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P Pradelles

Publications and source records attributed to P Pradelles.

At least 19 recordsLinked to original sources
PubMed2001

Use of free radical chemistry in an immunometric assay for 17 beta-estradiol.

BACKGROUND: We wished to develop an enzyme immunometric assay for 17 beta-estradiol (E2) in human serum using solid-phase immobilized epitope immunoassay (SPIE-IA) technology and free radical chemistry. METHODS: We used an anti-estradiol monoclonal antibody as capture antibody and Fenton-like reagents to cross-link it to E2. The same antibody, labeled with acetylcholinesterase, was used for detection. Serum was diluted 10-fold before assay. RESULTS: After correction by the dilution factor, the detection limit was 5 ng/L for human serum and intra- and interassay CVs were <7% and 15%, respectively, at concentrations of 169-2845 ng/L. No cross-reactivity was seen with other natural steroids. In comparison with a competitive commercial RIA performed on 88 undiluted human sera, the slope (SD) of the regression line was 1.05 (+/- 0.02) and the intercept was 47 (+/-27) ng/L (S(y/x) = 186 ng/L) at concentrations of 20-5000 ng/L (r(2) = 0.97). CONCLUSIONS: The use of Fenton-like chemistry in SPIE-IA technology allows a sensitive measurement of E2 in human serum and could be a new approach for the development of sensitive immunoassays.

Antibodies, Monoclonal↗
PubMed1999

A solid-phase immobilized epitope immunoassay (SPIE-IA) permitting very sensitive and specific measurement of angiotensin II in plasma.

We have developed a new enzyme immunometric assay for angiotensin II (AII) based on SPIE-IA technology (solid-phase immobilized epitope-immunoassay). A monoclonal antibody with optimal properties (mAb3 131) was selected from a series of 19 anti-AII mAbs. The mAb had to be purified from ascitic fluid in a specific manner in order to remove endogenous AII from the antibody-binding sites. We established a sensitive (minimum detectable concentration 0.5 pg/ml) and precise (CV below 15% in the 2-100 pg/ml range) SPIE-IA. Using different AII-related peptides, we observed that this new assay has a specificity profile that compares favourably with the corresponding competitive immunoassay. We have used the assay to measure AII in 42 plasma samples, and demonstrated a good correlation with values obtained using a commercial radioimmunoassay. Assay specificity was supported by HPLC fractionation experiments, confirming the absence of interference induced by endogenous AII-related products.

Acetylcholinesterase↗
PubMed1999

Cross-linking of 17 beta-estradiol to monoclonal antibodies by direct UV irradiation: application to an enzyme immunometric assay.

Ultraviolet irradiation was used to cross-link 17 beta-estradiol directly to monoclonal anti-17 beta-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradiation energy and wave-length. The best results were obtained at 254 (10 J/cm2, 45-min irradiation) and 312 nm (40 J/cm2, 160-min irradiation). The irradiation fully denatured both individual molecules (i.e., 17 beta-estradiol and monoclonal anti-17 beta-estradiol antibody), but 17 beta-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17 beta-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has considerable potential. We used this original approach to develop a new enzyme immunometric assay of 17 beta-estradiol based on our previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which uses chemical agents to cross-link haptens via amino groups to specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensitive (detection limit of 38 pg/mL in human serum). It correlated well with two competitive commercial immunoassays when tested on 40 human sera.

Antibodies, Monoclonal↗
PubMed1999

Pharmacokinetic analysis of 6-monoamino-beta-cyclodextrin after intravenous or oral administration to rats using a specific enzyme immunoassay.

