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An enzyme-linked immunosorbent assay (ELISA) for the olive fruit fly pheromone, Bactrocera oleae Gmelin, was developed. The assay uses polyclonal antibodies, raised in rabbits, against (+/-)-beta-[3-(1,7-dioxaspiro[5.5]undecane)]propionic acid, 2 (hapten I), conjugated to the KLH (keyhole limpet hemocyanin) by the carbodiimide method. A second hapten, (+/-)-delta-[3-(1,7-dioxaspiro[5.5]undecane)]butylamine, 3 (hapten II), after conjugation to a biotin moiety, was used for indirect immobilization onto ELISA microwells precoated with the glycoprotein avidin. The developed ELISA method measures the synthetic olive fruit fly pheromone in concentrations ranging between 0.08 and 10 microg/mL and shows great promise for practical applications for pheromone detection in environmental and biological samples. The results obtained strongly indicate that this technique, to our knowledge the first insect pheromone enzyme-linked immunosorbent assay so far reported, is a fast, sensitive, inexpensive, and highly convenient method for the analysis of a volatile and low molecular weight compound such as 1,7-dioxaspiro[5.5]undecane, 1.

A new pentadecapeptide bombesin analogue was prepared by Fmoc synthesis, purified by HPLC and identified by electron ionization mass spectrometry. The biological activity of the new peptide was tested on isolated human colonic muscle cells and compared to native bombesin. Labelling of the new biomolecule with Tc-99m yielded a single radioactive species which remained stable at room temperature for eight hours. In a binding assay, the radiolabelled peptide showed high affinity for oat-cell carcinoma (Kd = 9.8 nM) and colorectal adenocarcinoma (Kd = 27.2 nM). Biodistribution studies, performed in normal rodents, indicated uptake by organs that normally express bombesin receptors, such as liver, intestines and kidneys. Scintigraphic studies, performed in nude mice transplanted with small cell lung carcinoma and colon cancer cells, showed significant tumor uptake two hours p.i. The new synthetic pentadecapeptide appears to have promise for several malignancies, including oat-cell lung carcinoma, colorectal cancer and gastroenteropancreatic (GEP) tumors.

Bombesin-like peptides are neurotransmitters and cancer growth factors. Several tumors, breast cancer among them, show one or more than one of the three known bombesin receptors. We have synthesized and labeled with technetium 99m a new pentadecapeptide, analogue to the leu13 amphibian bombesin (99mTc BN). Labeling yield was 83 +/- 4%. Prone Scintimammography was performed on five patients affected by breast cancers (T categorization: two T1b and three T1c), after injecting 0.7 mg, 185 to 296 MBq (5 to 8 mCi) of the peptide. Total body scan did not show free technetium biodistribution. No adverse reaction was observed. Prone Scintimammography with 99mTc Sestamibi (99mTc SM) was also performed few days later. 99mTc BN detected all 5 cancers, whereas 99mTc SM only four: all the T1c and one T1b cancer. Two of them showed axillary node invasion that was detected by both the radiotracers. A fibroadenoma present on contralateral breast to the one with cancer, was not detected neither by 99mTc SM nor by 99mTc BN. Tumor/breast normal tissue ratio (T/B) was constantly higher with 99mTc BN than with 99mTc SM. Maximal T/B was measured as 1.79 with 99mTc SM and 2.25 with 99mTc BN 5 min after fast i.v. administration. In conclusion our 99mTc BN is taken up by primary breast cancer showing higher T/B than 99mTc SM (p < 0.01). In our limited scale, 99mTc BN appears to be safe and, in our limited scale, even more accurate than 99mTc SM for detecting breast cancer.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

Biotin is an important vitamin for cellular function and growth and, therefore, essential for fetal development. The fetus is exclusively dependent on maternal biotin supply. Since biotin is not produced within the body, maternal biotin levels depend on dietary intake. In order to investigate the biotin status of the human fetus, we measured the plasma biotin levels in 15 pregnant women and their fetuses who underwent amniocentesis and fetal blood sampling at 18-24 weeks of gestation for prenatal diagnosis of thalassemia. Maternal biotin was found to be 131 +/- (SD) 102 ng/l and fetal biotin 784 +/- 327 ng/l (p < 0.0001). Our findings are indicative of an active transport mechanism of biotin through the placenta in favor of the fetus.

