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.

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.

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.

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.

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.

Evaluation of azoestriol antisera for estriol measurements in pregnancy plasma directly and after extraction.

2-(4'-carboxyphenylazo)-estriol antisera were employed to quantitate pregnancy plasma estriol in ether extracts by single phase radioimmune assay without chromatography. Utilizing antiserum which crossreacted minimally even with the monoglucosiduronate metabolites, unextracted plasma estriol measurements were identical statistically to ether extract determinations.

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.

2- and 4-iodinated estriol as indicator ligands for estriol radioimmunoassays with anti-estriol-C6 conjugate antiserum.

The suitability of 2- and 4-125I-estriol as indicator ligands for estriol determination in radioimmunoassays with anti-estriol-C6 conjugate antiserum was tested and compared to the one of 3H-estriol, estriol-6-(O-carboxymethyl)oxime-125I-histamine and a commercially available 125I-estriol derivative of unknown structure. An ideal radioiodinated tracer would react identically with the analyte and its tritiated analogue and the 4-monoiodo-estriol was found to fulfil these requirements as shown by the pattern of dilution and standard curves obtained with the various labeled ligands. This observation was corroborated by a comparison of the apparent estriol concentrations in human pregnancy sera determined with the different indicator ligands. The experimentally proven advantage of 4-monoiodo-estriol over other iodinated estriol derivatives verifies a hypothesis deduced previously from binding constants obtained with analogous estriol derivatives and the same antiserum.

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.

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.

Progesterone as an immunologic blocking factor in human pregnancy serum.

The influence of estriol and progesterone on lymphocyte reactivity was examined by testing the cytotoxic effect on human embryonic fibroblast cells of non-pregnant women's lymphocytes incubated with different concentrations of estriol and progesterone and with complete and progesterone-depleted third trimester pregnancy sera. Progesterone, but not estriol, had a significant inhibitory effect on lymphocyte cytotoxicity at concentrations comparable to those present in pregnancy serum. 95% depletion of progesterone from pregnancy sera caused an 80% loss of inhibitory activity on lymphocyte cytotoxicity. These data suggest that the blood level of progesterone in pregnancy is sufficient to depress lymphocyte reactivity and that progesterone is responsible for the greater part of the serum inhibitory activity in at least the later stages of pregnancy.

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.

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.

Homogeneous enzyme immunoassay of estriol in picogram amounts.

A homogeneous enzyme immunoassay for estriol has been described using estriol-glucose-6-phosphate dehydrogenase (E3G6PD) as enzyme conjugate. Antisera for estriol were raised by immunising rabbits with two different immunogens, estriol-6-(O-carboxymethyl oxime) bovine serum albumin (E36CMOBSA) and estriol-3-carboxymethyl ether bovine serum albumin (E33CMEBSA). A new method for preparation of estriol 3-O-carboxymethyl ether in high yield (90%) has also been described. Addition of anti-estriol antibodies to E3-G6PD conjugate resulted in inhibition of enzyme activity. In the presence of free estriol, the antibody induced inhibition of enzyme activity was reduced in a concentration dependent manner. The use of the heterologous combination between immunogen and enzyme conjugate, i.e. using anti-E33CMEBSA and E36CMOG6PD improves the sensitivity at the expense of specificity, the cross-reaction with other estrogenic hormone being 10-15%.

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.

Synthesis of new steroid haptens for radioimmunossay. Part IV. 3-O-Carboxymethyl ether derivatives of estrogens. Specific antisera for radioimmunossay of estrone, estradiol-17beta, and estriol.

The syntheses of 3-O-carboxymethyl ether derivatives of estrone, estradiol-17beta, and estriol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of estrone, estradiol-17beta, and estriol. The previous concept that specific antisera for estrogens cannot be obtained by employing estrogens derivatized at the 3-position is unfounded.