Human Fetuin-A / AHSG / FETUA ELISA Pair Set

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Target Name (Antigen)
  • ahs
  • a2hs
  • hsga
  • fetua
  • wu:fb63g02
  • zgc:103687
  • AHSG
  • MGC116429
  • A2HS
  • AHS
  • HSGA
  • Aa2-066
  • pp63
  • alpha-2-HS-glycoprotein
  • alpha 2-HS glycoprotein
  • alpha-2-HS-glycoprotein 1
  • alpha-2-HS-glycoprotein L homeolog
  • Cphamn1_1981
  • AHSG
  • ahsg
  • ahsg1
  • ahsg.L
  • Ahsg
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Purpose The ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utlizes monoclonal antibody specific for Fetuin-A (AHSG / FETUA) coated on a 96-well plate. Standards and samples areadded to the wells, and any Fetuin-A (AHSG / FETUA) present binds to the immobilized antibody. The wells arewashed and a biotinylated rabbit anti-Fetuin-A (AHSG / FETUA) polyclonal antibody is then added, producing anantibody-antigen-antibody "sandwich". To produces color in proportion to the amount of Fetuin-A (AHSG / FETUA) present in the sample strepavidin-HRP and TMB substrate solution are loaded. The absorbances of the microwell areread at 450 nm.
Sensitivity The minimum detectable dose of human Fetuin-A ( AHSG / FETUA ) was determined to be approximately 31.25 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Characteristics Pair Set
Components Capture Antibody - 0.5 mg/mL of mouse anti-Fetuin-A monoclonal antibody. Dilute to a workingconcentration of 2 μg/mL in CBS before coating.
Detection Antibody - Each vial contains 120 μg biotinylated rabbit anti-Fetuin-A polyclonalantibody. Reconstitute with sterile 1 mL distilled water. Dilute to a working concentration of 2 μg/mLin detection antibody dilution buffer before use.
Standard - Each vial contains 52ng of recombinant Fetuin-A. Reconstitute with 1 mL detectionantibody dilution buffer. A seven-point standard curve using 2-fold serial dilutions in sample dilutionbuffer, and a high standard of 2000 pg/mL is recommended.
Streptavidin-HRP - 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000 Dilution indetection antibody dilution buffer before use
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name AHSG
Background Human Fetuin-A, also known as α2-Heremans-Schmid glycoprotein (AHSG) , is a member of cystatin superfamilyof protease inhibitors. It is expressed and secreted mainly by the cell source hepatocytes, and can be found athigh levels in the serum as a growth promoter. As an antagonist of transforming growth factor-beta1 (TGF-β1) , fetuin-A blocks TGF-beta1 binding to cell surface receptors, suppresses TGF-beta signal transduction andepithelial-mesenchymal transition, suggesting that the systemic fetuin A is closely associated with tumorprogression and resistance to chemotherapy in established cancers, as well as host immune suppression. Accordingly, therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-beta. It has been shown that fetuin A inhibits both insulin receptor autophosphorylation and undesirablecalcification, and plays a role in bone development, calcium homeostasis, regulation of soft tissue calcification, and insulin sensitivity. Fetuin-A is a major inhibitor of ectopic calcium phosphate precipitation and an acute phasereactant. Its deficiency, common in end-stage renal disease, has been suggested to be associated withcardiovascular complications. Interleukin-18 (IL-18) and Fetuin-A have been implicated in atherosclerosis. Preliminary evidence suggests that ankylosing spondylitis (AS) is associated with an increased risk ofatherosclerosis. Fetuin-A and IL-18 levels seem to be increased in AS patients regardless of disease activity andtreatment type. Fetuin-A prevents tissue calcification by forming soluble complexes with calcium and phosphate. A pathological depletion of serum fetuin-A has been observed in children on dialysis or after renaltransplantation. Higher serum Fetuin-A concentrations are associated with type 2 diabetes longitudinally andgreater adiposity in cross-sectional analyses. The high serum Fetuin-A concentrations in preterm childrensuggest that Fetuin-A is of high physiologic impact for the fetal and the preterm-born organism, showingextensive tissue formation. This might point to a new mechanism contributing to organ damage in these patients, comparable with children on dialysis.
Application Notes Optimal working dilution should be determined by the investigator.

The human Fetuin-A ( AHSG / FETUA ) ELISA Pair Set is for the quantitative determination of human Fetuin-A.This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.

Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
    6. Repeat the aspiration/wash as in step 2 of plate preparation.
    7. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    8. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
Calculation of Results

Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Restrictions For Research Use only
Format Lyophilized
Precaution of Use The Stop Solution suggested for use with this Pair Set is an acid solution. Wear eye, hand, face, andclothing protection when using this material.
Handling Advice Avoid repeated freeze-thaw cycles.
Storage 4 °C/-20 °C/-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Detection Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Streptavidin-HRP: Store at 4°C and protect it from prolonged exposure to light. DO NOT FREEZE! It is stable for up to 6 months from date of receipt.
Expiry Date 6 months
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