An indirect enzyme-linked assay was developed for quantifying biotin concentrations in human sera. Biotin standard solutions or unknown samples are preincubated with streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and added to plates coated with biotinylated bovine IgG (B-IgGb). The concentration of the streptavidin-HRP is such that the streptavidin binding sites are sufficient to bind apparently all the biotin present in samples, whereas, the remaining sites are inversely proportional to the amount of biotin in analysed sample. These sites could subsequently interact with the immobilized B-IgGb providing signal. The assay demonstrated dynamic range 5 to 640 ng/L, detection limit 2 ng/L, intra- and interassay C.V., 1.6-3.9% and 3.7-7.2% respectively, recovery 100-114% and linear recovery 90-117%. Serum biotin determined: healthy individuals 66 to 600 ng/L, pregnant women (> or = 36 weeks) 60 to 360 ng/L, and patients under chronic haemodialysis 0.56 to 1.62 micrograms/L. The method described is among those few which have been experimentally evaluated for their capabilitity of assessing biotin in human sera.

We present theoretical and experimental data necessary for raising specific antibodies for thymosin beta 10, a 43-amino acid residues peptide occurring in human tissues. We postulate that thymosin beta 10 contains three major antigenic determinants (residues 2-8, 17-25, and 35-41). For antibody development, we synthesized the N-terminal fragment thymosin beta 10(1-16) as well as the C-terminal fragments thymosin beta 10(31-43) and thymosin beta 10(38-43), due to their putative antigenic properties and minimal structural similarity with the homologous peptide thymosin beta 4, which also occurs in humans. The putative antigenic determinant 17-25 is present in all beta-thymosins and was therefore not synthesized. All antisera raised against the above peptide fragments or the intact molecule of thymosin beta 10 were found capable of recognizing and binding synthetic or natural thymosin beta 10 with high specificity, showing minimal cross-reactivity with thymosin beta 4 isolated from bovine tissues or synthetic thymosin alpha 1. Due to its easy preparation and the highly specific affinity of the antibody raised against it for the intact peptide, the fragment thymosin beta 10(38-43) may be considered the antigen of choice for developing anti-thymosin beta 10 antibodies, which can eventually be applied to immunochemical studies.

A large-scale method for the isolation of thymosin beta 4 (up to 120 mg) and thymosin beta 9 (up to 40 mg) from bovine lung (up to 2 kg) was developed. The isolation protocol included tissue homogenization in 0.4 M HClO4, centrifugation, solid-phase extraction through LiChroprep RP-18 material, chromatofocusing on polybuffer exchanger PBE 94-modified Sepharose and dialysis against water. The isolated products were characterized by analytical isoelectric focusing, reversed-phase HPLC, electrospray ionization mass spectrometry and amino acid analysis. The method developed is rapid and convenient, requires no expensive equipment and can be used for the isolation of thymosin beta 4 and homologous peptides from various animal tissues.

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.

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.

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).

A new radioimmunoassay for determining diethylstilbestrol in serum using N-(4'-OH-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)-hex anamide as a radiotracer and a double antibody as a separation reagent is described. The radiotracer is prepared by synthesizing 6-(4-O-diethylstilbestryl)-hexanoic acid and coupling its succinimidyl ester with mono-[125I]tyramine in tetrahydrofuran (16 h, 20-22 degrees C). The standard curve is linear (semi-log transformation) and the assay is sensitive (< 0.022 pmol/tube), reproducible (intra- and interassay coefficient of variation values, 5.3 and 8.1%, respectively), and accurate (recovery values, 95-101%), with a non-specific binding less than 3.2%. Diethylstilbestrol concentrations measured in sera of nine patients with prostatic cancer by the proposed assay ranged from 0.170 to 2.517 mumol/l, which corresponded to an only three-fold dosage variation. In all cases tested, dosing was adequate to retain markers of prostatic cancer in serum within accepted limits; nevertheless, individualization of dosing may be necessary to minimize toxicity.

