Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	78.13-5000 pg/mL
Sensitivity:	46.88 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human CDKN1A in samples. No significant cross-reactivity or interference between Human CDKN1A 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 CDKN1A. 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 CDKN1A 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 CDKN1A, 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 CDKN1A. The concentration of Human CDKN1A in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	p21Cip1: a cell-cycle regulatory protein that Interacts with cyclin-CDK2 and -CDK4, inhibiting cell cycle progression at G1. Its expression is tightly controlled by p53, through which this protein mediates the p53-dependent cell cycle arrest at G1 phase.
UniProt Protein Details:	Protein type:Inhibitor; Cell cycle regulation Chromosomal Location of Human Ortholog: 6p21. 2 Cellular Component: cyclin-dependent protein kinase holoenzyme complex; cytosol; nucleoplasm; nucleus; perinuclear region of cytoplasm Molecular Function:cyclin binding; cyclin-dependent protein kinase activating kinase activity; cyclin-dependent protein kinase inhibitor activity; metal ion binding; protein binding; protein complex binding; ubiquitin protein ligase binding Biological Process: cell cycle arrest; cellular response to amino acid starvation; cellular response to extracellular stimulus; DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest; DNA damage response, signal transduction by p53 class mediator resulting in induction of apoptosis; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; G1/S transition of mitotic cell cycle; G2/M transition of mitotic cell cycle; innate immune response; mitotic cell cycle; negative regulation of apoptosis; negative regulation of cell growth; negative regulation of cell proliferation; negative regulation of cyclin-dependent protein kinase activity; negative regulation of phosphorylation; nerve growth factor receptor signaling pathway; organ regeneration; phosphoinositide-mediated signaling; positive regulation of B cell proliferation; positive regulation of fibroblast proliferation; positive regulation of programmed cell death; Ras protein signal transduction; regulation of cyclin-dependent protein kinase activity; regulation of protein import into nucleus, translocation; response to arsenic; response to corticosterone stimulus; response to DNA damage stimulus; response to drug; response to hyperoxia; response to organic nitrogen; response to toxin; response to X-ray
NCBI Summary:	This gene encodes a potent cyclin-dependent kinase inhibitor. The encoded protein binds to and inhibits the activity of cyclin-cyclin-dependent kinase2 or -cyclin-dependent kinase4 complexes, and thus functions as a regulator of cell cycle progression at G1. The expression of this gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. This protein can interact with proliferating cell nuclear antigen, a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of cyclin-dependent kinase2, and may be instrumental in the execution of apoptosis following caspase activation. Mice that lack this gene have the ability to regenerate damaged or missing tissue. Multiple alternatively spliced variants have been found for this gene. [provided by RefSeq, Sep 2015]
UniProt Code:	P38936
NCBI GenInfo Identifier:	729143
NCBI Gene ID:	1026
NCBI Accession:	P38936. 3
UniProt Secondary Accession:	P38936,Q14010, Q6FI05, Q9BUT4,
UniProt Related Accession:	P38936
Molecular Weight:	18,119 Da
NCBI Full Name:	Cyclin-dependent kinase inhibitor 1
NCBI Synonym Full Names:	cyclin-dependent kinase inhibitor 1A
NCBI Official Symbol:	CDKN1A
NCBI Official Synonym Symbols:	P21; CIP1; SDI1; WAF1; CAP20; CDKN1; MDA-6; p21CIP1
NCBI Protein Information:	cyclin-dependent kinase inhibitor 1
UniProt Protein Name:	Cyclin-dependent kinase inhibitor 1
UniProt Synonym Protein Names:	CDK-interacting protein 1; Melanoma differentiation-associated protein 6; MDA-6; p21
Protein Family:	P21 prophage-derived protein
UniProt Gene Name:	CDKN1A
UniProt Entry Name:	CDN1A_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 (pg/mL)	O.D	Average	Corrected
5000	2.483 2.519	2.501	2.42
2500	1.765 1.791	1.778	1.697
1250	1.02 1.004	1.012	0.931
625	0.526 0.528	0.527	0.446
312.5	0.28 0.262	0.271	0.19
156.25	0.2 0.184	0.192	0.111
78.13	0.135 0.139	0.137	0.056
0	0.076 0.086	0.081	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CDKN1A were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CDKN1A 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)	264.14	721.92	2372.51	263.00	781.41	2480.98
Standard deviation	15.19	31.19	83.75	14.25	44.70	80.14
C V (%)	5.75	4.32	3.53	5.42	5.72	3.23

Recovery

The recovery of Human CDKN1A 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)	92-107	99
EDTA plasma (n=5)	86-102	93
Cell culture media (n=5)	88-102	93

Linearity

Samples were spiked with high concentrations of Human CDKN1A 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 (%)	91-102	87-97	93-107
1:2	Average (%)	97	92	98
1:4	Range (%)	88-100	80-90	82-95
1:4	Average (%)	95	85	88
1:8	Range (%)	86-98	83-96	89-99
1:8	Average (%)	93	89	94
1:16	Range (%)	91-101	83-95	84-95
1:16	Average (%)	96	89	90

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.