EK-80ACE051201 concentration of the control serum is specified
), and place it into
AFP ELISA KIT
(REF: EK-80)
The AFP ELISA system provides direct
quantitative in vitro determination of human
alpha-foetoprotein (AFP) in human serum.
AFP can be assayed in the range of 0-100
IU/ml using 100 µl serum samples.
Introduction
Alpha-fetoprotein (AFP) is a glycoprotein with a
molecular mass of 65000. It is normally produced
in large amounts by the foetal liver. AFP level
increases progressively and reaches a peak at the
30th week in the maternal serum, thereafter, it dec-
reases gradually. An elevated maternal serum AFP
is associated with the neural tube defect (spina
bifida) and placental abnormalities, whereas
decreased AFP levels in both maternal serum and
amniotic fluid are related to foetal chromosomal
abnormalities (Down-syndrome). Patients affected
by liver carcinoma, testicular or ovarian
teratocarcinoma present very high AFP levels.
Although less frequently, an increased AFP
concentration may also be observed in gastric,
breast and bronchial tumours. To cover the very
large range of concentration associated with the
great variety of pathologic processes, the current
microtiter plate immunoassay (ELISA) is a simple
and powerful diagnostic tool for the detection of
AFP in the range 0 - 100 IU/ml.
Principle of method
The technology uses two high affinity
monoclonal antibodies in an immunometric
assay system. The two antibodies react simul-
taneously with the antigen present in
standards or samples This reaction leads to
the formation of a capture antibody - antigen -
signal antibody complex, also referred to as a
“sandwich”. In the standard solid-phase
ELISA system the reaction is carried out in a
microtiter plate (12 strips) which acts as the
binder of sandwich complex.
In the present product standards and samples
are incubated with the conjugate which
contains the horse radish peroxidase (HRPO)
labelled antibody at 37 oC for 2 hours, then
washed repeatedly. After the addition of a
ready-to-use tetramethyl-benzidine
(TMB)/peroxide substrate the signal is
measured in an ELISA photometer at 450 nm
wavelength.
The concentration of antigen is directly
proportional to the optical density measured
in the wells. The unknown concentration of
AFP in patient samples is read off a
calibration curve constructed by plotting
binding values against a series of calibrators
containing known amount of AFP.
Contents of the kit
1. 1 vial CONJUGATE (12 ml), ready to use,
containing anti-AFP antibodies in buffer with
blue dye 0,01 % merthiolate and 2 mg/ml
chloracetamide (CAA).
2. 5 vials STANDARD (1 ml per vial),
containing 0 (S0), 10 (S1), 25 (S2), 50 (S3),
100 (S4) IU/ml AFP (WHO 1st IRP 72/225) in
serum with 0.1% Kathon CG.
(1 IU/ml = 1.21 ng/ml)
3. 1 vial CONTROL SERUM. 1.0 ml human
serum with 0.1% Kathon CG. The
(take care of spill-out!
in the quality certificate enclosed.
4. 1 vial SUBSTRATE (25 ml) ready to use,
in brown plastic bottle. Do not expose to
direct light!
5. 1 piece MICROTITER PLATE, ready to
use. 12 strips, packed in an air-tight foil.
6. 1 bottle WASH BUFFER
CONCENTRATE (20 ml), 0.01 %
merthiolate. See Preparation of reagents.
7. 1 vial STOP REAGENT (6 ml) 1M sulfuric
acid
Plate map
Cover stick
Quality certificate
Pack leaflet
Materials, tools and equipment
required
Precision pipettes with disposable tips (50,
100, 200 and 300 µl), distilled water, vortex
mixer, shaker, plastic foil, absorbent tissue,
disposable plastic reagent-trays (separate for
each reagent), ELISA thermostate, ELISA
photometer
Recommended tools and equipment
Repeating pipettes, multi-channel ELISA
pipettes
Specimen collection and storage
Serum samples can be prepared according to
common procedures used routinely in clinical
laboratory practice. Samples can be stored at
2-8 °C if the assay is carried out within 24
hours, otherwise aliquots should be prepared
and stored deep frozen (-20°C). Frozen
samples should be thawed and thoroughly
mixed before assaying. Repeated freezing and
thawing should be avoided. Do not use
lipemic, hemolyzed or turbid specimens.
If in an initial assay the serum sample is
found to contain more than 100 IU/ml AFP,
the sample can be diluted 10-fold with S0
standard and reassayed as described in Assay
Procedure.
Preparation of reagents, storage
Add the wash buffer concentrate (20 ml) to
600 ml distilled water to obtain 620 ml wash
solution. Upon dilution store at 2-8°C until
expiry date.
Store the rest of reagents between 2-8°C after
opening. At this temperature each reagent is
stable until expiry date. The actual expiry date
