Questions and Answers

Contact with the skin with the Stop Reagent, which contains 5% sulfuric acid, may cause irritation. In this case, rinse the affected area with plenty of water. 3,5-tetramethylbenzidine, which is a part of the tetramethylbenzidine (TMB) substrate solution, is a potential carcinogen. All other components of the kit are non-toxic in the concentrations used.
A certain potential hazard to laboratory personnel is posed by reagents containing components of human origin, as well as by the tested serum and plasma samples, which should be considered as potentially infected material that may contain hepatitis, HIV, and other pathogens. Therefore, disposable gloves should be worn when handling the kit.

For each assay, it is necessary to generate an individual calibration curve and use a control serum (the concentration range of the analyte in the control serum is indicated in the Quality Control Data Sheet and on the vial label).

If pre-dilution of samples is a mandatory step in the analysis, for example, when determining aTPO or aTG, when all test samples are diluted 101 times as standard, then no correction of the data calculated on the calibration graph is required. This is because the calibration samples and control serum in such kits are also pre-diluted 101-fold, i.e., the dilution factor of the calibrators, control serum, and test samples is the same. However, if the analyte content of the pre-diluted test sample exceeds the upper limit of the concentration determined by the kit, it must be further diluted according to the Instructions for Use of the kit and reanalyzed. In this case, multiply the result by the additional dilution factor.
In most kits, pre-dilution of the test kits is not required. However, if the measured analyte content of the test sample exceeds the upper limit of detection, it should be diluted according to the instructions for use of the kit and the analysis should be repeated. In this case, multiply the result by the dilution factor.
The recommended dilution factor may depend on the type of sample being tested, for example, when determining hCG concentrations at different gestational ages. Detailed information is provided in the Instructions for Use of the kits.

The construction of a calibration curve and determination of the analyte content in the QUANTITATIVE determination can be performed manually, using linear or logarithmic scale paper, as well as using special computer programs.

Scratches, fingerprints, drips, and other contaminants on the bottom of the wells on both sides of the plate can change the optical density reading (sometimes quite significantly).
Measure the optical density of the wells no later than 5 minutes after adding the stop reagent. This is because the oxidation reaction of chromogen does not stop completely after the addition of acid, and the optical density of the solutions in the wells continues to increase.
Spectrophotometry should be performed only at the recommended wavelength since even slight changes in it can lead to a drop in the recorded optical density values.
The presence of air bubbles in the wells of the plate, which sometimes appear after adding a Stop Reagent to the wells, leads to light scattering and false overestimation of the optical density during spectrophotometry.

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.

The quality of the wells is very important for reliable results. In each washing cycle, the wells should be filled evenly and almost completely with solution. No residual liquid should be left in the wells after the Washing Solution has been removed.
Allowing the Washing Solution to remain in the wells for too long and increasing the number of wash cycles can lead to leaching of the bound conjugate and degrade the quality of the assay.
Any residual Washing Solution should be removed by tapping the inverted plate on a sheet of filter paper.

In this case, the incubation time with the tetramethylbenzidine (TMB) substrate solution may be reduced to 7-10 min. If the color intensity remains excessive despite the above steps, remove a part of the chromogenic solution from all wells and re-measure the optical density.
Do not incubate the plate near heating devices, as the temperature in different wells will vary and changes in the assay results will become unpredictable.

Attempts to compensate for the effect of low temperature by extending the incubation time are not always successful, so if the room is very cold, it is better to incubate in a thermostat that maintains the recommended temperature, including room temperature (+18…+25°C).

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.

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.

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.

To ensure the most complete quality control of laboratory tests, it is recommended to analyze calibrators, control serum, and test samples in duplicate.

Yes, if the device is designed for analysis in a standard 96-well plate and has “open” software.
For some kits, it is necessary that the analyzer includes a thermostatically controlled incubator that maintains a temperature of +37°C±1°C. Some automatic ELISA analyzers do not provide the same incubation time in different wells of the plate. This is due to the sequential and insufficiently fast introduction of the reagents of the kit into the wells. In this case, a drift of results will be observed, especially noticeable when analyzing a large number of test samples, as well as in kits with a short incubation (30 min). To assess the degree of drift, it is recommended to place the control serum wells at the beginning and end of the series of samples to be analyzed.

