Aging, cognitive resources, and declarative learning.

A battery of cognitive tasks designed to assess information-processing speed, working memory capability, and declarative learning was administered to a cross-sectional sample of 477 adults ranging in age from 17 to 86 years. Results showed significant age-related decrements in all three constructs. A variety of structural equation models was fit to the results. The preferred model on empirical and conceptual grounds was one that showed (a) working memory capability as the most important mediator of age effects in declarative learning; (b) working memory capability as the mediator for the effects of general processing speed on declarative learning; and (c) differentiation among verbal, numeric, and spatial processing speed and between verbal and spatial working memory capability.

Assessment of the potency of different standards in the immunoradiometric assay of ACTH.

There is no generally accepted human international reference preparation for ACTH. The different centres offering clinical ACTH measurement therefore select assay standards from the limited range of human-purified and synthetic preparations currently available. We have examined the relative potencies of three synthetic ACTH standards in comparison with the NIBSC human-purified 1-39 (code 74/555) in a two-site immunoradiometric assay (IRMA) for ACTH. All of the standards produced parallel curves in the IRMA but there was a wide variation in potency between the preparations giving highly significant differences in quality control and patients' ACTH results. Standards supplied by the National Hormone and Pituitary Programme, Bachem UK Ltd and Universal Biologicals Ltd gave lower ACTH results than results calculated using the NIBSC preparation. The potency differences between these standards emphasise the need for a reference preparation and, in the meantime, the necessity to define normal reference ranges for each laboratory.

Clinical evaluation of a two-site immunoradiometric assay for adrenocorticotrophin in unextracted human plasma using monoclonal antibodies.

We have developed a sensitive two-site immunoradiometric assay (IRMA) for intact ACTH and its precursors, pro-opiomelanocortin and 22 kDa peptide in unextracted human plasma. The assay uses two monoclonal antibodies. Antibody 1A12, specific for ACTH 10-18, is radiolabelled and antibody 2A3 specific for the C-terminal region (ACTH 24-39), is coupled to Sephacryl S300 for the solid-phase. Samples are incubated for 18 h with labelled antibody followed by 2 h with solid-phase antibody. Separation employs the sucrose layering technique. Using human pituitary ACTH 1-39 (code 74/555) in diluent containing 10% horse serum to standardize the assay, the sensitivity (upper 99% confidence limit of zero standard) is 3.5 +/- 0.8 ng/l (n - 7). The mean coefficient of variation is 5.9% within-assay and 6.7% between-assay and is less than 10% between 22 and greater than 5000 ng/l. Mean recovery of ACTH 1-39 added to dexamethasone-suppressed human plasma is 109% and endogenous ACTH behaves indistinguishably from standard ACTH on dilution. In normal subjects, mean plasma ACTH levels are 30 ng/l at 0730 h, and 15 ng/l at 1630 h at rest. ACTH concentrations are between 60 and 330 ng/l, 8-10.5 h after metyrapone (2 g orally at 2300 h), between 140 and 320 ng/l, 30-60 min after insulin-induced hypoglycaemia, and less than 4 ng/l, 8 h after dexamethasone (1.5 mg orally at 2300 h). In a range of pathological conditions ACTH concentrations accurately reflect the disorders of the pituitary-adrenal axis. Endogenous ACTH immunoactivity is stable in vitro at 22 degrees C for at least 1 h in whole blood and at least 4 h in plasma. It is concluded that this two-site IRMA for ACTH in unextracted plasma offers a reliable assay for clinical purposes.

Selection and optimisation of monoclonal antibodies for a two-site immunoradiometric assay for ACTH.

Four monoclonal antibodies with predominant specificities towards different sequences within the ACTH molecule were investigated in a 2-site immunoradiometric assay (IRMA) for human ACTH. Antibody 3H9 recognises the extreme N-terminal sequence, antibodies 1A12 and 1D1 are specific for the mid N-terminal sequence but differ in that the former cross-reacts with alpha MSH whereas the latter does not, and antibody 2A3 recognises the C-terminal sequence. Combinations of iodinated antibodies with antibodies covalently linked to Sephacryl S300 were tested for their compatibility and potential for a sensitive assay. Two antibody combinations (1D1 plus 3H9 or 1A12) gave no dose-response curve indicating severe steric inhibition, whereas other combinations yielded assays with widely different detection limits (2-2400 ng ACTH/l). The combination of labelled 1D1 and solid-phase 2A3 gave the most sensitive assay and when optimised for antibody concentrations and incubation times the working range was 10-5 X 10(4) ng/l (CV less than 20%). The optimised sequential 2-step IRMA involves incubation of standard or test sample with labelled 1D1 for 18 h at 4 degrees C followed by incubation with solid-phase 2A3 for 2 h at room temperature, after which the labelled complex is separated by the sucrose layering technique. The detection limit of this IRMA was several 100-fold lower than by RIA using the same antibodies. The IRMA detected large molecular weight precursors containing the full ACTH sequence (22 000, 31 000 and 34 000) but not ACTH fragments (1-18, 1-24, 18-39). It is concluded that selected monoclonal antibodies provide a sensitive and rapid 2-site IRMA for intact ACTH and its precursors.