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The shelf life of an unopened EIA kit is 18 months, starting from the date of manufacture, indicated on the label, under strict adherence to the recommended transportation and storage conditions.
«Unopened Kit» means that the caps of the vials have not been opened and the plates are under vacuum (the packaging is tightly attached to the plate).
The shelf life of the in-use kit under the same conditions is 2 months from the date of opening and is mainly limited by the shelf life of the calibrators and control serum. A shorter shelf life is the general rule for all in-use ELISA kits. This is due to the fact that during the analysis, reagent vials may contain foreign substances that, despite the presence of preservatives, may cause microbial or biochemical degradation of their contents.
To prevent wastage of in-use kits that are rarely used, some kits allow for one-time freezing and thawing of calibrators and control serum in aliquots, which must be specified in the IFU.

The EIA kit should be stored at the temperature of +2…+8°C until the expiration date indicated on the packaging and in the Quality Control Data Sheet. Single transportation at a temperature up to 25ºС for 5 days is acceptable.
It’s especially important to ensure proper storage of the plate if the refrigerator experiences significant temperature fluctuations (for example, due to overload), this may leads to condensation in the wells and uneven heating of the plate, which is very dangerous for its stable operation.
It is strictly forbidden to freeze or expose the entire EIA kit to high temperatures.

The proper pipetting technique is essential to achieve accurate results to minimize accidental errors in sample volume. Press the pipette plunger to the second stop, insert the tip into the sample, gently (not abruptly, to avoid swirling and splashing of the sample inside the tip) take the required volume, and remove the drop from the outside of the tip by touching the edge of the tube (the drop is usually formed when the tip is deeply immersed in the sample), insert the tip into the well and squeeze the sample to the first stop along the wall of the well. Make sure that the tip touches the surface of the liquid, otherwise a droplet will form and part of the volume will not be added.
Strictly follow the recommended order and duration of the analysis procedure described in the IFU of the kit.
All reagent and sample additions should be performed rhythmically and quickly, without significant interruptions.
Do not leave wells unfilled with reagents between assay steps or after washing. During a forced pause, place the plate with the wells down on a sheet of damp filter paper.
Keep the intervals between adding and removing solution during well washing to a minimum.
Follow the recommended well-washing procedure and do not spontaneously change the volume of liquid, number of cycles, or order of washing.

It is necessary to comply with the temperature and duration of the incubation stage with the tetramethylbenzidine (TMB) substrate solution recommended in the IFU of the kit.
It is recommended to monitor the color development in the wells visually. The plate incubation with the TMB solution should be carried out in the dark to avoid edge effects (brighter coloration of the edge wells) and, as a result, significant variation in the results.
Incubation in a thermostat allows to standardise of the color development process, which is important for reliable reproducible results. This technique is highly recommended if the room temperature is subject to significant fluctuations.

At low temperatures (below +18°C), all processes occurring at the incubation and enzymatic coloration stages are slowed down. This leads to a reduction of the optical density of solutions in the wells, a significant deterioration in the method’s performance, and sometimes incorrect results.
At high temperatures (above +25°C), all processes occurring during the analysis are accelerated, and the rate of enzymatic coloration increases, and that is why the optical density of solutions in the wells can significantly exceed 3.0, which significantly complicates the calculation of results and makes it impossible.

Performing an enzyme immunoassay in monoplicates is often used in laboratory practice to reduce the cost of one test. However, this is a risk factor for incorrect results. Analysis of calibrators, control serum, and test samples in monoplicates makes it impossible to estimate the amount of difference in parallels and to determine incorrect or questionable results based on them.
Calibration samples should always be run in duplicates, as an accidental outlier may result in a miscalculation of all samples falling within the corresponding curve area. It is also recommended that unknown samples should be analyzed in duplicate to allow for repeat analysis if there is a significant variation in results for the same sample.