Combinatory effects of phytoestrogens and 17beta-estradiol on proliferation and apoptosis in MCF-7 breast cancer cells

Phytoestrogens have been described to be weak estrogens, SERMs or exhibit antiestrogenic properties. However, information about their activity in presence of estrogens is limited. Therefore, we have analysed the dose dependent combinatory activity of the phytoestrogens genistein (Gen), daidzein (Dai) and coumestrol (Cou), and 17ß-estradiol (E2) on cell proliferation and apoptosis induction in human MCF-7 breast cancer cells. Neither additive nor antagonistic effects on proliferation could be observed, but in contrast all phytoestrogens possessed the ability to inhibit apoptosis in the presence of 17ß-estradiol.

In summary, our in vitro results demonstrate that Gen does not exhibit any antiestrogenic properties. The additive growth stimulatory effects of Gen, Dai and Cou in the presence of E2 are not the result of a stimulation of proliferation; these phytoestrogens, at least in MCF-7 cells, could be characterised as inhibitors of apoptosis.     

Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: Potent endocrine disruptors inducing infertility

Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-β estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E₂), progesterone (P₄), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.     

The development and application of an enzyme immunoassay for urinary estrone conjugates

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High correlation coefficients for values obtained from each assay indicate that EIA measurements provide information that is comparable to values obtained by radioimmunoassay. EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.     

Synthesis of optically pure chrysobactin and immunoassay development

Chrysobactin (-N-(2,3-dihydroxybenzoyl)-d-lysyl-l-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of -N-(2,3-dibenzyloxybenzoyl)--N-Cbz-d-lysine with l-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10^–8 to 10^–10 mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples.     

The use of particle concentration fluorescence immunoassay technology for the analysis of rDNA products

Summary The electrophoretic and immunological techniques typically used to detect potentially useful biopharmaceutical proteins are sensitive with detection limits in the nanogram range. However, quantitation of a recombinant protein can be cumbersome and involve large numbers of samples throughout process optimization schemes. Although electrophoretic methods (i.e., SDS-PAGE and Western blots) now avail themselves to quantitation by densitometry, these techniques are time consuming because of the lack of appropriate automated systems. Biological activity assays, when available, often require relatively pure material and are not suitable for analyzing and quantitating impure or semi-purified samples, typical of the fermentation milieu. The optimization of several rDNA-derived protein systems from both prokaryotic and eukaryotic hosts has been completed using PCFIA, a rapid, sensitive system with high throughput. The development of Particle Concentration Fluorescence Immunoassay (CFIA) procedures for several of these rDNA-derived proteins of interest as potential biopharmaceuticals (e.g., -1-antitrypsin, tPA, soluble CD4, and a malaria vaccine candidate) are discussed.     

Properties and application to immunoassay of monoclonal antibodies to neuron-specific γγ enolase

Two monoclonal antibodies to human and bovine neuron-specific γγ enolase have been produced in the isolated hybrid cell lines, which were obtained by fusion between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. The monoclonal antibody to human γγ enolase (E1-G3) and that to bovine γγ enolase (B1-D6) consisted of γ2a/κ and γl/κ immunoglobulin chains, respectively. Both antibodies could bind with the respective antigen with a molar ratio of about 1:1, and were found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ, rat γγ and αγ, and bovine γγ enolases. However, the antibodies did not cross-react with the α or β subunit of human and rat enolase isozymes. Both antibodies could partially inhibit the activity of γγ and αγ enolases. E1-G3 antibody inhibited γγ and αγ enolase activity by 70 and 30%, respectively, and B1-D6 antibody, by 90 and 40%, respectively. Both antibodies had no effect on the activity of αα and ββ enolases of human and rat origins. The applicability of E1-G3 and B1-D6 antibodies to the sandwich-type enzyme immunoassay for neuron-specific enolase (enolase γ subunit) was examined, and it was found that the assay system using E1-G3 and B1-D6 as the labeled antibodies were sufficiently sensitive for the assay of serum neuron-specific enolase concentrations.     

Plant derived coumestrol phytochemical targets human skin carcinoma cells by inducing mitochondrial-mediated apoptosis,cell cycle arrest,inhibition of cell migration and invasion and modulation of m-TOR/PI3K/AKT signalling pathway

The current study was undertaken to investigate anticancer activity of coumestrol phytoestrogen against human skin cancer. MTT assay was performed for cell viability assessment and clonogenic assay for cell colony formation assessment. Apoptosis was analysed by Annexin V/FITC staining, AO/EB staining and western blotting assays. Effects on the m-TOR/PI3K/AKT signalling pathway were investigated by western blotting. Results indicated that coumestrol induced significant toxicity in human skin cancer cells in contrast to mouse skin cancer cells. The proliferation rate in normal skin cells remained almost intact. Annexin V-FITC and AO/EB staining assays indicated coumestrol induced cytotoxicity in skin cancer cells is mediated through apoptosis stimulation. The apoptosis in skin cancer cells was mediated through caspase-activation. Cell migration and invasion was inhibited by coumestrol in human skin cancer cells via inhibition of MMP-2 and MMP-9 expressions. Moreover, m-TOR/PI3K/AKT signalling pathway in SKEM-5 cells was blocked by coumestrol.     

