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.

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing.

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.

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.

Multiple forms of aromatase and response of breast cancer aromatase to antiplacental aromatase II antibodies.

Two distinct aromatase-active protein complexes are solubilized by use of deoxycholate and separated by diethylamino-ethyl-cellulose chromatography from lyophilized powder of 900 X g precipitate fraction of human term placenta. Aromatase activity to produce estriol, the major estrogen of human pregnancy, was designated to be aromatase I activity and measured by estriol formation from 16 alpha-hydroxytestosterone. Aromatases II activity was the designation for that which produces estrone plus estradiol and was measured by androstenedione aromatization. Aromatases II and I are eluted with 0.25 M and 0.5 M Tris buffer, respectively, from diethylaminoethyl-cellulose column in an Mr 2 million soluble complex. Each has a minimum active Mr 135,000 subunit, which is isolated by Bio-Gel filtration in the presence of detergents, and consists of a reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (Mr 83,000) and a cytochrome P-450 (Mr 52,000). Aromatase II was found to be the major aromatase, containing approximately five times more aromatase activity, reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity, cytochrome P-450, and protein than did aromatase I. Antibodies raised in rabbits against aromatase II and its reductase suppressed aromatase II activity of breast cancer tissues, as well as of adult male lung tissue, placental microsomes, and solubilized aromatase. The breast carcinoma specimens responded to the antibodies in different degrees, but there was no response to antibodies against rat liver cytochrome P-450. The results indicate similar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450.

Estrogens in fetal and maternal plasma of the rhesus monkey,.

The quantities of estrone and estradiol were determined by radioimmunoassay in maternal and fetal plasma of the rhesus monkey from day 59 to 163 of gestation. A two way analysis of variance of data classified according to fetal sex and 3 pooled gestational ages for each hormone and for mother or fetus (4 analyses) revealed significant elevations in fetal estradiol and maternal estrone concentrations with age. All other comparisons were not significant by these analyses. The concentrations of estradiol were greater in maternal than in fetal plasma [769 +/- 64 (SE) pg/ml, N = 63 VS 57 +/- 6 (SE) pg/ml, N = 77, P less than 0.01] by a t test. Estrone, on the other hand, was similar in mother and fetus [265 +/- 30 (SE) pg/ml, N = 60 vs 318 +/- 37 (SE) pg/ml, N = 73, P greater than 0.05]. No sex differences in the concentrations of these hormones were observed except in the fetus after 150 days of gestation. At this time plasma from female fetuses contained significantly more estradiol than plasma from male fetuses [118 +/- 20 (SE) pg/ml, N = 7 vs 61 +/- 10 (SE) pg/ml, N = 19, P less than 0.01]. Except for estradiol in female fetuses, the concentrations of estrogen were significantly higher in the umbilical vein than in the umbilical artery, an indication that the placenta is a major source of fetal estrogen in this species. Estrone and estradiol were significantly correlated in both the fetal and maternal circulation, r = 0.58, P less than 0.001 and r = 0.39, P less than 0.01 respectively. The results provide quantitative data about the estrogen miliue in which the monkey fetus develops and suggest mechanisms for controlling fetal estrogen in this species.

A specific radioimmunoassay for estrone sulfate in plasma and urine without hydrolysis.

Estrone sulfate, quantitatively the most important estrogen in plasma, has previously been determined only after hydrolysis and chromatography. An antiserum raised against estrone glucosiduronate-bovine thyroglobulin was found to be suitable for the specific RIA of estrone sulfate both in plasma and urine. Plasma levels were measured after solvent extraction without hydrolysis or chromatography. The mean (+/-SE) was 972 +/- 79 pg/ml (range, 537-1581) in 15 women in the follicular phase, 1806 +/- 160 pg/ml (range, 814-3358) in 15 women in the luteal phase, and 922 +/- 62 pg/ml (range, 461-1238) in 13 men. The urinary excretion of estrone sulfate, measured after simple chromatographic separation, ranged from 0.8-7.9 micrograms/24 h in men and 5.1-18.7 micrograms/24h in nonpregnant women. This was generally one-seventh to one-half the simultaneous estrone glucosiduronate excretion rate. An approximate mean renal clearance of estrone sulfate calculated from the above values was 2.7 ml/min. The low clearance rate is taken to reflect extensive binding of estrone sulfate by plasma proteins. The splanchnic extraction of estrone sulfate measured in 6 patients undergoing hepatic vein catheterization for diagnostic purposes was 29.8 +/- 11.1%, indicating net uptake of this compound by the splanchnic area.

