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Biomedical subjects

F Kohen

Publications and source records attributed to F Kohen.

At least 91 records · Page 5Linked to original sources
PubMed1986

Direct chemiluminescence immunoassay for estradiol in serum.

In this simple, reliable, fast solid-phase chemiluminescence immunoassay for directly measuring (i.e., without prior extraction) estradiol-17 beta in serum, a monoclonal antibody is used that binds estradiol with high affinity (Ka = 10(10) L/mol), and does not bind other steroids tested, the highest cross reactivity observed being 0.1% for estradiol-17 alpha. In this system the monoclonal antibody is bound to the wells of microtiter plates via a second antibody directed against the monoclonal antibody. Fifty microliters of serum and estradiol-displacing agents are added, followed by 100 pg of estradiol-isoluminol conjugate, and the label is measured by luminometry after the binding reaction. The sensitivity of the assay is 180 pmol per liter of serum, and the effective working range at less than or equal to 10% CV is 270 to 6700 pmol/L. Analytical recovery of added estradiol averaged 99.7% (SD 6.5%). Within- and between-assay CVs ranged between 5 and 12.7%. Thirty-five unknown serum samples can be assayed within 4 h. Results correlated well with those obtained with a direct RIA: r = 0.94 (n = 149). This assay opens new perspectives for chemiluminescence immunoassays.

Antibodies, Anti-Idiotypic↗
PubMed1986

Direct solid-phase chemiluminescence immunoassay for salivary progesterone.

A simple, direct chemiluminescence immunoassay for progesterone in mixed, unstimulated saliva is described. We use purified polyclonal anti-progesterone antibodies covalently coupled to polyacrylamide beads and progesterone-11 alpha-hemisuccinyl-aminobutylethyl isoluminol as the chemiluminescent ligand marker. Bound and free ligand are separated by simple centrifugation. The detection limit of the assay is 1.5 pg per tube (38 pmol/L). Intra- and interassay coefficients of variation for low, medium, and high progesterone concentrations are 7.9, 6.8, 8.8% and 9.3, 6.9, 8.5%, respectively. Analytical recovery of added progesterone is 99.6%. Mean +/- SD progesterone concentrations (pmol/L) in saliva are 178 +/- 46 in the follicular phase, 313 +/- 90 in the periovulatory phase, and 658 +/- 166 in the luteal phase of the menstrual cycle. Correlation between progesterone concentrations in serum (assayed by RIA after extraction) and in saliva is good (r = 0.88, p less than 0.001, n = 96). The assay is simple, fast (4 h, including 1.5 h of incubation time), and reliable.

Adolescent↗
PubMed1985

Ovarian lipoxygenase activity and its regulation by gonadotropin in the rat.

In our previous study a dose-dependent blockage of follicular rupture at ovulation by inhibitors of lipoxygenase was demonstrated. Here the presence of 5-lipoxygenase activity in the whole ovary and in the Graafian follicle is estimated by a chemiluminescence assay using unlabeled arachidonic acid as substrate in the presence of luminol and by conversion of 14C-arachidonic acid into lipoxygenase products as separated by HPLC. Both approaches demonstrated lipoxygenase activity in whole ovarian homogenates and in homogenates of preovulatory Graafian follicles. Furthermore, within 6 h after stimulation in vivo with hCG, lipoxygenase activity was increased by 2-fold in the whole ovarian homogenate and by 5-fold in the follicular homogenate. These results confirm the presence of lipoxygenase in rat ovaries, and its stimulation by gonadotropin and thus corroborate the suggested involvement of lipoxygenase products in follicular rupture at ovulation.

8,11,14-Eicosatrienoic Acid↗
PubMed1984

The measurement of urinary LH, by a solid-phase chemiluminescence immunoassay.

Ovulation in women may be predicted or detected by a simple, solid-phase, chemiluminescence immunoassay for the measurement of urinary LH. An IgG fraction of a donkey antiserum to rabbit immunoglobulins is passively adsorbed to the walls of polystyrene tubes. Human chorionic gonadotrophin-succinyl-butyl-ethyl-isoluminol and a primary antibody (rabbit anti-human LH) is incubated with samples of early morning urine. After the binding reaction, the solution is removed by aspiration. The antibody-bound fraction is washed twice with buffer. Sodium hydroxide is added and the mixture incubated. After cooling, luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated. The method has been evaluated in terms of sensitivity, within- and between-batch precision, bias and parallelism. The time interval between the peak day of LH in EMU and maximum follicular diameter during 38 menstrual cycles was -24 h (11%), O h (50%), + 24 h (28%) and + 48 h (11%).

