reaction mixture. Tyramine was radio-iodinated according to a modified chloramine-T method. Mono- and diforms were separated from each other, as well as from free 125I and unreacted tyramine, by means of paper electrophoresis or thin-layer chromatography. Both 125I-tyramine derivatives finally obtained were pure and had high specific activities, which averaged 2500 Ci/mmol for mono-125I-tyramine and 5000 Ci/mmol for di-125I-tyramine. Due to their characteristics, these derivatives can be used for labelling various substances indirectly and preparing in vivo and in vitro tracers of high purity and specific activity, when conjugation labelling is required.

Iodine Radioisotopes↗