Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	1.56-100 ng/mL
Sensitivity:	0.94 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human Cys-C in samples. No significant cross-reactivity or interference between Human Cys-C 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 Cys-C. 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 Cys-C 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 Cys-C, 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 Cys-C. The concentration of Human Cys-C in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CST3: As an inhibitor of cysteine proteinases, this protein is thought to serve an important physiological role as a local regulator of this enzyme activity. Defects in CST3 are the cause of amyloidosis type 6 (AMYL6); also known as hereditary cerebral hemorrhage with amyloidosis (HCHWA), cerebral amyloid angiopathy (CAA) or cerebroarterial amyloidosis Icelandic type. AMYL6 is a hereditary generalized amyloidosis due to cystatin C amyloid deposition. Cystatin C amyloid accumulates in the walls of arteries, arterioles, and sometimes capillaries and veins of the brain, and in various organs including lymphoid tissue, spleen, salivary glands, and seminal vesicles. Amyloid deposition in the cerebral vessels results in cerebral amyloid angiopathy, cerebral hemorrhage and premature stroke. Cystatin C levels in the cerebrospinal fluid are abnormally low. Genetic variations in CST3 are associated with age- related macular degeneration type 11 (ARMD11). ARMD is a multifactorial eye disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid that lie beneath the retinal pigment epithelium and within an elastin-containing structure known as Bruch membrane. Belongs to the cystatin family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Inhibitor; Secreted Chromosomal Location of Human Ortholog: 20p11. 21 Cellular Component: extracellular space; extracellular region Molecular Function:protein binding; protease binding; beta-amyloid binding; endopeptidase inhibitor activity; cysteine protease inhibitor activity Biological Process: negative regulation of proteolysis; fibril organization and biogenesis; negative regulation of peptidase activity; defense response; regulation of tissue remodeling Disease: Macular Degeneration, Age-related, 11; Cerebral Amyloid Angiopathy, Cst3-related
NCBI Summary:	The cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins and the kininogens. The type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. The cystatin locus on chromosome 20 contains the majority of the type 2 cystatin genes and pseudogenes. This gene is located in the cystatin locus and encodes the most abundant extracellular inhibitor of cysteine proteases, which is found in high concentrations in biological fluids and is expressed in virtually all organs of the body. A mutation in this gene has been associated with amyloid angiopathy. Expression of this protein in vascular wall smooth muscle cells is severely reduced in both atherosclerotic and aneurysmal aortic lesions, establishing its role in vascular disease. In addition, this protein has been shown to have an antimicrobial function, inhibiting the replication of herpes simplex virus. Alternative splicing results in multiple transcript variants encoding a single protein. [provided by RefSeq, Nov 2014]
UniProt Code:	P01034
NCBI GenInfo Identifier:	118183
NCBI Gene ID:	1471
NCBI Accession:	P01034. 1
UniProt Related Accession:	P01034
Molecular Weight:
NCBI Full Name:	Cystatin-C
NCBI Synonym Full Names:	cystatin C
NCBI Official Symbol:	CST3
NCBI Official Synonym Symbols:	ARMD11; HEL-S-2
NCBI Protein Information:	cystatin-C
UniProt Protein Name:	Cystatin-C
UniProt Synonym Protein Names:	Cystatin-3; Gamma-trace; Neuroendocrine basic polypeptide; Post-gamma-globulin
Protein Family:	Cystatin
UniProt Gene Name:	CST3
UniProt Entry Name:	CYTC_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 (ng/mL)	RLU	Average	Corrected
100	35287 39821	37554	37521
50	15816 16666	16241	16208
25	8192 6892	7542	7509
12.5	3424 3940	3682	3649
6.25	1937 1813	1875	1842
3.13	1036 966	1001	968
1.56	546 598	572	539
0	32 34	33	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Cys-C 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)	4.71	15.33	42.47	5.18	16.11	44.90
Standard deviation	0.42	1.30	4.21	0.57	1.45	4.71
C V (%)	8.92	8.48	9.91	11.00	9.00	10.49

Recovery

The recovery of Human Cys-C 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)	91-107	99
EDTA plasma (n=5)	97-111	104
Cell culture media (n=5)	98-112	105

Linearity

Samples were spiked with high concentrations of Human Cys-C 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 (%)	96-110	92-102	88-100
1:2	Average (%)	104	97	94
1:4	Range (%)	95-111	94-108	101-115
1:4	Average (%)	101	102	109
1:8	Range (%)	91-108	97-113	88-101
1:8	Average (%)	98	103	93
1:16	Range (%)	95-109	89-101	101-117
1:16	Average (%)	101	95	109

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.