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Fax :+ 32 16 50 90 45
GENTAUR Europe
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B-1040 BRUSSELS
BELGIUM
GENTAUR France
tel 01 43 25
01 50
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75005 PARIS
FRANCE
GENTAUR Italy
tel 02 36 00
65 93
fax 02 36 00
65 94
20135 MILANO
ITALY
GENTAUR Germany
tel +49 241
6085 13140
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6085 33033
Forckenbeckstraße 6,
D-52074
Aachen
GERMANY
| |
Description
The AFP
ELISA system provides a direct quantitative determination of
alphafetoprotein in human serum. Alphafetoprotein can be assayed in the
range of 0-100 IU/ml using 100 µl serum samples. Each kit contains
materials sufficient for 96 determinations permitting the construction
of one standard curve and the assay of 42 unknowns in duplicate.
Introduction
Alphafetoprotein (AFP) is a glycoprotein with a
molecular mass of 65000. It is normally produced in large amounts by the
foetal liver. Alphafetoprotein level increases progressively and reaches
a peak at the 30th week in the maternal serum, thereafter, it
decreases gradually. An elevated maternal serum alphafetoprotein 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 alphafetoprotein concentration may also be
observed in gastric, breast and bronchial tumors. 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 the method
The technology uses two high affinity monoclonal
antibodies in an immunometric assay system. The two antibodies react
simultaneously 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 tracer
which contains the horse radish peroxidase (HRPO) labelled antibody at
37°C 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
alphafetoprotein.
Contents of the kit
|
1 vial |
TRACER, ready to use.
12 ml per vial buffer solution with blue dye and preservatives. |
|
5 vials |
STANDARDS, ready to use.
1 ml per vial, containing 0 (S0), 10 (S1), 25 (S2), 50 (S3), 100
(S4) IU/ml, in serum with preservative. Calibrated against the
WHO 72/225 international reference preparation. |
|
1 vial |
CONTROL SERUM, ready to use.
1 ml human serum containing preservative.
The expected concentration is specified in the quality
certificate enclosed. |
|
1 piece |
MICROTITER PLATE, ready to use.
12 strips, packed in an air-tight foil. |
|
1 vial |
SUBSTRATE, ready to use.
>=25 ml, in brown plastic bottle. Do not expose to direct light. |
|
1 vial |
WASH BUFFER CONCENTRATE,
20 ml per vial, containing 0.01 % mertiolat.
See Preparation of reagents |
|
1 vial |
STOP REAGENT
6 ml per vial 1M sulfuric acid |
|
1 piece |
Plate map |
|
1 piece |
Quality certificate |
|
1 piece |
Pack leaflet |
Materials, and equipment required
Assay
tubes, about 12 x 75 mm.
Precision micropipettes (100 and 200 µl)
Multi-channel ELISA pipettes
Disposable plastic reagent-trays (separate for each reagent)
ELISA thermostate
ELISA photometer
Shaker
Vortex mixer
distilled water
Preparation of reagents
Except the
wash buffer, each reagent is supplied in ready to use form.
Add the wash buffer concentrate (20 ml) to 600 ml distilled water to
obtain 620 ml washing solution.
For use in the assay allow all regents to equilibrate to room
temperature.
Specimen collection and storage
Serum samples
can be prepared according to common proce-dures 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.
Samples of measured, or expected, concentration >100 IU/ml can be
(re)assayed after dilution with physiologic saline.
Assay procedure
(For a
quick guide, refer to Table 1.)
|
1 |
Equilibrate all reagents and samples to room temperature. |
|
2 |
Label the plate map for duplicates of each standards (S0-S4),
control serum (C) and samples (Sx). |
|
3 |
Mix all reagents and samples thoroughly before use. Avoid
excessive foaming. Fill out the tracer into the ELISA tray. |
|
4 |
Pipette 100 µl each of standards, control serum and
samples into the appropriate wells. |
|
5 |
Pipette 100 µl of tracer solution into each well by the
8-channel pipette. |
|
6 |
Cover the plate by the enclosed foil, shake it on the
shaker for a few seconds (take care of spill-out!), and place it
into the ELISA thermostate. |
|
7 |
Allow to incubate for 2 hours at 37°C. |
|
8 |
Take the plate out of the machine. Remove the cover and
pour the liquid directly over the lab sink. Holding in the
up-side down position place the plate immediately on an
absorbent tissue. Pay special attention to crossing-over between
wells due to droplets backflow. |
|
9 |
Pipette 300 µl wash buffer into each well by using the
8-channel pipette. Remove the wash buffer by using the same
pipette tips. Repeat this step 6 times, and use new tips for
each step. |
|
10 |
Pour the substrate into its plastic tray, and pipette 200
µl to each well with the aid of the 8-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.) |
|
11 |
Pipette 50 µl stop reagent into each well, and shake
gently for 5 minutes. |
|
12 |
Measure in the ELISA photometer at 450 nm. |
|
13 |
Calculate the concentrations of the samples as described
in Calculation of results. |
Table 1.
