Determination of human granulocyte elastase by the immunoactivation method on the Hitachi 717 automated analyser.

This paper describes a fully mechanized homogeneous immunoassay using the immunoactivation method for the rapid and specific determination of human granulocyte elastase (EC 3.4.21.37) in plasma. The method uses anti-elastase antibody fragments from sheep, conjugated to horseradish peroxidase. These enzyme-antibody conjugates bind to the elastase-alpha 1-proteinase inhibitor complex present in plasma. A separate sample blank with non-specific sheep antibody fragments conjugated to horseradish peroxidase corrects for errors introduced by the sample matrix. Measurements were performed with the clinical chemistry analyser Hitachi 717. A single determination can be performed in 10 min, requiring 24 microliters sample volume. The measuring range is about 20 to 1000 micrograms/l elastase. For within-run precision the coefficients of variation are 4.77%, 4.48% and 1.85% for elastase concentrations of 45.7, 89.1 and 385.4 micrograms/l; for day-to-day precision the coefficients of variation are 15.81%, 7.19% and 4.12% for elastase concentrations of 31.1, 65.5 and 440.2 micrograms/l, respectively. Correlation (y = bx + a) of results with those from the heterogeneous immunoassay showed a good agreement (r = 0.93, b = 1.11, a = -27.0, N = 121). Interferences by endogeneous substances and by drugs at therapeutic doses were not observed. The reference interval, determined by using plasma from 215 healthy individuals (C-reactive protein less than 5 mg/l, leukocyte count 4-8 x 10(9)/l), was 9-56 micrograms/l (2.5th to 97.5th percentile), with a median of 27 micrograms/l.

Preparation of electrophoretic variants of corticosteroid-binding globulin (CBG) using liquid liquid partition chromatography.

Human corticosteroid-binding globulin (CBG) was purified to homogeneity by application of three different chromatographic methods. After fractionation of pregnancy serum with ammonium sulfate the 80%-pellet was used for affinity chromatography based on tresyl activated Sepharose (Pharmacia, Uppsala, Sweden). The affinity eluate was injected into a Mono Q anion exchange column (Pharmacia). Fractions containing CBG were finally purified by liquid liquid chromatography on LiParGel 750 (Merck, Darmstadt, F.R.G.). The purified protein was characterized by IEF and PAGE. This paper describes a method for the chromatographic separation of the two variants of CBG without a loss of binding activity towards steroids for each of the two characteristic bands of this protein.

An enzyme-linked immunosorbent assay (ELISA) for human sex hormone-binding globulin.

An enzyme-linked immunosorbent assay (ELISA) of the "sandwich-type" for sex hormone binding globulin (SHBG) has been developed. A rabbit anti-SHBG antibody (RAb) is immobilized to the microtitre plate. After incubation with standards and samples a second monospecific rabbit anti-SHBG antibody, labelled with alkaline phosphatase is added (RAb). Following further washing substrate is added, colour developed and the plate read at 405 nm wavelength on a standard ELISA plate reader. The assay is not influenced by the presence of steroids at the binding site, and shows good agreement to SHBG binding capacity assay and commercially available IRMA. Its sensitivity, specificity and precision allows its use in the routine laboratory. The SHBG ELISA has been used to measure SHBG concentrations in sera of normal men, women, pregnant women, and women receiving high-dose medroxyprogesterone acetate as a treatment of metastatic breast cancer.

Application of liquid-liquid partition chromatography in the simultaneous purification of sex-hormone-binding globulin and corticosteroid-binding globulin.

Two human serum proteins, corticosteroid-binding globulin (CBG) and sex-hormone-binding globulin (SHBG), were purified to homogeneity by the application of a combination of three different modes of chromatography. Human pregnancy serum was fractionated with ammonium sulphate. SHBG (50% pellet) and CBG (80% pellet) were then purified by affinity chromatography on tresyl-activated Sepharose with 15-aminopentadecanoic acid (for SHBG) and 1,12-diaminododecane (for CBG) as spacers and 17 zeta-aminoethyl-5 alpha-androstan-3 beta,17-diol (for SHBG) and 17 alpha-hydroxy-4-androsten-3-one-17 beta-carboxylic acid (for CBG) as specific ligands for these two proteins. The eluate was injected into a Mono Q anion-exchange column. Fractions containing SHBG or CBG were finally purified by liquid-liquid chromatography on Lipar-Gel 750. This chromatographic sequence clearly separated SHBG and CBG from other proteins, mainly serum albumin, without a loss of protein or binding activity.

(Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)-4-estren-3-one: a new specific gamma-emitting ligand for determination of progesterone receptor.

An iodine-125 labeled ligand for progesterone receptor determination was synthesized: (Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)-4-estren-3-one ([125I]SH-D 510). The ligand is stable chemically as well as under the conditions of a receptor assay. The relative binding affinity of the nonradioactive compound towards human uterine progesterone receptor was 7.0 for the Z-isomer (promegestone (R5020) 1.0) and 0.95 for the E-isomer. 4 S and 8 S receptor forms were obtained on sucrose density gradient analysis. Progesterone receptors were assayed in 103 human mammary tumour cytosols, using either [3H]promegestone or [125I]SH-D 510. The coefficient of correlation was r = 0.951.

Radioimmunoassay of 5-hydroxy-3-indole acetic acid.

A direct radioimmunoassay of the methyl ester of urinary and serum 5-hydroxy-3-indole acetic acid is described. The antiserum, raised in a rabbit against a conjugate of bovine serum albumin with 5-hydroxytryptamine hemisuccinamide, contained two antigenic fractions, one binding N-acyl 5-hydroxytryptamine, and the other binding methyl ester of 5-hydroxy-3-indole acetic acid, and N-acyl 5-hydroxytryptamine. The N-acyl 5-hydroxytryptamine binding fraction was removed by affinity chromatography on a N-acyl 5-hydroxytryptamine agarose gel in the presence of excess methyl ester of 5-hydroxy-3-indole acetic acid. The antibody methyl ester of 5-hydroxy-3-indole acetic acid complexes were dissociated and this affinity-purified antiserum was used in all experiments. Polyethylene glycol in combination with goat anti-rabbit IgG was used to separate bound and unbound 125I-labeled Bolton-Hunter reagent- 5-hydroxytryptamine conjugate. Sample preparation (esterification of 5-hydroxy-3-indole acetic acid to its methyl ester) was performed with trimethylsilyldiazomethane in dioxane. In the analysis of urine, the reagents used in the methylation served as diluents, contributing to the final dilution of 1:1100. In the analysis of serum, a deproteination step (ethanol precipitation) prior to methylation was necessary to obtain reproducible results. The methylated 5-hydroxy-3-indole acetic acid was then extracted with ethyl acetate and the extract redissolved in assay buffer. The minimal detectable concentration of methyl ester of 5-hydroxy-3-indole acetic acid was 1.1 mumol/l (0.21 mg/l 5-hydroxy-3-indole acetic acid) urine or 100 fmol/tube.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of immunocytochemical estrogen receptor assay, estrogen receptor enzyme immunoassay, and radioligand-labeled estrogen receptor assay in human breast cancer and uterine tissue.

Determination of estrogen receptor content in 82 breast cancer specimens with immunocytochemical estrogen receptor assay (ER-EIA) (Abbott) was compared with our routinely used binding assay using 125I-estradiol as radioligand with Scatchard plot analysis of the binding data. Although the estrogen receptor content measured with the ER-EIA was approximately 2-fold higher compared with the binding assay, the immunochemical method proved to be a useful alternative for estrogen receptor determination. Furthermore, it is possible to detect estrogen receptors in FPLC Superose 12 (size exclusion column) eluates or in the fractions obtained after sucrose density centrifugation using the ER-EIA. Forty breast cancer samples were analyzed utilizing the immunocytochemical technique (ER-ICA) for visualization of the estrogen receptor content in frozen tumor tissues in relationship to the quantitative results obtained with the ER-EIA assay. Specific staining for estrogen receptor was confined only to the cell nucleus, was distributed irregularly among the tumor cells, and was variable in intensity. The staining intensity and the percentage of positively stained cells increased with increasing level of cytosolic estrogen receptor. In 27 of 40 cases the immunocytochemical results correlated well with the ER-EIA assay. Nine cases were ER-ICA negative with positive ER-EIA, and four were ER-ICA positive with negative ER-EIA.

