Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification

Abstract

Study objectives The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cut-off of 110 pg/mL and the inter-and intra-assay variability. Methods Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established, defined as the lowest converted concentration with a CV<25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs. Results The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n=41, range 1.6-17.0%), which was significantly higher than in RIAs with higher specific activity (n=36; median 3.2%, range 0.4-11.6%, p=.013). The CV around the 110 pg/mL cut-off was <7%. Conclusions Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cutt-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as “undetectable” when measured with the Phoenix Pharmaceuticals RIA.

Progress in Procalcitonin Detection Based on Immunoassay

“…RIA was developed to address these challenges by introducing a radioisotope-labeled antigen and unlabeled antigen to competitively bind antibodies [ 138 , 139 ]. Hence, the intensity of radiation is related to the amount of antigen measured.…”

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“…Currently, the radioimmunoassay technique is also performed with certain adaptations in a range of areas of health, for example, there are methods of quantifying hypocretin and thyroid hormones, in order to respectively diagnose type I narcolepsy (sleep disorder characterized by reduced levels of hypocretin) and thyroid disorders. In particular on type I narcolepsy, the method is based on the collection of cerebrospinal fluid (CSF) (containing hypocretin), such a sample is inserted into an environment with anti-hypocretin antibodies and radioactive (I-125) labeled hypocretin-1, which will compete with the CSF hypocretin by binding to the antibody, based on the radioactive concentration, the value of hypocretin is estimated, in a similar logic to the RIA with the measurement of insulin (Hoeven et al, 2022;Wu, 2022).…”