Buffers↗
PubMed1984

Measurement of choriogonadotropin by chemiluminescence immunoassay and immunochemiluminometric assay: 1. Use of isoluminol derivatives.

We have developed immunoassays, monitored by the detection of chemiluminescence, for measuring choriogonadotropin in human urine. These methods involve the use of derivatives of isoluminol and include: (a) a labeled antigen with a second antibody covalently linked to polyacrylamide beads as the solid-phase reagent (i.e., solid-phase chemiluminescence immunoassay); and (b) an excess concentration of a specific antibody passively adsorbed onto the walls of polystyrene tubes and a labeled antibody of different specificity (i.e., two-site immunochemiluminometric assay). After the respective binding reactions, the solutions are aspirated, the antigen- or antibody-bound fractions are washed twice with 500 microL of buffer, sodium hydroxide (2 mol/L; 200 microL) is added, and the mixture is incubated for 60 min at 60 degrees C. After cooling, the label is oxidized with microperoxidase/hydrogen peroxide and the resulting chemiluminescence signal is measured for 10 s. We have evaluated the methods in terms of their sensitivity, precision, and clinical utility, and we compare results with values obtained by radioimmunoassay.

Body Temperature↗
PubMed1984

Solid-phase chemiluminescence immunoassay for progesterone in unextracted serum.

We describe a simple, solid-phase chemiluminescence immunoassay for progesterone in 10 microL of unextracted serum ("direct" assay). Danazol at pH 8.0 is included (100 ng per tube) to displace progesterone from binding proteins in serum. A progesterone-11 alpha-hemisuccinyl-aminobutylethyl isoluminol conjugate serves as the chemiluminescent ligand marker and homologous antiprogesterone IgG covalently coupled to "Immunobeads" is the immunoadsorbant. After the binding reaction, bound and free ligand are separated by centrifugation and the chemiluminescence yield of the bound label is determined. The sensitivity, specificity, precision, and accuracy of the method are similar to those of a conventional radioimmunoassay for progesterone in which a radioligand of tritiated progesterone and serum extraction are used. Progesterone values obtained by this procedure agreed well (r = 0.987) with those obtained by radioimmunoassay. We conclude that the chemiluminescence immunoassay for progesterone in unextracted serum is analytically valid and offers a convenient alternative to radioimmunoassay.

Binding, Competitive↗
PubMed1983

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors.

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent KD of 0.02 nM for [3H]-spiroperidol. These antibodies bind also to other butyrophenones with IC50 values three to four orders of magnitude higher than the IC50 obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.

Animals↗
PubMed1983

Development of chemiluminescence monitored immunoassays for steroid hormones.

Solid-phase chemiluminescence based immunoassay methods for the measurement of steroids and urinary steroid conjugates are described. The assays incorporate the following features: (i) a steroid-chemiluminescent marker conjugate serves as the labeled ligand; (ii) polyclonal or monoclonal antibodies covalently coupled to polymer beads are utilized as the specific immunoadsorbent; and (iii) a centrifugation step after the binding reaction is used to remove interferring luminescent compounds along with unbound steroid. These assays proved satisfactory with respect to sensitivity, precision and accuracy and offer significant advantages over existing methods in speed, safety and economy. Moreover, the use of monoclonal antibodies provides optimal specifity and rigorous standardization.

Animals↗
PubMed1983

Possible involvement of lipoxygenase products of arachidonic acid pathway in ovulation.