A thymosin beta 4 ELISA was developed in which thymosin beta 4, absorbed on microwells, competed with thymosin beta 4 in solution for the binding sites of an anti-thymosin beta 4 antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin beta 4 antibodies were raised in rabbits against intact thymosin beta 4 as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin beta 4[1-14] and thymosin beta 4[1-11]. The antibody against thymosin beta 4[1-14] was used in the thymosin beta 4 ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin beta 9 as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin beta 9, was fast, easy to perform and exhibited good assay characteristics.

The synthesis of a radioiodinated diethylstilbestrol (DES) derivative is described. This derivative was prepared by coupling the previously synthesized active ester of 6-(4-O-diethylstilbestryl)hexanoic acid with mono-[125I]iodotyramine in dry tetrahydrofuran (20 to 22 C, 16 hours). The mono-[125I]iodotyramine was prepared using a chloramine-T method and purified by paper electrophoresis. The final product, N-(4'-hydroxy-[3'-125I]iodophenethyl)-6-(4-O- diethylstilbestryl)hexanamide, was separated by thin-layer chromatography (cyclohexane/ethanol/NH4OH 2.5 N/acetone; 40:50:5:20, v/v/v/v); it was stable for 2 months in ethanol at 4 C and had a specific activity higher than 540 Ci/mmol. The [125I]DES amide synthesized was found to retain the immunoreactivity of DES, since it competed with [3H]DES or DES in an in vitro radioimmunoassay system for the binding sites of a rabbit anti-DES antibody; thus, it seems to be capable of replacing the tritiated tracer used so far in DES radioimmunoassays.

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.

A radioimmunoassay method for the measurement of diethylstilbestrol directly on 50 microliters of human serum was established using a tritium-labeled radioligand and a double antibody as a separation reagent. The assay was sensitive (less than 0.17 micrograms/L), reproducible (intra-assay and interassay coefficient of variation values, 8.14% and 8.25%, respectively), and accurate (recovery up to 95%). Factors influencing the assay are identified and discussed.

1. Human peripheral lymphocytes, normal and stimulated by phytohemagglutinin or concanavalin A. were investigated with respect to their ability to biosynthesize neutral and acidic glycosphingolipids from D-[U-14C]glucose, D-[U-14C]galactose and D-[U-14C]glucosamine as precursors. 2. Galactose and glucosamie are taken up selectively, in the presence of excessive glucose concentrations. Labeling of total neutral glycosphingolipids from D-[U-14]galactose in normal cells decreases after the first 6 h while in stimulated cells there is a fourfold and a sevenfold increase after 6 and 12 h of incubation respectively. Under similar conditions stimulation with concanavalin A gives a fourfold increase after 12 h of incubation. 3. Analysis of individual glycosphingolipids biosynthesized from [U-14C]galactose shows that lactosylceramide is the major radioactive neutral glycosphingolipid and that stimulation by phytohemagglutinin yields an almost sevenfold and 13-fold increase in the radioactivity incorporated within 6 and 12 h of incubation respectively. Glucosylceramide shows about an eightfold increase, globotetraosylceramide a threefold increase and globotriaosylceramide a fourfold increase. The rate of incorporation into glucosylceramide of stimulated lymphocytes declines after 6 h of incubation, accompanied by a concomitant increase of incorporation into lactosylceramide. 4. At 12 h of incubation the ratios of radioactivities incorporated into neutral glycosphingolipids of phytohemagglutinin-stimulated cells compared to normal cells were 1.0 for D-[U-14C]glucose 7.0 for D-[U-14C]galactose and 2.5 for D-[U-14C]glucosamine. Respective ratios for lactosylceramide are 1.0 for [U-14C]glucose and 13.0 for [U-14C]galactose. These differences did not arise from changes of the uptake of the glycosyl precursor by the cell due to stimulation. 5. Incorporation of D-[U-14C]galactose into II3-N-acetylneuraminosyllactosylceramide by cells stimulated by phytohemagglutinin and concanavalin A is enhanced 14-times and 15-times respectively. With [U-14C]glucosamine as precursor, this increase in the labeling is much more impressive, 80-fold after 12 h of incubation by the phytohemagglutinin-stimulated lymphocytes. Neuraminidase treatment and gas radiochromatographic analysis of the labeled compound derived from [U-14C]-galactose as precursor indicate that 89% of the radioactivity was incorporated into the glucosyl and galactosyl moieties, in a ratio 1:1. With [U-14C]glucosamine as precursor, a selective labeling of the sialyl moiety of the II3-N-acetylneuraminosyllactosylceramide was indicated. 6. The pattern of complex gangliosides (more complex than II3-N-acetylneuraminosyllactosylceramide) which are biosynthesized after phytohemagglutinin stimulation of the cells, show no significant differences when compared to the patterns obtained from normal cells.

