Mouse FVIII (Coagulation Factor VIII) ELISA Kit (MOES00873)

SKU:
MOES00873
€299

Description

ELISA Kit Technical ManualMSDS

Mouse FVIII (Coagulation Factor VIII) ELISA Kit

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 Mouse FVIII . 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 Mouse FVIII 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 Mouse FVIII, 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 Mouse FVIII. The concentration of Mouse FVIII in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityMouse
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Mouse FVIII in samples. No significant cross-reactivity or interference between Mouse FVIII and analogues was observed.

Typical Data

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
202.469
2.497
2.4832.424
101.543
1.585
1.5641.505
50.952
0.92
0.9360.877
2.50.442
0.454
0.4480.389
1.250.267
0.265
0.2660.207
0.630.171
0.153
0.1620.103
0.310.104
0.118
0.1110.052
00.057
0.061
0.059--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse FVIII were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.033.179.681.012.9310.02
Standard deviation0.050.150.480.060.170.53
C V (%)4.854.734.965.945.805.29

Recovery

The recovery of Mouse FVIII 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)96-109102
EDTA plasma (n=5)90-10196
Cell culture media (n=5)88-10193

Linearity

Samples were spiked with high concentrations of Mouse FVIII 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)
1:2Range (%)90-10086-10086-101
Average (%)959392
1:4Range (%)88-10184-9785-98
Average (%)959092
1:8Range (%)89-10479-9389-102
Average (%)958594
1:16Range (%)89-10381-9388-101
Average (%)948793

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Mouse FVIII (Coagulation Factor VIII) ELISA Kit (MOES00873) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Mouse FVIII (Coagulation Factor VIII) ELISA Kit (MOES00873) Protein Information

UniProt Protein Function:F8: Factor VIII, along with calcium and phospholipid, acts as a cofactor for factor IXa when it converts factor X to the activated form, factor Xa. Defects in F8 are the cause of hemophilia A (HEMA). A disorder of blood coagulation characterized by a permanent tendency to hemorrhage. About 50% of patients have severe hemophilia resulting in frequent spontaneous bleeding into joints, muscles and internal organs. Less severe forms are characterized by bleeding after trauma or surgery. Of particular interest for the understanding of the function of F8 is the category of CRM (cross-reacting material) positive patients (approximately 5%) that have considerable amount of F8 in their plasma (at least 30% of normal), but the protein is non- functional; i.e. the F8 activity is much less than the plasma protein level. CRM-reduced is another category of patients in which the F8C antigen and activity are reduced to approximately the same level. Most mutations are CRM negative, and probably affect the folding and stability of the protein. Belongs to the multicopper oxidase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide

Cellular Component: extracellular space; extracellular region

Molecular Function:copper ion binding; metal ion binding; serine-type endopeptidase activity; oxidoreductase activity

Biological Process: platelet activation; hemostasis; acute-phase response; proteolysis; blood coagulation; blood coagulation, intrinsic pathway

UniProt Code:Q06194
NCBI GenInfo Identifier:238624180
NCBI Gene ID:14069
NCBI Accession:NP_032003.2
UniProt Secondary Accession:Q06194,A2AN88,
UniProt Related Accession:Q06194
Molecular Weight:
NCBI Full Name:coagulation factor VIII isoform 1
NCBI Synonym Full Names:coagulation factor VIII
NCBI Official Symbol:F8  
NCBI Official Synonym Symbols:Cf8; Cf-8; FVIII  
NCBI Protein Information:coagulation factor VIII; Factor VIII; procoagulant component
UniProt Protein Name:Coagulation factor VIII
UniProt Synonym Protein Names:Procoagulant component
Protein Family:Coagulation factor
UniProt Gene Name:F8  
UniProt Entry Name:FA8_MOUSE
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Additional Information

Product type:
ELISA
Reactivity:
Mouse
ELISA Type:
Sandwich
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