1

Human Alpha fetoprotein ELISA
Cat. No.: BA1003
Enzyme Immunoassay for the quantitative determination of Alpha
fetoprotein (AFP) in human serum and plasma.
Alpha-fetoprotein (AFP) is a 70-kDa tumor-associated fetal mammalian
glycoprotein which belongs to the gene family of serum albumin. AFP is
involved with both ontogenic and oncogenic growth, as well as
immunoregulatory processes.1 AFP is synthesized in the yolk sac, fetal liver,and gastrointestinal tract during pregnancy but is re-expressed in multiple
adult tumours of mixed mesodermal/endodermal origin.2 Native AFP during
pregnancy as well as tumour AFP stimulates cell and tissue growth, thustAFP supports cancerogenesis. Serum levels of AFP are elevated in patientswith liver cancer as well as other types of cancer (breast, esophagus,cervical, pancreatic, endometrial, gastric, lung, rectum), and noncancer
diseases cirrhosis, nephrotic syndrome, and gastritis. On the other hand,whereas patients suffering from multiple myeloma, Wilms' tumour, andother 22 types of noncancer diseases had significantly decreased medianserum AFP levels than that of healthy controls.3
Alpha-fetoprotein is a shuttle protein that delivers nutrients throughreceptor-mediated endocytosis to embryotic cells. In adults, alphafetoprotein can shuttle drugs into alpha-fetoprotein receptor-positive
myeloid-derived suppressor, regenerating and cancer cells.4
PRINCIPLE OF AFP ELISA
The microtiter plate is coated with the antibody specifically binding Alpha
fetoprotein. The human serum and plasma are incubated in the plate withthe capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith biotin-labelled detection antibody. Following another washing step,
Streptavidin-HRP conjugate is added into the well.
Unbound reagent is then washed out. Horseradish peroxidase (HRP) boundin the complex reacts with the chromogenic substrate (TMB) creating theblue colour. The reaction is stopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of AFP in the specimen. The
concentration of AFP in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Kit Contents
Item
Antibody Coated Microtiter Plate
Biotin-labelled Antibody
Streptavidin-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution

Qty.
96 wells
13 mL
13 mL
1 vial1 vial1 vial
13 mL
50 mL
13 mL
13 mL

MATERIAL REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ± 2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS

Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ± 2oC.

Preparation of Standards
Reconstitute lyophilized Human AFP Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for15 min. The concentration of human AFP in Master Standard is
20 ng/mL.
Prepare set of Standard solution as follows:
Volume of Standard

v_01

Std1

Standard 20 ng/mL (lyophilized)

Dilution
BufferSee CofA

Concentration

Std2300 µL of Std1300 µL

10 ng/mL

Std3300 µL of Std2300 µL

5 ng/mL

Std4300 µL of Std3300 µL

2.5 ng/mL

Std5300 µL of Std4300 µL

1.25 ng/mL

Std6300 µL of Std5300 µL

0.625 ng/mL

Std7300 µL of Std6300 µL

0.3125 ng/mLBlank-300 µL

0 ng/mL

20 ng/mL2

Use the Master Standard for serial dilution (as below). Mix each tube
thoroughly before the next transfer. The Dilution Buffer serves as Blank.

3.

Accuracy

Dilution linearity

Preparation of Quality Control A and B

SampleReconstitute the lyophilized human serum Quality Controls withdeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:10 in Dilution
Buffer, prior to use, see Preparation of samples.

Dilution

Measuredconcentration(ng/mL)
55.5
28.4
14.0
6.6
58.3
28.1
14.2
6.6

Expected
concentration(ng/mL)
27.7
13.9
6.9
29.2
14.6
7.3

Yield(%)

Measuredconcentration(ng/mL)
6.2
50.0
27.3
16.5

Expected
concentration(ng/mL)
56.2
31.2
18.7

Yield(%)

12x4x8xPreparation of Wash Buffer 1x2

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at
4oC for two weeks or at -20oC for long term storage.

2x4x8x102
101
96
96
98
90

Preparation of samples
Human EDTA plasma may be used with this assay. For long-term storage thesamples should be frozen at minimum -70oC. Lipemic or haemolytic samples
may cause false results.

Spiking Recovery

Recommended dilution of samples is 1:10, i.e., for singlets 20 µL of sample+ 180 µL of Dilution Buffer, for duplicates 30 µL of samples + 270 µL ofDilution Buffer, respectively.

SampleSpike(ng/mL)

150
25
12.5

Do not store the diluted samples.
ASSAY PROCEDURE

Typical standard curve

Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate for
1 hour at 25oC ±2oC, shaking at 300 rpm.
3. Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
4. Pipette 100 µL of Biotin-labelled Antibody into each well.
Incubate for 1 hours at 25oC ±2oC, shaking at 300 rpm.
5. Wash the wells as described in point 3.
6. Pipette 100 µL of Streptavidin-HRP into each well. Incubate for
30 min at 25oC ±2oC, shaking at 300 rpm.
7. Wash the wells as described in point 3.
8. Pipette 100 µL Substrate solution, incubate for 10 min,at 25oC ±2oC. Avoid exposure to the light during this step.
9. Pipette 100 µL of STOP solution.
10. Read the signal at 450 or 450/630 nm within 15 min.

The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standardpoints properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

Human AFP Standard Curve
3.5
3Absorbance (450/630 nm)

1.
2.

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:10 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

10.3125 0.625 1.25

2.5

5

1020

Concentration (ng/mL)

RESOURCES
Mean (ng/mL)
54.8
79.6

SD
4.1
3.8

CV (%)
7.5
4.8

SD
0.16
0.02

CV (%)
2.8
5.4

Inter-assay (Run – to – run)v_01

1.501Sample
122Precision

Intra-assaySample
12

2.5

0.5

Sensitivity

The limit of detection, defined as a concentration of human AFP giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 0.08 ng/mL of sample.
2.

89
8888

Mean (ng/mL)
5.8
3.6

Adigun OO, Yarrarapu SNS, Zubair M, Khetarpal S. Alpha Fetoprotein. 2022 Nov 23. In: StatPearls[Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID: 28613501.
2Wong RJ, Ahmed A, Gish RG. Elevated alpha-fetoprotein: differential diagnosis - hepatocellular
carcinoma and other disorders. Clin Liver Dis. 2015 May;19(2):309-23. doi:
10.1016/j.cld.2015.01.005. Epub 2015 Feb 27. PMID: 25921665.
3He Y, Lu H, Zhang L. Serum AFP levels in patients suffering from 47 different types of cancers andnoncancer diseases. Prog Mol Biol Transl Sci. 2019;162:199-212. doi: 10.1016/bs.pmbts.2019.01.001.
Epub 2019 Mar 6. PMID: 30905450.
4
Pak VN. The use of alpha-fetoprotein for the treatment of autoimmune diseases and cancer. TherDeliv. 2018 Jan;9(1):37-46. doi: 10.4155/tde-2017-0073. PMID: 29216804.