In Vitro Diagnostic Reagents Products
Alpha-fetoprotein (AFP) assay kit (ELISA) CE Approved

Alpha-fetoprotein (AFP) assay kit (ELISA)


[Product Name]:

Generic Name: Alpha-fetoprotein (AFP) assay kit (ELISA)


[Pack size]:
48 tests / kit, 96 tests / kit


[Intended Use]:
The Alpha-fetoprotein (AFP) assay kit is intended for quantitative determination of the Alpha-fetoprotein (AFP) concentration in human serum.


[Introduction]:
Alpha-fetoprotein (AFP) is a kind of embryonal single polypeptide which synthesized by embryo liver, yolk sac and gastrointestinal tract. Human AFP belongs to the alpha globulin, composed of 18 kinds of amino acids. The molecular weight of AFP is about 64 KD ~ 72 KD with a half-life of about 5d. Part of AFP can enter into the maternal blood circulation as amniotic fluid, AFP levels in body fluids of pregnant women all have a different enhanced. Normally, non-pregnancy women have lower concentration of serum AFP.
Liver cancer is one of common reasons that induce abnormally increase of AFP. Elevated serum AFP concentrations are observed in 70% ~ 95% of adult patients with liver cancer. AFP has a relative specificity for diagnosis of the liver cancer, human serum AFP concentration determination can be served as assistant in diagnosis of liver cancer, combined with other markers detection and imaging examination
. Elevation of serum AFP to abnormally high values also occurs in several malignant diseases including germline embryonal tumor and digestive tract tumor.


[Principle of test]:
This assay kit is based on an enzyme-linked immunosorbent sandwich assay (ELISA). Standards, samples and anti-AFP-HRP conjugate are added to the wells coated with Anti-AFP monoclonal antibody. AFP in the serum binds to antibody coated on the well, and then the anti-AFP-HRP antibody binds to AFP. Unbound proteins are washed off. Upon the addition of the tetramethyl-benzidine (TMB) substrate, the optical density (OD) is proportional to the concentration of AFP in the samples. A standard curve is prepared by plotting option density (OD) to the concentration of the AFP. Then read-off unknown sample values on the standard curve.

[Kit Contents]:

Contents Pack size 48 tests /kit 96 tests /kit
Micro well plate 1 ×48-well plate 1 ×96-well plate
AFP standard ( 0ng/mL) 1 ×0.3mL/vial 1 ×0.5mL/vial
AFP standard (5ng/mL) 1 ×0.3mL/vial 1 ×0.5mL/vial
AFP standard (20ng/mL) 1 ×0.3mL/vial 1 ×0.5mL/vial
AFP standard (50ng/mL) 1 ×0.3mL/vial 1 ×0.5mL/vial
AFP standard (200ng/mL) 1 ×0.3mL/vial 1 ×0.5mL/vial
AFP standard (500ng/mL) 1 ×0.3mL/vial 1 ×0.5mL/vial
anti-AFP-HRP conjugate 1 ×6mL/vial 1 ×12mL/vial
AFP diluent 1 ×6mL/vial 1 ×12mL/vial
Wash buffer(20×) 1 ×10mL/vial 1 ×20mL/vial
TMB substrate solution 1 ×6mL/vial 1 ×12mL/vial
Stop solution 1 ×3mL/vial 1 ×6mL/vial
Closureplate membrane 2pcs 2pcs
Instruction for use 1pc 1pc


Note:
1. Micro well plate coated with purified AFP monoclonal antibody.
2. AFP standard have been calibrated by international standard substances (WHO IS 72/225) for AFP immunoassay, AFP concentration unit 1 IU= 1.21 ng.
3. Mouse anti-AFP-horseradish peroxidase (HRP) is in anti-AFP-HRP conjugate reagent.
4. Do not mix regents with those from other LOTs.


[Storage and Expiry date]:
1. Store the kit at 2°C ~ 8°C. Do not freeze.
2. It is valid for
12 months in the storage conditions.
3. The kit must be used within
3 months if the reagents were opened.


[Equipment required but not supplied with the kit]:
1. ELISA analyzer (with a wavelength of 450 nm , 630 nm).
2. Precision pipettes (pipettes with disposable plastic tips for delivery of 50, 100 µL is applicable, and it is alternative for other applicable automatic sampler).
3. Thermotank (37°C incubator, alternative for other thermostatic equipment).

4. Wash device (automatic washing machine is recommended).
5. Microplate shaker.


[Specimen collection and handling]:
Collect blood by venipuncture and separate the serum by centrifugation at room temperature. Samples can be stored at 2-8°C for 7 days. For longer periods it is recommended to store the samples at –18°C or below. Avoid repeated freezing and thawing of the samples more than 3 times. No preservatives should be added to the serum.


