Biomedical subjects

F Kohen

Publications and source records attributed to F Kohen.

At least 55 records · Page 3Linked to original sources

PubMed1995

A direct non-competitive idiometric enzyme immunoassay for serum oestradiol.

We report a novel non-competitive enzyme immunoassay for oestradiol based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary anti-oestradiol idiotypic antibody (Ab1). The first anti-idiotype, the betatype, competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the second anti-idiotype, the alphatype, binds to the Ab1 in the presence of analyte but does not bind to the betatype/Ab1 complex because of steric hindrance. In the present format the biotinylated alphatype was captured onto anti-biotin IgG which was adsorbed on the surface of microtitre wells. Reaction mixtures containing the Ab1 complexed sequentially with an enzyme labelled second antibody reagent, with oestradiol standards or serum samples and with the betatype anti-idiotypic antibody were then allowed to react with the immobilized alphatype anti-idiotypic antibody. The enzyme activity of the bound fraction measured at 405 nm increased with increasing oestradiol concentrations over the range 0.06-2.5 ng/ml. The detection limit of the assay was 28 pg/ml. The intra-assay variation ranged from 3.5 to 12.4%, and inter-assay variation from 6 to 13.4%. The results obtained by the colorimetric idiometric immunoassay correlated well with those obtained by a direct radioimmunoassay (n = 85, r = 0.97). This non-competitive immunoassay, termed idiometric assay, for haptens permits the development of sensitive immunoassays with a wide working range, and a variety of end-point determinations depending on the label used (e.g., enzyme, chemiluminescent or fluorogenic compound).

Animals↗

PubMed1995

Novel heterocyclic ligands for the thiophilic purification of antibodies.

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.

Antibodies↗

PubMed1995

The measurement of progesterone in serum by a non-competitive idiometric assay.

A novel non-competitive idiometric time-resolved fluoroimmunoassay for the determination of serum progesterone was developed, based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary antiprogesterone antibody. The first anti-idiotype, the betatype, competes with progesterone for an epitope of the primary antiprogesterone antibody at the binding site. The second anti-idiotype, the alphatype, binds to the antiprogesterone antibody in the presence of progesterone, but does not bind to the betatype antiprogesterone complex due to epitope proximity. In the present configuration, the biotinylated alphatype was captured onto anti-biotin IgG which was immobilized on microtiter wells. Reaction mixtures containing europium-labeled antiprogesterone antibody complexed sequentially with progesterone in standards or serum samples and with the betatype anti-idiotypic antibody were then reacted with the immobilized alphatype anti-idiotypic antibody. After 30 min of incubation, the fluorescence of europium is measured by time-resolved fluorescence and is proportional to the concentration of progesterone over the range 0-320 nmol/mL. The method demonstrates good sensitivity, precision, and comparability with a direct competitive radioimmunoassay. The idiometric assay for progesterone is suitable for dipstick technology and biosensors.

Antibodies, Anti-Idiotypic↗

PubMed1995

The measurement of oestrone-3-glucuronide in urine by non-competitive idiometric assay.

We report a novel non-competitive idiometric assay for the measurement of oestrone-3-glucuronide (EG) in diluted urine. The method is based on the use of two types of anti-idiotypic antibody, the beta-type and alpha-type, that recognize different epitopes within the hypervariable region of the primary anti-EG antibody (Ab1). The beta-type anti-idiotypic antibody is analyte sensitive and competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the alpha-type is analyte insensitive, but does not bind the Ab1 in the presence of the beta-type due to epitope proximity. In the present format, reaction mixtures containing the europium labelled Ab1 are reacted sequentially with EG standards or diluted urine samples, with the beta-type anti-idiotypic antibody and biotinylated alpha-type anti-idiotypic antibody on immobilized streptavidin coated microtiter plates. After 1 h incubation, the fluorescence of europium is measured by a time-resolved fluorescence and is proportional to the concentration of EG over a range of 0-10 nmol/l. The method demonstrates good sensitivity, precision and comparability with an alternative competitive fluorescent immunoassay. The idiometric assay for EG may be applied for the monitoring of ovarian function in women and is suitable for dipstick technology.

Animals↗

PubMed1995

Menopause is associated with a significant increase in blood monocyte number and a relative decrease in the expression of estrogen receptors in human peripheral monocytes.

