Antibodies conjugated with new highly luminescent Eu3+ and Tb3+ chelates as markers for time resolved immunoassays. Application to simultaneous determination of clenbuterol and free cortisol in horse urine.

Highly luminescent Eu(3+) and Tb(3+) complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu(3+) is a subset of 1 and Tb(3+) is a subset of 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lanthanide chelates are very stable and highly luminescent in aqueous solution and allowed to reach 10 microg L(-1) and 40 microg L(-1) sensitivities for CL and for HC, respectively. Application to the horse urine, that is a very complex matrix, has a considerable interest in the control of illegal use of these compounds.

Time-resolved fluoroimmunoassay for quantitative determination of ampicillin in cow milk samples with different fat contents.

In this paper, we have reported an immunoassay with time-resolved revelation system for ampicillin in raw milk samples. Immunological methods appear to be a promising approach in the analysis of beta-lactam compounds, because they do not need previous sample pre-treatments. In fact, beta-lactam ring is not very stable in extensive sample pre-treatment procedures requested in conventional analytical techniques. Specimens were collected from lactating cows bred in various conditions and assayed for the fat contents. Ampicillin was assayed in samples with different fat concentrations. The assay was performed using ampicillin-specific polyclonal antibody raised in rabbit; the immunogen was synthesized using bovine thyroglobulin conjugated to ampicillin by glutaraldehyde reaction; as fluorescent marker we used goat anti-rabbit IgG conjugated with a chelating molecule complexed with Eu(3+). Bovine serum albumin (BSA) conjugated with ampicillin was synthesized and used to prepare a solid phase on polystyrene microtiter plates. The use of a lanthanide chelate as label allowed to achieve 1 ng mL(-1) sensitivity, which is four times more sensitive than limits requested from European Community. Fat contents did not affect the assay performance.

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis.

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.

Quantification of glycoalkaloids in tomato plants by time-resolved fluorescence using a europium chelator entrapped in liposomes.

In the control of tomato transgenic cultivars it is important to determine the glycoalkaloid content, which requires the screening of many samples. We have developed a simple method for the extraction and determination of glycoalkaloids in the leaf and fruit extracts of tomatoes using a europium chelator entrapped in phosphatidylcholine-cholesterol containing liposomes. The concentration values were quantified from liposome lysis effected by glycoalkaloid action. The fluorescent signal is linear between 1 and 10 micrograms of tomatine, which makes the method useful for the analysis of tomato samples.

Selectively conjugated melittins for liposome time-resolved fluoroimmunoassay of theophylline in serum.

Theophylline (Th) has been selectively conjugated to the four amino groups of melittin (Mel) by solid phase peptide synthesis. The cytolytic activity of the resultant Th-Mel compounds was tested on liposomes trapping the bovine serum albumin (BSA) conjugate with 4,7-bis(chlorosulfophenyl)-1,10-phenanthrol ine-2,9-dicarboxylic acid (BCPDA). The loss of lytic activity was the highest for Th-K7-Mel. Th-G1-Mel retains almost the same lytic activity as Mel. A homogeneous liposome time-resolved fluoroimmunoassay (LITRFIA) of Th in serum has been carried out with Th-G1-Mel between 5 ng and 10 microg.

Time-resolved fluoroimmunoassay for delta(9)-tetrahydrocannabinol as applied to early discrimination of Cannabis sativa plants.

In the cultivations of Cannabis, it is important to be able to distinguish fiber-type plants from drug-type plants by an easy observation of their phenotype. This study required the screening of many samples for their cannabinoid content. A simple and highly sensitive time-resolved fluoroimmunological method was developed for the determination of Delta(9)-tetrahydrocannabinol in the leaf extracts. The useful range of the calibration curve was between 10 pg and 25 ng of standard. Matrix effects were minimized by a high dilution of samples.

Evaluation of three immunoassays for detection of Toxoplasma-specific immunoglobulin G and M.

A time-resolved immunofluorometric assay of the IgG specific for Toxoplasma gondii has been compared with two commercial methods, one automated enzyme linked fluorescent assay (VIDAS) and one automated colorimetric enzyme immunoassay (BEIA). The coefficients of variation were 3.4% and 7.4% within-assay (n = 12), and 9.2% and 8.1% between-assay (n = 10) for two sera at low and high concentrations of specific IgG. The regression lines obtained with 96 samples were y = 1.04x + 2.1 and y = 0.98x - 1.1. We also compared a time-resolved capture fluoroimmunoassay for Toxoplasma-specific IgM with a commercial immunoenzymatic assay (BEIA TOXO-M). The sensitivity and specificity were 100%, calculated from assays of 78 samples.

Comparison of time-resolved fluoroimmunoassay and immunoenzymometric assay for clenbuterol.

A time-resolved fluoroimmunoassay (TR-FIA) for the direct determination of clenbuterol residues in horse urine using a highly specific monoclonal antibody has been compared with an immunoenzymometric assay (IEMA). The sensitivity of both methods was 10 pg; the calibration curve was linear between 10 and 10(5) pg for the TR-FIA and between 10 and 10(4) pg for the IEMA.

Fractionation of an antiserum to progesterone by affinity chromatography: effect of pH, solvents and biospecific adsorbents.

