Alpha-Fetoprotein (AFP) Enzyme Immunoassay Kit

Catalog Number: ART-IA1001
$420.00

Product Attachments

 

Alpha-Fetoprotein (AFP) Enzyme Immunoassay Kit

Cat# ART- IA1001

Name

AFP ELISA

Full name

Human AFP (Alpha Fetoprotein) ELISA Test Kit

Category Name

Cancer ELISA kits

Catalog number

ART-IA1001

Test

96

Method

ELISA method: Enzyme Linked Immunosorbent Assay

Principle

Sandwich Complex

Detection Range

0-300 ng/mL

Sample

20ul serum, plasma with EDTA/heparin, cell culture supernates

Specificity

98.5%

Sensitivity

2.0 ng/mL

Total Time

~80min

Shelf Life

12 Months from the manufacturing date

 

Intended Use

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

Background

Alpha-fetoprotein (AFP) is a glycoprotein with a molecular weight 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.

Entrez Gene IDs  

174 (Human); 11576 (Mouse); 24177 (Rat)

Alternate Names        

AFP; Alpha-1-fetoprotein; alpha-fetoglobulin; alpha-Fetoprotein; DSCAM2; FETA; HP; HPAFP

NCBI Accession Number

NP_001125.1

Swiss Prot Number

P02771

Subcellular location

Secreted

 

  1. Secure the desired number of coated wells in the holder.
  2. Dispense 20µl of standard, specimens, and controls into appropriate wells.
  3. Dispense 100 µl 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, or using a plate

    washer.

  1. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
  2. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets. 9. Dispense 150µl of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
  3. Incubate at room temperature for 30 minutes.
  4. Remove the incubation mixture by flicking plate contents into a waste container.
  5. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
  6. Strike the wells sharply onto absorbent paper to remove residual water droplets.
  7. Dispense 100µl TMB substrate into each well. Gentle mix for 5 seconds.
  8. Incubate at room temperature for 20 minutes.
  9. Stop the reaction by adding 100µl of stop solution to each well.
  10. Gently mix for 30 seconds to make sure that the blue color changes to yellow color completely.
  11. Read optical density at 450nm 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.

 

Results

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 450nm 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.

 AFP ELISA

Sample 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.

Performance

I Accuracy:

Comparison between our Kits and Commercial Available Kits provides the following data:

N = 79
Correlation Coefficient = 0.985
Slope = 1.038 Intercept = 0.729
Mean (Our) = 55.12
Mean (Abbott) = 52.24

II Precision:

1) Intra-assay

Concentrations

N

Mean

S.D.

%CV

Group1

24

31.04

1.45

4.66

Group2

24

126.8

5.2

4.1

Group3

24

270.8

12.09

4.78

2) Inter-assay

Concentrations

N

Mean

S.D.

%CV

Group1

24

30.58

1.88

6.1

Group2

24

125.1

7.08

5.7

Group3

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 102.2 %.

IV Recovery

Various patient serum samples of known AFP levels were mixed and assayed in duplicate. The average recovery was 101.1 %.

V Cross-reactivity

The following human materials were tested for crossreactivity of the assay.

Limitation of the assay

There are some limitations of the assay:

  1. As with all diagnostic grade tests, a definite clinical diagnosis should not be based on the results of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated.
  2. Studies have implicated possible interference in immunoassay results in some patients with known rheumatoid factor and antinuclear antibodies. Serum samples from patients who have received infusions containing mouse monoclonal antibodies for diagnostic or therapeutic purposes, may contain antibody to mouse protein (HAMA). Although we have added some agents to avoid the interferences, we cannot guarantee it will eliminate all the effects of that.

Storage

  1. Unopened test kits should be stored at 2-8oC upon receipt.
  2. The microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. 3. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
  3. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-2 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

 

Assay Solution's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.
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