is given on the package label and in the
quality certificate.
Assay procedure
(For a quick guide , refer to Table 1.)
1. Equilibrate reagents and samples to room
temperature before use. Homogenize all
reagents and samples by gentle mixing to
avoid foaming.
2. Label the plate map for duplicates of
each standard (S0-S4), control serum (C)
and samples (Sx).
3. Pipette 100 µl each of standards, control
serum and samples into the appropriate
wells.
4. Pipette 100 µl of conjugate solution into
each well by the multi-channel pipette.
5. Cover the plate by the enclosed foil,
shake it on the shaker for a few seconds
the ELISA thermostate. Allow to
incubate for 2 hours at 37 oC.
6. Take the plate out. Remove the cover and
pour the liquid directly over the lab sink.
Holding in the upside down position
place the plate immediately on an
absorbent tissue. Pay special attention to
crossing-over between wells due to
droplets backflow.
7. Pipette 300 µl wash buffer into each well
by using the multi-channel pipette.
Remove the wash buffer by using the
same pipette tips. Repeat this step 5
times, and use new tips for each step
8. Pour the substrate into its plastic tray,
and pipette 200 µl to each well with the
aid of the multi-channel pipette. Place the
plate into the dark for 30 minutes. (If less
than the whole volume is used in one
assay, do not pipette directly from the
bottle, and never fill the unused reagent
back into its original bottle).
9. Pipette 50 µl stop reagent into each well,
and shake gently for 5 minutes.
10. Measure in the ELISA photometer at 450
nm.
11. Calculate the concentrations of the
samples as described in Calculation of
results.
Table 1. Assay Protocol, Pipetting Guide (all
volumes in microlitres)
Standard Control Sample
Standard 100
Control 100
Sample 100
Conjugate 100 100 100
Incubate for 2 hours at 37 oC
Decant the fluid and blot on filter paper
Wash buffer 300 300 300
Decant the fluid and blot on filter paper
Repeat the washing step 5 times
Substrate 200 200 200
30 minutes at room temperature
Stop reagent 50 50 50
5 minutes at room temperature
Measurement
Calculate the results
Calculation of results
The calculation is illustrated using
representative data. Data obtained should be
similar to those shown in Table 2.
Manual calculation
Calculate the average OD for each pair of
duplicates. Subtract the mean NSB from all
(standars and samples) mean OD-s. Draw the
standard curve on a lin-lin graph paper by
plotting calculated OD of each standard level
(ordinate) against the respective concentration
(abscissa). Obtain sample values by
interpolation of sample OD on the standard
curve.
Data evaluation using normalized binding
For computerised calculations and/or quality
assessment normalised specific binding
values, rather than A.U. values are used.
Specific binding values can be calculated for
each standard and sample according to the 2 15 15.7 4.83 necessary volume of substrate into a separate
following equation: container for use.
3 15 23.7 7.68
S1-4/Mx (OD) –S0(OD)
B/Bmax (%) = ---------------------------------- x 100 4 15 28.1 7.11 -Discard the excess TMB substrate after use.
S4 (OD) –S0(OD), 5 15 43.1 9.83 Additional information
S0 is the zero-binding, or, non-specific 6 15 66.3 7.52 Components from various lots or from kits of
binding (NSB), Mx= sample. different manufacturers should not be mixed or
Reproducibility interchanged.
Table 2. Typical assay data To determine inter-assay precision 6 patient
OD OD B/Bmax, samples were measured in duplicates in 15 Precaution
mean % independent assays by 2 operators using
S0 0.057 different kit batches. Values obtained are Biohazard
0.053 2.50 Human blood products used in the kit have been
0.048 shown below.
S1 0.214 obtained from healthy human donors. They were
0.231
0.223 8.21 Sample Number Mean CV% tested individually by using approved methods
of runs value (EIA, enzyme immunoassay), and were found to be
S2 0.574 negative, for the presence of both Human
0.601 26.5
0.628 1 15 6.72 11.5 Immunodeficiency Virus antibody (Anti-HIV-1)
S3 1.091 2 15 16.00 5.90 and Hepatitis B surface Antigen (HBsAg).
1.112 51.2
1.133 Care should always be taken when handling human
S4 2.159 3 15 23.55 4.88 specimens to be tested with diagnostic kits. Even if
2.124 100 the subject has been tested, no method can offer
2.088 4 15 28.74 3.83
complete assurance that Hepatitis B Virus, Human
C 1.089
1.045 30.9 5 15 46.56 4.53 Immunodeficiency Virus (HIV-1), or other
1.000 infectious agents are absent. Human blood samples
6 15 67.77 4.89 should therefore be handled as potentially infectious
2,5 Recovery materials.