Combinational biosynthesis of dual-functional streptavidin-phycobiliproteins for high-throughput-compatible immunoassay

The development of fluorescent tools with desired fluorescence and efficient targeting is of great importance in the high-throughput immunoassay. Here we report combinational biosynthesis of new dual-functional streptavidin-phycobiliproteins (SA-PBPs) in Escherichia coli by fusing streptavidin with phycobiliproteins. These recombinant proteins can achieve a purity over 95% after one-step purification, and their maximum absorption and fluorescence emission wavelength are at 556 nm and 568 nm for SA-PCA-PEB (streptavidin-phycocyanin α subunit-phycoerythrin), and 624 nm and 646 nm for SA-PCA-PCB (streptavidin-phycocyanin α subunit-phycocyanobilin), respectively. The potential application of these dual-functional SA-PBPs in immunoassay was evaluated using “sandwich” ELISA method for detection of two biomarkers of liver cancers, i.e. α-fetoprotein (AFP) and carcinoembryonic antigen (CEA). As a result, both SA-PCA-PCB and SA-PCA-PEB showed a good linear function with AFP & CEA within 0–50 ng/ml concentrations. Their limits of detection (LODs) for AFP and CEA were 0.25 ng/mL and 0.28 ng/ml using SA-PCA-PEB, and 1.01 ng/ml and 1.12 ng/ml using SA-PCA-PCB respectively. These results indicate that these novel dual-functional SA-PBPs are useful tools for a wide variety of immunoassay, and may have the advantages in their higher expandability and compatibility with existing and future immunoassay technologies.     

Novel poly-substituted aryl acridinium esters and their use in immunoassay

A new series of stable acridinium ester conjugates have been developed for use as non-isotopic labels in immunoassay. They have proved to be a flexible alternative to radioimmunoassay. We present data showing the successful development of immunoassays in sandwich, competitive and receptor formats. In addition, hydrophilic acridinium ester analogues have been synthesized, encapsulated in liposomes, and utilized as labels in immunoassay. The potential of this technology is discussed.     

Synthesis, characterization, and evaluation of ferrocene-theophylline conjugates for use in electrochemical enzyme immunoassay

A series of 8-(ferrocenylalkyl)theophylline conjugates were synthesized for evaluation in a homogeneous, competitive electrochemical immunoassay for theophylline with amperometric detection of the ferrocene label at +320 mV. The electrical signal was amplified via redox cycling with the glucose oxidase/glucose system. The resulting catalytic current was strongly inhibited upon binding of the conjugates to anti-theophylline antibodies such that a large excess of theophylline was required to achieve complete reversal leading to an assay with poor sensitivity in the clinical range. A study of the nonspecific interaction of the antibodies with various ferrocene derivatives indicated that this was reduced when a charged functional group was present on the metallocene ring. Consequently, a conjugate was synthesized with a quaternary ammonium group which when incorporated into the assay resulted in improved sensitivity.     

Quantitative immunoassay of biotoxins on hydrogel-based protein microchips

Three-dimensional gel-based microchips with immobilized proteins were used for quantitative immunoassay of a series of plant (ricin and viscumin) and bacterial (staphylococcal enterotoxin B, tetanus and diphtheria toxins, and lethal factor of anthrax) toxins. It was shown that different types of immunoassays (direct, competitive, and sandwich type) could be carried out on gel microchips. As shown by confocal microscope studies, antigen-antibody interactions involving the formation of tertiary antibody-antigen-antibody complex occur in the whole volume of microchip gel elements. Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection (0.1 ng/ml for ricin). Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays were recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin.     

