Test principle
All AlerTox ELISA tests work on the principle of a quantitative sandwich ELISA. An antibody directed
against the target antigen (protein) is bound on the surface of a microtiter plate. Antigen-containing
samples or standards are given into the wells of the microtiter plate. After 20 minutes incubation at room
temperature (15 - 25 ºC/59 - 77 ºF), the wells are washed with diluted washing solution to remove unbound
material. A peroxidase-conjugated second antibody directed against the same antigen is given into the
wells and after 20 minutes of incubation the plate is washed again. A substrate solution is added and
incubated for 20 minutes, resulting in the development of a blue color. The color development is inhibited
by the addition of a Stop Solution, and the color turns yellow. The yellow color is measured photometrically
at 450 nm. The concentration of the antigen is directly proportional to the color intensity of the test sample
Supplied Materials
1）Breakable strips of 8 wells each coated with antigen-specific antibodies：12 strips
2）5 AlerTox Standards. Ready to use.：5 x 3 mL
2a） 5 AlerTox Standards; 100x concentrated
(only AlerTox ELISA Casein & AlerTox ELISA Milk)：5 x 1 mL
3） Conjugate solution. Ready to use：1 x 15 mL
4） Substrate solution (TMB). Ready to use：1 x 15 mL
5） Stop Solution, containing H2
SO4
. Ready to use： 1 x 15 mL
6） Extraction & Sample Dilution Buffer 10x：4 x 30 mL
6a） Extraction & Sample Dilution Buffer 5x
(only AlerTox ELISA Casein & AlerTox ELISA Milk)： 4 x 60 mL
7） Washing Solution 10x：2 x 60 mL
Storage advice
• All kit components should be kept at 2 – 8 °C (36 - 46 °F) in the dark. DO NOT FREEZE.
• Return all reagents to 2 – 8 °C (36 - 46 °F) immediately after use.
• The diluted Washing Solution concentrate can be used for 4 weeks, when stored at
2 – 8 °C (36 - 46 °F).
• The diluted Extraction & Sample Dilution Buffer can be used for 1 week, when stored at
2 – 8 °C (36 - 46 °F). When crystals precipitate while storing, warm up to 37 °C (99 °F) for
15 min. before use.
• The Sample Extracts are stable for at least 24 hours at 2 – 8 °C (36 - 46 °F), or frozen for
longer storage.
Sample preparation
a) Wine samples:
1. Add 0.5 ml of wine to 9.5 ml of extraction buffer 1x and mix.
2. Use directly 100 µl in the ELISA plate. Centrifugation is normally not necessary as long as the
mixture is clear. If not, centrifuge as described under b) point 4. For other liquid samples proceed
with b) point 3.
b) Other kinds of samples:
1. To maximize homogeneity and representativeness of the sample drawing, a minimum of 5 g
sample should be pulverized finely in a mortar, impact mill or a similar device.
2. 0.5 g of the homogenized mixture is suspended in 10 ml of extraction buffer 1x.
3. Incubate the mixture for 15 min in a pre-heated water bath at 60 °C (140 °F) (AlerTox ELISA
Crustacean 40 °C (104 °F)). To ensure good homogeneity, the samples should be shaken every
two minutes.
4. Centrifuge the mixtures for 10 minutes at 2000 x g. If it is not possible to separate the supernatant
from the precipitate completely, then filter the supernatant after centrifugation. Cool down the
filtrate to room temperature (15 - 25 ºC/59 - 77 ºF).
(For AlerTox ELISA Casein and AlerTox ELISA Milk kits see also special hints on point 9.
Special Hints).
5. 100 µl of this solution are applied per well. If the results of a sample are out of the measuring
range, further dilution with the pre-diluted Extraction and Sample dilution buffer is necessary. The
additional dilution must be considered when calculating the concentration.