The antigenic epitopes of human grown hormone as mapped by monoclonal antibodies.

The antigenic epitopes of human GH (hGH) have been investigated using monoclonal antibodies (Mabs) to hGH. A panel of 12 Mabs was incorporated into two-site binding assays in order to investigate the antigenic surface of hGH. Nine reaction patterns were observed. The Mabs were further characterized in terms of their cross-reactivity with human placental lactogen, human PRL, with recombinant hGH molecules (MetLeu8hGH, Met14hGH), and with fragments derived from enzymatic or chemical digestion of hGH. These studies provided information on the antigenic sites that are shared with human placental lactogen and on the N-terminal and C-terminal regions of the hGH molecule. Based upon these findings, we tentatively suggest the epitope model for hGH comprising at least 10 antigenic sites (epitopes) which may be grouped into five antigenic regions.

Chemiluminescence immunoassay of plasma progesterone, with progesterone-acridinium ester used as the labeled antigen.

This simple solid-phase chemiluminescence immunoassay for measurement of progesterone in extracts of venous plasma has sensitivity and precision similar to that of conventional radioimmunoassay with use of a tritiated antigen. The labeled antigen, 11 alpha-progesteryl-2-succinoyltyramine-4-(10-methyl)-acridini um-9-carboxylate, and a monoclonal antibody to progesterone-11 alpha-succinyl-bovine serum albumin are incubated with a 100-microL aliquot of plasma extract (equivalent to 20 microL of plasma) and 50 microL of a suspension of an IgG fraction of a donkey antiserum to mouse immunoglobulins, covalently attached to cellulose particles. After the antibody-binding reaction (60 min at 4 degrees C), 1 mL of phosphate buffer is added to each tube, the tubes are centrifuged (5 min, 1500 X g), and the supernatant fluid is aspirated. The washing step is repeated and diluted hydrochloric acid (50 mmol/L, 50 microL) is added to the pellet. Luminescence is initiated by oxidation with dilute sodium hydroxide/hydrogen peroxide. The signal is integrated over 10 s. The light yield is inversely proportional to the progesterone concentration in the standard or sample.

The measurement of urinary LH, by a solid-phase chemiluminescence immunoassay.

Ovulation in women may be predicted or detected by a simple, solid-phase, chemiluminescence immunoassay for the measurement of urinary LH. An IgG fraction of a donkey antiserum to rabbit immunoglobulins is passively adsorbed to the walls of polystyrene tubes. Human chorionic gonadotrophin-succinyl-butyl-ethyl-isoluminol and a primary antibody (rabbit anti-human LH) is incubated with samples of early morning urine. After the binding reaction, the solution is removed by aspiration. The antibody-bound fraction is washed twice with buffer. Sodium hydroxide is added and the mixture incubated. After cooling, luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated. The method has been evaluated in terms of sensitivity, within- and between-batch precision, bias and parallelism. The time interval between the peak day of LH in EMU and maximum follicular diameter during 38 menstrual cycles was -24 h (11%), O h (50%), + 24 h (28%) and + 48 h (11%).

Measurement of choriogonadotropin by chemiluminescence immunoassay and immunochemiluminometric assay: 1. Use of isoluminol derivatives.

We have developed immunoassays, monitored by the detection of chemiluminescence, for measuring choriogonadotropin in human urine. These methods involve the use of derivatives of isoluminol and include: (a) a labeled antigen with a second antibody covalently linked to polyacrylamide beads as the solid-phase reagent (i.e., solid-phase chemiluminescence immunoassay); and (b) an excess concentration of a specific antibody passively adsorbed onto the walls of polystyrene tubes and a labeled antibody of different specificity (i.e., two-site immunochemiluminometric assay). After the respective binding reactions, the solutions are aspirated, the antigen- or antibody-bound fractions are washed twice with 500 microL of buffer, sodium hydroxide (2 mol/L; 200 microL) is added, and the mixture is incubated for 60 min at 60 degrees C. After cooling, the label is oxidized with microperoxidase/hydrogen peroxide and the resulting chemiluminescence signal is measured for 10 s. We have evaluated the methods in terms of their sensitivity, precision, and clinical utility, and we compare results with values obtained by radioimmunoassay.

Development of a simple, low-cost, continuous-flow system for the automated radioimmunoassay of steroid glucuronides.

A novel liquid-phase immunoassay is described which involves: competitive binding of the analyte and labelled antigen to specific antibodies; enzymic transformation of the free ligand to a more hydrophobic derivative; and separation of the antibody-bound and free ligand by partition of the reactants into an organic and aqueous phase. The principles of the method, described as ligand differentiation immunoassay, have been used in conjunction with a tritiated antigen to establish a continuous-flow system for the measurement of oestriol-16 alpha-glucuronide in urine. Conventional autoanalyser (AAII) modules have been used, and the pump tubes and glass connections are standard accessories. The equipment is relatively inexpensive (pounds 1500 + detector) and the reagents are readily available. Each sample takes 55 minutes to be processed, and 30 results are obtained per hour.

