Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	78.13-5000 pg/mL
Sensitivity:	46.88 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human CTSC in samples. No significant cross-reactivity or interference between Human CTSC 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 CTSC. 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 CTSC 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 CTSC, 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 CTSC. The concentration of Human CTSC in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CTSC: Thiol protease. Has dipeptidylpeptidase activity. Active against a broad range of dipeptide substrates composed of both polar and hydrophobic amino acids. Proline cannot occupy the P1 position and arginine cannot occupy the P2 position of the substrate. Can act as both an exopeptidase and endopeptidase. Activates serine proteases such as elastase, cathepsin G and granzymes A and B. Can also activate neuraminidase and factor XIII. Defects in CTSC are a cause of Papillon-Lefevre syndrome (PLS); also known as keratosis palmoplantaris with periodontopathia. PLS is an autosomal recessive disorder characterized by palmoplantar keratosis and severe periodontitis affecting deciduous and permanent dentitions and resulting in premature tooth loss. The palmoplantar keratotic phenotype vary from mild psoriasiform scaly skin to overt hyperkeratosis. Keratosis also affects other sites such as elbows and knees. Defects in CTSC are a cause of Haim-Munk syndrome (HMS); also known as keratosis palmoplantaris with periodontopathia and onychogryposis or Cochin Jewish disorder. HMS is an autosomal recessive disorder characterized by palmoplantar keratosis, onychogryphosis and periodontitis. Additional features are pes planus, arachnodactyly, and acroosteolysis. Defects in CTSC are a cause of aggressive periodontititis type 1 (AP1); also known as juvenile periodontitis (JPD) and prepubertal periodontitis (PPP). AP1 is characterized by severe and protracted gingival infections, leading to tooth loss. AP1 inheritance is autosomal dominant. Belongs to the peptidase C1 family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3. 4. 14. 1; Endoplasmic reticulum; Protease Chromosomal Location of Human Ortholog: 11q14. 2 Cellular Component: Golgi apparatus; extracellular space; membrane; lysosome; endoplasmic reticulum Molecular Function:identical protein binding; protein binding; protein self-association; chaperone binding; serine-type endopeptidase activity; apoptotic protease activator activity; chloride ion binding; cysteine-type peptidase activity; phosphatase binding Biological Process: response to organic substance; apoptosis; immune response; proteolysis; T cell mediated cytotoxicity; aging Disease: Periodontitis, Aggressive, 1; Papillon-lefevre Syndrome; Haim-munk Syndrome
NCBI Summary:	The protein encoded by this gene, a member of the peptidase C1 family, is a lysosomal cysteine proteinase that appears to be a central coordinator for activation of many serine proteinases in immune/inflammatory cells. It is composed of a dimer of disulfide-linked heavy and light chains, both produced from a single protein precursor, and a residual portion of the propeptide acts as an intramolecular chaperone for the folding and stabilization of the mature enzyme. This enzyme requires chloride ions for activity and can degrade glucagon. Defects in the encoded protein have been shown to be a cause of Papillon-Lefevre syndrome, an autosomal recessive disorder characterized by palmoplantar keratosis and periodontitis. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P53634
NCBI GenInfo Identifier:	88683003
NCBI Gene ID:	1075
NCBI Accession:	AAI13851. 1
UniProt Secondary Accession:	P53634,Q2HIY8, Q53G93, Q71E75, Q71E76, Q7M4N9, Q7Z3G7 Q7Z5U7, Q8WY99, Q8WYA7, A8K7V2, B5MDD5,
UniProt Related Accession:	P53634
Molecular Weight:	51,854 Da
NCBI Full Name:	CTSC protein
NCBI Synonym Full Names:	cathepsin C
NCBI Official Symbol:	CTSC
NCBI Official Synonym Symbols:	JP; HMS; JPD; PLS; CPPI; DPP1; DPPI; PALS; DPP-I; PDON1
NCBI Protein Information:	dipeptidyl peptidase 1; cathepsin J; dipeptidyl peptidase I; dipeptidyl transferase; dipeptidyl-peptidase I
UniProt Protein Name:	Dipeptidyl peptidase 1
UniProt Synonym Protein Names:	Cathepsin C; Cathepsin J; Dipeptidyl peptidase I; DPP-I; DPPI; Dipeptidyl transferaseCleaved into the following 3 chains:Dipeptidyl peptidase 1 exclusion domain chain; Alternative name(s):; Dipeptidyl peptidase I exclusion domain chainDipeptidyl peptidase 1 heavy chain; Alternative name(s):; Dipeptidyl peptidase I heavy chainDipeptidyl peptidase 1 light chain; Alternative name(s):; Dipeptidyl peptidase I light chain
Protein Family:	Cathepsin
UniProt Gene Name:	CTSC
UniProt Entry Name:	CATC_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
5000	61045 61467	61256	61233
2500	33043 35665	34354	34331
1250	19490 16848	18169	18146
625	9294 9490	9392	9369
312.5	5069 4597	4833	4810
156.25	2699 2323	2511	2488
78.13	1334 1344	1339	1316
0	22 24	23	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CTSC 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)	269.21	663.95	2234.25	245.86	726.13	2401.14
Standard deviation	21.73	50.26	161.54	31.13	72.76	231.23
C V (%)	8.07	7.57	7.23	12.66	10.02	9.63

Recovery

The recovery of Human CTSC 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)	86-98	93
EDTA plasma (n=5)	84-97	91
Cell culture media (n=5)	94-108	99

Linearity

Samples were spiked with high concentrations of Human CTSC 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 (%)	92-106	87-100	87-101
1:2	Average (%)	100	92	93
1:4	Range (%)	101-118	85-97	86-101
1:4	Average (%)	109	90	93
1:8	Range (%)	100-114	90-106	98-112
1:8	Average (%)	107	97	103
1:16	Range (%)	101-116	83-95	89-104
1:16	Average (%)	108	90	96

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.