Wallac 1470 wizard gamma counter

Radiolabelling of A1M with ¹²⁵I (Perkin Elmer Life Sciences) was done using the chloramine T method. Protein-bound iodine was separated from free iodide by gel-chromatography on a Sephadex G-25 column (PD10, GE Healthcare, Stockholm, Sweden). A specific activity of around 0.1–0.2 MBq/μg protein was obtained. Radioimmunoassay (RIA) was performed by mixing goat antiserum against human A1M (“Halvan”; diluted 1:6000) with ¹²⁵I-labelled A1M (appr. 0.05 pg/ml) and unknown patient samples or calibrator A1M-concentrations. After incubating overnight at RT, antibody-bound antigen was precipitated by adding bovine serum and 15% polyethylene glycol, centrifuged at 2500 rpm for 40 minutes, after which the ¹²⁵I-activity of the pellets was measured in a Wallac Wizard 1470 gamma counter (Perkin Elmer Life Sciences).

Plasma Mel was assayed using a total melatonin radioimmunoassay (RIA) kit (RE29301, IBL International, Hamburg, Germany), with preceding extraction procedure according to the method presented by Kulczykowska et al. (2007 (link)) and modified by Guellard et al. (2019 (link)). The solid-phase extraction was carried out on Octadecyl C18 Speedisk Column, 10 μm (J.T. Baker, Phillipsburg, USA). Mel labelled with iodine-125 (¹²⁵I) was used as a tracer for this analysis. All samples in duplicate were counted for 1 min in a Wallac Wizard 1470 gamma counter (Perkin Elmer Life Science, Waltham, USA). The detection limit was 3.0 pg/mL of plasma. The intra-assay coefficients of variation were 6.5%. The inter-assay variation was not determined because all samples were measured in the same assay.

17β-Oestradiol was measured in plasma using a Spectria Estradiol radioimmunoassay (RIA) kit (68633; Orion Diagnostica, Finland) according to the method described previously by Kulczykowska et al. (2015) (link). Concentrations of E₂ were measured directly from 100 µl plasma without extraction. Iodinated E₂ with ¹²⁵I was used as a tracer. A standard curve was prepared using six standard dilutions of 50, 150, 500, 1500, 5000 and 15,000 pmol l^–1. The assay was performed according to the kit manufacturer's instructions with slight modifications. The samples were added to RIA tubes that had been precoated with polyclonal anti-rabbit antiserum. After vortexing for 10 s, the tubes were incubated for 2 h at 37°C, decanted, washed with 1 ml Tween 20 solution and decanted again. The radioactivity in each tube was measured for 1 min using a Wallac Wizard 1470 gamma counter (PerkinElmer Life Science, Shelton, CT, USA). The detection limit of the assay was 37 pmol l^–1. The intra-assay coefficient of variation was 6.5%. The inter-assay variation was not determined because all samples were measured in the same assay. The mean E₂ concentrations in the plasma of round gobies during the spawning-capable phase and the regressing phase were 10±1.2 ng ml^–1 and 5±1.6 ng ml^–1, respectively.

Plasma T₄ was measured by a total thyroxine RIA kit (OCPG07-T4, Cisbio Bioassays, Codolet, France) without preceding extraction procedure according to the method presented by Kulczykowska et al. (2007 (link)). The T₄ labelled with ¹²⁵I was used as a tracer for RIA. All samples were assayed in duplicate and counted for 1 min in a Wallac Wizard 1470 gamma counter (Perkin Elmer Life Science, Waltham, USA). The detection limit was 1.1 ng/mL of plasma. The intra-assay coefficients of variation were 5.6%. The inter-assay variation was not determined because all samples were measured in the same assay.

The A1M concentration of RPTEC protein extracts was determined using RIA, as described previously [67 (link)]. Briefly, human urine A1M (purified as previously described) [68 (link)] was labelled with ¹²⁵I, according to the chloramine T-method [69 (link)]. Separation of iodine-labelled proteins from free iodide was achieved by gel chromatography on a Sephadex G-25 column (PD-10, GE Healthcare, Chicago, IL, USA). Standard A1M concentrations or unknown samples were mixed with goat anti-A1M-serum, diluted 1:6000 in RIA buffer (0.1 M phosphate, pH 7.4, 0.1% BSA, 0.02% NaN₃), and ¹²⁵I-labelled A1M (0.1–0.2 MBq/μg protein). Following incubation (overnight, room temperature), bovine serum and 15% polyethylene glycol in RIA buffer was added and the samples were centrifuged (2500× g, 40 min). The ¹²⁵I activity was measured using a Wallac Wizard 1470 gamma counter (Perkin-Elmer Life Sciences).