Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	9.38-600 ng/mL
Sensitivity:	5.63 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human PT in samples. No significant cross-reactivity or interference between Human PT 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 PT. 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 PT 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 PT, 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 PT. The concentration of Human PT in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	Function: Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing. Ref. 12
UniProt Protein Details:	Catalytic activity: Selective cleavage of Arg-\|-Gly bonds in fibrinogen to form fibrin and release fibrinopeptides A and B. Subcellular location: Secreted extracellular space. Tissue specificity: Expressed by the liver and secreted in plasma. Post-translational modification: The gamma-carboxyglutamyl residues, which bind calcium ions, result from the carboxylation of glutamyl residues by a microsomal enzyme, the vitamin K-dependent carboxylase. The modified residues are necessary for the calcium-dependent interaction with a negatively charged phospholipid surface, which is essential for the conversion of prothrombin to thrombin. Involvement in Disease: Defects in F2 are the cause of factor II deficiency (FA2D) [ MIM:613679]. It is a very rare blood coagulation disorder characterized by mucocutaneous bleeding symptoms. The severity of the bleeding manifestations correlates with blood factor II levels. Ref. 2 Ref. 30 Ref. 31 Ref. 32 Ref. 33 Ref. 34 Ref. 35 Ref. 36 Ref. 37 Ref. 38 Ref. 39 Ref. 40Genetic variations in F2 may be a cause of susceptibility to ischemic stroke (ISCHSTR) [ MIM:601367]; also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors. Ref. 43Defects in F2 are a cause of susceptibility to thrombosis (THR) [ MIM:188050]. It is a multifactorial disorder of hemostasis characterized by abnormal platelet aggregation in response to various agents and recurrent thrombi formation. Note=A common genetic variation in the 3-prime untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis. Ref. 32 Ref. 41 Pharmaceutical use: The peptide TP508 also known as Chrysalin (Orthologic) could be used to accelerate repair of both soft and hard tissues. Miscellaneous: Prothrombin is activated on the surface of a phospholipid membrane that binds the amino end of prothrombin and factors Va and Xa in Ca-dependent interactions; factor Xa removes the activation peptide and cleaves the remaining part into light and heavy chains. The activation process starts slowly because factor V itself has to be activated by the initial, small amounts of thrombin. It is not known whether 1 or 2 smaller activation peptides, with additional cleavage after Arg-314, are released in natural blood clotting. Thrombin can itself cleave the N-terminal fragment (fragment 1) of the prothrombin, prior to its activation by factor Xa. The cleavage after Arg-198, observed in vitro, does not occur in plasma. Sequence similarities: Belongs to the peptidase S1 family. Contains 1 Gla (gamma-carboxy-glutamate) domain. Contains 2 kringle domains. Contains 1 peptidase S1 domain.
NCBI Summary:	Coagulation factor II is proteolytically cleaved to form thrombin in the first step of the coagulation cascade which ultimately results in the stemming of blood loss. F2 also plays a role in maintaining vascular integrity during development and postnatal life. Mutations in F2 leads to various forms of thrombosis and dysprothrombinemia. [provided by RefSeq]
UniProt Code:	P00734
NCBI GenInfo Identifier:	135807
NCBI Gene ID:	2147
NCBI Accession:	P00734. 2
UniProt Secondary Accession:	P00734,Q4QZ40, Q53H04, Q53H06, Q7Z7P3, Q9UCA1, B2R7F7 B4E1A7,
UniProt Related Accession:	P00734,Q15253,Q16505,Q69EZ7,Q69EZ8,Q86WA1,Q8TD58
Molecular Weight:	70,037 Da
NCBI Full Name:	Prothrombin
NCBI Synonym Full Names:	coagulation factor II (thrombin)
NCBI Official Symbol:	F2
NCBI Official Synonym Symbols:	PT
NCBI Protein Information:	prothrombin; serine protease; OTTHUMP00000197117; OTTHUMP00000233661; prothrombin B-chain
UniProt Protein Name:	Prothrombin
UniProt Synonym Protein Names:	Coagulation factor II
Protein Family:	Prothrombin
UniProt Gene Name:	F2
UniProt Entry Name:	THRB_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 (ng/mL)	O.D	Average	Corrected
600	2.473 2.483	2.478	2.39
300	1.663 1.665	1.664	1.576
150	0.986 0.954	0.97	0.882
75	0.546 0.564	0.555	0.467
37.5	0.316 0.304	0.31	0.222
18.75	0.199 0.187	0.193	0.105
9.38	0.134 0.15	0.142	0.054
0	0.083 0.093	0.088	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human PT 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)	28.24	72.85	262.16	30.55	68.88	263.52
Standard deviation	1.52	4.14	14.39	1.96	3.35	13.70
C V (%)	5.38	5.68	5.49	6.42	4.86	5.20

Recovery

The recovery of Human PT 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)	87-103	94
EDTA plasma (n=5)	91-106	97
Cell culture media (n=5)	93-106	99

Linearity

Samples were spiked with high concentrations of Human PT 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 (%)	90-104	89-103	86-97
1:2	Average (%)	96	96	91
1:4	Range (%)	92-107	85-97	84-94
1:4	Average (%)	99	91	89
1:8	Range (%)	92-107	82-95	83-97
1:8	Average (%)	98	88	90
1:16	Range (%)	86-100	88-98	81-92
1:16	Average (%)	93	93	87

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.