Colloidal gold-based immunochromatographic strip assay for the rapid detection of three natural estrogens in milk.

In this study, we developed highly sensitive and specific monoclonal antibodies (mAbs) against estrone (E1), 17ß-estradiol (17ß-E2), and estriol (E3). The half-maximal inhibitory concentration values of anti-E1, anti-17ß-E2, and anti-E3 mAbs were 0.46, 0.36, and 0.39 ng/mL, respectively, based on competitive enzyme-linked immunosorbent assay (ic-ELISA) results. A rapid colloidal gold-based immunoassay strip assay was developed for the determination of E1, 17ß-E2, and E3 residues in milk samples. The assay had a visual cut-off value of 5 ng/mL, and required 10 min to assess with the naked eye. The results obtained from the immunochromatographic strip assay were consistent with those obtained from ic-ELISA and gas chromatography-mass spectrometry. The immunochromatographic strip assay is useful and rapid for the detection of E1, 17ß-E2, and E3 in milk.

Immunoanalytical characteristics of unconjugated estriol: indications and analytical performances.

After a short description of the structure and the physiological roles of unconjugated estriol, this paper points out pre-analytical conditions and performances of the serum unconjugated estriol immunoassay, essentially used for Down syndrome screening.

Enhancing Antibody Response against Small Molecular Hapten with Tobacco Mosaic Virus as a Polyvalent Carrier.

Virus nanoparticles (VNPs) have been applied as carrier proteins for effective vaccine development. In this paper, we report the usage of tobacco mosaic virus (TMV) as a carrier for the display of the small molecule estriol (E3), a weakly immunogenic hapten. A highly efficient copper (I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) was performed for the conjugation of E3 onto TMV capsid at tyrosine (Tyr) 139, by which the antigen density could be controlled. The immune properties of these constructs were evaluated in mice. We found that a strong and long-term antibody response was elicited by conjugating a high density of small molecular haptens on TMV through an oligo(ethylene glycol) (OEG) linker, likely due to the effective activation of B-cells. This study suggests that TMV can serve as a promising platform to induce strong humoral immune responses and that the optimized conjugation strategy was critical to produce high quality antibodies.

[Establishment of the dot immunoenzyme filtration assay for quantitative detection of estriol].

OBJECTIVE: To make anti-estriol (E3) monoclonal antibody (mAb) with high specificity, and develop a rapid, simple and sensitive method for the detection of E3 in county-level institutions. METHODS: The compounds of E3 were conjugated to horseradish peroxidase (HRP), bonine serum albumin (BSA) and ovalbumin (OVA) respectively by the method of active ester, and the hybridoma cell lines secreting specific anti-E3 mAb were developed via monoclonal antidody technology. The dot immunoenzyme filtration assay in competitive-inhibition format was established with nitrocellulose membrane as solid-phase carrier and anti-E3 mAb as coating antibody. RESULTS: 5 hybridoma cell lines secreting specific anti-E3 mAb were obtained with E3-mAb titers in the range of 1×10(5); to 5×10(5); and the affinity constant (Ka) from 5.1×10(8); L/mol to 5.2×10(9); L/mol. The limit of detecting (LOD) value for E3 was 2 ng/mL and the detection range was 2-200 ng/mL. CONCLUSION: The dot immunoenzyme filtration assay with high-speed detection, easy accessibility and high sensitivity was successfully established.

Development of competitive ELISAs for 17beta-estradiol and 17beta-estradiol +estrone+estriol using rabbit polyclonal antibodies.

Estrogens are a family of feminizing hormones that are excreted by vertebrates. It has been documented that their presence in surface waters, even in the ng/L range, can have detrimental impacts on fish reproduction. Two competitive enzyme-linked immunosorbent assays using rabbit polyclonal antibodies were developed: one for 17beta-estradiol and a second one for 17beta-estradiol (E2)+estrone (E1)+estriol (E3). Two different conjugates were synthesized using the Mixed-anhydride (for the 17beta-estradiol ELISA) and the Mannich (for the E1 + E2 + E3 ELISA) reactions. The 17beta-estradiol ELISA was highly specific with an IC(50) of 243 ng/mL for 17beta-estradiol. The E1 + E2 + E3 ELISA exhibited cross-reactivity with estrone (85%) and estriol (62%) with an IC(50) of 18 ng/mL for 17beta-estradiol. Cross-reactivity was tested against 13 chemically related compounds and both immunoassays showed significant cross-reactivity with two estradiol conjugates: beta estradiol-17-valerate and beta estradiol-3-benzoate (from 57 to 84 %) for which, to our knowledge, there are currently no commercially available ELISA. Characteristics (sensitivity, inter and intra assay variation, and cross-reactivity) of the E1 + E2 + E3 ELISA were further compared to those from a commercial Estriol ELISA. The commercial ELISA was more specific, sensitive and its inter-assay variation was less (9.5% compared to 10% for the E1 + E2 + E3 ELISA) but the E1 + E2 + E3 ELISA had less intra-assay variation (4% compared to 5% for the commercial ELISA). Finally, a solid-phase extraction method compatible with the E1 + E2 + E3 immunoassay demonstrated that this combined approach of extraction and immunoassay had good potential for determining estrogen concentrations in environmental samples such as surface water in urban and agricultural ecosystems.

