Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human PROC in samples. No significant cross-reactivity or interference between Human PROC 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 PROC. 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 PROC 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 PROC, 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 PROC. The concentration of Human PROC in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	PROC: Protein C is a vitamin K-dependent serine protease that regulates blood coagulation by inactivating factors Va and VIIIa in the presence of calcium ions and phospholipids. Defects in PROC are the cause of thrombophilia due to protein C deficiency, autosomal dominant (THPH3). A hemostatic disorder characterized by impaired regulation of blood coagulation and a tendency to recurrent venous thrombosis. However, many adults with heterozygous disease may be asymptomatic. Individuals with decreased amounts of protein C are classically referred to as having type I protein C deficiency and those with normal amounts of a functionally defective protein as having type II deficiency. Defects in PROC are the cause of thrombophilia due to protein C deficiency, autosomal recessive (THPH4). A hemostatic disorder characterized by impaired regulation of blood coagulation and a tendency to recurrent venous thrombosis. It results in a thrombotic condition that can manifest as a severe neonatal disorder or as a milder disorder with late-onset thrombophilia. The severe form leads to neonatal death through massive neonatal venous thrombosis. Often associated with ecchymotic skin lesions which can turn necrotic called purpura fulminans, this disorder is very rare. Belongs to the peptidase S1 family.
UniProt Protein Details:	Protein type:Protease; Apoptosis; EC 3. 4. 21. 69 Chromosomal Location of Human Ortholog: 2q13-q14 Cellular Component: endoplasmic reticulum lumen; Golgi lumen; extracellular region Molecular Function:protein binding; serine-type endopeptidase activity; calcium ion binding Biological Process: cellular protein metabolic process; negative regulation of blood coagulation; blood coagulation; proteolysis; post-translational protein modification; peptidyl-glutamic acid carboxylation; leukocyte migration; negative regulation of apoptosis Disease: Thrombophilia Due To Protein C Deficiency, Autosomal Recessive; Thrombophilia Due To Protein C Deficiency, Autosomal Dominant
NCBI Summary:	This gene encodes a vitamin K-dependent plasma glycoprotein. The encoded protein is cleaved to its activated form by the thrombin-thrombomodulin complex. This activated form contains a serine protease domain and functions in degradation of the activated forms of coagulation factors V and VIII. Mutations in this gene have been associated with thrombophilia due to protein C deficiency, neonatal purpura fulminans, and recurrent venous thrombosis. [provided by RefSeq, Dec 2009]
UniProt Code:	P04070
NCBI GenInfo Identifier:	131067
NCBI Gene ID:	5624
NCBI Accession:	P04070. 1
UniProt Secondary Accession:	P04070,Q15189, Q15190, Q16001, Q53S74, Q9UC55, B4DPQ7
UniProt Related Accession:	P04070
Molecular Weight:	461
NCBI Full Name:	Vitamin K-dependent protein C
NCBI Synonym Full Names:	protein C (inactivator of coagulation factors Va and VIIIa)
NCBI Official Symbol:	PROC
NCBI Official Synonym Symbols:	PC; APC; PROC1; THPH3; THPH4
NCBI Protein Information:	vitamin K-dependent protein C; prepro-protein C; autoprothrombin IIA; anticoagulant protein C; blood coagulation factor XIV
UniProt Protein Name:	Vitamin K-dependent protein C
UniProt Synonym Protein Names:	Anticoagulant protein C; Autoprothrombin IIA; Blood coagulation factor XIVCleaved into the following 3 chains:Vitamin K-dependent protein C light chain; Vitamin K-dependent protein C heavy chain; Activation peptide
Protein Family:	Vitamin K-dependent protein
UniProt Gene Name:	PROC
UniProt Entry Name:	PROC_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
1000	49854 57496	53675	53650
500	20696 24282	22489	22464
250	10681 9741	10211	10186
125	4500 5302	4901	4876
62.5	2493 2413	2453	2428
31.25	1314 1248	1281	1256
15.63	688 728	708	683
0	25 25	25	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PROC 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)	53.61	130.21	458.17	49.14	129.61	446.75
Standard deviation	5.50	15.51	29.28	4.98	9.38	36.81
C V (%)	10.26	11.91	6.39	10.13	7.24	8.24

Recovery

The recovery of Human PROC 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)	88-99	93
EDTA plasma (n=5)	89-102	96
Cell culture media (n=5)	89-103	94

Linearity

Samples were spiked with high concentrations of Human PROC 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 (%)	89-104	99-117	99-114
1:2	Average (%)	96	106	107
1:4	Range (%)	95-108	99-111	98-115
1:4	Average (%)	101	105	105
1:8	Range (%)	90-106	86-101	89-99
1:8	Average (%)	97	93	94
1:16	Range (%)	92-106	98-112	85-96
1:16	Average (%)	97	103	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 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.