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alpha-Fetoprotein (AFP) ELISA Kit

Details for Product No. ABIN612701
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Target Name (Antigen)
Synonyms FETA, HPAFP
Reactivity
Alternatives Human
Kits with alternative reactivity to:
(5), (4), (4), (1), (2), (3), (15), (2), (8), (6), (1), (2), (6), (1), (1), (1)
Methode Type Sandwich ELISA
Minimum Detection Limit 50 pg/mL
Application
ELISA
Pubmed 8 references available
Quantity 96 tests
Options
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Catalog No. ABIN612701
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Purpose The AssayMax Alpha-Fetoprotein ELISA kit is designed for detection of human Alpha- Fetoprotein in plasma, serum and cell culture supernatants
Sample Type Plasma, Cell Culture Supernatant
Detection Method Colorimetric
Specificity This assay recognizes both natural and recombinant human Alpha-Fetoprotein. Reference Value: The normal blood level of Alpha-Fetoprotein in adult individual is averaged 1-5 ng/ml.
Components Alpha-Fetoprotein Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human Alpha-Fetoprotein. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Alpha-Fetoprotein Standard: Human Alpha-Fetoprotein in a buffered protein base (80 ng, lyophilized). 1 Biotinylated Alpha-Fetoprotein Antibody (100x): A 100-fold biotinylated polyclonal antibody against Alpha-Fetoprotein (80µl). MIx Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (90µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water.
Alternative Name Alpha-Fetoprotein
Background Alpha-Fetoprotein (AFP) is a fetal-specific glycoprotein with a molecular weight of around 70 kDa. It is expressed in the embryonic liver, by cells of the vitelline sac and by the fetal intestinal tract in the first trimester of pregnancy. After birth the synthesis of alpha-fetoprotein decreases rapidly. AFP level in adults is low but detectable. Alpha-fetoprotein has no known function in healthy adults. High level of alpha-fetoprotein in adult individual may be associated with Hepatocellular carcinoma (HCC), malignant tumor of the liver. Thus, the concentration of alpha-fetoprotein in serum can be measured as a first step in HCC diagnosis. Moreover the elevated level of alpha-fetoprotein has been observed in lung cancer , gastric cancer , yolk sac tumor and adenocarcinoma.
Research Area Serum/Plasma Proteins
Sample Volume 50 µL
Assay Time < 4 h
Plate Pre-coated,Strips (12 x 8)
Protocol This assay employs a quantitative sandwich enzyme immunoassay technique, which measures Alpha-Fetoprotein in less than 4 hours. A polyclonal antibody specific for Alpha-Fetoprotein has been pre-coated onto a microplate. Alpha-Fetoprotein in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for Alpha-Fetoprotein, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.
Reagent Preparation Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. MIx Diluent Concentrate (10x): Dilute the MIx Diluent Concentrate 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Alpha-Fetoprotein Standard: Reconstitute the 160 ng of human Alpha-Fetoprotein Standard with 4 ml of MIx Diluent to generate a stock solution of 40 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. The stock solution can be further dilute 1:4 with MIx Diluent to produce 10 ng/ml standard solution. Prepare triplicate standard points by serially diluting the Standard solution (10 ng/ml) 1:2 2 with MIx Diluent to produce 5, 2.5, 1.25, 0.625, 0.313 and 0.156 ng/ml. Sample Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [AFP] (ng/ml) P1 1 part Stock (40 ng/ml) + 3 part MIx Diluent 10.00 P2 1 part P1 + 1 part MIx Diluent 5.000 P3 1 part P2 + 1 part MIx Diluent 2.500 P4 1 part P3 + 1 part MIx Diluent 1.250 P5 1 part P4 + 1 part MIx Diluent 0.625 P6 1 part P5 + 1 part MIx Diluent 0.313 P7 1 part P5 + 1 part MIx Diluent 0.156 P8 MIx Diluent 0.000 Biotinylated Alpha-Fetoprotein Antibody (100x): Dilute the antibody 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate1: 20 with reagent grade water. SP Conjugate (100x): Dilute the conjugate 1:100 with MIx Diluent. Any remaining solution should be frozen at -20°C.
Sample Collection Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Dilute samples 1:4 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Remove serum and assay. Dilute samples 1:4 into MIx Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 2000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20°C or below. Avoid repeated freeze-thaw cycles.
Assay Procedure Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated Alpha-Fetoprotein Antibody to each well and incubate for one hour. Wash a microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 10 minutes or till the optimal color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some 3 unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.
Calculation of Results Calculate the mean value of the duplicate or triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is used for illustration only. A standard curve should be generated each time the assay is performed.
Assay Precision Intra-assay and inter-assay coefficients of variation were 4.7 % and 7.1 % respectively.
Restrictions For Research Use only
Handling Advice The kit should not be used beyond the expiration date.
Storage 4 °C/-20 °C
Storage Comment Store kit at 2-8°C or -20°C upon arrival up to the expiration date. Opened MIx Diluent may be stored for up to 1 month at 2-8°C. Store reconstituted reagents at -20°C or below. Opened unused strip wells may return to the foil pouch with the desiccant pack, reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator.
Product cited in: Blohm, Vesterling-Hörner, Calaminus et al.: "Alpha 1-fetoprotein (AFP) reference values in infants up to 2 years of age." in: Pediatric hematology and oncology, Vol. 15, Issue 2, pp. 135-42, 1998 (PubMed).

Hiroshima, Iyoda, Toyozaki et al.: "Alpha-fetoprotein-producing lung carcinoma: report of three cases." in: Pathology international, Vol. 52, Issue 1, pp. 46-53, 2002 (PubMed).

Chen, Röcken, Treiber et al.: "Clinical implications of alpha-fetoprotein expression in gastric adenocarcinoma." in: Digestive diseases (Basel, Switzerland), Vol. 21, Issue 4, pp. 357-62, 2004 (PubMed).

Sell: "Alpha-fetoprotein, stem cells and cancer: how study of the production of alpha-fetoprotein during chemical hepatocarcinogenesis led to reaffirmation of the stem cell theory of cancer." in: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, Vol. 29, Issue 3, pp. 161-80, 2008 (PubMed).

Kelleher, Colum, Doyle et al.: "Alpha fetoprotein in metastatic gastric carcinoma." in: Gut, Vol. 15, Issue 5, pp. 401-3, 2010 (PubMed).

Gomaa, Khan, Leen et al.: "Diagnosis of hepatocellular carcinoma." in: World journal of gastroenterology : WJG, Vol. 15, Issue 11, pp. 1301-14, 2009 (PubMed).

Chen, Ho, Lee et al.: "Enhanced detection of early hepatocellular carcinoma by serum SELDI-TOF proteomic signature combined with alpha-fetoprotein marker." in: Annals of surgical oncology, Vol. 17, Issue 9, pp. 2518-25, 2010 (PubMed).

Pauniaho, Tatti, Lahdenne et al.: "Tumor markers AFP, CA 125, and CA 19-9 in the long-term follow-up of sacrococcygeal teratomas in infancy and childhood." in: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, Vol. 31, Issue 4, pp. 261-5, 2010 (PubMed).

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