25oh Vitamin D Total

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1 Measurement of 25-Hydroxyvitamin D with a novel and unique ELISA assay 1 % ALL-IN-ONE Simple automation START STOP E N G I N E 25oh Total elisa by Powered by patented DIAsource Monoclonal Antibodies

2 Based on a vast majority of clinical studies to define normal circulating 25 OH VIT D levels e.g. US National health and Nutrition Examination Survey (US NHANES-study) Dietary sources of 2 and D 3 Since many years the role of vitamin D in bone and mineral metabolism was recognized in bone-related diseases. Clinical applications of 25-Hydroxyvitamin D measurements were merely related to the diagnosis and monitoring of therapy for rickets (children),osteomalacia, postmenopausal osteoporosis, and renal osteodystrophy. As a results of more recent studies a link between deficiency and many other diseases is suggested. These include cancer, cardiovascular disease, autoimmune diseases, diabetes, depression. Clinical aspects: UVB SKIN 7-dehydrocholesterol Pre 3 Heat There are two forms of in the human body namely Lipo- 3 Protein DBP 3 (cholecalciferol) and 2 (ergocalciferol), which 25-OHase DBP 25(OH)D P, Ca are structurally very similar. 3, the main form in humans, is 2+, & other factors CIRCULATION 24-OHase 1-OHase 1-OHase Urine Calcitroic acid 1,25(OH) 2 D 1,25(OH) 2 D Preosteoclast produced in the skin from 7-dehydrocholesterol in response to direct RANKL RANK PTH sunlight and can also be obtained in small amounts from animalbased foods (oily fish, primarily salmon and mackerel). Osteoblast PATIENT ng/ml of VITAMIN D RELATED STATUS 25(OH) Vit D* DISEASES: PTH Vit D Deficiency <1 Rickets in Children Vit D Insufficiency 1- Osteoporosis, Osteomalacia Calcium & Ca Ca AND HPO AND HPO 4 RELEASE 4 phosphorus Mature Vit D Sufficiency >-1 Cancer ABSORPTION Osteoclast Blood Type II Diabetes Risk for Toxicity >1 Bone 2 can be obtained in small amounts from plant-based * Peripheral Artery Disease Metabolic Neuromuscular mineralization functions functions Auto Immune Diseases Parkinson s disease foods (some vegetables, yeast and fungi). 3 and D2 are metabolized in the liver to their respective 25-Hydroxyvitamin D3 and D2 forms which are converted in the kidneys to the active forms (1.25-dihydroxyvitamin D3 and D2). Determining Status - + The measurement of 25-Hydroxyvitamin D concentration in the serum or plasma is the best indicator of vitamin D nutritional status. It is generally accepted that serum 25-Hydroxyvitamin D levels reflect the body s storage levels of vitamin D and correlate with the clinical symptoms of vitamin D deficiency. There is no consensus about the optimal 25-Hydroxyvitamin D level, but many publications suggest a range ng/ml (>nmol/l) as optimal. The most widely used intervals are indicated in table 1 Table 1 : Suggested Reference Values for Adults Status 25-Hydroxyvitamin D Total (ng/ml) Deficiency < 1 Insufficiency 1 - Sufficiency Toxicity >1 Paediatric reference intervals have not been established, but the American Association for Paediatrics (AAP) recommends a value of 2 ng/ml for healthy children. Several population studies have identified widespread 25-Hydroxyvitamin D insufficiency (> % of the population) in apparent healthy populations. About percent of the United States population has inadequate levels of vitamin D. Groups that are at higher risk for inadequate levels of 25 Hydroxyvitamin D levels include, individuals with limited sun exposure,the elderly, darkskinned individuals and breast-feeding infants. Measurements of 25-Hydroxyvitamin D levels can be used in: Diagnosing insufficiency or deficiency, to help identifying individuals who may benefit from supplementation to reach optimal levels. Monitoring response to supplements for bone-related diseases e.g. rickets (children), osteomalacia, postmenopausal osteoporosis, and renal osteodystrophy or non-bone related diseases Diagnosing toxicity, e.g. patients with suspected toxicity ( hypercalcemia)

