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Human Factor VIII / F8 ELISA Kit

SKU:
HUFI00820
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P00451
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
F8, Coagulation Factor ?, F8C, Antihemophilic factor, AHF, Procoagulant component
Reactivity:
Human
Research Area:
Immunology
€599
Frequently bought together:

Description

Human Factor VIII/F8 ELISA Kit

The Human Factor VIII (F8) ELISA Kit is specifically designed for the precise measurement of Factor VIII levels in human samples such as serum and plasma. This kit boasts exceptional sensitivity and specificity, guaranteeing consistent and accurate results for various research purposes.Factor VIII is a key protein in the blood clotting cascade, essential for the proper formation of blood clots. Dysregulation of Factor VIII levels can lead to bleeding disorders such as hemophilia A.

The Human Factor VIII ELISA Kit enables researchers to monitor Factor VIII levels in biological samples, aiding in the diagnosis and management of bleeding disorders.Whether investigating hemostasis disorders or assessing the efficacy of treatments targeting Factor VIII, this ELISA kit provides a reliable tool for researchers in the field of coagulation disorders. Advance your research with the Human Factor VIII ELISA Kit from Assay Genie.

Product Name:Human Factor VIII / F8 ELISA Kit
Product Code:HUFI00820
Size:96 Assays
Alias:F8, Coagulation Factor 8, F8C, Antihemophilic factor, AHF, Procoagulant component
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human F8 concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human F8 and the recovery rates were calculated by comparing the measured value to the expected amount of Human F8 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10394
EDTA plasma(n=5)85-10295
UFH plasma(n=5)89-10496
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human F8 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-104%86-98%85-105%
EDTA plasma(n=5)89-100%82-91%84-95%
UFH plasma(n=5)87-100%89-99%80-91%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotP00451
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, signal peptide; Secreted

Chromosomal Location of Human Ortholog: Xq28

Cellular Component: extracellular space; extracellular region; plasma membrane

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

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

Disease: Hemophilia A; Factor Viii Deficiency

NCBI Summary:This gene encodes coagulation factor VIII, which participates in the intrinsic pathway of blood coagulation; factor VIII is a cofactor for factor IXa which, in the presence of Ca+2 and phospholipids, converts factor X to the activated form Xa. This gene produces two alternatively spliced transcripts. Transcript variant 1 encodes a large glycoprotein, isoform a, which circulates in plasma and associates with von Willebrand factor in a noncovalent complex. This protein undergoes multiple cleavage events. Transcript variant 2 encodes a putative small protein, isoform b, which consists primarily of the phospholipid binding domain of factor VIIIc. This binding domain is essential for coagulant activity. Defects in this gene results in hemophilia A, a common recessive X-linked coagulation disorder. [provided by RefSeq, Jul 2008]
UniProt Code:P00451
NCBI GenInfo Identifier:119767
NCBI Gene ID:2157
NCBI Accession:P00451.1
UniProt Secondary Accession:P00451,Q14286, Q5HY69,
UniProt Related Accession:P00451
Molecular Weight:24,641 Da
NCBI Full Name:Coagulation factor VIII
NCBI Synonym Full Names:coagulation factor VIII, procoagulant component
NCBI Official Symbol:F8
NCBI Official Synonym Symbols:AHF; F8B; F8C; HEMA; FVIII; DXS1253E
NCBI Protein Information:coagulation factor VIII; factor VIII F8B; antihemophilic factor; coagulation factor VIIIc
UniProt Protein Name:Coagulation factor VIII
UniProt Synonym Protein Names:Antihemophilic factor; AHF; Procoagulant componentCleaved into the following 4 chains:Factor VIIIa heavy chain, 200 kDa isoform; Factor VIIIa heavy chain, 92 kDa isoform; Factor VIII B chain; Factor VIIIa light chain
Protein Family:Factor VIII intron 22 protein
UniProt Gene Name:F8
UniProt Entry Name:FA8_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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