[Testing procedure] (Allow all reagents to reach room temperature before use):
1. Dilute wash buffer (20× ) (1:19) with distilled water.
2. Label or mark the micro well strips to be used on the plate.
3. Pipet 50 µL of AFP standards(0, 5, 20, 50, 200,500 ng/mL), AFP diluent (users as supplied), and serum samples into appropriate wells, mixed well, cover the plate and incubated at 37°C for 30min.
4. Aspirate and wash the wells
3 times with wash buffer. We recommend using an automatic ELISA plate washer for better consistency.
5. Add 100µL of anti-AFP-HRP conjugate reagent into each well. Cover the plate and incubate at 37°C for 30 min.
6. Aspirate and wash the wells
3 times with wash buffer as above.
7. Dispense 100 µL HRP substrate solution into each well. Mix gently, cover the plate and incubate for 10 min at 37°C.
8. Add 50 µL stop solution into each well and mixed gently.
9. Read optical density (OD) value at 450 nm in Elisa Analyzer within 30 minutes after addition of the stop solution.
10. The standard curve and regression equation were established by AFP standards color intensity, then calculate the AFP concentration in samples (Correlation coefficient(r) ≥ 0.9900).


[Normal reference range]:
Normal reference range is ≤ 20ng/mL. Recommend the laboratories to establish their own normal reference rangethe above data is only for reference.


[Result Interpretation]:
If the sample concentration is higher than detection limit, it is feasible to dilute it with AFP diluent. Detection Concentration ×Dilute Multiples = Original Concentration


[The limitations of testing procedure]:
It is not applicable for the whole blood samples detection, serving as assistant in vitro diagnosis, the result should not be as absolute evidence.


[Performance characteristics]:

NO. Performance Characteristics
1 Detection limit Detection limit ≤ 5.0 ng/mL;
2 LinearityAFP concentration- OD value curve correlation coefficient(r) ≥
0.9900 in the detection range;
3 AccuracyThe relative deviation of detected concentration to target
concentration ≤ 15.0%;
4 Precision coefficient of variation (CV) ≤ 15.0% in the detection range;
5 SpecificityNo less than 10 serum samplesall the detected concentration is ≤
20.0ng/mL.


Note:
1. Appearance: Visual method is used, in the natural light bright visual packaging components should be complete, complete, clear identification, reagent without leakage.
2. Detection limit: Based on ten replicate determinations of the zero standard, the detection limit of AFP is ≤ 5.0ng/mL. The detection limit is defined according to formula: X+2SD (X: the concentration corresponding to the mean of the absorbance values of the AFP calibrator 0, SD: standard deviations).
3. Linearity: Concentration of AFP standards set as the X-axis, optical density(OD) measured correspond to the AFP standards set as Y-axis, using four parameters fitting the standard curve, X-Y curve correlation coefficient (r) should be no less than 0.9900.
4. Accuracy: Determination of two different concentrations AFP standards from different nation (international) or enterprise standards that had been standardized by the nations, and repeat 3 times, relative deviation(B) is calculated according to the formula: B=X-T/ T×100% (X: average determination , T: the target value). The relative deviation is no more than 15.0%.
5. Precautions:
5.1 Intra-assay precision:
Two quality control products with different concentration from the same batch run in 10 replicates, calculating the coefficient of variation (CV), CV= SD / X×100% ( X: average of 10 times determination, SD: standard deviation). The detection results of three batches show that the products have good intra-assay precision (CV ≤ 15.0%).
5.2 Inter-assay precision: Two quality control products with different concentration from the same batch were run in at least three independent assays. The detection of three batches demonstrated the products have good inter-assay precision (CV ≤ 20.0%).
5.3 Inter-batch precision: Two quality control products with different concentration from the three batches AFP assay kits run in 10 replicates, calculating the average of 30 times determination results of (X) and standard deviation (SD), CV is no more than 20.0%.
6. Specificity: Normal serum samples is no less than 10 pcs, the result is ≤ 20.0ng/ml.
7. Stability: Put the AFP assay kits at 37oC for7 days, the test results complied with 1 ~ 6 (except 5.3) performance characteristics.
8. High dose hook effect: AFP concentration of 50,000 IU/mL did not cause any hook effect.

[Notice]:
This test kit should be used within expiry time according to introduction for use. The manual operation should be paid attention, so as to not affect the accuracy of determination. Pipetting suction is just used once so as to avoid cross contamination.
If there is any crystallization in wash buffer, it can be used after dissolved in 37
oC. Abandoned reagents and samples should be disposed as the potential infectious material.


[Caution]:
Material used in the preparation of human source reagent has been tested and found to be Non-Reactive for HIV-1/2 Antibody, Hepatitis B Surface Antigen (HBs-Ag) and HCV Antibody. Since no method can completely rule out the presence of blood borne diseases, the handling and disposal of human source reagents of this product should
be made if they were potentially infected.

Specifications
48 tests/box 96 pcs/box
Applicable scope

The Alpha-fetoprotein (AFP) assay kit is intended for quantitative determination of the Alpha-fetoprotein (AFP) concentration in human serum.


The Alpha-fetoprotein (AFP) assay kit is intended for quantitative determination of the Alpha-fetoprotein (AFP) concentration in human serum.