Several progesterone-AH Sepharose 4B matrices were prepared as biospecific adsorbents suitable for affinity chromatography to fractionate antibodies of different affinity and specificity from a polyclonal antiserum to progesterone-11 alpha-hemisuccinate-BSA. From an affinity column of progesterone-11 alpha-hemisuccinate-AH Sepharose 4B no antibodies can be eluted, even with glycine buffer (pH 2.6) and 30% of 2-methoxyethanol. The use of biospecific adsorbents, prepared by coupling with AH Sepharose 4B progesterone derivatives [5-pregnene-3,20-dione di(ethyleneacetal)-11 alpha-ol-11 alpha-hemisuccinate; 4-pregnene-11,20 beta-diol-3-one-11 alpha-hemisuccinate 20 beta-benzoate; progesterone-3-carboxymethyloxime] having a low cross-reactivity with the antiserum, makes the elution of various antibody fractions of variable affinity and specificity possible. 2-Methoxyethanol or N,N-dimethylformamide gradients, in acetate or TRIS buffer, were equally efficient for fractionating the antiprogesterone serum, while a decreasing pH gradient was less effective and eluted antibody fractions that were further separated into various binding components by a solvent gradient. Antibodies eluted from the affinity columns by an eluent containing a high solvent concentration have affinities higher than antibodies eluted at lower solvent concentration.

Determination of aflatoxin B1 in agricultural commodities by time-resolved fluoroimmunoassay and immunoenzymometric assay.

A time-resolved fluoroimmunoassay (TR-FIA) and an immunoenzymometric assay (IEMA) were applied for the measurement of aflatoxin B1 in soya seeds, dried figs and raisins. The extraction procedure was simple and no clean-up was found to be necessary. Limits of detection were 0.5 microgram kg-1 for TR-FIA (range of linearity of calibration graph 2.5-5 x 10(4) pg; inter-assay relative standard deviation Sr < 5%) and 0.2 microgram kg-1 for IEMA (linear range 1.0-5 x 10(3) pg; Sr < 11%).

Comparison of a time-resolved immunofluorimetric assay with two immunoenzymatic methods for alpha-foetoprotein in human serum.

We compared two commercial methods with a time-resolved immunofluorimetric assay for alpha-foetoprotein in human serum using the europium complex of 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid as label. The correlation coefficients were r1 = 0.94 and r2 = 0.96.

Comparison of two time-resolved fluoroimmunoassays (TR-FIA), as applied to oestriol in human serum and progesterone in bovine milk.

We compared two time-resolved fluoroimmunoassay systems for measuring free oestriol in human serum and progesterone in bovine milk. By reading the fluorescence of europium complex of 4,7-bis(chlorosulphophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid in solution, the measuring range is increased for both oestriol (10-50,000 ng/l instead of 25-50,000 ng/l) and for progesterone (10-50,000 ng/l instead of 25-10,000 ng/l). In addition, the interassay coefficients of variation were lowered from 9.5 to 5.7% for oestriol and from 7.5 to 5.4% for progesterone, at the smaller hormone concentrations detectable by each method.

Affinity of europium-labelled proteins A and G for immunoglobulin G from seven mammalian species.

Proteins A and G were each labelled with two different europium chelates (p-isothiocyanatophenyl-ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid). Their affinities for IgG from rabbit, goat, horse, sheep, mouse, pig, and rat were then measured using time-resolved fluorescence. Protein G labelled with the second chelate was found to be especially effective in binding to goat and horse IgG.

Direct time-resolved fluoroimmunoassay of estriol in serum.

A rapid, direct, solid-phase, time-resolved fluoroimmunoassay for free estriol in serum, using Europium-chelate-protein A as a label, is described. The coefficient of correlation with the results of RIA was 0.983.

Time-resolved fluoroimmunoassay of progesterone in milk.

A direct, solid-phase, time-resolved fluoroimmunoassay for progesterone in cow's and goat's milk, using europium-chelate-protein A as a label, is described. The coefficients of correlation with the results by RIA were 0.987 and 0.989.

Evaluation of time-resolved fluoroimmunoassay with Eu-labelled protein-A for serum progesterone.

Progesterone has been assayed in several serum samples by a time-resolved fluoroimmunoassay (TR-FIA). The solid phase was 11 alpha-hydroxyprogesterone hemisuccinate bound covalently to ovalbumin and adsorbed on wells of polystyrene. The assay was based on competitive reaction of solid phase-bound hormone and samples with specific antibody labelled in situ with protein-A prelabelled with europium. The bound Eu was dissociated from the solid phase by an enhancement solution and measured by time-resolved fluorometry. The coefficient of correlation between TR-FIA and RIA was 0.97.

Influence of functional groups on homologous antibody affinity of 11 alpha-hydroxyprogesterone derivatives. I. Synthesis and affinity evaluation.

Syntheses and cross-reactivities with progesterone toward the same specific antibody are reported for a series of amides of 11 alpha-hydroxyprogesterone 11-hemisuccinate. Some hypotheses are made regarding the effects of the chemical structure of the substituents on the immunological properties of derivatives.

Two new fluorescent derivatives of progesterone to use in fluoroimmunoassay.

Synthesis, fluorometric and immunological properties of two new fluorescent derivatives of progesterone are reported. Both compounds were obtained from 11 alpha-hydroxyprogesterone 11-hemisuccinate; the fluorescent molecules were joined to the steroid by bifunctional arms. The first of these is cysteamine whose thiol group was reacted with N-(3-fluoranthenyl) maleimide, and the second is tyramine whose phenolic group was reacted with 1-nitroso-2 naphthol.

Radioimmunoassay of progesterone and estriol in plasma using antibodies immobilized onto protein A-Sepharose CL-4B.

Protein A-Sepharose CL-4B was used as a solid phase for antibodies in the radioimmunoassay of progesterone and estriol. The method was fast and easily standardizable. Immobilized antibodies had the same binding capacity as free antibodies and gave good correlation curves (r = 0.996 for progesterone and r = 0.989 for estriol). Sensitivity was 12.5 pg/tube for progesterone and 8.0 pg/tube for estriol. Comparison of progesterone radioimmunoassay with chemically immobilized antibody onto Sepharose CL-4B was also carried out.