[Monoclonal antibodies for the immunohistochemical detection of estrogen receptor status in tissue sections of primary breast cancers]. / Monoklonale Antikörper zum immunhistochemischen Nachweis des Ostrogenrezeptor-Status am Gewebeschnitt primärer Mammakarzinome.

The article reports on first experiences with immunohistochemical staining methods (ER-ICA and ER-D5) using monoclonal antibodies for determination of oestrogen receptor status in tissue slides. 95 primary breast cancers were analysed by ER-ICA; in addition, a subgroup of 31 cases was stained for ER-D5. The results of the two immunohistochemical procedures revealed a large measure of agreement with the standard ligand binding assay. The evaluation of the immunohistochemical staining reaction led to the formation of a staining Score: Score points of ER-ICA, not those of ER-D5, revealed good correlation with oestrogen receptor concentrations in cytosol, determined by standard ligand binding assay. The used staining Score was examined for intra- and inter-individual differences of evaluation and proved to be well reproducible. Further clinical studies have to prove how far immunohistochemical features can convey clinically relevant information not yielded by the steroid binding assay.

An automatic multidimensional chromatography system for purification of human uterine progesterone receptor and induction of polyclonal antibodies.

This paper reports on the synthesis of Org2058-bonded microparticulate silicas and their use in affinity chromatography as the first step for the purification of human progesterone receptor. The development of microprocessor-controlled instruments allows all the various steps to be performed automatically. The various steps used for the purification of human progesterone receptor were carried out with the FPLC system: affinity chromatography, desalting of eluate on Sephadex G-25, anion-exchange chromatography using a Mono Q column. With this procedure the receptor was purified approx. 10,000-fold within 24 h. The yield of receptor was generally 85-95%. Investigations with induced anti-progesterone receptor antibodies obtained after the fourth immunization show their immunoreactive behaviour towards progesterone receptor in crude cytosol, which was proved by sucrose density gradient centrifugation and by gel filtration on the FPLC system using a Sepharose 12 column. This implies that progesterone receptor was efficiently purified by our purification procedure.

A simple, low-cost computer program for Scatchard plot analysis of binding data in steroid hormone receptor assays.

This paper presents a relatively simple computer program for Scatchard plot analysis of binding data in steroid hormone receptor determination. The program is written in BASIC and adapted to a low-cost desktop calculator. The program is designed to permit data input without demanding extensive technical knowledge of either computer systems or mathematical background of receptor binding assays.

3H-labelled RU 38486: characterization of binding sites in human uterine cytosol.

The behaviour of the antifertilizing synthetic steroid RU 38486 towards human uterine progestin receptor was investigated. RU 38486 competed in the same order of magnitude as progesterone for the [3H]R 5020 binding site of progestin receptor, whereas R 5020 was unable to compete against [3H]RU 38486. This apparent contradiction could be explained by means of HPLC-chromatography. HPLC-chromatography with an anion exchange column (MonoQ, Pharmacia, Uppsala, Sweden) showed that [3H]RU 38486 forms at least two stable complexes with uterine cytosol, on one hand with serum albumin, which presents almost 90% of bound radioactivity, and on the other hand with the two native progestin receptor forms, corresponding to 4 S and 8 S receptor forms in sucrose density gradient analysis. Whether reduced binding of salt-activated RU 38486 receptor complexes to DNA-cellulose is due to reduced activation is still uncertain and remains to be further investigated.

Characterization of human spleen tumor glucocorticoid receptors using [3H]cortisol as ligand.

The present report describes an assay system allowing the quantification and characterization of [3H]cortisol binding to glucocorticoid receptors in bloodrich human tissue. The essence of this assay lies in the selective binding of 17 beta-carboxylic acids of natural corticoids to corticosteroid binding globulin (CBG). In the presence of 1240 nmol/l 11 beta-hydroxy-3-oxo-4-androstene-17 beta-carboxylic acid only glucocorticoid receptors were detectable with the expected properties: high affinity for synthetic and natural glucocorticoids, but failure to bind to the respective 17 beta-carboxylic acids, apparent Kd's at 0-4 degrees C for [3H]cortisol of approx 30 nmol/l, Nmax similar to those determined with [3H]dexamethasone and the typical sequence of relative binding affinities (dexamethasone greater than cortisol greater than progesterone greater than 17 beta-methyl-testosterone, estradiol).