PROBLEM: The clinical significance of the differential expression of estrogen receptor (ER) in human monocytes was evaluated. METHOD: Two color flow cytometry analysis was used on peripheral blood samples of young and postmenopausal females and postmenopausal females treated with estrogen replacement therapy. In addition, the monocyte and lymphocyte counts and the blood estrogen levels of each patient were determine. RESULTS: During menopause there is a significant decrease in the percentage of ER positive monocytes, and an increase in blood monocyte number, which declines following estrogen replacement therapy to values of the young. CONCLUSIONS: These findings suggest that estrogen modulates the monocyte numbers and its effects may be mediated through the ER in the monocytes.

Adolescent↗

PubMed1995

Anti-idiotypic antibody as an oestrogen mimetic: removal of Fc fragment converts agonist to antagonist.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) caused an increase in uterine creatine kinase (CK) activity when administered in vivo to immature female rats, indicating that the antibody has oestrogenic-like activity. It was, therefore, of interest to investigate the structural requirements of clone 1D5 to act as an oestrogen mimetic in an in vitro model system. In the present study, the effect of clone 1D5 and its proteolytic fragments, F(ab')2, Fab' and Fc on CK activity was examined in cultured skeletal cells having functional oestrogen receptor (ER). Incubation of female-derived calvaria cells or epiphyseal cartilage cells with clone 1D5 (8.33 nM) or oestradiol (E2) (30 nM) for 24 h caused a significant increase in CK activity, indicating that clone 1D5 acted as an agonist. On the other hand, incubation of male-derived calvaria cells devoid of a functional ER with clone 1D5 or E2 did not have any effect on CK activity. Incubation of female-derived calvaria cells with clone 1D5 and E2 did not result in any further increase in CK activity, whereas dihydrotestosterone (DHT) did not alter the response to clone 1D5. The CK response to clone 1D5, in female-derived calvaria cells was time- and dose-dependent and could be inhibited in a dose-dependent manner by the oestrogen antagonist tamoxifen. In contrast, the proteolytic fragments of clone 1D5, the F(ab')2 dimer (12 nM) and the Fab' monomer (24 nM), and the Fc fragment (28 nM) did not have E2-like activity in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Assessment of estrogen receptor distribution in human endometrium by direct immunofluorescence.

OBJECTIVE: To use a direct immunofluorescence technique employing a fluorescein labeled anti-idiotypic antibody that recognizes the estrogen receptor (ER) order to assess the distribution of ER in the uteri of normal women throughout the normal menstrual cycle and of a woman exposed prenatally to diethylstilbestrol (DES). SUBJECTS: Included in the study were 25 women aged between 35 and 50 years and an amenorrheic patient diagnosed as "DES Syndrome". LOCALIZATION: Localization of ER expression in frozen sections of uterine tissue was achieved by direct immunofluorescence using a fluorescein labeled anti-idiotypic antibody that interacts with ER. RESULTS: Analysis of the immunofluorescence staining indicated that in the normal human endometrium the intensity of ER staining varied according to the phase of the cycle as well as according to the cell type. On the other hand, endometrial ER evaluation of the patient with DES syndrome showed minimal expression of ER and after treatment with conjugated estrogens, endometrial biopsy revealed a significant increase in ER expression. CONCLUSIONS: These findings indicate that the fluorescein labeled anti-idiotypic antibody can be used to detect ER in normal and pathological human endometrium and to monitor changes in ER expression in the endometrium during hormonal therapy.

Adult↗

PubMed1994

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide.

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Antibodies, Monoclonal↗

PubMed1993

Cloning of LL5, a novel protein encoding cDNA from a rat pituitary library.

While screening a rat pituitary cDNA library for a peptide hormone receptor, we identified a cDNA that represents a novel gene. The 3.8 kb cDNA has an open reading frame of 2.3 kb encoding a protein of 781 amino acids (M(r) = 87,507). Southern blot analysis of rat, mouse, bovine and human genomic DNAs revealed that a homologous gene is present in these species probably in a single copy. Northern blot analysis showed that in addition to the pituitary gland, the gene is also expressed in other rat tissues. Scanning of DNA and protein databanks revealed no significant homology to any other sequence. Thus, this gene encodes a heretofore unidentified protein.

Amino Acid Sequence↗

PubMed1993

Monoclonal anti-biotin antibodies simulate avidin in the recognition of biotin.

The sequence of the VH gene of a monoclonal anti-biotin antibody was determined. Biotin-binding motifs, similar to those in avidin and streptavidin, were identified in complementary determining regions 2 and 3, suggesting that natural selection of functional motifs may occur in unrelated protein types.