Assay Protocol, Pipetting Guide (all volumes in microliters)
Well
Reagents |
Standard |
Sample |
Control |
|
Standard |
100 |
|
|
|
Sample |
|
100 |
|
|
Control |
|
|
100 |
|
Tracer |
100 |
100 |
100 |
|
Incubate for 2 hours at 37°C |
|
Repeat in 6 cycles |
Wash buffer |
300 |
300 |
300 |
|
Decant the supernatant |
|
Substrate |
200 |
200 |
200 |
|
30 minutes at room temperature |
|
Stop reagent |
50 |
50 |
200 |
|
5 minutes at room temperature |
|
Measurement |
|
Calculation of results |
Table
2. Typical Assay Data
| |
AFP
IU/ml |
OD |
OD |
OD mean |
B/Bmax, % |
AFP,
IU/ml |
|
S0* |
0 |
0.057 |
0.048 |
0.053 |
2.50 |
- |
|
S1 |
10 |
0.214 |
0.231 |
0.223 |
8,212 |
- |
|
S2 |
25 |
0.574 |
0.628 |
0.601 |
26,471 |
- |
|
S3 |
50 |
1.091 |
1.133 |
1.112 |
51,155 |
- |
|
S4 |
100 |
2.159 |
2.088 |
2.124 |
100 |
- |
|
C |
- |
1.089 |
1,000 |
1,045 |
30.88 |
46,367 |
Figure 1
A typical standard curve
(Do not use to calculate unknown samples)
Calculation of results
The
calculation is illustrated using representative data. Data otained
should be similar to those shown in Table 2.
Manual calculation
Calculate the average OD for each pair of duplicates. Substract the mean
NSB from all (standars and samples) mean ODs. 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 counts 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 following equation:
|
B/Bmax (%) = |
S1-4/Mx (AU) – S0(AU) |
x 100 |
|
————————— |
|
S4 (AU) – S0(AU) |
S0
is the zero-binding, or, non-specific binding (NSB).
Characterisation of the assay
Assay
parameters
Bo/Bmax < 5 %
Sensitivity
A detection limit of 0.8 IU/ml has been obtained by assaying 15
replicates of the zero standard. The sensitivity has been determined as
the concentration corresponding to the sum of the mean cpm and its
double standard deviation.
Specificity
Monoclonal antibodies do not cross-react with human serum albumin.
Hook effect
There is no high dose "hook effect" up to the AFP concentration of 5000
IU/ml.
Precision and reproducibility
6 samples were assayed with 15 replicates within one run, and with
duplicates in 15 runs, to determine the intra-assay, and inter-assay
precision, respectively. Values obtained are shown below.
|
Intra-assay |
Inter-assay |
|
mean (IU/ml) |
CV % |
mean (IU/ml) |
CV % |
|
7.25 |
7.78 |
6.72 |
11.53 |
|
15.69 |
4.83 |
16.00 |
5.90 |
|
23.65 |
7.68 |
23.55 |
4.88 |
|
28.06 |
7.11 |
28.74 |
3.83 |
|
43.13 |
9.83 |
46.56 |
4.53 |
|
66.25 |
7.52 |
67.77 |
4.89 |
Linearity of dilution
A serial dilution of 12 individual serum samples was carried out with
the zero-standard. The equation for the expected (x) against the
measured (y) concentration was:
y =
0.9737 x – 0.2657, R = 0.9538, n = 12
Recovery
Recovery was defined as the measured increase expressed as per cent of
expected increase upon spiking serum samples with known amount of
alphafetoprotein. 86 ± 7,62 % (mean ± SD) was obtained for 12 serum
pools.
Normal values
Male: 0.47 – 5.39 IU/ml.
Female: 0.37 – 4.41 IU/ml.
Gestation, week 16: 25 IU/ml (1 MoM).
Pathological values
hepatocarcinoma: > 100 IU/ml
hepatitis: < 100 IU/ml
It is recommended that each laboratory establish its own reference
intervals.
The results obtained should only be interpreted in the context of the
overall clinical picture. None of in vitro diagnostic kits can be used
as the one and only proof of any disease or disorder.
Additional information
Storage
Store the reagents between 2-8 °C. At this temperature each reagent is
stable until expiry date. Unused reagents left from a partial working up
can be stored for a later use under the same conditions.
Availability
From stock.
Shelf life
The minimum shelf life of kit reagents is usually 16 weeks from the date
of manufacturing. The actual expiry date is given on the package label
and in the quality certificate. Components from various lots or from
kits of different manufacturers should not be mixed or interchanged.
Precautions and warnings
This kit should only be used for in vitro diagnostic purposes.
Potentially infectious materials
Human blood products provided as components of this product have been
obtained from donors tested individually and found negative for Human
Immunodeficiency Virus antibody (HIV-Ab) as well as for Hepatitis B
surface Antigen (HBsAg) using approved EIA methods.
Care
should always be taken when handling human specimens to be tested with
diagnostic kits. Even if the subject has been tested, no method can
offer complete assurance that Hepatitis B Virus, Human Immunodeficiency
Virus (HIV), or other infectious agents are absent, and all human blood
samples should be considered potentially infectious. |
AFP (alpha
fetoprotein) ELISA test 2
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