We have developed a highly sensitive enzyme immunoassay for 6-monoamino-beta-CD (mono(6-amino-6-deoxy)cyclomaltoheptaose) and its parent compound (beta-CD) with a detection limit in the 100 pg/mL range. The polyclonal antibodies obtained are highly specific for the beta-cyclodextrin core and do not recognize other cyclic cyclodextrins (i.e., alpha- and gamma-CD) or linear analogues. This enzyme immunoassay can be used to quantify 6-monoamino-beta-CD in rat urine and plasma. Using this immunoassay, we have evaluated the main pharmacokinetic parameters of 6-monoamino-beta-CD after iv administration to the rat of a 25 mg/kg dose. Since this method is strictly specific to the native beta-CD form, we have demonstrated that the molecule rapidly disappeared from plasma but is probably distributed in the tissues. The urinary route appears as the predominant way of elimination since almost all the administered drug is recovered in urine. Finally, analysis of the same molecule after oral administration to the rat (25 mg/kg) demonstrates low plasma levels and that about 1% of the administered dose is excreted in urine. These experiments demonstrate the high stability of the beta-CD core irrespective of the method of administration. This immunological method could provide relevant information on the fate of beta-CD and some derivatives for drug delivery using different modes of administration (oral, parenteral, transmucosal, or dermal).

Administration, Oral↗
PubMed1999

A new specific enzyme immunoassay allowing an efficient pharmacokinetic evaluation of gamma-cyclodextrin after intravenous administration to rats.

PURPOSE: Because of its ability to form complexes with drugs, gamma-cyclodextrin is of great potential value in pharmaceutical formulations. The biological fate of y-cyclodextrin must therefore be considered in safety evaluation, using sensitive and specific methods applicable to biological fluids. METHODS: Antibodies were raised against gamma-cyclodextrin, allowing the development of a new enzyme immunoassay. The analytical characteristics of this assay were evaluated. Rats were given a single intravenous 25 mg/kg dose of gamma-cyclodextrin. Plasma and urine samples were collected and assayed. RESULTS: This new enzyme immunoassay was sensitive (limit of detection close to 94 pg/mL) and suitable for quantification of gamma-cyclodextrin in urine and plasma after methanol extraction. The use of different linear and cyclic compounds demonstrated the high specificity of the assay. After i.v. administration, the concentration of gamma-cyclodextrin rapidly decreased in the plasma while the molecule was probably distributed into the tissues. Although urinary elimination predominates, only 50% of the injected gamma-cyclodextrin was recovered in urine, suggesting enzymatic degradation and/or tissular storage. CONCLUSIONS: This assay may provide important information on the fate of gamma-cyclodextrin inclusion complexes dedicated to drug-delivery using various modes of administration (oral, parenteral, transmucosal or dermal).

Acetylcholinesterase↗
PubMed1998

Endogenous nitric oxide inhibits bronchoconstriction induced by cold-air inhalation in guinea pigs: role of kinins.

Inhalation of cold air in guinea pigs increases total pulmonary resistance (RL), an effect that is mediated by kinins and tachykinins. Bronchoconstriction induced by bradykinin (BK) inhalation in guinea pigs is markedly inhibited by nitric oxide (NO) release from the airway epithelium. We investigated whether endogenous NO modulates the increase in RL induced by inhalation of cold air. In anesthetized and artificially ventilated guinea pigs pretreated with atropine, cold-air inhalation (13 degrees C in the trachea) for 5 min did not increase RL. Pretreatment with intravenous N(G)-nitro-L-arginine methyl ester (L-NAME) (but not with its inactive enantiomer, D-NAME) increased RL, an effect reversed by L-Arg. The increase in RL induced by cold air after L-NAME was abolished by the tachykinin NK2-receptor antagonist SR 48968 or the kinin B2-receptor antagonist, HOE 140. After administration of SR 48968, inhalation of cold air reduced baseline airway tone. However, after HOE 140, cold-air inhalation did not affect baseline airway tone. L-NAME exaggerated the bronchoconstriction induced by BK. However, L-NAME did not affect capsaicin-induced bronchoconstriction. BK increased cyclic guanosine monophosphate (cGMP) levels in strips of guinea pig trachealis muscle in vitro, whereas the selective tachykinin NK2-receptor agonist [betaAla8]neurokinin A (4-10) was without effect. The present data suggest that bronchoconstriction induced by cold-air inhalation and mediated by kinin and tachykinin release is inhibited by endogenous NO, and that kinins, but not tachykinins or cold air alone, release bronchorelaxant NO.

Air↗
PubMed1997

Competitive enzyme immunoassay for urinary vanillylmandelic acid.