Amino Acid Sequence↗

PubMed1993

Localization of estrogen receptors in long bones and vertebrae of human fetuses.

In order to investigate the possible role of estrogen in the development of cartilage and bone we studied by immunofluorescence immunohistochemistry and autoradiography 26 human embryos and fetuses 7-22 weeks in gestational age associated with pregnancy interrupted for non-medical reasons. In order to demonstrate the presence of estrogen receptors (ERs) in human fetal cartilage, cryostat sections of long bones and lumbar and thoracic vertebrae were prepared for (1) fluorescent immunocytochemistry using an antiidiotypic monoclonal antibody to anti-estradiol receptor monoclonal Ab labeled with fluorescein isothiocyanate (FITC), (2) immunohistochemistry using monoclonal antihuman estradiol receptor antibody, labeled with strept. A-B immunoperoxidase, and (3) autoradiographic localization of estradiol using labeled (3H) 17 beta estradiol. In fetuses aged 10 weeks or older, intranuclear and perinuclear localization of ER was demonstrated by all methods, mainly amongst chondrocytes of the proliferating and higher hypertrophic zones of the epiphyses and in the cartilage of vertebral bodies. These data suggest that estrogen acts directly on chondrocytes of human fetuses through an ER-mediated mechanism.

Antibodies, Monoclonal↗

PubMed1992

Characterization of an antiidiotypic antibody mimicking the actions of estradiol and its interaction with estrogen receptors.

The ability of a monoclonal antiidiotypic antibody (clone 1D5) directed against the binding site of a monoclonal antiestradiol antibody to interact with the estrogen receptor (ER) was investigated. The following lines of evidence indicate that clone 1D5 has the capacity of mimicking the actions of estradiol, and recognizes ER: 1) in binding experiments, clone 1D5 inhibited the binding of [3H]estradiol to porcine cytosolic 32-kilodalton ER fragment in a dose-dependent manner; irrelevant antibody had no effect; 2) in sucrose gradient density analysis, clone 1D5 abolished the specific peak of the [3H] estradiol-ER complex in the 4S region; 3) in immunoprecipitation experiments, clone 1D5 interacted with unoccupied ER, but not with estradiol-occupied ER; 4) in direct immunofluorescence studies clone 1D5 stained the nuclei of cultured rat epithelial cells and recognized estrogen binding sites in nuclear cryostat sections prepared from human, rat, and mouse estrogen-responsive tissues; and 5) When clone 1D5 was injected to immature female rats, it caused 46% increase in uterine creatine kinase activity, suggesting that clone 1D5 may possess estrogenic like activity. Under the same experimental conditions, estradiol caused 58% increase in creatine kinase activity. Collectively, these results suggest that clone 1D5 interacts with the steroid binding site of ER. Therefore, clone 1D5 can serve as a tool in the study of function and structure relationship of ER and to detect changes of ER levels in target cells of various species.

Animals↗

PubMed1992

Direct enzyme immunoassay of estradiol in serum of women enrolled in an in vitro fertilization and embryo transfer program.

We present a fast and simple direct enzyme immunoassay for the sexual steroid hormone estradiol-17 beta in serum of women enrolled in an in vitro fertilization and embryo transfer program. We added 25 microL of standards or samples and 125 microL of a mixture of monoclonal antibody and displacing agents to the wells of a microtiter plate previously coated with a second antibody. After 30 min 50 microL estradiol-horseradish peroxidase conjugate is added. Total incubation time is 75 min. The immunoassay is stopped by washing the microtiter plate. The amount of bound conjugate is then measured at 450 nm with hydrogen peroxide as a substrate and 3,3',5,5'-tetramethylbenzidine as chromogen. The detection limit is 0.24 nmol/L. The assay is thus limited in its application. Analytical recovery is 90.8% (range 80.4-102.9%); within- and between-assay CVs range between 1.3% and 15.4%. Total assay time, including preparation of reagents and data reduction, is only 2.5 h. Results of this enzyme immunoassay compare well with those obtained with four different commercial radioimmunoassays (r = 0.88-0.93).

Embryo Transfer↗

PubMed1991

Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach.

We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Ab1). In this format the primary Ab1 was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab1. Fluorescence was proportional to the concentration of oestradiol over the range 0-8 ng/ml. The sensitivity of the assay was 80 +/- 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand (n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays.

