A novel, fully-automated, chemiluminescent assay for the detection of 1,25-dihydroxyvitamin D in biological samples

BACKGROUND
1,25-Dihydroxyvitamin D (1,25-(OH)2D), the hormonal form of vitamin D, is difficult to measure because of its low circulating levels (pg/mL), and similarity to more abundant metabolites. Here a fully-automated chemiluminescent assay that accurately and precisely measures 1,25-(OH)2D is described.


METHOD
The novel 1,25-(OH)2D assay was conceived based on four pillars: (1) the VDR's ligand binding domain (LBD) as a capture molecule; (2) reaction conditions wherein 1,25-(OH)2D favors binding to LBD vs. the vitamin D binding protein; (3) exploitation of liganded-LBD's conformational change; (4) a monoclonal antibody specific to liganded-LBD. This specific, conformational, sandwich approach, unique for automated measurement of haptens, is superior to more cumbersome, conventional competitive formats.


RESULTS
Accuracy of the 1,25-(OH)2D assay was corroborated by its alignment against LC-MS/MS with fit Deming regression equations of y=0.98x + 1.93 (r=0.92), and y=1.07x+3.77 (r=0.94) for different methods from Endocrine Sciences, Laboratory Corporation of America® and the University of Washington, respectively. Good analytical precision was manifested by its low estimated limit of quantitation (1.57pg/mL), average intra-assay imprecision (3.5%CV; range 1.1-4.7%), and average inter-assay imprecision (4.5%CV; range 3.4-7.2%). Expected and measured recovery values were congruent (93.4% mean).


CONCLUSIONS
The novel 1,25-(OH)2D method exhibited excellent correlation with well validated LC-MS/MS assays from two laboratories. Significantly, its 65min turn-around time is quicker, and sample volume smaller (75μl) than current methods.


Introduction
The synthesis of vitamin D 3 in skin is a two-step process: 7dehydrocholesterol is converted to previtamin D 3 upon exposure to UV light; previtamin D 3 isomerizes to vitamin D [1]. Vitamin D 3 and vitamin D 2 (that originates from irradiation of ergosterol) may also be obtained in the diet. [1]. Upon entry into the circulation, vitamin D is readily hydroxylated in the liver at carbon-25 to produce 25-hydroxyvitamin D (25-(OH)D), the major circulating metabolite [2]. Vitamin D metabolites generally associate with the vitamin D binding protein (VDBP) to be carried through the bloodstream [3]. Although 25-(OH)D is inactive itself, its serum level affords the commonly accepted method for assessing vitamin D status of patients.
The classical role of vitamin D is the maintenance of plasma calcium and phosphate at near constant levels for proper neuromuscular function, bone mineralization, and the prevention of hypocalcemic tetany [4]. Under physiological conditions where calcium and phosphate homeostasis is disturbed, 25-(OH)D is converted in the kidney by 1a-hydroxylase to produce 1a,25dihydroxyvitamin D (1,25-(OH) 2 D), which acts on target organs to normalize plasma calcium and phosphate [4]. Most, if not all, of circulating 1,25-(OH) 2 D is produced in the kidney as revealed by the extremely low circulating levels in nephrectomized rats [5] or patients [6].
The functions of 1,25-(OH) 2 D are mediated through a nuclear receptor that binds to vitamin D response elements (VDREs) in target genes to regulate their transcription. The vitamin D receptor (VDR) is a member of the steroid/thyroid hormone nuclear receptor superfamily and contains characteristic functional domains including the DNA binding domain and the ligand binding domain (LBD) [7]. The structure of the LBD was recently solved by NMR studies, which depict a distinct, ligand-specific conformational change upon binding of 1,25-(OH) 2 D [8].
It has long been established that vitamin D deficiency causes bone diseases such as rickets, osteomalacia, and secondary hyperparathyroidism, but with the discovery of VDR in nonclassical target tissues, 1,25-(OH) 2 D is now known to have a broader spectrum of actions, and has been associated with increased risks for various chronic diseases including infectious and autoimmune, diabetes, cancer, cardiovascular ailments, hypertension and complications during pregnancy [9]. Clinical studies implicating vitamin D in the forgoing conditions have been based predominantly upon measurement of 25-(OH)D. Since 1,25-(OH) 2 D is the active hormone and does not correlate with 25-(OH)D status, important relationships have remained unexplored. Facile measurement of 1,25-(OH) 2 D has been difficult due to its extremely low circulating levels (pg/mL) [10]. Current methods, requiring laborious, sample consumptive, pre-analytical steps are significantly compromised at the low end of their measuring range because of their intrinsic imprecision [10]. In this manuscript, a new, fully-automated method for measurement of 1,25-(OH) 2 D in biological fluids is presented. The novel assay is accurate, precise throughout the entire assay range, fast, and requires only 75 ml of sample unencumbered by the need for pre-analytical enrichment through precipitation and subsequent reconstitution. The exquisite specificity and avidity of the VDR for its substrate was used to produce the first sandwich, automated chemiluminescent assay for a small molecule hapten like 1,25-(OH) 2 D.

