Population Range (pg/ml) Mean SD NHealthy Adults 5.3-91.6 32.6 pg/mL 23.4 pg/mL 118

1,25(OH)2 VITAMIN DELISA ASSAY

Fastest time to result in 1,25(OH)2 Vitamin D assessment with proven extraction method and breakable wells.

Fast one day protocol on ELISA automate.

CLINICAL ASPECTSVitamin D is mainly synthesized in the skin from 7- dehydrocholesterol and is partially from dietary origin. In the liver, Vitamin D is hydroxylated on carbon 25 to produce the obligatory intermediate 25OH Vitamin D. 25OH Vitamin D must be metabolized further before it can carry out the functions of Vitamin D on intestine, kidney and bone. This subsequent reaction takes place exclusively in the kidney in the non-pregnant mammal. Thus 25OH Vitamin D is further hydroxylated in the 1a-position to produce 1a,25 dihydroxyvitamin D (1,25(OH)2 Vitamin D).

1,25(OH)2 Vitamin D is the active form of Vitamin D with regard to the known functions whereas 25OH Vitamin D and Vitamin D itself can be excluded as being physiologically functional. Furthermore since 1,25(OH)2 Vitamin D is produced

in the kidney and has some of its functions in the bone and intestine, it must be considered as a hormone. This hormone stimulates the intestinal absorption of both calcium and phosphorus. It also stimulates bone resorption and mineralization thereby preventing the development of rickets and osteomalacia.

The levels of 1,25(OH)2 Vitamin D in plasma or serum is 100 to 1000 less than that of 25OH Vitamin D. Due to its low concentrations and the presence of many similar metabolites, the measurement of 1,25(OH)2 Vitamin D requires extraction and separation either by HPLC or by column chromatography preferable with the use of organic solvent. This extraction method is still recognized as the Gold Standard for 1,25(OH)2 Vitamin D.

1,25(OH)2 Vitamin D ELISA Art. Code KAP1921Format ELISA breakable wellsLabel HRPSize 96 testsSample Type SerumSample Volume 500 µL Controls 2 levelsRange 0-320 pg/mLSensitivity 1,7 pg/mLExtraction 60 min Incubation 1h, Overnight , 1,5 h

METHOD PRINCIPLE AND CHARACTERISTICSOnly samples and controls, not the calibrators, are extracted with a mixture of solvents and applied on cartridges to separate 1,25(OH)2 Vitamin D from the other Vitamin D metabolites. After elution samples and controls are incubated directly in microtiterplate coated with anti-1,25(OH)2 Vitamin D antibodies.

After an overnight incubation at 4°C, the microtiter plate is washed and the working conjugate solution is added and incubated for 1 hour at 4°C.

After washing the Chromogenic solution (TMB) is added and incubated for 15 minutes at room temperature. The reaction is stopped with the addition of Stop Solution and the microtiterplate is read at the appropriate wavelength. The amount of 1,25(OH)2 Vitamin D is determined colourimetrically by measuring the absorbance, which is inversely proportional to the 1,25(OH)2 Vitamin D concentration.

Graham D Carter (DEQAS Organizer):The successive abandonment of sample extraction, chromatography, [...] in immunoassays has undoubtedly contributed to the inconsistencies reported by Farrell et al., [...].25-Hydroxyvitamin D: A Difficult Analyte. Clinical Chemistry 2012, 58:3486–488.

CLINICAL APPLICATIONThe measurement of circulating 1,25(OH)2 Vitamin D is indicated in several disorders affecting calcium metabolism such as: ▪ Assessment of Kidney functions: Chronic kidney failure and Haemodialysis (after kidney transplants)▪ Hyper and hypo-parathyroidism,▪ Rickets disease▪ Tumor-associated hypercalcemia▪ Osteomalacia as a results of various types of Vit D metabolism disturbances

SPECIFICITYThe percentage of cross-reaction estimated by comparison of the concentration yielding a 50% inhibition are respectively:

Compound Cross-Reactivity (in %)1,25(OH)2 Vitamin D3 1141,25(OH)2 Vitamin D2 108

25OH Vitamin D3 0.000425OH Vitamin D2 0.0003

24,25(OH)2 Vitamin D3 0.002725,26(OH)2 Vitamin D3 0.007

Sample Dilution Theoretical Conc. (pg/mL)

