Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.63-40 ng/mL
Sensitivity:	0.38 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human CDKN2A in samples. No significant cross-reactivity or interference between Human CDKN2A and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human CDKN2A. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CDKN2A 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 CDKN2A, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human CDKN2A. The concentration of Human CDKN2A in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	Function: Acts as a negative regulator of the proliferation of normal cells by interacting strongly with CDK4 and CDK6. This inhibits their ability to interact with cyclins D and to phosphorylate the retinoblastoma protein. Ref. 11 Ref. 13
UniProt Protein Details:	Subunit structure: Heterodimer with CDK4 or CDK6. Predominant p16 complexes contained CDK6. Interacts (isoforms 1,2 and 4) with CDK4 (both 'T-172'-phosphorylated and non-phosphorylated forms); the interaction inhibits cyclin D-CDK4 kinase activity. Interacts with ISCO2. Ref. 13 Ref. 14 Subcellular location: Cytoplasm. Nucleus Ref. 14. Tissue specificity: Widely expressed but not detected in brain or skeletal muscle. Isoform 3 is pancreas-specific. Ref. 2 Post-translational modification: Phosphorylation seems to increase interaction with CDK4. Involvement in Disease: The association between cutaneous and uveal melanomas in some families suggests that mutations in CDKN2A may account for a proportion of uveal melanomas. However, CDKN2A mutations are rarely found in uveal melanoma patients. Melanoma, cutaneous malignant 2 (CMM2) [MIM:155601]: A malignant neoplasm of melanocytes, arising de novo or from a pre-existing benign nevus, which occurs most often in the skin but also may involve other sites. Note: Disease susceptibility is associated with variations affecting the gene represented in this entry. Ref. 24 Ref. 27 Ref. 29 Ref. 30 Ref. 31 Ref. 32 Ref. 34 Ref. 36 Ref. 38 Ref. 39 Ref. 41 Ref. 42Familial atypical multiple mole melanoma-pancreatic carcinoma syndrome (FAMMMPC) [MIM:606719]: An inherited cancer predisposition syndrome characterized by an increased risk of developing malignant melanoma and/or pancreatic cancer. Mutation carriers within families may develop either or both types of cancer. Note: The disease is caused by mutations affecting the gene represented in this entry. Li-Fraumeni syndrome (LFS) [MIM:151623]: Autosomal dominant familial cancer syndrome that in its classic form is defined by the existence of a proband affected by a sarcoma before 45 years with a first degree relative affected by any tumor before 45 years and another first degree relative with any tumor before 45 years or a sarcoma at any age. Other clinical definitions for LFS have been proposed (PubMed:8118819 and PubMed:8718514) and called Li-Fraumeni like syndrome (LFL). In these families affected relatives develop a diverse set of malignancies at unusually early ages. Four types of cancers account for 80% of tumors occurring in TP53 germline mutation carriers: breast cancers, soft tissue and bone sarcomas, brain tumors (astrocytomas) and adrenocortical carcinomas. Less frequent tumors include choroid plexus carcinoma or papilloma before the age of 15, rhabdomyosarcoma before the age of 5, leukemia, Wilms tumor, malignant phyllodes tumor, colorectal and gastric cancers. Note: The disease is caused by mutations affecting the gene represented in this entry. Ref. 35Melanoma-astrocytoma syndrome (MASTS) [MIM:155755]: Characterized by a dual predisposition to melanoma and neural system tumors, commonly astrocytoma. Note: The disease is caused by mutations affecting the gene represented in this entry. Ref. 37 Sequence similarities: Belongs to the CDKN2 cyclin-dependent kinase inhibitor family. Contains 4 ANK repeats. Sequence caution: The sequence AAB60645. 1 differs from that shown. Reason: Erroneous initiation.
NCBI Summary:	This gene generates several transcript variants which differ in their first exons. At least three alternatively spliced variants encoding distinct proteins have been reported, two of which encode structurally related isoforms known to function as inhibitors of CDK4 kinase. The remaining transcript includes an alternate first exon located 20 Kb upstream of the remainder of the gene; this transcript contains an alternate open reading frame (ARF) that specifies a protein which is structurally unrelated to the products of the other variants. This ARF product functions as a stabilizer of the tumor suppressor protein p53 as it can interact with, and sequester, the E3 ubiquitin-protein ligase MDM2, a protein responsible for the degradation of p53. In spite of the structural and functional differences, the CDK inhibitor isoforms and the ARF product encoded by this gene, through the regulatory roles of CDK4 and p53 in cell cycle G1 progression, share a common functionality in cell cycle G1 control. This gene is frequently mutated or deleted in a wide variety of tumors, and is known to be an important tumor suppressor gene. [provided by RefSeq, Sep 2012]
UniProt Code:	P42771
NCBI GenInfo Identifier:	3041660
NCBI Gene ID:	1029
NCBI Accession:	P42771. 2
UniProt Secondary Accession:	P42771,O95440, Q15191, Q5VVJ5, Q96B52, Q9NP05, A5X2G7 D3DRK1,
UniProt Related Accession:	P42771,Q8N726
Molecular Weight:
NCBI Full Name:	Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3
NCBI Synonym Full Names:	cyclin-dependent kinase inhibitor 2A
NCBI Official Symbol:	CDKN2A
NCBI Official Synonym Symbols:	ARF; MLM; P14; P16; P19; CMM2; INK4; MTS1; TP16; CDK4I; CDKN2; INK4A; MTS-1; P14ARF; P19ARF; P16INK4; P16INK4A; P16-INK4A
NCBI Protein Information:	cyclin-dependent kinase inhibitor 2A; CDK4 inhibitor p16-INK4; multiple tumor suppressor 1; cell cycle negative regulator beta; cyclin-dependent kinase 4 inhibitor A; cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)
UniProt Protein Name:	Cyclin-dependent kinase inhibitor 2A, isoforms 1/2/3
UniProt Synonym Protein Names:	Cyclin-dependent kinase 4 inhibitor A; CDK4I; Multiple tumor suppressor 1; MTS-1; p16-INK4a
UniProt Gene Name:	CDKN2A
UniProt Entry Name:	CD2A1_HUMAN

As the OD 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 (ng/mL)	O.D	Average	Corrected
40	2.473 2.487	2.48	2.412
20	1.576 1.582	1.579	1.511
10	0.884 0.866	0.875	0.807
5	0.457 0.493	0.475	0.407
2.5	0.238 0.22	0.229	0.161
1.25	0.176 0.158	0.167	0.099
0.63	0.116 0.124	0.12	0.052
0	0.067 0.069	0.068	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CDKN2A were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CDKN2A 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 (ng/mL)	2.08	6.28	16.73	1.92	6.51	15.76
Standard deviation	0.11	0.37	0.80	0.11	0.37	0.68
C V (%)	5.29	5.89	4.78	5.73	5.68	4.31

Recovery

The recovery of Human CDKN2A 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)	92-106	99
EDTA plasma (n=5)	95-107	101
Cell culture media (n=5)	86-99	91

Linearity

Samples were spiked with high concentrations of Human CDKN2A 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 (%)	95-112	84-97	86-98
1:2	Average (%)	103	91	92
1:4	Range (%)	94-106	80-89	83-95
1:4	Average (%)	99	85	90
1:8	Range (%)	90-102	81-96	87-98
1:8	Average (%)	96	87	93
1:16	Range (%)	90-103	79-93	86-99
1:16	Average (%)	96	86	93

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.