A radioimmunoassay (RIA) is described for the detection and quantitation of prothymosin alpha (ProT alpha), and its N-terminal fragments containing as a minimum the first ten amino acid residues. This range of peptides includes thymosins alpha 1 (T alpha 1) and alpha 11 (T alpha 11). Antibodies against T alpha 1 and the tracer T alpha 1(1-10)Tyr11(125I), an analogue of the major epitope, were utilized in this RIA. 50% displacement of the ligand was observed with 1.3 pmol of T alpha 1 and 6.4 pmol of ProT alpha. The partially homologous parathymosin alpha (ParaT alpha) showed less than 2% crossreactivity with ProT A. Sephacryl S-200 gel filtration separation of the peptides of calf thymus, chicken spleen and trout spleen extracts prepared by a method eliminating proteolysis, combined with the above RIA, showed the presence of a major immunoreactive peak. Its elution volume corresponded to that of rat ProT alpha (apparent mol. weight 36,000) for both calf (37,000) and chicken (35,000) tissues. In trout it corresponded to a significantly higher molecular weight (62,000). No detectable levels of shorter fragments, including T alpha 1, were observed in any of the above species. The levels of ProT alpha-like peptides in calf thymus, chicken spleen and trout spleen were found to be 246, 8.6 and 7.7 micrograms respectively, of rat ProT alpha equivalents per gram of fresh tissue. The significance of the presence of ProT alpha-like polypeptides in vertebrate species as distant as fish and mammals, the absence of short T alpha 1-like fragments, and the relative conservation of the N-terminus as suggested by the RIA is discussed.

The synthesis of two radioiodinated biotin derivatives with the biotin-ureido group intact is described. This synthesis was performed by coupling (pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to tyramine, which was radioiodinated prior to this using a modified chloramine-T method. Two derivatives were produced, the N-[beta-(4-OH-3-125I-phenyl)ethyl] and the N-[beta-(4-OH-3,5-di 125I-phenyl)ethyl] biotin amides, depending on the amount of tyramine used in the radioiodination reaction. The final products were separated by thin layer chromatography (n-butanol: 2N NH4OH: ethanol, 3:1:1, v/v/v). The radioiodinated derivatives that were synthesized or their resulting mixture were found to complete with biotin for the avidin-binding sites; thus, they were capable of being used as tracers in biotin radioassays. The specific activity of their mixture was high-greater than 350 Ci/mmol-and they were stable for 2 mo at 4 degrees C.

Antibodies against the N-terminus of rat parathymosin alpha have been raised in rabbits by conjugating parathymosin alpha (1-30) to hemocyanin. A radioimmunoassay for parathymosin alpha was established by utilizing antibodies against the above polypeptide and parathymosin alpha(1-12)[Tyr] as tracer. The useful range was 5-450 pmol for parathymosin alpha. An epitope was located in the amino acid sequence 1-12. The antiserum failed to crossreact with the same molar concentrations of the partly homologous thymosin alpha 1 or prothymosin alpha. With this radioimmunoassay, parathymosin alpha was isolated from calf thymus after separation from prothymosin alpha by reversed phase HPLC. Endogenous proteases did not appear to generate N-terminal fragments of parathymosin alpha in rat liver extracts in a similar fashion to that observed for prothymosin alpha. Parathymosin alpha has a ubiquitous distribution in the human tissues examined, with levels ranging from 93 (brain) to 1043 (liver) ng of parathymosin alpha(1-30) equivalents/g (wet weight).