All glassware and reagent dishes must be thoroughly washed and dried. Please note that even residual contamination from synthetic detergents and oxidizing agents can affect reagent stability and assay results. For this reason, rinse the dishes thoroughly with distilled water.
Separate containers (reagent dishes) and, if possible, separate pipettes with replaceable tips should be provided for the conjugate and tetramethylbenzidine (TMB) substrate solution.
It is recommended to use disposable reagent dishes and pipette tips. When putting the tips on the pipette, do not touch the working surfaces of the table, laboratory glassware, the packaging of the kit, the operator’s hands, or work clothes with the tips.

In addition to a set of reagents for the analysis, the laboratory must have a vertical scanning spectrophotometer (reader) that allows measuring the optical density of solutions in wells at 450 nm and 620-680 nm in the range from 0 to 3.0 optical density units.
In order to comply with the recommended conditions for analysis in the laboratory, the following equipment is required
– an air thermostat that maintains a temperature of +37°C±1°C, which does not provide shaking of the plate;
– a device for shaking the plates (shaker) that maintains room temperature (+18…+25°C) and a temperature of +37°C±1°C. The recommended shaking intensity is 300-800 rpm. In addition, some kits provide only incubation at room temperature (+18…+25°C) without shaking, in which case no additional equipment (“thermoshaker”) is required.
– pipettes with replaceable tips that allow for the collection of liquid volumes in the range from 5.0 µL to 1.0 µL;
– pipettes with replaceable tips that allow for volumes of up to 250 µL or a plate rinsing device;
– 500 mL measuring cylinder;
– distilled or deionized water;
– filter paper.
Carefully read the Instructions for Use of the kits that will be used by laboratory staff to create the necessary minimum equipment.
Note. It is also recommended to use the eight-channel pipette for simultaneously adding reagents to the wells of the plate ( EIA Buffer, Conjugate, Tetramethylbenzidine (TMB) substrate solution, and Stop Reagent).

The EIA kit components should be allowed to stand at room temperature (+18…+25°C) for at least 30 minutes before analysis.
The plate package should be opened only after the specified time, otherwise, moisture will condense on the cooled surface of the wells, which may lead to damage to the kit.

It is advisable that the number of independent series of experiments does not exceed four.

These signs are:
· visible damage to the unopened package with the plate and vials with reagents, calibrators, and control serum;
· presence of sediment, suspended particles, and mold in the solutions;
· the color of the reagents, including the tetramethylbenzidine (TMB) substrate solution, does not match the description.
Such components must not be used for the analysis.
Before starting work, make sure that all components of the EIA kit correspond to the description given in the IFU by visual characteristics.

After first opening, store the vial with the Stop Solution at +2…+8°C for the entire shelf life of the kit. If the color of the Stop Solution changes, its use is not recommended.

The vial with the tetramethylbenzidine (TMB) substrate solution must be tightly closed immediately after opening and stored at +2…+8°C during the entire shelf life of the kit.
Do not freeze the tetramethylbenzidine (TMB) substrate solution.
If stored in the light, or if the tetramethylbenzidine (TMB) substrate solution is contaminated with blood, oxidants, or metal ions, spontaneous blue or blue coloration may occur. This reagent should not be used for analyzes due to increased background during spectrophotometry, decreased sensitivity, and range of detectable analyte concentrations.

After first opening, store the vial with the conjugate at +2…+8°C for no longer than 2 months.
It is strictly forbidden to freeze the conjugate. This will lead to its spoilage.

After first opening, store the vial of Wash Solution Concentrate at +2…+8°C for the entire shelf life of the kit. Store the ready-to-use Washing Solution at room temperature (+18…+25°C) for no longer than 5 days or at +2…+8°C for no longer than 14 days.

After opening the vials, store the calibrators and control serum at +2…+8°C for no longer than 2 months.
It is strictly forbidden to freeze calibrators and control serum unless specified in the IFU of the kit.

The remaining strips should be immediately resealed in the bag along with the silica gel, closed with the zip-lock, if such a lock is not available, carefully sealed with the plate sealing tape, supplied with the kit, and placed in the original packaging with the silica gel.
It is strictly forbidden to freeze the plate. This applies to both the plate in its original vacuum packaging and the in-use kit.