Recovery was defined as the measured Chemical hazard

2 increase expressed as per cent of expected
increase upon spiking serum samples with
known amount of AFP. 86 ± 7,62 % (mean ±
1,5
SD) was obtained for 12 serum pools.
Components contain Kathon CG as an
antimicrobial agent. The total amount of Kathon
CG present in each pack is 6.5 mg.
Use by Control

1
Dilution test (linearity)
A serial dilution of 12 individual serum Batch code Standard
samples was carried out with the zero- Caution, consult
0,5
standard. The equation for the expected (x) accompanying Coated plate
documents
against the measured (y) concentration was:
0 y=0.9737 x – 0,2657, R = 0.9538, n= 12 Biological risk Connjugate
0 10 20 30 40 50 60 70 80 90 100
Expected Values
Consult operating
Wash buffer
Figure 1: A typical standard curve Healthy male: 0.47 – 5.39 IU/ml. instructions
(Do not use to calculate unknown samples!) Healthy female: 0.37 – 4.41 IU/ml In vitro diagnostic
Substrate
medical device
At 16-week gestation: 30 IU/ml (1 MOM)
Characterization of assay
Hepatocarcinoma: >100 IU/ml Manufacturer Stop reagent

Typical assay parameters Hepatitis: <100 IU/ml

Temperature
limitation
Bo/Bmax < 5 % It is recommended that each laboratory Catalogue number
determine a reference range for its own Store between 2-8oC
Sensitivity patient population, since this may vary in
For the analytical sensitivity 0.08 IU/ml has different laboratories or regions.
been obtained by assaying 15 replicates of the
zero standard. The sensitivity has been Procedural notes
determined as the concentration Website: http://www.izotop.hu
corresponding to the sum of the mean OD and Technical e-mail: immuno@izotop.hu
its double standard deviation. 1) The wash procedure is critical. Insufficient Commercial e-mail: commerce@izotop.hu
washing will result in poor precision and
Hook effect falsely elevated absorbance readings.
There is no high dose “ hook effect ” up to the
AFP concentration of 5000 IU/ml . 2) The TMB substrate is sensitive to certain INSTITUTE OF ISOTOPES Ltd.
handling and storage conditions. Please note 1535 Budapest. Pf.: 851.
Specificity Phone: (36-1)392-2577, Fax: (36-1)395-9247
the following precautions:
The monoclonal antibodies used in this IRMA
kit are specific for AFP. No cross reactivity -TMB is very light sensitive and direct
was found with human albumin. expsosure to light (especially sunlight) should
be avoided.
Precision
6 patient samples were assayed in 15 -Do not leave the cap off of the storage bottle
replicates to determine intra-assay precision. for prolonged periods of time.
Values obtained are shown below.
-To avoid contaminating the entire bottle of
Sample Number of Mean CV% substrate, never pipette directly from the
replicates value substrate bottle. Always pour just the
1 15 7.25 7.78