The concentration of atrial natriuretic peptides (ANP) in body tissues and fluids is generally measured by RIA. However, there is no agreement on the best procedure for the measurement of ANP. In fact, the assay of plasma ANP with RIA techniques still presents some difficult or unsolved methodological problems. We tested the analytical performance of some RIA methods or commercial RIA kits for the assay of ANP, each using different antisera and tracers. Five laboratories with many years' experience in the ANP assay by RIA took part in the study. The analytical performance of four commercial RIA kits for ANP assay (supplied by Peninsula Laboratories INC, Belmont, CA, USA, INCSTAR Corporation, Stillwater, MI, USA, and Amersham International plc, Buckinghamshire, UK) was evaluated employing a special set of lyophilized reference materials and plasma samples from normal subjects and patients with cardiac failure. None of the RIA kits tested showed sensitivity and precision at an acceptable level (CV < 15%) for the measurement of plasma ANP concentrations in normal subjects. Precision and sensitivity were improved by using "fresh" (or purified) preparations of tracer, and by adding PEG to the separation step. As far as the accuracy is concerned, the comparisons between the results obtained with the RIA techniques indicated that the antisera employed show different specificities. The direct assay (without preliminary purification) overestimated plasma levels owing to interference by blood constituents and is therefore not recommended. Four different procedures for purification of plasma samples which use extraction with Sep-Pack C18 cartridges, and an alternative procedure by immunoextraction, were evaluated. The extraction step reduced precision and increased the complexity of the assay. Moreover, the quantitative recovery of ANP added to plasma samples after extraction with Sep-Pak cartridges was about 50-70%, whereas the recovery by immunoextraction was 70-90%.
Evaluation of the Analytical Performance of Ria Methods For Measurement of Atrial Natriuretic Peptides - A Multicenter Study
OPOCHER, GIUSEPPE;MANTERO, FRANCO;
1991
Abstract
The concentration of atrial natriuretic peptides (ANP) in body tissues and fluids is generally measured by RIA. However, there is no agreement on the best procedure for the measurement of ANP. In fact, the assay of plasma ANP with RIA techniques still presents some difficult or unsolved methodological problems. We tested the analytical performance of some RIA methods or commercial RIA kits for the assay of ANP, each using different antisera and tracers. Five laboratories with many years' experience in the ANP assay by RIA took part in the study. The analytical performance of four commercial RIA kits for ANP assay (supplied by Peninsula Laboratories INC, Belmont, CA, USA, INCSTAR Corporation, Stillwater, MI, USA, and Amersham International plc, Buckinghamshire, UK) was evaluated employing a special set of lyophilized reference materials and plasma samples from normal subjects and patients with cardiac failure. None of the RIA kits tested showed sensitivity and precision at an acceptable level (CV < 15%) for the measurement of plasma ANP concentrations in normal subjects. Precision and sensitivity were improved by using "fresh" (or purified) preparations of tracer, and by adding PEG to the separation step. As far as the accuracy is concerned, the comparisons between the results obtained with the RIA techniques indicated that the antisera employed show different specificities. The direct assay (without preliminary purification) overestimated plasma levels owing to interference by blood constituents and is therefore not recommended. Four different procedures for purification of plasma samples which use extraction with Sep-Pack C18 cartridges, and an alternative procedure by immunoextraction, were evaluated. The extraction step reduced precision and increased the complexity of the assay. Moreover, the quantitative recovery of ANP added to plasma samples after extraction with Sep-Pak cartridges was about 50-70%, whereas the recovery by immunoextraction was 70-90%.Pubblicazioni consigliate
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