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Human FG alpha (Fibrinogen Alpha) ELISA Kit (HUES03019)

SKU:
HUES03019
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P02671
Sensitivity:
3.75ng/mL
Range:
6.25-400ng/mL
ELISA Type:
Sandwich
Reactivity:
Human
Sample Type:
Serum, plasma and other biological fluids
Research Area:
Immunology
€599
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Description

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Assay type:Sandwich
Format:96T
Assay time:4.5h
Reactivity:Human
Detection Method:Colormetric
Detection Range:6.25-400 ng/mL
Sensitivity:3.75 ng/mL
Sample Volume Required Per Well:100µL
Sample Type:Serum, plasma and other biological fluids
Specificity:This kit recognizes Human FG alpha in samples. No significant cross-reactivity or interference between Human FG alpha 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 FG alpha. 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 FG alpha 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 FG alpha, 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 FG alpha. The concentration of Human FG alpha in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:FGA: Fibrinogen has a double Function: yielding monomers that polymerize into fibrin and acting as a cofactor in platelet aggregation. Defects in FGA are a cause of congenital afibrinogenemia (CAFBN). This is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. The majority of cases of afibrinogenemia are due to truncating mutations. Variations in position Arg-35 (the site of cleavage of fibrinopeptide a by thrombin) leads to alpha- dysfibrinogenemias. Defects in FGA are a cause of amyloidosis type 8 (AMYL8); also known as systemic non-neuropathic amyloidosis or Ostertag-type amyloidosis. AMYL8 is a hereditary generalized amyloidosis due to deposition of apolipoprotein A1, fibrinogen and lysozyme amyloids. Viscera are particularly affected. There is no involvement of the nervous system. Clinical features include renal amyloidosis resulting in nephrotic syndrome, arterial hypertension, hepatosplenomegaly, cholestasis, petechial skin rash. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 4q28

Cellular Component: extracellular space; cell surface; fibrinogen complex; plasma membrane; extracellular region; cell cortex; vesicle; external side of plasma membrane

Molecular Function:protein binding, bridging; protein binding; cell adhesion molecule binding; structural molecule activity; receptor binding

Biological Process: protein polymerization; platelet activation; extracellular matrix organization and biogenesis; positive regulation of heterotypic cell-cell adhesion; cell-matrix adhesion; signal transduction; cellular protein complex assembly; platelet degranulation; positive regulation of protein secretion; positive regulation of vasoconstriction; innate immune response; blood coagulation; response to calcium ion; positive regulation of exocytosis

Disease: Amyloidosis, Familial Visceral; Afibrinogenemia, Congenital; Dysfibrinogenemia, Congenital

NCBI Summary:The protein encoded by this gene is the alpha component of fibrinogen, a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots. In addition, various cleavage products of fibrinogen and fibrin regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types. Mutations in this gene lead to several disorders, including dysfibrinogenemia, hypofibrinogenemia, afibrinogenemia and renal amyloidosis. Alternative splicing results in two isoforms which vary in the carboxy-terminus. [provided by RefSeq, Jul 2008]
UniProt Code:P02671
NCBI GenInfo Identifier:1706799
NCBI Gene ID:2243
NCBI Accession:P02671. 2
UniProt Secondary Accession:P02671,Q4QQH7, Q9BX62, Q9UCH2, A8K3E4, D3DP14, D3DP15
UniProt Related Accession:P02671
Molecular Weight:69,757 Da
NCBI Full Name:Fibrinogen alpha chain
NCBI Synonym Full Names:fibrinogen alpha chain
NCBI Official Symbol:FGA
NCBI Official Synonym Symbols:Fib2
NCBI Protein Information:fibrinogen alpha chain; fibrinogen, A alpha polypeptide
UniProt Protein Name:Fibrinogen alpha chain
Protein Family:Fibrinogen
UniProt Gene Name:FGA
UniProt Entry Name:FIBA_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.DAverageCorrected
4002.351
2.359		2.3552.299
2001.66
1.71		1.6851.629
1000.955
0.925		0.940.884
500.463
0.497		0.480.424
250.273
0.251		0.2620.206
12.50.171
0.147		0.1590.103
6.250.105
0.113		0.1090.053
00.055
0.057		0.056--

Precision

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

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

Intra-assay PrecisionInter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)19.9654.13167.4019.5152.54175.95
Standard deviation1.292.978.651.282.538.97
C V (%)6.465.495.176.564.825.10

Recovery

The recovery of Human FG alpha spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample TypeRange (%)Average Recovery (%)
Serum (n=5)85-9590
EDTA plasma (n=5)91-10999
Cell culture media (n=5)87-10395

Linearity

Samples were spiked with high concentrations of Human FG alpha 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 (%)97-110100-11196-112
Average (%)103105103
1:4Range (%)86-10084-9587-99
Average (%)939094
1:8Range (%)88-10282-9488-102
Average (%)938794
1:16Range (%)90-10481-9486-101
Average (%)978793

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
  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.

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