Improved production of oestradiol antibodies of high affinity and specificity by hormonal manipulation of the immunised animals and affinity chromatography of the antisera.

The titre, affinity and specificity of oestradiol antisera were increased by hormonal manipulation of immunised rats. Castration was as effective as daily injection of the aromatase inhibitor, 1,4,6-androstene-3,17-dione. Chromatography on the affinity matrix oestrone-3-CME/Sepharose-4B was less successful.

Factors influencing the measurement of oestrone sulphate by dipstick particle capture immunoassay.

Factors influencing the performance of a dipstick competitive particle capture immunoassay (PCI) for the steroid oestrone sulphate (OS) were investigated. Appropriate 'blocking' of the nitrocellulose dipstick membrane was necessary for the upward flow of microsphere particles. Traditional protein blocking agents including BSA, gelatin and casein were unsatisfactory while synthetic polymers and surfactants were effective in promoting microsphere movement. A simple buffer consisting of 1% aqueous NaCl containing 0.05% Tween 20 was suitable for carrying the components up the dipstick and facilitating the antibody-antigen interactions. Increasing microsphere diameters from 0.3 to 0.8 microm allowed the microsphere antibody coating concentration to be reduced which enabled lower concentrations of OS to be measured. However, upward flow rate and the maximum signal attainable was compromised as a consequence. Enlarging the dipstick membrane nominal pore size from 3 to 12 microm increased the speed of test dot development, but assay sensitivity suffered as a result in some instances. Changing the capture antigen markedly influenced the dose-response lines. No dose-response was achieved with OG-BSA as the capture antigen while OHS-BSA and OCMO-BSA as capture antigens produced dose-response lines with means +/- S.E.M. EC(50) values of 140 +/- 16 and 19 +/- 1 ng/ml, respectively.

Relevance of oestrone presentation to the specificity of the elicited antisera, as revealed by affinity separation.

Polyclonal antisera raised against two different azobenzoyl-oestrone derivatives were analysed to investigate both the latency/intensity relationship of the immune response and the influence of antigen presentation on the specificity of the antisera elicited. Elongation of the azo-bridge of the hapten ([p(carboxyphenyl)-azo]-1,3,5[10]- oestratrien-3 ol-17 one) with a short aliphatic chain (4-amino-n-butyric acid) resulted in a marginal increase in the antibody yield, without affecting the time required to attain the maximum titre. The increased flexibility and mobility of the extended azo-bridge was shown to result in the appearance of antisera which cross-reacted with oestrogens with D ring structures different to that of oestrone. Antiserum fractionation by affinity chromatography through a stationary phase exposing the carrier protein determinants, as modified by the addition of the coupling bridge and the phenol ring, resulted in a reduction in its specificity. These findings are discussed with regard to the phenomena underlying the specificity of a polyclonal antiserum.

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.

An electro-active system of immuno-assay (EASI assay) utilising self assembled monolayer modified electrodes.

Most immunoassays currently rely on optical methods for signal generation e.g. in ELISA and rapid assay formats. It has become apparent as in the Glucose sensor market that there is a need for simple direct electrical immuno-sensors. We have investigated the novel use of organic conducting monolayers used as a direct electrochemical detection support for an immuno-reaction. It was found that antibodies raised to a carbazole dimer monolayer could increase the charge movement across that monolayer surface. Antibody fragments were taken from a specific anti-carbazole antibody fragment library and combined with an antibody fragment directed to the hormone estrone 3 glucuronide (E3G), the target antigen to form a bispecific antibody fragment. The device utilised these specific antibody fragments and incorporated them on the top plate of a capillary fill format as the immuno-assay components. The immuno-reaction utilised a competition assay. Free E3G analyte in the sample displaced the bispecific antibody fragment from the immuno-surface leaving it free to bind the carbazole monolayer surface. There the binding was detected using amperometric or coulometric methods. By combining all there element it was possible to develop a sensitive immuno-assay that could detect E3G in a reproducible calibrated fashion down to 10 ng/ml.

Evaluation of antibody- and antigen-coated enzymeimmunoassays for measuring oestrone-3-glucuronide concentrations in urine.