An enzyme immunoassay for urinary vanillylmandelic acid (VMA) using polyclonal antiserum and VMA-acetylcholinesterase conjugate as enzymatic tracer is described. Two different strategies for immunogen preparation were developed and enantioselectivity was demonstrated. Selected EIA allowed direct measurement of urinary VMA using D(-)-VMA as standard with good sensitivity (MDC = 0.1 mumol/l) and precision (CV less than 7% in 0.2-2.25 mumol/l range). Cross-reactivity with homovanillic acid (HVA) was 0.8% and less than 0.4% with other structurally related catecholamine metabolites. Intra- and inter-assay repeatability were less than 10% and recovery was 97.3% +/- 3%. Good correlation was obtained for EIA and HPLC analysis with normal and pathologic human urine samples (EIA = 0.895 HPLC-7.085, r2 = 0.98, n = 47).

Acetylcholinesterase↗
PubMed1997

Competitive enzyme immunoassay with monoclonal antibody for homovanillic acid measurement in human urine samples.

A fast competitive enzyme immunoassay (EIA) for measuring homovanillic acid in human urine samples was developed with a monoclonal antibody and acetylcholinesterase as enzyme label. Enzyme detection was performed by an easy colorimetric assay. Monoclonal antibodies were screened on the basis of sensitivity, specificity, and correlation studies. EIA has a detection limit of 0.5 micromol/L, a CV <10% in the 1.25-10 micromol/L range, and intra- and interassay CVs of <10%. Cross-reactivity with vanillylmandelic acid was 0.5% and <8% for other structurally related catecholamine metabolites. Parallelism of the EIA was shown in dilution studies and the correlation with routine HPLC assay in 62 normal and pathologic samples was EIA = 1.492 (HPLC) - 3.46, S(y/x), = 47.52, range = 4-1800 micromol/L, r2 = 0.977. Additional data concerning the validity of this assay were provided by HPLC analysis of urinary immunoreactive material.

Acetylcholinesterase↗
PubMed1996

Enzyme immunometric assay of thyroliberin (TRH).

An enzyme immunometric assay of thyroliberin (TRH) using monoclonal antibodies and a derivatization procedure is described. This assay, named SPIE-IA, involves a four step procedure after chemical derivatization of TRH and biological samples by diazotized APEA. Step 1: derivatized TRH was immunocaptured by a monoclonal anti-TRH antibody coated on a 96-well microtiter plate. Step 2: after washing, derivatized TRH was cross-linked via its amino group to the wells using glutaraldehyde. Step 3: washing and treatment with NaOH. Step 4: measurement of bound TRH using a monoclonal anti-TRH antibody labeled with acetylcholinesterase. The minimal detectable concentration was 0.1 pmol/ml: with a coefficient of variation less than 10% in the 0.156-10 pmol/ml range. This assay is 26-fold more sensitive and more specific than the competitive enzyme immunoassay using the same monoclonal capture antibody, derivatized TRH and TRH-acetylcholinesterase conjugate as tracer. Good correlation was observed between SPIE-IA and a sensitive competitive enzyme immunoassay using polyclonal antibodies.

Animals↗
PubMed1996

Enzyme immunoassays for bromocriptine and its metabolites.

We have developed two bromocriptine enzyme immunoassays with different specificities for applications in human and animal pharmacokinetic studies. The first assay uses antibodies directed against the cyclopeptide structure of bromocriptine, and is specific for untransformed bromocriptine. The second assay uses antibodies directed against the bromolysergic part of the molecule and allows the measurement of both bromocriptine and its metabolites. Enzymatic tracers were obtained by covalent coupling of bromocriptine analogs to acetylcholinesterase from the electric eel Electrophorus electricus. Both assays have a limit of detection of 10 pg/ml and a limit of quantification of 50 pg/ml. The specificity of the assays was determined following fractionation by high-performance liquid chromatography of rat samples obtained after administration of bromocriptine.

Administration, Oral↗
PubMed1996

Evidence that epithelium-derived relaxing factor released by bradykinin in the guinea pig trachea is nitric oxide.

