alpha Fetoprotein ELISA Kit

Catalog No. ABIN996898

Human alpha Fetoprotein ELISA Kit, Colorimetric assay for quantification of Human alpha Fetoprotein.

Quick Overview for alpha Fetoprotein ELISA Kit (ABIN996898)

Target: alpha Fetoprotein (AFP) (alpha-Fetoprotein (AFP))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 0-300 ng/mL

Application: ELISA

Sample Type: Serum

Product Details alpha Fetoprotein ELISA Kit

Minimum Detection Limit: 0 ng/mL

Purpose: AFP Enzyme Immunoassay test kit is intended for the quantitative determination of AFP concentration in human serum.

Analytical Method: Quantitative

Specificity: 97%

Sensitivity: 2.0 ng/mL

Material not included: 1. Precision pipettes: 0.0 mL , 0. 4 till approx. 0.2 mL .
2. Disposable pipette tips.
3. Distilled water.
4. Vortex mixer or equivalent.
5. Absorbent paper or paper towel.
6. Graph paper.
7. Microtiter plate reader.

Application Details

Sample Volume: 20 μL

Assay Time: 1 - 2 h

Plate: Pre-coated

Reagent Preparation:

1. All reagent should be brought to room temperature (18-22 °C) before us.
   2. Dilute 1 volume of Wash Buffer (50x) with 49 volumes of distilled water. For example, Dilute 15 mL of Wash Buffer (50x) into distilled water to prepare 750 mL of washing buffer (1x). Mix well before use.

Sample Preparation:

Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. This kit is for use with serum samples without additives only.

Assay Procedure:

1. Secure the desired number of coated wells in the holder.
   2. Dispense 20 mL of standard, specimens, and controls into appropriate wells.
   3. Dispense 100 mL of zero buffer into each well.
   4. Thoroughly mix for 10 seconds. It is very important to have complete mixing in this setup.
   5. Incubate at room temperature (18-22 °C) for 30 minutes.
   6. Remove the incubation mixture by flicking plate content into a waste container.
   7. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
   8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
   9. Dispense 150 mL of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
   10. Incubate at room temperature for 30 minutes.
   11. Remove the incubation mixture by flicking plate contents into a waste container.
   12. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
   13. Strike the wells sharply onto absorbent paper to remove residual water droplets.
   14. Dispense 100 mL TMB substrate into each well. Gentle mix for 5 seconds.
   15. Incubate at room temperature for
   2. minutes.
   16. Stop the reaction by adding 100 mL of stop solution to each well.
   17. Gently mix for 30 seconds to make sure that the blue color changes to yellow color completely.
   18. Read optical density at 450 nm with a microtiter reader within 15 minutes.
   
   Important Note:
   The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

Calculation of Results:

I. Accuracy: Comparison between Our Kits and Commercial Available Kits provides the following data N = 79 Correlation Coefficient = Slope =
1.038 Intercept = 0.729 Mean (Our) = 55.12 Mean (Abbott) = 5
2.24 0.985 II. Precision. 1]. Intra-Assay: Concentrations Replicates Mean S.D. % CV Level I 24 3
1.04
1.45
4.66 Level II 24 126.8
5.20
4.10 Level III 24 270.8 1
2.93
4.78 2]. Inter-Assay: Concentrations Replicates Mean S.D. % CV Level I 24 30.58
1.88
6.1 Level II 24 125.1
7.08
5.7 Level III 24 268.3 14.06
5.2 III. Linearity Two patient sera were serially diluted with 0 ng/mL standard in a linearity study. The average recovery was 10
2.2 %. AFP (ng/mL) Absorbance (450nm) 0 0.012 5 0.127 20 0.455 50 0.952 150
2.150 300
2.932 Sample Dilution Expected Observed % Recov. undiluted 27
1.20 27
1.20 2x 135.60 137.12 10
1.1 4x 67.80 69.45 10
2.4 8x 3
3.90 35.16 10
3.7 16x 16.95 17.20 10
1.5 32x
8.48
9.31 109.9 Average Recovery: 10
2.2 % IV. Recovery Various patient serum samples of known AFP levels were mixed and assayed in duplicate. The average recovery was 10
1.1 %. Conc. of AFP (ng/mL) Expected Concentration Observed Concentration % Recovery 9
2.70 94.20 10
1.6 77.63 79.31 10
2.2 2
3.24 2
3.10 99.4 156.18 159.23 10
2.0 206.06 210.10 10
2.0 148.94 150.52 10
1.1 149.73 148.97 99.50 Average Recovery: 10
1.1 %
Calculate the mean absorbance value (A450) for each set of reference standards, specimens, controls and patient samples. Constructed a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in ng/mL on graph paper, with absorbance values on the vertical or Y-axis and concentrations on the horizontal or X-axis. Use the mean absorbance values for each specimen to determine the corresponding concentration of AFP in ng/mL from the standard curve. Example of standard curve Results of typical standard run with optical density reading at 450 nm shown in the Y-axis against AFP concentrations shown in the X-axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve. Expected values and sensitivity In high-risk patients, AFP values between 100 and 350 ng/mL suggest a diagnosis of hepatocellular carcinoma, and levels over 350 ng/mL usually indicate the disease. Approximately 97 % of the healthy subjects have AFP levels less than 8.5 ng/mL. It is recommended that each laboratory establish its own normal range. The minimum detectable concentration of AFP by this assay is estimated to be 2.0 ng/mL.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Expiry Date: 12-14 months

Target Details for alpha Fetoprotein

Target: alpha Fetoprotein (AFP) (alpha-Fetoprotein (AFP))

Alternative Name: AFP (Alpha Fetoprotein)

Background: Alpha-fetoprotein (AFP) is a glycoprotein with a molecular weigh of approximately 70,000 daltons. AFP is normally produced during fetal and neonatal development by the liver, yolksac, and in small concentrations by the gastrointestinal tract. After birth, serum AFP concentrations decrease rapidly, and by the second year of life and thereafter only trace amounts are normally detected in serum. Elevation of serum AFP to abnormally high values occurs in several malignant diseases, most notably nonseminomatous testicular cancer and primary hepatocellular carcinoma. In the case of nonseminomatous testicular cancer, a direct relationship has been observed between the incidence of elevated AFP levels and the stage of disease. Elevated AFP levels have also been observed in patients diagnosed with seminoma with nonseminomatous elements, but not in patients with pure seminoma. In addition, elevated serum AFP concentrations have been measured in patients with other noncancerous diseases, including ataxia telangiectasia, hereditary tyrosinemia, neonatal hyperbilirubinemia, acute viral hepatitis, chronic active hepatitis, and cirrhosis. Elevated serum AFP concentrations are also observed in pregnant women. Therefore, AFP measurements are not recommended for use as a screening procedure to detect the presence of cancer in the general population.

Pathways: C21-Steroid Hormone Metabolic Process