Unique effect of the pregnancy hormone estriol on antigen-induced production of specific antibodies in female BALB/c mice

We investigated the modulatory effect of estriol (E(3)), an estrogen predominantly produced during human pregnancy, on the antigen-induced production of specific antibodies in female BALB/c mice, and its effect was compared with 17beta-estradiol (E(2)). Estriol (E(3)) had a very different effect than E(2) on the antigen-induced production of specific antibodies in animals immunized with two different antigens, i.e., the bovine serum albumin (BSA) and pneumococcal polysaccharide serotype-14 (PPS-14). While E(2) strongly stimulated the production of BSA-specific antibodies (mostly IgG1), E(3) had little or no effect on their production. In comparison, when the bacterial PPS-14 was the immunogen, E(3) and E(2) both strongly increased the production of PPS-14-specific antibodies (mostly IgM). E(3) and E(2) also had a similar effect on the thymus weight reduction and on the spontaneous antibody production in these animals. Our results provided an example demonstrating that the pregnancy hormone E(3) has a distinctly different profile of modulatory actions in the immune system compared to E(2), while the former strongly enhanced the body's ability to produce bacteria-specific IgM antibodies, it had no effect on the production of specific antibodies against a soluble protein. This differential effect of E(3) may be beneficial for reducing the risk of developing antibody-mediated immune attack against the maternal and fetal elements during pregnancy.     

Binding characteristics of estrone,estradiol and estriol to the human myometrial estrogen receptor

The human myometrial estrogen receptor in cytosol from pre-menopausal uterine samples has been characterized. At 0° estradiol (K_D 0.38 × 10⁻¹⁰M) has the highest affinity to the receptor followed by estrone (K_D 0.76 × 10⁻¹⁰M) and estriol ((K_D 1.33 × 10⁻¹⁰M). The association rate constant is 2.8 × 10⁵M⁻¹s⁻¹ for estradiol, 2.1 × 10⁵M⁻¹s⁻¹ for estrone and 0.79 × 10⁵M⁻¹s⁻¹ for estriol. The dissociation constants and the association rate constants increase with temperature. The calculated thermodynamic parameters indicate a positive change in entropy for the formation of the estrogen receptor complex.The cytoplasmic estrogen receptor has a sedimentation coefficient of 4 s in low salt sucrose gradients. In buffer containing diisopropylfluorophosphate (DFP) to inhibit proteolytic activity the estrogen receptor complex sediments solely as an 8 s peak if [³H]-estradiol is added to the buffer prior to homogenization and the tissue sample is used immediately after hysterectomy. Estrogen receptor complexes that sediment at 4 s and 8 s are found if [³H]-estradiol is omitted from the homogenization buffer and instead added after the cytosol preparation. Most likely a protease is involved the activity of which is not completely inhibited by DFP.Addition of low concentrations of Cu²⁺ (10 μM) to the cytosol increases the dissociation constant and decreases the estrogen-binding capacity of the receptor. The rate of association is reduced in the presence of 20 μM Cu²⁺. The estrogen receptor complexes do not show any change in their sedimentation profiles in the presence of Cu²⁺.     

Preparation of specific antiserum to estriol 3-sulfate 16-glucuronide

The preparation and antigenic properties of estriol 3-sulfate 16-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Coupling of 6-oxoestriol 3-sulfate 16-glucuronide acetate-methyl ester 6-(O-carboxymethyl)oxime with BSA by the activated ester method followed by removal of the protecting groups with alkali provided the desired conjugate. The antisera raised against the conjugate in rabbits were highly specific to the double conjugate, estriol 3-sulfate 16-glucuronide, discriminating from ring A or D monoconjugated and unconjugated estrogens. The specificity of antisera elicited has been discussed on the basis of stereochemistry of the hapten-[C-6]-BSA conjugate.     

Factors involved in the production of banded structures in mammalian chromosomes

Dozens of reagents were tested for their ability to produce bands in Chinese hamster chromosomes by incubating air-dried preparations in aqueous solutions of these reagents for a definite period prior to the Giemsa staining. Acids were found to be without effect on the band production. Many of salts were able to induce bands if their pH was alkaline. Strong bases were also found to be potent band inducing reagents. They produced bands only in a few seconds. Protein denaturants such as urea, guanidine-HCl and several surface active compounds were also effective in the band production. — In the light of these results, it seems very likely that the solubilization or extraction of some chromosomal proteins, probably of acid nature, would be the primary cause of the appearance of the banded structure in chromosome arms.Contribution No. 885 from the National Institute of Genetics, Japan.     

A method for the production of highly specific polyclonal antibodies

Two polyclonal antibodies directed against paramyosin and tropomyosin from Owenia fusiformis (a marine polychete annelid) were obtained using a new method of immunization. After purification by two-dimensional gel electrophoresis, proteins were transferred onto a nitrocellulose sheet using the Western blot technique. The proteins bound to their cellulose support were injected into rabbits without Freund's adjuvant and without solubilization of nitrocellulose with dimethyl sulfoxide. Highly specific polyclonal antibodies were generated.     

Preparation of specific antiserum to estradiol 3-glucuronide

The preparation and antigenic properties of estradiol 3-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is coupled to the carrier protein through the aminomethyl group at C-6 by glutaraldehyde, have been described. Antibody raised against the immunogen in the rabbit possessed high affinity (K_A= 3.7 × 10⁹M⁻¹) and excellent specificity to estradiol 3-glucuronide, exhibiting no significant cross-reactivities with estrogen glucuronides except for estriol 3-glucuronide (3.64%) and no cross-reactions with free estrogens, their sulfates and other steroids.     

Production of monoclonal antibodies to estriol and their application in the development of a sensitive nonisotopic immunoassay

The development of highly specific monoclonal antibodies to estriol and a nonisotopic immunoassay (EIA) for unconjugated estriol based on the use of these monoclonal antibodies have been described. The monoclonal antibodies show little cross reactivity with other steroids and steroid conjugates and can be used directly in immunoassays without any purification. The EIA described here can be performed in 96-well microtiter plates or polystyrene tubes that have been coated with estriol-bovine serum albumin conjugate. In this assay, estriol in the standard or clinical samples (serum or saliva) competes with the immobilized steroid on the plate or the tube for binding with the antibody. The assay shows good agreement with radioimmunoassay (RIA) and is highly sensitive and reliable. Since no prior processing or extraction of the clinical samples is necessary, the method is potentially applicable for routine use in fetal monitoring as well as in a steroid laboratory.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.