Specimens
To evaluate the LIAISON 1 XL 1,25-Dihydroxyvitamin D assay in biological fluids, 78 human serum samples routinely submitted to Laboratory Corporation of America for 1,25-(OH) 2 D testing were anonymized and used to make pools with concentrations covering the analytical measuring range of the assay and parsed into multiple aliquots for analysis by multiple methods. The protocol for this study was determined to be exempt under existing regulations by the Institutional Review Board.

Method comparison
Method comparison of 1,25-(OH) 2 D measurement included two different LC-MS/MS methods using immune-extraction to enrich for 1,25-(OH) 2 D as a pre-analytical step; the first method was from Endocrine Sciences, Laboratory Corporation of America (Calabasas Hills, CA), and the second LC-MS/MS analysis was performed at the University of Washington and is outlined by Strathmann et al. [11]. A commercially available immunoassay (Method A) was also compared to the LIAISON 1 XL 1,25-dihydroxyvitamin D assay (DiaSorin, Stillwater, MN).

Performance evaluation
Imprecision, recovery, limit of quantitation (LOQ), limit of blank (LOB), linearity, and analytical specificity were evaluated according to CLSI guidelines. The reference range was verified on a subset of 41 apparently healthy blood donor volunteers.

Statistical analysis
All data were analyzed using EP Evaluator 1 (Data Innovations, LLC). Linear regressions were performed by regular and Deming methods using Analyse-it TM for Microsoft Excel.

The novel 1,25-dihydroxyvitamin D assay
Specimens were tested to evaluate the novel 1,25-(OH) 2 D method using the CE marked, Investigational Use Only DiaSorin LIAISON 1 XL 1,25-Dihydroxyvitamin D assay (Stillwater, MN). The novelty and efficacy of this 1,25-(OH) 2 D assay depend upon four key design pillars: (1) use of the ligand binding domain (LBD) of the VDR as a capture molecule; (2) use of reaction conditions where binding of 1,25-(OH) 2 D to the LBD is favored over binding to the VDBP by $200-fold. At those same conditions, inactive metabolites of vitamin D, including 25(OH)D, 24,25-dihydroxyvitamin D, and 25,26-dihydroxyvitamin D, preferentially bind to the VDBP by 10-100-fold creating a binding differential of 2000-20,000-fold (which greatly minimizes interference from the 100-1000-fold higher physiological concentrations of inactive metabolites compared to 1,25(OH) 2 D); (3) exploitation of the LBD conformational change induced upon 1,25-(OH) 2 D binding as delineated by NMR analysis [8]; (4) the use of a monoclonal antibody that selectively recognizes the unique LBD conformation produced when 1,25-(OH) 2 D is bound to the LBD.
Monoclonal antibodies (Mab) against the LBD-1,25-(OH) 2 D complex screened by ELISA were characterized by Dual Polarization Interferometry, DPI (AnaLight 4D, Farfield Group, UK). DPI measures real time binding events on the face of a biosensor chip. The selected clone 11B4 accumulated 8.9 ng of the LBD-1,25-(OH) 2 D complex within the first 400 sec as opposed to a minimal amount of unbound, control apo-LBD during the same time frame (Fig. 1), showing the high specificity of the 11B4 Mab for the LBD-1,25-(OH) 2 D complex. Following a second wash step to remove unbound conjugate, starter reagents are added triggering a chemiluminescent reaction. The light flash emitted, measured by a photomultiplier tube, affords a signal directly proportional to the amount of 1,25-(OH) 2 D present in the sample.
The above comparisons indicate that the LIAISON 1 XL 1,25-Dihydroxyvitamin D assay is comparable to LC-MS/MS, which is considered a higher order method with respect to accuracy.
Recovery studies were performed to determine agreement between expected and measured amounts. Three sets of low and high samples were mixed at different ratios (3:1, 1:1 and 1:3) and tested. Table 1 depicts neat, expected, and measured concentrations, as well as the percent recovery which was calculated from the measured and expected values. The assay had a mean recovery of 93.4%.