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing.

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.

Synovial fluid estrogens in rheumatoid arthritis.

Experimental and clinical evidence suggest that immune reactivity is modulated by gender. Immune reactivity is greater in females than in males and lymphocytes and monocytes from female subjects shows higher antigen presenting activity and mitogenic responses. Steroid hormones can be converted along defined pathways to downstream hormones in the periphery. The conversion of dehydroepiandrosterone (DHEA) in target macrophages leads to an increase of downstream effector hormones (including estrogens), which may be an important factor for local immunomodulation at least in RA synovitis. The presence in the RA synovial fluids (SF) of an altered sex hormone balance resulting in lower immunosuppressive androgens and higher immunoenhancing estrogens, might determine a favorable condition for the development of the immuno-mediated RA synovitis and synovial hyperplasia. The increased estrogen concentration observed in RA SF of both sexes are characterized by the hydroxylated forms, in particular 16alpha-hydroxyestrone, that is a mitogenic and proliferative endogenous hormone. In contrast to 16alpha-hydroxylated estrogens, the 2-hydroxylated forms inhibit growth promoting effects of E2 and were found low in RA SF. Therefore, dose-related conversion to pro- or anti-inflammatory downstream metabolites of estrogens might support the dual role of estrogens (pro or anti-inflammatory) for example during estrogen replacement therapy, depending on local concentration (i.e. SF in RA) of 16alpha-hydroxyestrone or 2-hydroxyestrogens.

Combinational use of antibody affinities in an immunoassay for extension of dynamic range and detection of multiple analytes.

Here, we describe the coordinated use of two antibodies with different affinities in a single immunoassay to extend the dynamic range and to enable detection of multiple analytes. The combination of dual antibodies was permitted with a flow-based assay at the antibody concentration below the dissociation constant, enabling affinity to govern the antibody-antigen binding. Both high and low affinity antibodies to estriol were used in combination to extend the range. The binding of each antibody was mutually independent and individually occurred over concentration ranges of 10 pM(-1) nM and 100 pM(-1) microM. The wide dynamic range of 10 pM(-1) microM was thus achieved as summation of the proportional signals to the total binding. When a combination of antibodies toward different antigens was used, it effectively detected multiple analytes within a mixture. In simultaneous analysis of a mixture of estradiol and estriol, the total signal was the sum of the binding signals from anti-estradiol and anti-estriol antibodies. In a further refinement, the individual antibodies were flowed through the flow cell sequentially, allowing the quantification of each binding signal within the combination. With this sequential format, measurement of the individual hormones in the range of 1.6 pM(-1) nM was shown. Furthermore, the same flow format was successfully applied to assay estriol and estradiol hormones in mixtures of six related compounds.

Quantitative measurement of 17 beta-estradiol and estriol in river water by time-resolved fluoroimmunoassay.

A sensitive method for detecting 17 beta-estradiol (E2) and estriol (E3) in river water has been developed, based on the time-resolved fluoroimmunoassay by using a fluorescent europium chelate label, 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6"-yl)- chlorosulfo-o-terphenyl (BHHCT)-Eu3+. In the E2 assay, microtiter plates were coated with the E2-bovine serum albumin (BSA) conjugate. The anti-17 beta-estradiol antibody, the biotinylated goat anti-rabbit IgG antibody and the BHHCT-Eu3+ labeled streptavidin (SA)-BSA conjugate were used. In the E3 assay, the goat anti-rabbit IgG antibody was coated on a microtiter plate. The anti-estriol antibody and the BHHCT-Eu3+ labeled E3-BSA conjugate were used. The detection limits for E2 and E3 were 2.3 pg/ml and 4.3 pg/ml, respectively, and the analytical recoveries were 95-120%. Quantitative measurement of estrogens in river water was carried out for Kanda River (Tokyo, Japan) by using the method. The E2 and E3 levels were 32 pg/ml and 5.5 pg/ml, respectively. The detection limits of the present method are in the same orders of magnitude as those of ELISA for E2, and are 1-2 orders of magnitude better for E3.