3 DIAsource 25-Hydroxyvitamin D Total ELISA Method principle and Characteristics 25 OH Total ELISA (KAP1971) The DIAsource 25-Hydroxyvitamin D Total ELISA is Art. Code KAP1971 a competitive ELISA with a novel pre-treatment step performed inside the ELISA MT-plate. During a first 2 hours Format ELISA MT incubation step, at room temperature, 25-Hydroxyvitamin Size 96 tests D (D2 and D3) present in calibrators, controls and samples is dissociated from binding serum proteins to fix on binding Sample Type Serum sites of a specific monoclonal antibody (Patent Pending). Sample Volume µl After a washing step a fixed amount of 25-Hydroxyvitamin D labelled with biotin in presence of horseradish peroxidase Controls 2 levels (HRP), competes with unlabelled 25-Hydroxyvitamin D2 Range -1 ng/ml and 25-Hydroxyvitamin D3 present on the binding sites of the specific monoclonal antibody. After a minutes Sensitivity 1.5 ng/ml incubation at room temperature, the MT-plate is washed to Total Analysis Time < 4 hours stop the competition reaction. The Chromogenic solution (TMB ) is added and incubated Incubation for 15 min. The reaction is stopped with the addition Wavelength 4 nm of Stop Solution and the MT-plate is then read at the appropriate wavelength. The amount of substrate turnover is determined colorimetrically by measuring the absorbance, which is inversely proportional to the total 25-Hydroxyvitamin D (D2 and D3) concentration. Calibration The DIAsource 25-Hydroxyvitamin D Total ELISA is calibrated against the reference method ID-LC/MS-MS* 1 DIAsource Elisa (ng/ml) A correlation was performed with 94 serum samples comparing the DIAsource 25-Hydroxyvitamin D Total ELISA to LC/MS-MS. The regression analysis demonstrated a slope of.914, an intercept of 2.33 ng/ml (Figure 1) and a correlation of R= y =.9137x R =.92 n = LC/MS-MS (ng/ml) Figure 1 : Correlation of DIAsource ELISA with LC/MS-MS This LC/MS-MS methodology used for the correlation studies of the DIAsource 25-Hydroxyvitamin D Total ELISA assay shows a high level of traceability to the ID-LC/MS-MS reference methodology (Correlation Coefficient R>.97). *Candidate Reference Measurement Procedures for Serum 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 by Using Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC/MS-MS),Clinical chemistry, 211,Vol.57,3,pag

4 Method Comparison 1 1 DIAsource ELISA (ng/ml) 2 y = 1.746x R =.93 n = 74 Diasource ELISA (ng/ml) 2 y = 1.634x R =.93 n = DiaSorin Liaison (ng/ml) IDS ELISA (ng/ml) Figure 2 :Method comparison with DiaSorin Liaison Figure 3 :Method comparison with IDS Elisa A further correlation was performed with 74 serum samples comparing the DIAsource 25-Hydroxyitamin D Total ELISA assay with the commercially available and FDA approved DiaSorin 25-Hydroxyvitamin D Liaison assay. The regression analysis demonstrated a slope of 1.75, an intercept of.158 ng/ml (Figure 2) and a correlation of R=.93 In addition to the comparison with DiaSorin Liaison, a correlation was performed in 118 serum samples with the commercially available IDS 25-Hydroxyvitamin D ELISA assay. The regression analysis shows a slope of 1.63, an intercept of.831 ng/ml (Figure 3) and a correlation of R=.93 Assay performances Compound Cross-Reactivity (%) 25OH OH ,25(OH)2-Vitamin.D3 1,25(OH)2-Vitamin.D2 <.2 3 <.2 2 <.2 24,25(OH)2-Vitamin.D3 >1 25,26(OH)2-3 >1 Added 25 OH-Vit.D3 (ng/ml) Recovery (%) Added 25 OH-Vit.D2 (ng/ml) Recovery (%) epi-25 hydroxy vitamin D3 <.2 The DIAsource 25-Hydroxyvitamin D Total ELISA assay demonstrated very low cross-reactivity to other metabolites of 3 and D2. The assay performance is not affected by hemolysis (5 g/l haemoglobin tested), bilirubinemia (.25 g/l bilirubin tested) or triglycerides (5 g/l tested). The DIAsource 25-Hydroxyvitamin D Total ELISA assay demonstrated an excellent recovery : the assay showed an equimolar detection of both abundant 25-Hydroxyvitamin D forms (D2 and D3) in the human body.

