Stability of serum retinol-binding protein at −20⁰C

Dear Editor,

Retinol-binding protein 4 (RBP4) is a low-molecular-weight protein of 21 kDa that transports retinol (vitamin A alcohol) from the liver to target peripheral tissues.^1,2 RBP4 is a member of the lipocalin family of proteins and circulates in the blood bound to transthyretin (TTR, also known as thyroxine-binding protein and pre-albumin). This complex increases the serum half-life of RBP4 by reducing its glomerular filtration and renal catabolism, resulting in highly regulated and constant concentrations, except in instances of vitamin A deficiencies and some disease states.^1–4

RBP4 is easily measured in serum to provide information on retinol and vitamin A status. Retrospective/small-scale clinical research studies may store samples for long durations prior to RBP4 quantitation. We evaluated the stability of RBP4 concentrations during prolonged (up to 36 months) storage at −20⁰C.

Blood collected into serum separator tubes (Becton Dickinson, Plymouth, United Kingdom) were allowed to clot for 30 minutes at room temperature. Samples were centrifuged for 15 minutes at 1000xg and stored immediately at ≤ −20°C.

RBP4 measurement utilises a solid phase ELISA which incorporates a quantitative sandwich enzyme immunoassay technique (R&D Systems, Abingdon, United Kingdom). A monoclonal antibody specific for human RBP is pre-coated onto a 96-well microplate. Prior to analysis, serum samples are diluted 1000-fold. Standards, samples and quality controls are pipetted into the wells and incubated for 1 hour at room temperature on a shaker (500rpm). Following a series of washing steps, a monoclonal antibody, specific for human RBP4 and conjugated to horseradish peroxidase, is added and incubated for a further hour at room temperature. After a series of washing steps, a substrate solution containing equal volumes of stabilised hydrogen peroxide and stabilised chromogen (tetramethylbenzidine) is added to the wells and allowed to incubate for 30 minutes at room temperature protected from light. Colour develops in proportion to the amount of RBP4 bound in the initial step. The colour development is stopped using 2-N-sulphuric acid, which turns the colour of the wells from blue to yellow. The optical density of each well is determined using a microplate reader (Thermo Scientific™ Varioskan™) at wavelength 450 nm. The raw data is corrected by the software, SoftMax® Pro, to account for background noise by subtracting the zero standard optical density from all other results obtained. A four-parameter logistic standard curve fit is generated, and all concentrations are quantified and multiplied by the dilution factor and reportable in mg/L.

A total of 29 serum samples stored at −20⁰C were analysed, and the results compared with the original results. Five samples each were stored for 6, 9, 12, 18 or 36 months; a further four were stored for 30 months.

Analyse IT (version 5.2) was used for statistical analysis. Data were analysed by t-test and linear regression analysis. A p value  < 0.05 was considered statistically significant.

All serum samples had measurable RBP4. The immunoassay had a mean minimum detectable concentration of 0.000224 mg/L, with precision studies showing a < 10% inter- and intra-assay coefficient of variance. The concentrations of RBP4 in the samples analysed ranged 15.80–48.11 mg/L. Manufacturer-quoted reference range was 18–50 mg/L.

All 29 serum sample results were comparable and showed no difference compared with the original values. After storage for each of the following intervals, paired comparisons were 6 months (r = 0.97, p = 0.18), 9 months (r = 0.94, p = 0.45), 12 months (r = 0.99, p = 0.19), 18 months (r = 0.80, p = 0.27), 30 months (r = 0.96, p = 0.27) and 36 months (r = 0.98, p = 0.35).

We have demonstrated that RBP4 concentrations are stable for up to 36 months when stored at −20⁰C. To our knowledge, this is the first study to establish the stability of RBG4 protein for up to 36 months.