Intended use 
This immunoassay kit allows for the in vitro quantitative determination of General Calcitriol concentrations in serum, Plasma, tissue homogenates and Cell culture supernates and Other biological fluids. 
Test principle 
The ELISA is based on the competitive binding enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to General Calcitriol, During the reaction, General Calcitriol in the sample or standard competes with a fixed amount of biotin-labeled General Calcitriol for sites on a pre-coated Monoclonal antibody specific to General. Excess conjugate and unbound sample or standard are washed from the plate. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of General Calcitriol in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Precision

Intra-assay Precision (Precision within an assay):Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.

Intra-Assay CV: 4.7%

Inter-assay Precision (Precision between assays):Three samples of known concentration were tested in five separate assays to assess inter-assay precision.

Inter-Assay CV: 8.6%

Recovery

Recovery was determined by spiking various levels of Calcitriol into serum and plasma.

Recovery was determined by spiking various levels of Calcitriol into serum and plasma .

Sample Type	Average(%)	Recovery  Range (%)
Serum	90	88-98
Plasma	84	79-93

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Calcitriol and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1：2	1:4	1:8	1:16
serum(n=5)	110-119%	100-110%	100-110%	108-117%
EDTA plasma(n=5)	114-123%	91-101%	85-97%	107-117%
heparin plasma(n=5)	107-116%	105-115%	100-110%	103-113%

 

General Annotation

 

Function:

Calcitriol, also called 1,25-dihydroxycholecalciferol or 1,25-dihydroxyvitamin D3, is the hormonally active form of vitamin D with three hydroxyl groups (abbreviated 1,25-(OH)2D3 or simply 1,25(OH)2D). It increases the level of calcium (Ca2+) in the blood by (1) increasing the uptake of calcium from the gut into the blood, (2) decreasing the transfer of calcium from blood to the urine by the kidney, and (3) increasing the release of calcium into the blood from bone.

 

Location:

Calcitriol is produced in the cells of the proximal tubule of the nephron in the kidneys by the action of 25-hydroxyvitamin D3 1-alpha-hydroxylase, a mitochondrial oxygenase and an enzyme which catalyzes the hydroxylation of 25-hydroxycholecalciferol (calcifediol). The activity of the enzyme is stimulated by PTH. The reaction is an important control point in Ca2+ homeostasis. The production of calcitriol is also increased by prolactin, a hormone which stimulates lactogenesis (the formation of breast milk), a process which requires large amounts of calcium. It is decreased by high levels of serum phosphate and by an increase in the production of the hormone FGF-23 by osteocyte cells in bone.

 

Sequence:C27H44O3

 

 

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