Radioimmunoassay

In a placebo-controlled, double-blind study we evaluated the safety and kinetics of a new narcotic antagonist, nalmefene, after 2, 6, 12, and 24 mg intravenous doses to healthy men. At each dose level four subjects received active drug and two received placebo. The drug was well ...

The prothoracicotropic hormone (PTTH) of Drosophila melanogaster is a modulator of ecdysteroid (molting hormone) synthesis and was isolated and characterized from extracts of whole larvae (approximately 4 x 10(5) larvae). The purification protocol included delipidation, salt-extraction, heat treatment, conventional column chromatography, and HPLC, and yielded about 50 microg of pure hormone. Biological activity was followed using a ring gland in vitro assay in which ecdysteroidogenesis by control ring glands as measured by radioimmunoassay was compared with ring gland incubations containing active fractions. The molecular weight of the purified PTTH was 45 kDa and N-terminal amino acid sequence analysis indicated that those analyzed sequences displayed no significant homology with known peptides or peptide hormones, including PTTH from the silkmoth, Bombyx mori. Western blot analysis indicated that the native form of Drosophila PTTH was a single 66-kDa polypeptide with N-linked carbohydrate chains and intrachain disulfide bonds. The purified 45-kDa peptide is the deglycosylated form, a result of glycosidase activity present during preparation of the PTTH extract. The deglycosylated form shows heterogeneity, presumably as a result of varying degrees of deglycosylation at the N terminus.

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z′ value of 0.82±0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous...

The cell wall degrading enzyme polygalacturonase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in “Never ripe” mutants and the lack of activity in “ripening inhibitor” mutants can be correlated to the levels of immunologically detectable polygalacturonase protein.

An antiserum against α-amino-(4-hydroxy-6-benzothiazolyl)propionic acid (AHBP), a major product obtained after hydriodic acid hydrolysis of pheomelanin, was raised in rabbits immunized with AHBP coupled to bovine serum albumin (BSA). The antiserum was used to develop a radioimmunoassay (RIA) to AHBP. The limit of detection of the RIA is 0.1 ng of AHBP. The antiserum does not crossreact with other major hydriodic acid degradation products, and the assay has been used to estimate the amounts of AHBP in synthetic and natural melanins.