Measured Conc. (pg/mL)

Slope Y-intercept (pg/mL)

R2 Recovery (%)

1/1 118.9 118.9

0.99 1.12 0.99

1001/2 59.4 60.7 1021/4 29.7 29.3 991/8 14.8 16.6 112

LINEARITY

METHOD COMPARISONA method correlation was performed with 11 serum samples comparing the DIAsource 1,25(OH)2 Vitamin D ELISA to the Quest Diagnostics LC-MS/MS assay. The linear regression analysis demonstrated a slope of 0.97, an intercept of 8.3 pg/mL and a correlation coefficient of R = 0.92. A correlation was performed with 118 serum samples comparing the DIAsource 1,25(OH)2 Vitamin D ELISA to the DIAsource 1,25(OH)2 Vitamin D RIA. The Passing & Bablok regression analysis demonstrated a slope of 0.99 and an intercept of 8.2 pg/mL.

ASSAY COMPARISON

DIAsource ELISA ELISA Competitor A ELISA Competitor BMicroplate Breakable wells Non-breakable wells Non-breakable wellsSample volume 500 µL 500 µL 1000 µLCalibrators 6 7 7Controls 2 2 2Range 0-320 pg/mL 0-150 pg/mL 0-300 pg/mLProtocol steps 1/18/1 h 1.5/0.5/18/1.5/1 h 0.5/1/20/1/0.5 hNumber of steps (separation + ELISA) 12 + 16 24 + 19 13 + 22

DIA

sour

ce E

LIS

A

(ng/

ml)

0

15

30

45

60

75

90

LC-MS/MS (ng/ml)0 15 30 45 60

DIA

sour

ce E

LIS

A

0

20

40

60

80

100

120

140

DIAsource RIA0 20 40 60 80 100 120 140

AUTOMATION ON OPEN ELISA PLATFORMA specific and fast protocol is validated for the automated application of 1,25(OH)2 Vitamin D ELISA measurement on the Stratec Gemini open ELISA pltaform. The incubation with extracted samples takes place at 30°C for 1 hour. This protocol allows same day results and can also be used on other open ELISA platforms.

PRACTICAL ADVANTAGES OF THE DIASOURCE 1,25(OH)2 VITAMIN D ELISA ASSAY - TIMELINEThe timetable represent a graphic overview of the protocol handling time. One can notice that the DIAsource 1,25(OH)2 Vitamin D ELISA assay has the fastest TAT (Turn Around Time) compared with 2 other commercially available ELISA assays.

Hour of the day 9 10 11 12 13 14 15 16 17 9 10 11 12ELISA kit DIAsource

ELISA Kit Competitor A

ELISA Kit Competitor B

DIAsource Automated

Extraction of sample Isolation of 1,25(OH)2 Vitamin D by specific separation techniqueELISA assay incubation ELISA assay separation and absorbance reading

DIASOURCE 1,25(OH)2 VITAMIN D ELISA: KEY FEATURES▪ Gold Standard extraction protocol ensuring no interference ▪ Limited number of incubation steps▪ Breakable wells format▪ 108% cross-reactivity against 1,25(OH)2 Vitamin D2▪ Very low Limit of Detection (1.7 pg/mL)▪ Good correlation with LC-MS/MS▪ DEQAS samples accurately measured▪ Automated one day protocol available (applicable on any open ELISA platform)

Ord

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Description Article code Format Size1,25(OH)2 Vitamin D ELISA KIP1929 ELISA - HRP 96 wells1,25(OH)2 Vitamin D Extraction Cartridges 1102491 For KAP1921 42 cartridges1,25(OH)2 Vitamin D Extraction Kit (Solvents, ready-to-use)

3019700 For KAP1921 2 x 42 extractions

16:00h - 10:00h overnight incubation ↲

Manufactured by: DIAsource ImmunoAssays SA Rue du Bosquet 2 BE 1348 Louvain-La-Neuve Tel. 32 10 84 99 00 Fax 32 10 84 99 96

Distributed by:

For more information: visit www.diasource-diagnostics.com

REFERENCE VALUESThese values are given only for guidance (reference values dependent of the season). The observed ranges are based on 2.5% to 97.5% percentiles.

Population Range (pg/ml) Mean SD NHealthy Adults 5.3-91.6 32.6 pg/mL 23.4 pg/mL 118