The possible involvement of products of the lipoxygenase pathway of arachidonic acid cascade in ovulation was tested by intrabursal injection of nordihydroguaiaretic acid (NDGA); 5, 8, 11-eicosatriynoic acid (5, 8, 11-ETYA), 3 amino-1-(3 trifluromethyphenyl)-2-pyrazoline hydrochloride (BW755c) and (FPL 55712). All these drugs reduced the number of ova released from the treated ovaries in a dose-dependent manner, without affecting ovulation from contralateral ovaries. NDGA was most potent since it completely blocked ovulation from the treated ovaries in 17/38 rats receiving a dose higher than 0.15 mg/bursa. This effect of NDGA cannot be ascribed to its inhibition of ovarian PGE synthesis. Conversion of labeled arachidonic acid via the lipoxygenase pathway by preovulatory rat follicles was demonstrated by TLC chromatography. Collectively, these results suggest the involvement of products of lipoxygenase pathway of arachidonic acid in ovulation in the rat.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1983

Studies on human milk macrophages: effect of activation on phagocytosis and secretion of prostaglandin E2 and lysozyme.

Breast milk macrophages cultured in vitro synthesized and secreted increasing amounts of protein, lysozyme, and prostaglandin E2(PGE2) into the extracellular medium. These cells were also shown to actively phagocytose labeled zymosan particles in culture. Morphologic characteristics, phagocytosis, and secretory responses of the macrophages were altered depending on the presence of various stimuli in the culture. Concanavalin A, endotoxin and zymosan particles, but not latex particles, all resulted in an increased PGE2 secretion into the medium. Although total protein synthesis was not altered by any of these stimuli, Concanavalin A and endotoxin resulted in a decreased lysozyme concentration in the extracellular medium. Concanavalin A enhanced, whereas endotoxin and prior phagocytosis of latex particles inhibited phagocytosis of labeled zymosan particles. These findings indicate that phagocytosis and secretions of milk macrophages may be altered depending on the nature of the stimulating agent.

Concanavalin A↗
PubMed1983

Decrease in blood and ovarian 3 alpha- and 3 beta-androstanediol levels in rats induced to ovulate with pregnant mare serum gonadotropin.

Several hours before the first ovulation progesterone metabolism in the rat ovary, in vitro, is shifted from the production of 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) as the major metabolite toward the production of 4-ene-3-oxosteroids. In the present paper, changes in levels of 3 alpha-diol and its 3 beta-epimer as well as testosterone in blood and ovaries around the time of the first ovulation have been studied in immature PMSG-treated rats. Forty-eight hours after PMSG, a considerable increase in blood and ovarian testosterone concentration was observed, whereas the concentrations of both androstanediols in blood decreased sharply. At 52 h, the level of ovarian 3 beta-diol was only one third of the control level and continued to fall. The decrease in ovarian 3 alpha-diol was less pronounced, but reached about half, or less, of the control value. In PMSG-treated rats in which the LH surge was blocked by pentobarbitone, the decrease in blood diols was delayed but not prevented. It is concluded that the decrease in production of the androstanediols preceding the first ovulation observed previously in isolated ovaries, also occurs in the intact rat. The decrease in androstanediols occurs very shortly before an ovulation induced with an injection of PMSG and is dependent on the occurrence of an LH surge. Since it is assumed that the androstanediols have a prepubertal role in inhibiting uterine and ovarian growth and in preventing cyclic LH release, it is essential that their concentration decrease several hours before the first ovulation.

Androstane-3,17-diol↗
PubMed1983

Solid-phase chemiluminescence immunoassay for plasma estradiol-17 beta during gonadotropin therapy compared with two radioimmunoassays.

Estradiol in plasma was determined with a simple solid-phase immunoassay based on chemiluminescence in patients undergoing ovulation induction. In the assay, we use a purified monoclonal antibody to estradiol-17 beta, with estradiol-6-carboxymethyl oxime-aminobutylethyl isoluminol as the chemiluminescent marker. The antibody is covalently coupled to polymer beads. Bound and free ligand are separated by simple centrifugation. Values so determined were compared with those determined with two different radioimmunoassay (RIA) methods, one involving the same monoclonal solid-phase antibody plus iodinated estradiol, 125I-RIA, the other a polyclonal antibody and tritiated estradiol, 3H-RIA. In the latter RIA, dextran-coated charcoal was used to separate bound and free ligand. Values determined by the three methods agreed well: for chemiluminescence vs 125I-RIA, r = 0.93; for chemiluminescence vs 3H-RIA, r = 0.94; for 125I-RIA vs 3H-RIA, r = 0.94. The three methods are similar in sensitivity, specificity, and precision. The present method is simple and reliable, taking advantage of the specificity and sensitivity intrinsic to the use of an antibody without the inconveniences associated with use of an isotopically labeled ligand.