5 Automation : Application on open ELISA platform Stratec Gemini The DIAsource 25-Hydroxyvitamin D Total ELISA assay is a competitive enzyme-immunoassay for the quantitative determination of 25-Hydroxyvitamin D Total (D2 + D3) in human serum. It makes use of a novel pre-treatment step which eliminates the use of solvent precipitation and centrifugation. This sample pre-treatment step is performed inside the sample well of the ELISA microtiter plate which highly facilitates the automation on different open ELISA platforms.. Assay Protocol add µl sample, Standard Control and add 1 µl Incubation Buffer Incubate 12 min at RT with shaking Wash Plate add 2 µl conjugate Incubate min at RT with shaking Wash Plate add 1 µl TMB Incubate 15 min at RT with shaking add 1 µl STOP Read Absorbance at 4 nm Automation : Validated application on Stratec Gemini open ELISA platform The novel approach of the DIAsource 25-Hydroxyvitamin D Total ELISA assay, makes the assay easy to program on any open ELISA platform without the need for manual sample pre-treatment outside the ELISA platform. The DIAsource 25-Hydroxyvitamin D Total ELISA has extensively been validated on the Stratec Gemini. A validated protocol is available and permits larger laboratories to easily automate their 25-Hydroxyvitamin D determinations. A in-house correlation was performed with 54 serum samples comparing the DIAsource 25-Hydroxyitamin D Total ELISA assay manually performed and automated on the Stratec Gemini, an OPEN ELISA automate. The regression analysis demonstrated a slope of.98, an intercept of 2.29 ng/ml and a correlation of R=.98 1 Automated on Stratec Gemini (ng/ml) 2 Stratec Gemini, 2-plate open ELISA system Distributed by DIAsource ImmunoAssays 1 y =.9811x R = Manually (ng/ml)

6 DIAsource (art. Code KAP1971) Competitor Assay 1 (ELISA) Competitor Assay 2 (ELISA) Pretreatment AIO * In the assay well Outside the assay well Outside the assay well Transfer Pre-treated sample into MT NOT NECESSARY YES YES 1 % Automatable Specificity 1 % D3 & 83 % D2 1 % D3 & 75 % D2 1 % D3 & 1% D2 Vortex NO Each sample 1 sec (pre-treatment) NO Analytical sensitivity 1,5 ng/ml 2 ng/ml 2,2 ng/ml N of steps Total Incubation time overnight Total Analysis time < 2 > 2 > 11 (2 days) User friendly *AIO = ALL IN ONE, means that pre-treatment and ELISA are performed in the same MT-plate, which does not require a pre-treatment step in a second MT-plate or tubes before to transfer pre-treated samples to the MT-plate. Highlights: The DIAsource 25-Hydroxyvitamin D Total ELISA (art. code KAP1971) assay shows very competitive sensitivity, precision and performance characteristics to all other immunoassays in the market but uses a novel and extremely easy pre-treatment step directly in the microtiter plate. The assay is calibrated to the ID-LC/MS-MS reference method. It shows a high correlation with other commercially available Chemiluminescence assays, with LC/MS-MS and with other 25-hydroxyvitamin D ELISA assays. The novel approach of the DIAsource assay, makes it extremely easy to program on any open ELISA platform without the need for a manual sample pre-treatment outside the ELISA automate as is the case in other commercially available ELISA assays. A validated assay protocol is available on the Stratec Gemini open ELISA automate and permits larger laboratories to easily automate their 25-hydroxyvitamin D determinations. Ordering Information: Article Description Size KAP Hydroxyvitamin D Total ELISA 96 test LIT2 DIAsource ImmunoAssays S.A. Tel Fax Technical Inquiries: tech.support@diasource.be To order: Belgium Free Phone BeLux Tel +32 () Fax +32 () ordering@diasource.be France Free Phone Free Fax ordering@diasource.be