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 F7 in samples. No significant cross-reactivity or interference between Human F7 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 F7. 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 F7 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 F7, 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 F7. The concentration of Human F7 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	F7: Initiates the extrinsic pathway of blood coagulation. Serine protease that circulates in the blood in a zymogen form. Factor VII is converted to factor VIIa by factor Xa, factor XIIa, factor IXa, or thrombin by minor proteolysis. In the presence of tissue factor and calcium ions, factor VIIa then converts factor X to factor Xa by limited proteolysis. Factor VIIa will also convert factor IX to factor IXa in the presence of tissue factor and calcium. Defects in F7 are the cause of factor VII deficiency (FA7D). A hemorrhagic disease with variable presentation. The clinical picture can be very severe, with the early occurrence of intracerebral hemorrhages or repeated hemarthroses, or, in contrast, moderate with cutaneous-mucosal hemorrhages (epistaxis, menorrhagia) or hemorrhages provoked by a surgical intervention. Finally, numerous subjects are completely asymptomatic despite very low factor VII levels. Belongs to the peptidase S1 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3. 4. 21. 21; Protease; Apoptosis; Secreted; Secreted, signal peptide; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 13q34 Cellular Component: endoplasmic reticulum lumen; extracellular region; Golgi lumen; plasma membrane Molecular Function:glycoprotein binding; protein binding; serine-type peptidase activity Biological Process: blood coagulation; blood coagulation, extrinsic pathway; ER to Golgi vesicle-mediated transport; peptidyl-glutamic acid carboxylation; positive regulation of cell migration; positive regulation of leukocyte chemotaxis; positive regulation of positive chemotaxis; positive regulation of protein kinase B signaling cascade; signal peptide processing Disease: Factor Vii Deficiency; Myocardial Infarction, Susceptibility To
NCBI Summary:	This gene encodes coagulation factor VII which is a vitamin K-dependent factor essential for hemostasis. This factor circulates in the blood in a zymogen form, and is converted to an active form by either factor IXa, factor Xa, factor XIIa, or thrombin by minor proteolysis. Upon activation of the factor VII, a heavy chain containing a catalytic domain and a light chain containing 2 EGF-like domains are generated, and two chains are held together by a disulfide bond. In the presence of factor III and calcium ions, the activated factor then further activates the coagulation cascade by converting factor IX to factor IXa and/or factor X to factor Xa. Defects in this gene can cause coagulopathy. Alternative splicing results in multiple transcript variants encoding different isoforms that may undergo similar proteolytic processing to generate mature polypeptides. [provided by RefSeq, Aug 2015]
UniProt Code:	P08709
NCBI GenInfo Identifier:	119766
NCBI Gene ID:	2155
NCBI Accession:	P08709. 1
UniProt Secondary Accession:	P08709,Q14339, Q5JVF1, Q5JVF2, Q9UD52, Q9UD53, Q9UD54 B0YJC8,
UniProt Related Accession:	P08709
Molecular Weight:	49,320 Da
NCBI Full Name:	Coagulation factor VII
NCBI Synonym Full Names:	coagulation factor VII
NCBI Official Symbol:	F7
NCBI Official Synonym Symbols:	SPCA
NCBI Protein Information:	coagulation factor VII
UniProt Protein Name:	Coagulation factor VII
UniProt Synonym Protein Names:	Proconvertin; Serum prothrombin conversion accelerator; SPCA
Protein Family:	F7-1 fimbrial protein
UniProt Gene Name:	F7
UniProt Entry Name:	FA7_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	50294 57056	53675	53650
500	21586 23392	22489	22464
250	10673 9749	10211	10186
125	4659 5143	4901	4876
62.5	2574 2332	2453	2428
31.25	1303 1259	1281	1256
15.63	678 738	708	683
0	24 26	25	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human F7 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human F7 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)	53.63	105.07	393.82	55.69	101.27	363.99
Standard deviation	4.37	8.80	27.92	6.20	10.84	24.53
C V (%)	8.15	8.38	7.09	11.13	10.70	6.74

Recovery

The recovery of Human F7 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)	95-109	101
EDTA plasma (n=5)	90-106	97
Cell culture media (n=5)	91-103	97

Linearity

Samples were spiked with high concentrations of Human F7 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 (%)	101-117	88-103	85-100
1:2	Average (%)	109	94	91
1:4	Range (%)	102-116	101-114	88-101
1:4	Average (%)	109	107	95
1:8	Range (%)	91-105	86-97	103-117
1:8	Average (%)	98	92	109
1:16	Range (%)	85-95	101-118	101-116
1:16	Average (%)	90	109	108

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.