Regulatory effects of estriol on T cell migration and cytokine profile: inhibition of transcription factor NF-kappa B.

The protective role of pregnancy in autoimmune conditions, such as multiple sclerosis (MS), is potentially associated with immune regulation by sex hormones produced during pregnancy. This study was undertaken to examine the regulatory effects of estriol on the T cell functions, including transmigration and the cytokine production. The results revealed for the first time that estriol significantly inhibited T cell transmigration at a concentration range typical of pregnancy, which correlated with decreased T cell expression of matrix metalloproteinase-9. Estriol was also found to alter the cytokine profile of T cells toward Th2 phenotype by up-regulating the production of IL-10 and inhibiting TNFalpha secretion of T cells. However, the inhibitory effects of estriol on T cells were not antigen-dependent. Further characterization indicated that estriol inhibited nuclear transcription factor kappa B (NF-kappa B), which controls a variety of immune-related genes. This study provides new evidence that estriol is a potent regulator for the T cell functions potentially through its interaction with the NF-kappa B signaling pathway.

An immunoassay for small analytes with theoretical detection limits.

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated antispecies secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical limits of detectability. The optimum achievable dynamic range of this immunoassay was 4-300 pM. Because the proportion of free anti-estriol antibody in the mixture was controlled by the Kd of the antibody-estriol interaction, when the concentration of the antibody was below the Kd, the smallest detectable estriol concentration approached the theoretical limit of detectability achievable with this antibody.

Progesterone and estradiol secretion by porcine luteal cells is influenced by individual and combined treatment with prostaglandins E2 and F2alpha throughout the estrus cycle.

The present experiments were conducted to test whether the ratio of PGE2:PGF2alpha affects steroid secretion by porcine luteal cells. We examined the effect of separate and combined treatment with PGE2 and PGF2alpha on progesterone and estradiol secretion. Luteal cells were collected at three different stages of the luteal phase (1-3 days after ovulation; 10-12 days after ovulation and 14-16 days after ovulation). PGE2 alone in a dose dependent manner increased progesterone production by cells collected from mature corpora lutea. On the other hand, PGF2alpha in a dose dependent manner decreased progesterone secretion by cells of the same origin. Progesterone secretion by cells isolated from mature and regressing corpora lutea and treated with both prostaglandins increased in comparison to PGF2alpha-treated cultures. However, in cells collected from regressing corpora lutea PGE2 and PGF2alpha in a ratio of 2:1 and 4:1 increased estradiol production when compared to control and both ratios increased estradiol secretion in comparison to PGF2alpha-treated cells. These data 1) confirm the luteotropic effect of PGE2 and the luteolytic effect of PGF2alpha; 2) demonstrate that when the ratio of PGE2 to PGF2alpha changed from 1:1 to 2:1 or 4:1 cells were protected against the inhibitory effects of PGF2alpha on progesterone secretion by cells collected during the mid- and late luteal phase; and 3) suggest that elevated estradiol production by luteal cells, isolated during late luteal phase, under the influence of increased doses of PGE2 may serve as an additional source of estradiol to blastocysts, during early pregnancy in the pig.

Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis.

OBJECTIVE: To evaluate the use of estriol in the treatment of experimental autoimmune encephalomyelitis (EAE) and other cell mediated autoimmune diseases. BACKGROUND: Experimental autoimmune encephalomyelitis is a T helper 1 (Th1)-mediated autoimmune demyelinating disease that is a useful model for the study of immune responses in MS. Interestingly, both EAE and MS have been shown to be ameliorated during late pregnancy. METHODS: Estriol, progesterone, and placebo pellets were implanted in mice during the effector phase of adoptive EAE. Disease scores were compared between treatment groups, and autoantigen-specific humoral and cellular responses were examined. RESULTS: Estriol treatment reduced the severity of EAE significantly compared with placebo treatment whereas progesterone treatment had no effect. Estriol doses that induced serum estriol levels that approximated estriol levels during late pregnancy were capable of ameliorating disease. Estriol-treated EAE mice had significantly higher levels of serum antibodies of the immunoglobulin (Ig) G1 isotype specific for the autoantigen myelin basic protein (MBP). Further, MBP-specific T-lymphocyte responses from estriol-treated EAE mice were characterized by significantly increased production of the Th2 cytokine interleukin 10 (IL-10). T lymphocytes were shown to be the primary source of IL-10 within antigen-stimulated splenocyte populations. CONCLUSIONS: Estriol as a hormone involved in immune changes during pregnancy may provide a basis for the novel therapeutic use of estriol for MS and other putative Th1-mediated autoimmune diseases that improve during late pregnancy.