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 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.

Contact with the skin with the Stop Reagent, which contains 5% sulfuric acid, may cause irritation. In this case, rinse the affected area with plenty of water. 3,5-tetramethylbenzidine, which is a part of the tetramethylbenzidine (TMB) substrate solution, is a potential carcinogen. All other components of the kit are non-toxic in the concentrations used.
A certain potential hazard to laboratory personnel is posed by reagents containing components of human origin, as well as by the tested serum and plasma samples, which should be considered as potentially infected material that may contain hepatitis, HIV, and other pathogens. Therefore, disposable gloves should be worn when handling the kit.

For each assay, it is necessary to generate an individual calibration curve and use a control serum (the concentration range of the analyte in the control serum is indicated in the Quality Control Data Sheet and on the vial label).

If pre-dilution of samples is a mandatory step in the analysis, for example, when determining aTPO or aTG, when all test samples are diluted 101 times as standard, then no correction of the data calculated on the calibration graph is required. This is because the calibration samples and control serum in such kits are also pre-diluted 101-fold, i.e., the dilution factor of the calibrators, control serum, and test samples is the same. However, if the analyte content of the pre-diluted test sample exceeds the upper limit of the concentration determined by the kit, it must be further diluted according to the Instructions for Use of the kit and reanalyzed. In this case, multiply the result by the additional dilution factor.
In most kits, pre-dilution of the test kits is not required. However, if the measured analyte content of the test sample exceeds the upper limit of detection, it should be diluted according to the instructions for use of the kit and the analysis should be repeated. In this case, multiply the result by the dilution factor.
The recommended dilution factor may depend on the type of sample being tested, for example, when determining hCG concentrations at different gestational ages. Detailed information is provided in the Instructions for Use of the kits.

The construction of a calibration curve and determination of the analyte content in the QUANTITATIVE determination can be performed manually, using linear or logarithmic scale paper, as well as using special computer programs.

Scratches, fingerprints, drips, and other contaminants on the bottom of the wells on both sides of the plate can change the optical density reading (sometimes quite significantly).
Measure the optical density of the wells no later than 5 minutes after adding the stop reagent. This is because the oxidation reaction of chromogen does not stop completely after the addition of acid, and the optical density of the solutions in the wells continues to increase.
Spectrophotometry should be performed only at the recommended wavelength since even slight changes in it can lead to a drop in the recorded optical density values.
The presence of air bubbles in the wells of the plate, which sometimes appear after adding a Stop Reagent to the wells, leads to light scattering and false overestimation of the optical density during spectrophotometry.

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.

The quality of the wells is very important for reliable results. In each washing cycle, the wells should be filled evenly and almost completely with solution. No residual liquid should be left in the wells after the Washing Solution has been removed.
Allowing the Washing Solution to remain in the wells for too long and increasing the number of wash cycles can lead to leaching of the bound conjugate and degrade the quality of the assay.
Any residual Washing Solution should be removed by tapping the inverted plate on a sheet of filter paper.

In this case, the incubation time with the tetramethylbenzidine (TMB) substrate solution may be reduced to 7-10 min. If the color intensity remains excessive despite the above steps, remove a part of the chromogenic solution from all wells and re-measure the optical density.
Do not incubate the plate near heating devices, as the temperature in different wells will vary and changes in the assay results will become unpredictable.

Attempts to compensate for the effect of low temperature by extending the incubation time are not always successful, so if the room is very cold, it is better to incubate in a thermostat that maintains the recommended temperature, including room temperature (+18…+25°C).

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.

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.

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.

To ensure the most complete quality control of laboratory tests, it is recommended to analyze calibrators, control serum, and test samples in duplicate.

Yes, if the device is designed for analysis in a standard 96-well plate and has “open” software.
For some kits, it is necessary that the analyzer includes a thermostatically controlled incubator that maintains a temperature of +37°C±1°C. Some automatic ELISA analyzers do not provide the same incubation time in different wells of the plate. This is due to the sequential and insufficiently fast introduction of the reagents of the kit into the wells. In this case, a drift of results will be observed, especially noticeable when analyzing a large number of test samples, as well as in kits with a short incubation (30 min). To assess the degree of drift, it is recommended to place the control serum wells at the beginning and end of the series of samples to be analyzed.