Antibodies, Monoclonal↗
PubMed1982

Preparation of monoclonal antibodies able to discriminate between testosterone and 5 alpha-dihydrotestosterone.

A procedure for production of monoclonal antibodies to testosterone is described. The method involves immunization of rats with a bovine serum albumin conjugate of testosterone 3-(0-carboxymethyl) oxime followed by polyethylene glycol induced hybridization of the immune lymphocytes with mouse myeloma cells. The resulting hybridomas were cloned and the antibodies produced by each clone were characterized. All the antibodies obtained showed high affinity for testosterone, (Ka = 10(10) 1/mol), but clones differed widely in the degree of cross-reaction of the antibodies with other steroids, such as 5 alpha-dihydrotestosterone (range 2-100%) and androstenedione (less than 0.1-4%). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.

Animals↗
PubMed1982

The effect of human monocytes and macrophages on lymphocyte proliferation.

Human monocytes suppressed both the phytohaemagglutin (PHA) or antigen-induced lymphocyte proliferative response, when the monocyte: lymphocyte ratio was increased or when the monocytes were stimulated with zymosan or endotoxin. The effect of monocytes on autologous lymphocyte proliferation was compared with that of macrophages obtained by culturing monocytes in vitro for 7 days. The lymphocyte proliferative responses were increased in the presence of macrophages, however, neither increasing their number nor stimulation by zymosan or endotoxin altered the autologous lymphocyte proliferative response to PHA or purified protein derivative (PPD). The PGE2 concentration in the medium of both the cultured monocytes or macrophages activated by zymosan and endotoxin rose markedly without a corresponding suppressor effect on lymphocyte proliferation in the presence of macrophages in the culture. Thus it seems that while PGE2 is a useful marker of mononuclear phagocyte activation, other molecular species are of importance in determining the lymphocyte proliferative response to mitogens and antigens.

Candida↗
PubMed1982

Measurement of plasma estradiol-17 beta by solid-phase chemiluminescence immunoassay.

We describe a simple, solid-phase chemiluminescence immunoassay for the measurement of estradiol-17 beta in extracts of peripheral venous plasma. The method has similar sensitivity, specificity, precision, and accuracy to a conventional radioimmunoassay with a tritiated antigen. An immunoglobulin G fraction od monoclonal antibodies to estradiol-6-carboyxmethyl oxime-bovine serum albumin is passively adsorbed onto the walls of polypropylene tubes. The labeled antigen is estradiol-6-carboxymethyl oxime-aminobutylethyl isoluminol. After the binding reaction (1 h at 22 degrees C), the solution is removed by aspiration (400 microliters). Sodium hydroxide (5 mol/L, 300 microliters) is added and the mixture incubated for 30 min at 37 degrees C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 s. The light yield is inversely proportional to the concentration of estradiol in the standard or sample.

Adult↗
PubMed1981

Evaluation of different progesterone-isoluminol conjugates for chemiluminescence immunoassay.

The effect of varying the length of the alkyl bridge linking the chemiluminescent label isoluminol to progesterone on the light yield and binding affinity of progesterone chemiluminescent-marker conjugates was investigated. For this purpose five different derivatives of isoluminol, aminoethyl isoluminol (AEI), aminoethylethyl isoluminol (AEEI), aminobutyl isoluminol (ABI), aminobutyl-ethyl isoluminol (ABEI) and aminoethyl-ethyl isoluminol (AHEI) were covalently linked through a peptide bond to progesterone-11 alpha -hemisuccinate (P-11-HS). The resulting progesterone chemiluminescent-marker conjugates were then evaluated as potential labels for the development of an immunoassay based on monitoring chemiluminescence. These conjugates were able to compete with tritiated progesterone for the binding sites of an antibody raised against progesterone-11 alpha-HS bovine serum albumin, and they showed higher affinity than unaltered progesterone. These conjugates produced light upon oxidation by a hydrogen peroxide-microperoxidase system. The chemiluminescent reaction was optimized in terms of pH, concentration of oxidant, catalyst and conjugate design. Under optimal conditions, all the conjugates were detectable at femtomolar levels. The lowest detection limit was obtained using P-11-HS-ABEI (0.1 fmol). These results indicated that immunoassay techniques based on chemiluminescence can be developed with these labels.

Animals↗