Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Tissue homogenates,cell lysates and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human CYP24A1 in samples. No significant cross-reactivity or interference between Human CYP24A1 and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human CYP24A1. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CYP24A1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human CYP24A1, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human CYP24A1. The concentration of Human CYP24A1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CYP24A1: Has a role in maintaining calcium homeostasis. Catalyzes the NADPH-dependent 24-hydroxylation of calcidiol (25- hydroxyvitamin D(3)) and calcitriol (1-alpha,25-dihydroxyvitamin D(3)). The enzyme can perform up to 6 rounds of hydroxylation of calcitriol leading to calcitroic acid. It also shows 23- hydroxylating activity leading to 1-alpha,25-dihydroxyvitamin D(3)-26,23-lactone as end product. Defects in CYP24A1 are the cause of hypercalcemia infantile (HCAI). HCAI is a disorder characterized by abnormally high level of calcium in the blood, failure to thrive, vomiting, dehydration, and nephrocalcinosis. Belongs to the cytochrome P450 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 1. 14. 13. 126; Mitochondrial; Oxidoreductase Chromosomal Location of Human Ortholog: 20q13 Cellular Component: mitochondrial inner membrane Molecular Function:iron ion binding; heme binding; 25-hydroxycholecalciferol-24-hydroxylase activity; oxidoreductase activity; 1-alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) 24-hydroxylase activity Biological Process: osteoblast differentiation; steroid metabolic process; response to vitamin D; vitamin metabolic process; xenobiotic metabolic process; vitamin D metabolic process; vitamin D catabolic process Disease: Hypercalcemia, Infantile
NCBI Summary:	This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This mitochondrial protein initiates the degradation of 1,25-dihydroxyvitamin D3, the physiologically active form of vitamin D3, by hydroxylation of the side chain. In regulating the level of vitamin D3, this enzyme plays a role in calcium homeostasis and the vitamin D endocrine system. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	Q07973
NCBI GenInfo Identifier:	19862747
NCBI Gene ID:	1591
NCBI Accession:	Q07973. 2
UniProt Secondary Accession:	Q07973,Q15807, Q32ML3, Q5I2W7,
UniProt Related Accession:	Q07973
Molecular Weight:	59kDa
NCBI Full Name:	1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial
NCBI Synonym Full Names:	cytochrome P450, family 24, subfamily A, polypeptide 1
NCBI Official Symbol:	CYP24A1
NCBI Official Synonym Symbols:	CP24; HCAI; CYP24; P450-CC24
NCBI Protein Information:	1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial; 1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial; 24-OHase; cytochrome P450 24A1; cytochrome P450-CC24; vitamin D 24-hydroxylase; exo-mitochondrial protein; vitamin D(3) 24-hydroxylase; 1,25-dihydroxyvitamin D3 24-hydroxylase; cytochrome P450, subfamily XXIV (vitamin D 24-hydroxylase)
UniProt Protein Name:	1,25-dihydroxyvitamin D(3) 24-hydroxylase, mitochondrial
UniProt Synonym Protein Names:	Cytochrome P450 24A1; Cytochrome P450-CC24
Protein Family:	1,25-dihydroxyvitamin D(3) 24-hydroxylase
UniProt Gene Name:	CYP24A1
UniProt Entry Name:	CP24A_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
2000	53199 58243	55721	55693
1000	21681 26267	23974	23946
500	11716 10348	11032	11004
250	4992 5596	5294	5266
125	2668 2548	2608	2580
62.5	1330 1292	1311	1283
31.25	637 711	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CYP24A1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CYP24A1 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	105.39	206.82	902.96	96.54	197.55	857.08
Standard deviation	11.45	15.02	84.61	12.38	16.38	64.45
C V (%)	10.86	7.26	9.37	12.82	8.29	7.52

Recovery

The recovery of Human CYP24A1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	95-109	100
EDTA plasma (n=5)	89-101	94
Cell culture media (n=5)	98-111	105

Linearity

Samples were spiked with high concentrations of Human CYP24A1 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	91-105	89-105	91-101
1:2	Average (%)	98	96	96
1:4	Range (%)	87-98	88-101	95-109
1:4	Average (%)	93	95	102
1:8	Range (%)	92-107	98-114	92-105
1:8	Average (%)	99	105	98
1:16	Range (%)	86-98	101-118	93-104
1:16	Average (%)	92	109	99

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.