Antibody fragment Fv4155 bound to two closely related steroid hormones: the structural basis of fine specificity.

BACKGROUND: The concentration of steroid glucuronides in serial samples of early morning urine (EMU) can be used to predict the fertile period in the female menstrual cycle. The monoclonal antibody 4155 has been used as a convenient means of measuring the concentration of steroid glucuronides in EMU, as it specifically recognises the steroid hormone estrone beta-D-glucuronide (E3G), with very high affinity, and the closely related hormone estriol 3-(beta-d-glucuronide) (EI3G), with reduced affinity. Although 4115 binds these hormones with different affinities, EI3G differs from E3G only in the addition of a hydroxyl group and reduction of an adjacent carbonyl. To investigate the structural basis of this fine binding specificity, we have determined the crystal structures of the variable fragment (Fv) of 4155 in complex with each of these hormones. RESULTS: Two crystal forms of the Fv4155-EI3G complex, at resolutions of 2.1 A and 2.5 A, and one form of the Fv4155-E3G complex, at 2.1 A resolution were solved and refined. The crystal structures show the E3G or EI3G antigen lying in an extended cleft, running form the centre of the antibody combining site down one side of the variable domain interface, and formed almost entirely from residues in the heavy chain. The binding cleft lies primarily between the heavy chain complementarity determining regions (CDRs), rather than in the interface between the heavy and light chains. In both complexes the binding of the glucuronic sugar, and rings A and B of the steroid, is specified by the shape of the narrow cleft. Analysis of the Fv structure reveals that five of the six CDR regions can be assigned to one of the predefined canonical structural classes. CONCLUSIONS: The difference in the binding affinity of Fv4155 for the two steroid hormones is accounted for by a subtle combination of a less favoured hydrogen-bond geometry, and a minor rearrangement of the water molecule network around the binding site. The rearrangement of water molecules results from the burial of the additional hydroxyl group of the EI3G in a hydrophobic environment.

Enhancement of the sensitivity of a chemiluminescent immunoassay for estradiol based on hapten heterology.

OBJECTIVE: To develop a highly sensitive chemiluminescent immunoassay (CLIA) that measures the serum estradiol (E2) levels in postmenopausal women. METHODS: The previously developed competitive CLIA for E2 consisted of an immobilized antigen and a labeled antibody with an N-functionalized acridinium ester that was modified to enhance its sensitivity. The modifications were changing the immobilized antigen from E2 to its analogues with its expected effect on hapten heterology and selecting optimal incubation conditions. RESULTS: The hapten heterology in which estriol was used as the immobilized antigen instead of E2 enhanced the sensitivity of the CLIA about 3-fold. A low incubation temperature and a long incubation time also effectively increased the sensitivity of the CLIA. The combination of these modifications enhanced sensitivity about 10-fold. The proposed CLIA with a 16 pmol/L detection limit, was about 5-fold more sensitive than commercially available immunoassay kits for E2. CONCLUSION: The proposed CLIA is sensitive enough to measure serum E2 levels in postmenopausal women.

Highly specific polyclonal antisera against estriol: cross-reactivity restriction following affinity chromatography.

An immunosorbent technique was developed to attenuate cross-reactivity of a polyclonal antiserum against a 4(2) (rho-carboxyphenylazo)-1,3,5[10]-estratrien-3,16 alpha,17 beta-triol-bovine serum albumin conjugate. The chromatographic separation of antiserum through stationary phases having either rho(carboxymethyl)phenylazo-phenol or rho(carboxymethyl)-phenylazo-2-naphthol side residues reduced the antiserum avidity, while increasing the apparent antiserum affinity and decreasing the residual cross-reactivities against heterologous ligands. The highly specific antiserum obtained allowed the development of a competitive binding assay over an extended analytical range, which opens up the possibility of direct measurement of estriol from the early pregnancy to delivery. The significance of the attenuation of antiserum cross-reactions after affinity chromatography is discussed with reference to epitope-paratope interaction in the case of small endogenous molecules like estrogens.