Imprecision and sensitivity
The LIAISON 1 XL 1,25-Dihydroxyvitamin D assay was evaluated for intra-assay imprecision using 2 levels of control and 4 patient sample pools. Twenty replicates of each of the controls and 4 patient pools were assayed. Inter-assay precision was performed in 5 assay runs with at least 4 replicates of each of the 2 levels of control and 4 sample pools. The average intra-assay imprecision was 3.5%CV (range 1.1-4.7%CV), and inter-assay imprecision average was 4.5%CV (range 3.4-7.2%CV) ( Table 2).
One of the beneficial characteristics of this assay, is its extremely low imprecision, even at low 1,25-(OH) 2 D concentrations (<5 pg/mL). A LOQ study was performed by measuring samples with concentrations ranging from 0.342 to 16.5 pg/mL for 10 days. In Fig. 5 EP Evaluator was used to derive the estimated LOQ (1.57 pg/mL) based on a fitted curve. As shown %CV remain relatively constant down to sample concentrations as low as 1-2 pg/mL, while other methods routinely exhibit higher imprecision especially at concentrations below 15 pg/mL [11][12][13][14][15].
The limit of blank was determined with a total of 10 replicates of the zero standard. The lowest non-zero concentration that could be differentiated from zero was calculated as 0.071 pg/mL (95% confidence) using EP Evaluator 1 , which is well below the manufacturer's claim of 0.35 pg/mL.
Interfering substances were evaluated for the LIAISON XL 1,25-dihydroxyvitamin D assay using the Assurance Interference Test Kit from Sun Diagnostics New Gloucester, ME. The effect of hemolysis and icteric and lipemic matrix constituents on measurement were evaluated based on CLSI EP7-A. No significant interference was observed for triglycerides (3000 mg/dL), hemolysate (500 mg/dL), conjugated bilirubin (20 mg/dL) and unconjugated bilirubin (20 mg/mL).

Reference range
Forty-one ostensibly "normal" patient samples (samples from healthy volunteers) were assayed to verify the reference range for serum samples as stated by the manufacturer per package insert (25.0-86.5 pg/mL). The central 95% interval revealed by the data for the 41 points assayed was 29.7-88.8 pg/mL. The mean of the samples tested was 54.4 pg/mL.

Conclusions
A novel method was developed for the measurement of 1,25-(OH) 2 D in biological samples. The unique design of this assay makes it the first fully-automated, small-molecule, sandwich chemiluminescent assay available and allows for the measurement of a molecule that is present at 1000x lower concentrations than other quite similar metabolites. The assay is extremely accurate when compared to LC-MS/MS and has low imprecision, especially near its limit of quantitation. The assay has a rapid time to first result (65 min) with a throughput of 50-80 tests/hr thereafter, requires significantly smaller sample volume (75 ml), and performs well in terms of accuracy, imprecision, sensitivity, linearity and specificity. The novel assay does not require any preanalytical extraction or purification steps, which greatly diminishes its imprecision which was shown to be very low in this evaluation. Gold-standard methods like LC-MS/MS, however, still require pre-analytical processing steps which can contribute to higher imprecision, attributable to extraction in addition to measurement, rather than just direct measurement.
The assay is robust, and its close alignment with LC-MS/MS standardization is supported by its performance in the vitamin D External Quality Assessment Scheme (DEQAS), where multiple labs perform very well against the target value and with very small variation between participants (data not shown). van Helden and Weiskirchen also reported on their evaluation of the assay and concluded that the novel 1,25-(OH) 2 D assay is highly robust [16].
The simplicity of use, and its ability to accurately measure even small concentrations of 1,25-dihydroxyvitamin D make this assay a valuable tool for future prospective and retrospective studies in patients with not only bone and mineral metabolism diseases, but also in other disease states such as infectious and autoimmune diseases, diabetes, cancer, cardiovascular ailments, hypertension and complications during pregnancy, where associations to 25(OH)D have been previously shown, but may need to be revisited to determine the role of the active form of vitamin D, 1,25-(OH) 2 D in subjects/patients with low circulating 25(OH)D.   Table 1 Recovery of 1,25-(OH) 2 D in serum using the novel 1,25-(OH) 2 D assay. Three separate specimens (low samples) were assayed to determine circulating levels of 1,25-(OH) 2 D using the novel assay. Each sample was mixed with different concentrations of another patient sample (high sample) through sample mixing at ratios of 3:1, 1:1 and 1:3 (low sample: high sample) and assayed. The high samples were also assayed neat. Percent recoveries were calculated from measured and expected values.  Table 2 Intra-and Inter-assay imprecision for the novel 1,25-(OH) 2 D assay. The 1,25-(OH) 2 D assay was evaluated for intra-assay imprecision using 2 levels of control and 4 patient sample pools. Twenty replicates of each of the controls and 4 patient pools were assayed. The inter-assay imprecision was performed in 5 assay runs on different days with at least 4 replicates of each of the 2 levels of control and 4 sample pools.  5. Sensitivity: Limit of Quantitation (LOQ). Eight samples around the limit of quantitation indicated by the manufacturer (5 pg/mL) were measured for 10 days and %CVs were determined. EP Evaluator 1 was used to estimate the LOQ, the point at which the fitted curve crosses the 20% CV line. The estimated LOQ is 1.57 pg/mL.

Previous presentation
The method was presented at the 2014 IFCC World Lab in Instanbul, Turkey.