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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.

The water specimens were collected from wells and irrigation ditches in the agricultural area to the south of Milan and from Olona River and Mantua Lake and analyzed for paraquat detection. The assay was performed using a specific polyclonal antibody raised in sheep and rabbit anti-sheep IgG conjugated with a chelating molecule 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid complexed with Eu3+ as a fluorescent marker. Bovine serum albumin conjugated with 5-(1‘-methyl[4,4‘]bipyridinyl-1-yl)pentanoic acid was used in solid phase preparation. The sensitivity achieved was 20 ng L-1. The recovery in samples spiked with three different PQ concentrations was between 88 and 108%. Keywords: Paraquat; bipyridylium herbicides; immunoassay; europium chelate; time-resolved fluorescence

An antigenic derivative of dimethylphthalate has been synthesized and used to raise an antibody that selectively recognizes o-phthalate esters. With this antibody we have standardized a direct time-resolved fluoroimmunoassay for these esters in water. Its sensitivity is 0.5 pmol/ml.

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.

Protein A was covalently labelled with 4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid in several molar ratios. The binding affinity for IgG and the delayed fluorescence of the corresponding complexes with Eu(III) were determined for each labelled compound. The immunoaffinity decreased slowly with increase in ratio of the label. A better immunoaffinity/labelling ratio was obtained by coupling protein A to the chelate via bovine serum albumin. A suitable ratio of chelating agent to protein A for producing a universal marker to be used in time-resolved fluoroimmunoassays can be determined.

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.

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.

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.

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.

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.

A fluoroimmunoassay for progesterone is described. It involves a labelled antigen synthesized by linking phenanthroimidazole-2-amine† to progesterone-11α-hemisuccinate and specific antiserum (rabbit anti-progesterone- 11α-hemisuccinate-bovine serum albumin) with a heterogeneous procedure.

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.

We have studied the clarifying activity of a purified peptide fraction of gametic origin in senile cataract of the dog. The active substance, administered intramuscularly, had significant and durable clarifying effects on the cortical area of the lens.

The s.c. administration for 25 days of the inhibin-like factor extracted from bull spermatozoa to immature 20-day-old male rats reduced serum FSH levels by about 60% and serum LH and testosterone levels by about 50%. However, neither tubular nor interstitial modifications of the histological appearance of the testis were found and there were no changes in the activity of delta 5-3 beta-hydroxysteroid dehydrogenase. The assay described could be usefully employed for evaluating the activities of inhibin-like factors.

A standard curve for progesterone using a solid phase time-resolved fluoroimmunoassay was obtained. The 11α-hydroxyprogesterone hemisuccinate bonded to ovalbumin was adsorbed to the wells of polystyrene microtiter strips. After incubation of scalar quantities of free progesterone (from 12 to 5000 pg) with specific antibody and an excess of Eu-labelled protein-A, the wells were washed and the europium bound to the solid phase was measured as a 2-naphthoyltrifluoroacetone chelate using a time resolved fluorometer.