A monoclonal antibody to oestrone-3-glucuronide (OG) was generated and incorporated into antigen- and antibody-coated competitive enzymeimmunoassays (EIAs) using OG-, 6-ketoestrone-6-O-carboxymethyl-oxime (OCMO) and oestrone-3-hemisuccinate (OHS) as the steroid coating antigens or 'tracers' in each format respectively. In the coated-antigen format, standard curves with the lowest mean values (pg/well) for sensitivity (1.1 +/- 0.1), mid-point (ED50; 8.2 +/- 0.7) and high-point (ED20; 31 +/- 2) were obtained using OCMO coupled to bovine serum albumin (BSA) as the coating antigen, and horseradish peroxidase (HRP) as the enzyme label coupled directly to the monoclonal antibody. Standard curves generated using enzyme-labelled OG, OCMO and OHS as 'tracers' in the antibody-coated EIA format were all similar, but had higher mean sensitivity, ED50 and ED20 values than those obtained in the optimal coated-antigen format. In both EIA formats alkaline phosphatase (AP) was found to be inferior to HRP as an enzyme label. Measurement of OG concentrations in early morning urine samples from women with natural, regular menstrual cycles, using the antigen-coated EIA, demonstrated the characteristic elevation in OG concentrations associated with the onset of the urinary LH surge. This technically straightforward and robust antigen-coated EIA may be of interest to laboratories with a requirement to measure OG concentrations in urine, or other biological samples.

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.

Antisera reactive directly to estrone sulfate.

Derivatives of estrone were prepared and linked to bovine serum albumin or its methyl-esterified form to produce immunogens which were effective in raising antisera to estrone sulfate. The most effective was estrone-3-methylphosphonothioate, electrostatically complexed with methylated bovine serum albumin. The ionically combined hapten functioned as an antigenic determinant as do covalently bound haptens when administered to sheep in emulsions with Freund's complete adjuvant. Estrone-3-phosphate covalently or electrostatically linked to bovine serum albumin also produced antisera reactive to estrone sulfate. Estrone sulfate itself, after electrostatically complexing to methylated bovine serum albumin and administration with Freund's complete adjuvant to sheep, was ineffective in producing antisera. The sera which had workable titres to estrone sulfate showed considerable cross-reaction with free estrone but was otherwise highly specific with little or no reaction with other steroid sulfates, glucosiduronates or other free steroids. Radioimmunoassay curves using [6,7-3H]-estrone sulfate were highly sensitive and were effective in the range of 5-250 pg estrone sulfate.

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.

Specificity studies of monoclonal and rabbit antibodies raised against steroid glucuronides.

Monoclonal and rabbit antibodies raised against estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide have been studied with respect to their ability to bind free estrone and its conjugates or free pregnanediol and its conjugates, respectively. High titre and high specificity were observed with monoclonal antibodies produced against pregnanediol-3 alpha-glucuronide, whereas the monoclonal antibodies produced against estrone-3-glucuronide were not so specific when compared with the corresponding rabbit antibodies. Both monoclonal and rabbit antibodies had affinity constants in the range of 10(9)--10(10) liter/mole.

Equol: a contributor to enigmatic immunoassay measurements of estrogen.

The efficacy of radioimmunoassay (RIA) for the measurement of estradiol-17 beta (E2) in murine plasma was investigated. When Sephadex LH-20 or celite column chromatography was used to separate E2 from estrone (E1) and other cross-reacting compounds, the results were erratic if small volumes of mouse plasma were resolved. Assay of a diethyl ether extract of plasma (500 microL) was the most practical method for estimating the concentration of estradiol-17 beta in mice. This method was used to determine the pattern of estrogen secretion during the estrous cycle, on the day of implantation and during pregnancy. No convincing change in estrogen secretion was observed in the diestrous/proestrous mouse. By comparison, estrogen levels were elevated during pregnancy. Taken together, these results implied that cross-reactive components in plasma masked low levels of endogenous estrogen. Further evaluation of mouse plasma and urine using a co-chromatography technique to examine estrogen elution from a reverse-phase HPLC system followed by GC/MS analysis indicated the presence of equol [7-hydroxy-3-(4-hydroxyphenyl)chroman], a phytoestrogen metabolite with a ring structure similar to estradiol-17 beta. Equol and possibly other cross-reactive components of plasma may account for the apparent lack of increased estrogen secretion during the mouse estrous cycle and on the day of implantation as determined by the radioimmunoassay of ether extracts of plasma.

Synthesis of new immunogens for estrone and estradiol-17 beta and antisera evaluation.

Syntheses of the 6 alpha-O-carboxymethyl ether derivatives of estrone and estradiol-17 beta and the preparation of their bovine serum albumin conjugates are described. The generation and evaluation of antisera produced from these conjugates is discussed.