Two-site and competitive chemiluminescent immunoassays.
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Animals↗
Biomedical subjects
F Kohen
Publications and source records attributed to F Kohen.
Chemiluminescent labelled streptavidin (STAV) as a universal marker in steroid and peptide immunoassays.
The tetrameric structure of streptavidin and its exceptionally strong affinity to biotin (Ka = 10(15)M 1) can be exploited to achieve an amplification of the signal in immunoassays. In the approach described here streptavidin (STAV) labelled with aminobutylethyl-isoluminol (ABEI) served as a universal marker in immunoassays for both haptens and big antigens. The advantageous features of streptavidin can be applied to any immunoassay using biotinylated antibodies as the primary probe. In two-site immunometric assays for larger antigens the liquid phase 'tracer' antibody is biotinylated. In hapten assays the solid phase antigen technique (Wood et al., 1982) is employed, in which sample-antigen and solid phase-antigen compete for a biotinylated antibody. In this paper we demonstrate the use of STAV-ABEI as a universal chemiluminescent label in steroid assays and in an immunometric assay using human growth hormone (hGH) as an example.
Antigens↗
The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites.
We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 microliters) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.
Estrone↗
A thiophilic adsorbent for the one-step high-performance liquid chromatography purification of monoclonal antibodies.
Thiophilic adsorption chromatography, developed originally by Porath and colleagues (1985, FEBS Lett. 185, 306-310) for conventional chromatographic techniques, was transformed to the HPLC mode upon preparation of new and improved sulfur-containing silica beads. The new thiophilic adsorbent is of high capacity and is suitable for the rapid and single-step purification of all subclasses of monoclonal and polyclonal antibodies. Due to its broad specificity, the thiophilic silica column is an efficient, stable, and inexpensive substitute for protein A and protein G columns used today to purify antibodies.
Adsorption↗
Indication of different lactogen and somatogen binding sites in the human growth hormone molecule as probed with monoclonal antibodies.
The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction has been investigated using as probes monoclonal antibodies (Mabs) to hGH of defined epitope specificity profile. Seven high affinity Mabs were studied for their ability (i) to inhibit the binding of 125I-hGH to Nb2-SP rat lymphoma cells and to IM-9 human lymphocytes possessing lactogen and somatogen type receptors, respectively; and (ii) to interfere with the hormone (hGH or Met8Leu hGH)-induced proliferation in Nb2-11C lymphoma cells. The ability of these Mabs to inhibit the 125I-hGH binding and the hormone-induced proliferation in Nb2-11C cells was negatively correlated with the ability of these Mabs to cross-react with met14 hGH. Furthermore, Mabs Nos. 3 and 7, which cross-reacted minimally (0.2-0.4%) with Met8Leu hGH, were unable to interfere with the mitogenic activity of Met8Leu hGH in Nb2-11C cells. These results indicate that the first 13 amino acids of the N-terminal region of hGH are necessary for its lactogen activity. The inhibition of 125I-hGH binding to IM-9 cells by these Mabs was similar to those observed in Nb2-SP cells, except for Mabs Nos. 19 and 1. These Mabs inhibited more strongly the binding of 125I-hGH to IM-9 than to Nb2 cells and recognized antigenic epitopes close to the C-terminal part of the molecule. These results suggest that the somatogen receptor binding site of hGH may be located on two sites, one at the N-terminal and the other one close to the C-terminal, while the lactogen receptor is mainly confined to the N-terminal part.
Animals↗
The antigenic epitopes of human grown hormone as mapped by monoclonal antibodies.
The antigenic epitopes of human GH (hGH) have been investigated using monoclonal antibodies (Mabs) to hGH. A panel of 12 Mabs was incorporated into two-site binding assays in order to investigate the antigenic surface of hGH. Nine reaction patterns were observed. The Mabs were further characterized in terms of their cross-reactivity with human placental lactogen, human PRL, with recombinant hGH molecules (MetLeu8hGH, Met14hGH), and with fragments derived from enzymatic or chemical digestion of hGH. These studies provided information on the antigenic sites that are shared with human placental lactogen and on the N-terminal and C-terminal regions of the hGH molecule. Based upon these findings, we tentatively suggest the epitope model for hGH comprising at least 10 antigenic sites (epitopes) which may be grouped into five antigenic regions.
Animals↗
Somatotropin as measured by a two-site time-resolved immunofluorometric assay.