An automated microfluidic-based immunoassay cartridge for allergen screening and other multiplexed assays

A microfluidic cartridge and system for multiplexed immunoassays is described. The passive microfluidic cartridge was composed of three layers of injection molded plastic sealed together using a thermal staking technique. Using this platform technology, a specific immunoglobulin E (IgE) panel assay was constructed. Allergen extract targets, positive and negative controls, and IgE calibration standards were immobilized within the cartridge as a microarray. A computer-controlled solenoid array provided the necessary actuation force for pumping reagents within the cartridge to perform an automated, chemiluminescent indirect immunoassay. A 20-target allergen extract panel was demonstrated on the device with a total analysis time of 27 min. Allergen screening results showed 84% agreement for 3 house dust mites (N = 300) compared with a commercial test and 80% agreement overall (N = 978). Average coefficients of variation (N = 80) were measured as 20.5% for low/medium levels and 20.4% for medium/high levels. The average limit of detection (N = 160) was measured at 0.535 AU, and cutoff levels of 1.0 AU were estimated at less than 1 IU/ml (2.4 ng/ml). Such a system has potential applications in decentralized allergen screening as well as in other near-patient diagnostic immunoassays where multiplexed analysis, ease of use, and short analysis time are critical.     

Measuring antibody affinity and performing immunoassay at the single molecule level

Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.     

Solid-phase and bead-based cytokine immunoassay: a comparison

Cytokines and chemoattractive cytokines (chemokines) are present in a wide variety of body fluids such as plasma, cerebrospinal fluid, bronchoaveolar fluid, amniotic fluid, synovial fluid, middle ear effusion fluid, and urine. Cytokines can be detected using classical solid-phase sandwich immunoassays such as enzyme-linked immunosorbent assay (ELISA) or with a bead based multiplex immunoassay (MIA). The physical chemical properties of the different body fluids (such as pH and total protein content) differ, which may have an impact on the outcome of the cytokine assay. Both ELISA as well as MIA cytokine detection systems are constructed by sandwiching the protein of interest between a capture and reporter antibody. When the biological sample contains heterophilic antibodies (such as in patients with auto-immune diseases), these non-specific antibodies can cause false positive results. During pathological conditions, cytokines may be found over a wide concentration range; likewise have to cover this dynamic range in a similar fashion. The correct (statistical) analysis of standard curves and (multiplexed) data are critical for proper interpretation. Classical ELISA based cytokine assays are robust, easy to use and very well suited for measurement of single cytokines. Due to an increased interest in the integral approach to understand biological processes (the omics era), multiplex immunoassays for detection of cytokines and the interpretation of these assays are gaining popularity.     

Bioluminescent immunoassay of thyrotropin and thyroxine using obelin as a label

Solid-phase bioluminescent immunoassay of thyroid hormones, human thyrotropin (hTSH) and two forms of thyroxine (T4), whose determinations are vitally important for diagnostics of thyroid diseases and the efficiency of treatment, is described. The recombinant obelin, a Ca(2+)-regulated photoprotein originally derived from the luminous marine hydroid Obelia longissima, is employed as a bioluminescent label. To produce obelin conjugates with anti-hTSH, anti-T4 immunoglobulins (IgG), and T4, additional SH groups are introduced into the obelin molecule using Traut's reagent (2-iminothiolane) and then obelin possessing extra SH groups is conjugated with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate-activated IgGs or T4. The total yield of obelin conjugates determined by luminescent activity is 60-65% after all chemical and purification procedures. The obtained conjugates are stable to lyophilization and in solution for at least 9 months at 4 degrees C, with loss of activity not exceeding 10%. The application of obelin conjugates for determination of the hTSH, total T4, and free T4 in standard, control, and patient sera displays high sensitivity and reproducibility of results. The results of bioluminescent immunoassays are closely comparable to those obtained by the radioimmunoassay method (R=0.95-0.99).     

Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide.

Tri- or dinitrophenylated pneumococcal polysaccharide type III (TNP- or DNP SIII)) induced a primary 19S anti-TNP response without generating immunological memory to the hapten in LAF₁ mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. abortus organisms (DNP-BA) induced both 19S and 7S primary responses and memory to the hapten. Spleen cells from mice immunized with TNP-KLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLH-primed spleen cells for as short as 5 min at 0 °C with 10 μg of TNP-SIII per milliliter virtually abolished their ability to give 19S and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.     

Mass spectrometric immunoassay for quantitative determination of transthyretin and its variants

Transthyretin (TTR, or prealbumin) is a tetrameric protein found in plasma and cerebrospinal fluid. Its major role is to transport thyroid hormones (thyroxin-T4) and retinol (through association with retinol-binding protein). TTR has been studied extensively due to the great number of point mutations that result in sequence heterogeneity. Many of these variants are associated with pathological conditions that result in extracellular deposition of amyloid fibers in tissues. In this work, we have developed a rapid mass spectrometric immunoassay for determination and quantification of TTR and its variants from human serum and plasma samples. The assay was fully characterized in terms of its precision, linearity and recovery characteristics. The new assay was also compared with a conventional TTR ELISA. Furthermore, we have applied the optimized method to analyze TTR and its modifications in 44 human plasma samples, and in the process optimized a method for TTR proteolytic digestion and identification of point mutations.