Bradykinin, applied locally to the airways, is a weak bronchoconstrictor agent in guinea pigs in vivo and it may cause constriction or dilatation of guinea pig airways smooth muscle in vitro. We examined the motor effect of bradykinin perfused through the lumen of isolated guinea pig tracheal tubes with or without nitric oxide (NO) synthase inhibitors. In the presence of NG-nitro-D-arginine methyl ester (D-NAME) or NG-monomethyl-D-arginine (D-NMMA) intraluminal bradykinin caused a moderate concentration-dependent relaxation. In contrast, in the presence of NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) tracheas developed a sustained increase in tone, and bradykinin caused a marked, concentration-dependent contraction, both effects being reversible by pretreatment with L-arginine, but not with D-arginine. The ability of bradykinin to relax (in the presence of D-NAME) or contract (in the presence of L-NAME) guinea pig tracheal tubes was not affected by indomethacin. Bradykinin contracted epithelium-denuded tracheas in the presence of either L-NAME or D-NAME. Both contraction and relaxation by bradykinin were blocked by the kinin B2 receptor antagonist, HOE 140. Baseline production of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in strips of guinea pig trachealis in vitro was markedly reduced by L-NAME, but not by D-NAME. Bradykinin increased baseline cyclic GMP concentration. These results indicate that bradykinin releases NO or a NO-related molecule, which, possibly by increasing cyclic GMP concentrations, mediates relaxation and opposes contraction induced by bradykinin itself, and further, that bradykinin releases NO from the tracheal epithelium.

Adrenergic beta-Antagonists↗
PubMed1996

Two different approaches for developing immunometric assays of haptens.

To improve immunoassays of small haptens, we developed two different approaches for their measurement in a non-competitive format. We first devised two-site immunometric assays for small peptides (8-11 amino acids) by selecting two sets of antibodies specifically directed against C- and N-terminal moieties of the peptides. In each case, assay sensitivity improved substantially over that of the corresponding competitive assays. More interestingly, all of these new immunometric assays were much more specific than the competitive assays. In a second approach, we developed a new procedure, solid-phase-immobilized epitope immunoassay (SPIE-IA), in which a single monoclonal antibody uses the same epitope for capture and tracer binding and the hapten is covalently cross-linked to solid-phase proteins. To date, SPIE-IA have been successfully applied to the determination of haptens bearing primary amino groups, including substance P, thyroxine, leukotriene C4, endothelin, and angiotensin II. In each case, assay sensitivity was significantly improved.

Amino Acid Sequence↗
PubMed1995

Enzyme immunometric assay for L-thyroxine using direct ultraviolet irradiation.

A new enzyme immunometric assay for L-thyroxine using uv irradiation as a cross-linking procedure is described. L-Thyroxine in plasma samples is immunocaptured by a monoclonal anti-L-thyroxine antibody coated on 96-well microtiter plates. After uv irradiation and methanol treatment, the covalently linked L-thyroxine is measured using the same monoclonal anti-L-thyroxine antibody labeled with acetylcholinesterase. A minimal detectable concentration of 4.8 nmol/liter was observed with a coefficient of variation less than 16% in the 20-320 nmol/liter. Specificity of the assay was very satisfying and a good correlation (r = 0.959) was noted for 33 human plasma samples between this assay and a commercial competitive radioimmunoassay.

Antibodies, Monoclonal↗
PubMed1995

Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay.

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.

Amino Acid Sequence↗
PubMed1995

Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays.

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.

Amino Acid Sequence↗
PubMed1995

Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

Amino Acid Sequence↗
PubMed1995

AcSDKP serum concentrations vary during chemotherapy in patients with acute myeloid leukaemia.

AcSDKP is a physiological negative regulator of cell proliferation in mammals. In Ara-C-treated mice its plasmatic concentrations decrease while the CFU-S start cycling. Infusion of AcSDKP protects these animals from death by blocking the proliferation of primitive haemopoietic cells. We measured AcSDKP serum concentrations in 20 AML patients during the course of high-dose cytoreductive treatment. We observed an early and sharp increase of AcSDKP during the induction treatment in 12 patients, reaching a peak during the initial 3 d of treatment in nine of them. These results are contrary to those observed in mice treated with high doses of Ara-C. They encourage further clinical investigation, and suggest that treatments with synthetic AcSDKP (Seraspenide) will perhaps have to be adjusted to the type of disease and the schedule of chemotherapy in order to optimize its myeloprotective effect.

Acute Disease↗