All glassware and reagent dishes must be thoroughly washed and dried. Please note that even residual contamination from synthetic detergents and oxidizing agents can affect reagent stability and assay results. For this reason, rinse the dishes thoroughly with distilled water.
Separate containers (reagent dishes) and, if possible, separate pipettes with replaceable tips should be provided for the conjugate and tetramethylbenzidine (TMB) substrate solution.
It is recommended to use disposable reagent dishes and pipette tips. When putting the tips on the pipette, do not touch the working surfaces of the table, laboratory glassware, the packaging of the kit, the operator’s hands, or work clothes with the tips.

In addition to a set of reagents for the analysis, the laboratory must have a vertical scanning spectrophotometer (reader) that allows measuring the optical density of solutions in wells at 450 nm and 620-680 nm in the range from 0 to 3.0 optical density units.
In order to comply with the recommended conditions for analysis in the laboratory, the following equipment is required
– an air thermostat that maintains a temperature of +37°C±1°C, which does not provide shaking of the plate;
– a device for shaking the plates (shaker) that maintains room temperature (+18…+25°C) and a temperature of +37°C±1°C. The recommended shaking intensity is 300-800 rpm. In addition, some kits provide only incubation at room temperature (+18…+25°C) without shaking, in which case no additional equipment (“thermoshaker”) is required.
– pipettes with replaceable tips that allow for the collection of liquid volumes in the range from 5.0 µL to 1.0 µL;
– pipettes with replaceable tips that allow for volumes of up to 250 µL or a plate rinsing device;
– 500 mL measuring cylinder;
– distilled or deionized water;
– filter paper.
Carefully read the Instructions for Use of the kits that will be used by laboratory staff to create the necessary minimum equipment.
Note. It is also recommended to use the eight-channel pipette for simultaneously adding reagents to the wells of the plate ( EIA Buffer, Conjugate, Tetramethylbenzidine (TMB) substrate solution, and Stop Reagent).

The EIA kit components should be allowed to stand at room temperature (+18…+25°C) for at least 30 minutes before analysis.
The plate package should be opened only after the specified time, otherwise, moisture will condense on the cooled surface of the wells, which may lead to damage to the kit.

It is advisable that the number of independent series of experiments does not exceed four.

These signs are:
· visible damage to the unopened package with the plate and vials with reagents, calibrators, and control serum;
· presence of sediment, suspended particles, and mold in the solutions;
· the color of the reagents, including the tetramethylbenzidine (TMB) substrate solution, does not match the description.
Such components must not be used for the analysis.
Before starting work, make sure that all components of the EIA kit correspond to the description given in the IFU by visual characteristics.

After first opening, store the vial with the Stop Solution at +2…+8°C for the entire shelf life of the kit. If the color of the Stop Solution changes, its use is not recommended.

The vial with the tetramethylbenzidine (TMB) substrate solution must be tightly closed immediately after opening and stored at +2…+8°C during the entire shelf life of the kit.
Do not freeze the tetramethylbenzidine (TMB) substrate solution.
If stored in the light, or if the tetramethylbenzidine (TMB) substrate solution is contaminated with blood, oxidants, or metal ions, spontaneous blue or blue coloration may occur. This reagent should not be used for analyzes due to increased background during spectrophotometry, decreased sensitivity, and range of detectable analyte concentrations.

After first opening, store the vial with the conjugate at +2…+8°C for no longer than 2 months.
It is strictly forbidden to freeze the conjugate. This will lead to its spoilage.

After first opening, store the vial of Wash Solution Concentrate at +2…+8°C for the entire shelf life of the kit. Store the ready-to-use Washing Solution at room temperature (+18…+25°C) for no longer than 5 days or at +2…+8°C for no longer than 14 days.

After opening the vials, store the calibrators and control serum at +2…+8°C for no longer than 2 months.
It is strictly forbidden to freeze calibrators and control serum unless specified in the IFU of the kit.

The remaining strips should be immediately resealed in the bag along with the silica gel, closed with the zip-lock, if such a lock is not available, carefully sealed with the plate sealing tape, supplied with the kit, and placed in the original packaging with the silica gel.
It is strictly forbidden to freeze the plate. This applies to both the plate in its original vacuum packaging and the in-use kit.

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 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.