To date, many of the current criteria for diagnosis of somatotropin (growth hormone, GH) deficiency have been based upon measurement of this hormone by competitive radioimmunoassay (RIA) with use of polyclonal antibodies. In recent years, however, the development of hybridoma technology has led to the generation of various monoclonal antibodies (Mabs) to GH with different affinities and epitope specificities. Subsequently, these reagents have been used in the development of noncompetitive two-site immunometric assays (e.g., immunoradiometric assay; IRMA). In general, the values obtained for serum GH by IRMA have been lower than those obtained by RIA, because of the epitope-specificity profile of the Mabs in the IRMA. Attempting to obtain GH values numerically similar to those by RIA, we used a combination of Mabs to GH in developing and evaluating a two-site time-resolved immunofluorometric assay (IFMA) based on the streptavidin-biotin interaction. Fluorescence is proportional to concentration of analyte and is linearly related to concentration over the range 0.3 to 40 micrograms/L. The assay was satisfactory with respect to sensitivity, accuracy, and precision (CV less than 10% over the entire working range). In addition, the concentration of GH was determined by the IFMA and a competitive RIA in serum obtained from GH deficient and acromegalic patients. The pairing of antibodies in the IFMA gave numerical values that agreed well with those by RIA (r = 0.97; n = 100).
Acromegaly↗
Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay.
We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.
Antibodies, Monoclonal↗
Immunometric assay for lutropin (hLH) based on the use of universal reagents for enzymatic labelling and magnetic separation and monitored by enhanced chemiluminescence.
A solid-phase immunometric assay of human lutropin (hLH) is described. Two different anti-hLH antibodies were utilized as capture antibodies, and anti-IgG antibodies covalently coupled to magnetic particles and horseradish peroxidase, respectively, served as 'universal' detection reagents. An anti-hLH antibody raised in rabbits was incubated with a goat anti-rabbit IgG covalently bound to magnetic particles. The resulting complex was added to a separately incubated mixture of hLH and monoclonal anti-hLH antibody. Following incubation, the immunocomplex was sedimented in a magnetic field and the supernatant discarded. Finally a sheep anti-mouse antibody (F(ab')2 fragment) conjugated to horseradish peroxidase as label was added. Following a further incubation, the particles were sedimented in the magnetic field and washed. The hLH content of the sample was quantitated by measuring 'enhanced chemiluminescence'. The sensitivity of the assay was 2.5 +/- 0.9 IU/l (mean +/- SD), the within-run variation ranged from 7.9 to 11%, the between-run variation from 12.9 to 19.8%. Cross-reaction with hFSH or hTSH could not be detected, but was approximately 0.1% with hCG. The results correlated well with those obtained by radioimmunoassay (r = 0.84).
Antibodies, Anti-Idiotypic↗
The avidin-biotin reaction as a universal amplification system in immunoassays.
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Adolescent↗
Antitumor effects of inhibitors of arachidonic acid cascade on experimentally induced intestinal tumors.
The antitumor action of inhibitors of cyclooxygenase (indomethacin) and lipoxygenase activity (nordihydroguaiaretic acid) of arachidonic acid cascade was investigated in the chemically induced large bowel tumors in Sprague-Dawley rats. Indomethacin treatment completely prevented the carcinogenic effect of methylazoxymethanol. Thus, no tumors were found in the 14 rat test group, compared with 13 of 14 tumor-bearing rats in the untreated control group. Although nordihydroguaiaretic acid treatment does not abolish prostaglandin synthesis, it does reduce the effect of the carcinogen and tumors were found in only five of 14 treated rats. From this study it can be postulated that not only is reduction in prostaglandin formation responsible for the inhibition of tumor growth, but also leukotrienes may play some role.
Animals↗
Recent advances in chemiluminescence-based immunoassays for steroid hormones.
Several formats of solid-phase separation techniques for the measurement of steroids and urinary steroid conjugates using chemiluminescence as an end point are described. These formats include: (1) immunoadsorption of second antibodies directed against the first antibody on solid support; (2) specific immunoadsorbents consisting of primary antibodies covalently coupled to polymer beads; (3) second antibody coupled to a polymer containing magnetic particles. In these assays a steroid-chemiluminescent marker conjugate serves as the labelled ligand, and highly specific homologous monoclonal antibodies are used to provide optimal specificity and rigorous standardization. These techniques enabled the direct measurement of steroid glucuronides in diluted urine and of steroids in plasma.
Animals↗
Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils.
Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca2+ concentration ([Ca2+]i) and induces production of leukotriene C4 (LTC4). This model was used to examine the role of Ca2+ in LTC4 formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2 x 10(-7) M), selectively prevented the stimulatory effect of the antigen on LTC4 production whereas the response to calcium ionophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 micrograms/ml) did not overcome the inhibitory action of HC. An elevated [Ca2+]i is known to be essential for the activation of both 5-lipoxygenase and phospholipase A2. The stimulatory effect of the antigen on LTC4 production was abolished when the cells were incubated in Ca2+-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca2+. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca2+ concentration of 150 microM and 40 microM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca2+]i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of 45Ca2+ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca2+]i, and this may be associated with the inhibitory action of HC on LTC4 formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.