A novel pseudohomogeneous radioimmunoassay for the measurement of plasma testosterone.

A description is given of a novel liquid phase immunoassay for the measurement of testosterone in peripheral venous plasma from men and women. The procedure involves: (i) competitive binding of the analyte and tritiated antigen to specific antibodies; (ii) enzymatic conjugation of the free ligand with glucuronic acid; and (iii) separation of the antibody-bound and free ligand by partition of the reactants into an organic and aqueous phase. The technique has been called ligand differentiation immunoassay (LIDIA). The mean sensitivity was 5 pg/tube (equivalent to 0.35 nmol/l female plasma; 0.87 nmol/l male plasma). The mean reagent blank (+/- SD) was 0.24 (0.12) nmol/l female plasma; 0.61 (0.30) nmol/l male plasma. A precision profile gave values less than 50%; the within batch variation was less than 8.3% and the between batch variation over three months was 12.3%. An accuracy profile between the second and penultimate points on the calibration curve gave values between 77 and 103%. The correlation coefficient 'r' between LIDIA and a heterogeneous radioimmunoassay was 0.92 when applied to plasma from men and 0.95 to samples from women.

Multiple immunoassay: the simultaneous measurement of two urinary steroid glucuronides as an index of ovarian function.

Ovarian function in women may be monitored effectively by a simple, solid-phase, multiple immunoassay for the simultaneous measurement of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide in diluted urine. IgG fractions of the respective antisera are passively absorbed to the walls of polypropylene tubes. The labelled antigens are estrone-3-glucuronyl-5-aminoethyl-ethyl-isoluminol and [6,9-3H]-pregnanediol-3 alpha-glucuronide. Daily samples of early morning urine are diluted in buffer (1:200; v/v), and 200 microliters removed, in duplicate, for assay. After the binding reaction (18 h at 4 degrees C), the solution is removed by aspiration. The antibody-bound fraction is washed twice with buffer (300 microliters) is added and the mixture incubated for 50 min at 22 degrees C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 s. Subsequently, liquid scintillation fluid (4 ml) is added to the tube and the radioactivity measured. The unknown values are determined from appropriate calibration curves. The combined method has similar sensitivity, accuracy, precision and clinical utility to the values obtained from the separate measurement of the two analytes.

Measurement of serum thyroxine by solid-phase chemiluminescence immunoassay.

Descriptions are given of two solid-phase chemiluminescence immunoassays for the measurement of total thyroxine in serum. The antibodies were either attached to small uniform plastic microspheres (method 1) or passively adsorbed to antibody-coated tubes (method 2). The labelled antigen was thyroxine-aminobutyl ethyl isoluminol. After the antibody-binding reaction the antibody-bound fraction was washed, sodium hydroxide was added, and the mixture was incubated. Luminescence was initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 seconds. The light yield is inversely proportional to the concentration of thyroxine in the standard or sample. Both methods have similar sensitivity and precision to that obtained by a conventional radioimmunoassay.

Measurement of plasma estradiol-17 beta by solid-phase chemiluminescence immunoassay.

We describe a simple, solid-phase chemiluminescence immunoassay for the measurement of estradiol-17 beta in extracts of peripheral venous plasma. The method has similar sensitivity, specificity, precision, and accuracy to a conventional radioimmunoassay with a tritiated antigen. An immunoglobulin G fraction od monoclonal antibodies to estradiol-6-carboyxmethyl oxime-bovine serum albumin is passively adsorbed onto the walls of polypropylene tubes. The labeled antigen is estradiol-6-carboxymethyl oxime-aminobutylethyl isoluminol. After the binding reaction (1 h at 22 degrees C), the solution is removed by aspiration (400 microliters). Sodium hydroxide (5 mol/L, 300 microliters) is added and the mixture incubated for 30 min at 37 degrees C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 s. The light yield is inversely proportional to the concentration of estradiol in the standard or sample.

Hysterectomy, hormones, and behaviour. A prospective study.

60 premenopausal women were assessed before and after hysterectomy for menorrhagia or fibroids or both. Their mood, sexual functioning, and plasma oestrogens and gonadotrophins were regularly assessed for a period of up to 3 years. Patients were randomly assigned to receive either oestrone sulphate or placebo tablets during the trial. No evidence was found that this group of patients showed depression or sexual difficulties related to the hysterectomy. In comparison with their baseline gynaecological condition, they showed improved mood and vigour and unimpaired sexual activity.