High-performance and ion-exchange chromatography and chromatofocusing of the human uterine progesterone receptor: its application to the identification of 21-[3H]dehydro Org 2058-labelled receptor.

Two independent lines of evidence were used to identify the human uterine progesterone receptor. First, three differently tritiated progestogens (Org 2058, R 5020, progesterone) were used for reversible labelling of the receptor. Secondly, the highly potent affinity label 21-[3H]dehydro Org 2058 was used to label covalently the steroid-specific binding site of the receptor. The labelled cytosols were chromatographed on a Mono Q high-performance anion-exchange column in the absence or presence of a high molar excess of the respective unlabelled competitor steroids. In the case of 21-[3H]dehydro Org 2058, Org 2058 was used as the unlabelled competitor. After elution with a NaCl gradient, the radioactivity was determined in each fraction and the elution profiles (absorption, A at 280 nm; radioactivity, dpm) were superimposed. Free steroid was eluted with the washing buffer. When the NaCl gradient was performed, two peaks of radioactivity were located. The specifically protein-bound radioactivity was eluted at 0.08 M NaCl. Two non-specific steroid-binding entities were eluted at 0.1 and 0.22 M NaCl, the second of which was identified as albumin. The elution profiles of tritiated progesterone, R 5020, Org 2058 and the affinity label 21-dehydro Org 2058 were identical. In a second set of experiments, Org 2058- and 21-dehydro Org 2058-labelled cytosols were subjected to high-performance liquid chromatography on a Mono P high-performance chromatofocusing column in the absence or presence of a high molar excess of unlabelled Org 2058. After elution with Polybuffer 74, only one specifically labelled protein (pH 6.4) was detected. When the Mono P-purified receptor was submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis, two labelled polypeptides with Mr = 45,000 and 27,000 were detectable.

17 beta-carboxamide steroids: highly effective inhibitors of the phytohaemagglutinin mediated blastogenesis of normal human peripheral lymphocytes.

Several novel 17 beta-carboxamide analogues of dexamethasone were synthesized. The common precursor, 9-fluoro-16 alpha-methyl-11 beta,17-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxylic acid, did not bind to the glucocorticoid receptors of rat liver and human spleen tumours. In addition, no inhibition of the mitogen-induced blastogenesis of cultured human peripheral lymphocytes was observed. The 17 beta-carboxamide analogues, however, bound with similar affinities to the glucocorticoid receptors of both tissues. They inhibited the mitogen-induced blastogenesis of peripheral lymphocytes, showing the same potency and same order of binding affinity as the natural glucocorticoids.

Synthesis of biotin-labelled dexamethasone derivatives. Novel hormone-affinity probes.

A new, general methodology for 'sandwich' affinity chromatography of steroid hormone receptors is proposed, the part purification of the human spleen tumor glucocorticoid receptor is quoted as an illustration. 9-Fluoro-16 alpha-methyl-11 beta, 17-dihydroxy-1,4-androstadiene-3-one-17 beta-carboxylic acid was coupled to biotin using pentamethylenediamine (BioDex 1) as a spacer. The bifunctional derivative binds to glucocorticoid receptors and avidin-Sepharose and efficiently protects the glucocorticoid receptor against inactivation when previously added during homogenisation. We have standardized the capacity and optimum conditions for elution of receptor-BioDex-1 complexes which are bound to avidin-Sepharose. Receptor purification of several thousand fold can be obtained with good yield.

Synthesis of a new disulfide affinity adsorbent for purification of human uterine progesterone receptor.

For purification of the human uterine progesterone receptor, an affinity adsorbent was synthesized in which the specific ligand (16 alpha-ethyl-3-oxo-19nor-androst-4-ene 17 beta-carboxylic acid) was bound to derivatized celulose using a disulfide-group-containing spacer. The purified receptor protein, isolated by reductive cleavage of the disulfide bond, bound the synthetic gestagen R5020 with high affinity (Kd 12.2 nmol/l). The affinity gel was highly efficient. A 24000-fold purification of progesterone receptor with a recovery of 40% could be achieved in a single step within 6 h. By means of dodecyl sulphate/polyacrylamide gel electrophoresis two main polypeptides with molecular weights of about 43000 and 108000 could be demonstrated.