Animals↗

PubMed1991

Idiometric assay, the third way: a noncompetitive immunoassay for small molecules.

A novel, noncompetitive immunoassay applicable to the measurement of small molecules including ovarian steroids is described. Using monoclonal antibodies with the ability to recognize both beta-typic and alpha-typic binding sites, a new simplified, sensitive, and specific immunoassay system has been developed. The initial work of this development is presented, along with preliminary results for a novel immunoassay for estradiol.

Antibodies, Anti-Idiotypic↗

PubMed1990

Pharmacological effects of the continuous administration of an LHRH agonist in the ageing male rat: comparison with orchidectomy.

The age-related changes in tissue response to chronic treatment for 1 month with a potent LHRH agonist were investigated in the ageing male rat, and the observed pharmacological effects were compared with orchidectomy. In both young (4 months) and old (22 months) rats, treatment resulted in a significant decrease in the weights of prostates and testes, a decrease in plasma LH and testosterone levels, a loss of LH receptors in the testes and in a complete depletion of prostatic nuclear androgen receptors, reaching levels observed after castration. In young rats, treatment with an LHRH agonist or orchidectomy induced a three- or sixfold increase in prostatic creatine kinase (CK) activity which may have been induced by the local stimulatory effect of oestradiol arising from the conversion of precursor steroids secreted by the adrenal. On the other hand, in old rats, 7 days after orchidectomy or after treatment with an LHRH agonist a twofold increase or no change was induced in prostatic CK activity respectively. SDS gel electrophoresis patterns of cytosolic prostatic proteins of young rats treated with an LHRH agonist or young rats orchidectomized 7 days previously revealed the presence of several intensified proteins, two of them having apparent molecular weight of 67 kDa and 43 kDa, whereas in the old rats treated with LHRH agonist or old rats castrated 7 days previously, these two proteins were not intensified. The results of this study confirmed that continuous treatment with an LHRH agonist to young and old rats induces medical castration since the pharmacological effects observed were the same as those induced with surgical castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1990

Idiometric assay: noncompetitive immunoassay for small molecules typified by the measurement of estradiol in serum.

We report here a novel noncompetitive immunoassay applicable to the measurement of small-molecular-mass compounds and typified by the direct measurement of estradiol in serum. Two types of anti-idiotypic antibody recognize different epitopes within the hypervariable region of the specific primary antibody (e.g., anti-estradiol). The first anti-idiotype (betatype) recognizes an epitope at the unoccupied binding site, which is masked in the presence of the analyte (e.g., estradiol). The second (alphatype) recognizes an epitope close to the binding site and is unaffected by the presence or absence of the analyte, but is sterically hindered from binding to the primary antibody in the presence of the betatype. The use of these matched antibodies (primary antibody, alphatype, and betatype) has enabled the development of a method for determining antibody occupancy that is not based on a conventional two-site assay. An excess amount of purified monoclonal antibody against estradiol is immobilized onto the walls of microtiter wells. After the capture of analyte, the unoccupied antibody sites are blocked by the addition of an excess amount of betatype. Subsequently, analyte occupancy is determined by the addition of excess europium-labeled alphatype, incubation, washing, and time-resolved fluorometry. The method demonstrates good sensitivity, precision, and comparability with alternative competitive immunoassays.

Antibodies, Monoclonal↗

PubMed1990

Direct chemiluminescence immunoassay of estradiol in saliva.

A sensitive and simple direct solid-phase chemiluminescence immunoassay is described for estradiol in saliva. In this assay, a second antibody is bound to the wells of microtiter plates. Either buffer with standards or saliva (100 microL) is incubated in these wells with monoclonal anti-estradiol antibody and with estradiol-isoluminol conjugate. Incubation time is 2 h. Chemiluminescence of the bound fraction is measured in a manually operated luminometer (Biocounter). The assay has a detection limit of 3.8 pmol/L; analytical recovery of added estradiol is 96.8% (SD 7.0%); within- and between-assay CVs range between 2.5% and 12.7%. Forty unknown saliva samples can be assayed and results calculated within 4.5 h. Results of a slightly modified procedure-with black microtiter plates and a prototype of an automated plate reader (Luminoskan)--compare well with those of the described method (r = 0.97). Because steroid-binding globulins have been found in saliva, the effect of displacing agents on the results of the direct chemiluminescence assay is described.

Estradiol↗