Animals↗
Gonadotropin-induced differentiation of granulosa cells is associated with the co-ordinated regulation of cytoskeletal proteins involved in cell-contact formation.
The gonadotropin-induced differentiation of granulosa cells in culture was studied, with particular attention being given to the organization and expression of cytoskeletal proteins involved in the formation of cell contacts, as well as to progesterone production. Gonadotropin-treated granulosa cells formed clusters of spherical cells containing few vinculin-containing focal contacts, exhibited a diffuse distribution of actin, and had few adherens junctions but more gap junctions than cells grown without the hormone. In gonadotropin-treated cells, the levels of synthesis of the cytoskeletal proteins, vinculin, alpha-actinin, and actin, were dramatically reduced, but the synthesis of the tubulins and vimentin was unaffected. Decreased levels of synthesis of these cytoskeletal proteins were also observed in an in vitro translation assay using poly(A)+ RNA from gonadotropin-treated cells. The hybridization of cytoplasmic RNA with cloned actin and vimentin cDNAs revealed a marked decrease in actin-RNA levels, but no change in vimentin-RNA levels in these cells. Such alterations in cytoskeletal-protein expression were also observed in cells treated with compounds that cause elevated cellular cAMP levels by acting at a stage beyond gonadotropin receptor stimulation. Furthermore, by keeping the cells in a spherical configuration in suspension culture, or by treating the cells with cytochalasin B, similar changes in the synthesis of these cytoskeletal proteins were observed. During this process, there was a concomitant increased in the production of progesterone (although to a much lesser extent in suspension culture) that occurred in parallel with the appearance of large mitochondria with lamellar-tubular cristae and a well-developed smooth endoplasmic reticulum, these features being characteristic of granulosa-lutein cells in vivo. Our results suggest that changes in cell shape and contact, together with the regulation of cytoskeletal elements that determine cellular morphogenesis, are part of the gonadotropin-controlled differentiation program in granulosa cells and may also occur during the maturation of these cells in vivo.
Animals↗
Amplified bioluminescence assay using avidin-biotin technology.
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Antibodies, Monoclonal↗
Surface chemiluminescent immunoassays of steroids.
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Digoxin↗
Characterization of a prolactin-daunomycin ligand as a probe for drug targeting.
Conjugates of ovine prolactin and daunomycin were prepared for use as affinity-labelled drug carriers in cancer cells carrying the prolactin receptor. The binding affinity of the conjugates to prolactin receptors in rat liver membrane preparations and in viable granulosa cells derived from estradiol- and pregnant mare serum gonadotropin (PMSG)-treated immature female rats was less than an order of magnitude lower than prolactin. The toxicity of the conjugate in cultured granulosa cells was dependent upon the concentration of the daunomycin present in the culture. The cytotoxic effect of the ligand was abolished by the addition of free prolactin or NH4Cl to the granulosa cell cultures. These conjugates may be useful probes in drug targeting against hormone-sensitive cancer.
Animals↗
Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells.
The effect of bovine corneal extracellular matrix (ECM) on gonadotropin-primed rat granulosa cells in vitro was studied by examining the following parameters: 1) rate of cell attachment to culture dishes; 2) modulation of cell morphology; 3) specific binding of [125I]human(h)CG to LH/hCG receptors; 4) cAMP response to hCG stimulation; and 5) basal and hCG stimulated progesterone production. Attachment of cells to culture dishes occurred significantly earlier on ECM, as compared with uncoated dishes (6 h vs. 24 h). Cells grown on ECM were epitheloid and organized in multilayer aggregates, closely resembling their organization in the intact wall of the ovarian follicle. In contrast, cultures on uncoated dishes grew as a monolayer of markedly flattened cells. A 2-fold increase in number of LH/hCG receptors occurred on ECM within 48 h, probably due to de novo synthesis. Scatchard analysis revealed no change in hormone affinity to the receptor during the culture period [association constant (Ka) = 2.5 X 10(10)M-1 for hCG]. Cells grown on ECM had a parallel increase in cAMP responsiveness to hCG stimulation. Cells grown in serum-free medium on ECM-coated dishes preserved only 50% of LH/hCG receptors and cAMP responsiveness after 48 h. Cells cultured on ECM showed a marked elevation in progesterone production even in the absence of gonadotropin stimulation, whereas cells grown on uncoated dishes almost completely lost their ability to produce progesterone both in the presence and absence of hCG. These results indicate that ECM plays a substantial role in the maintenance and further propagation of granulosa cell differentiation in vitro.
Animals↗