United States
Immunoassay Kits Trouble Shooting
Weak Color Development
- Was substrate added at the correct point in the assay?
See the assay procedure provided in the instruction manual. - Were the antibody and conjugate added at the correct time?
See the assay procedure provided in the instruction manual. The antibody and conjugate provided are often color-coded for convenience and to help reduce laboratory errors. - Did all of the components belong to the specific kit being used?
When customers order more than one type of kit, they can sometimes confuse the reagents between kits. - How long was the substrate incubation?
It is possible that Stop Solution was added to the plate without allowing the full substrate incubation. - What were the conditions of the substrate incubation?
If a plate is left to incubate on a cold lab bench or under a drafty area during ambient incubations, signal values (e.g. optical density) may be lower than expected. - Were reagents brought to room temperature prior to use?
It is important to ensure that all reagents are brought to room temperature prior to use, or as mentioned in the product specific instruction manual. Usually leaving the kit out on the bench top at ambient temperature for about half an hour prior to setting up the assay will be sufficient, when the reagents can be stored at 4°C. Frozen volumes take a little more time to come to room temperature. Do not thaw frozen reagents in a water bath. If a different standard/sample diluent is used (such as culture media) this must also be warmed. - Was the plate read at the correct wavelength?
See the assay procedure provided in the instruction manual to ensure you’re reading the plate at the correct wavelength. It may be necessary to check the filters in your plate reader and the program using during reading. If others are using the instrument, they may make changes to the settings for their experiment. - Were the proper volumes of reagents added?
See the assay procedure provided in the instruction manual. - What were the conditions of the incubations?
If the incubation times and temperatures are not observed, this can lead to lower than expected signal values (e.g. optical density). Pay attention that in air-conditioned rooms the temperature does not drop below 21°C. - How was the plate shaken during incubations (if required)?
If customers do not have a plate shaker, they will often use an orbital flask shaker or some other piece of equipment. This is not a problem as long as the liquid is vigorously displaced about 3/4 of the way up the sides of the wells without coming out. It is very important that the plate is secured into place. If the plate is not shaken and it is required in the procedure, a longer incubation may be necessary to bring the reagents to equilibrium. - How long after the addition of Stop Solution was the plate read?
The plate needs to be read at the correct wavelength as soon as possible after the addition of the Stop Solution. We generally recommend that the plate be read within 10 minutes.
High Background
- How was the plate washed?
It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. - What were the incubation times and temperatures?
If the plate was incubated for too long or at a higher than recommended temperature, high background could result.
Drift
- Were reagents brought to room temperature prior to use?
If the reagents are not at a constant temperature prior to their addition into the wells, the results from one side of the plate to the other can differ depending on the temperatures at addition. - Was the set-up of the assay interrupted?
If the assay is interrupted at any point during the addition of reagents, it is possible that differing results will be seen before the interruption versus after. The wells that had reagents added before the interruption will have been incubating for longer than those after.
Poor Precision
- Were the wells washed properly?
All wells receive the same treatment during the wash step. If some are washed less than others, this can translate to poor precision. It is important that the plate is washed thoroughly. If plate washing is troublesome, a squirt bottle can be filled with diluted Wash Buffer and all of the wells completely filled from this. The plate contents can be dumped into the sink and shaken to remove excess buffer. This should be repeated for the number of times recommended in the instruction manual. It is important to remember that adding too little Wash Buffer can result in high background, while adding too much is not a problem. The contents of the wells should be aspirated and the plate tapped dry on lint-free paper towels. - Were the wells aspirated sufficiently after the wash steps?
It is very important that as little Wash Buffer as possible remains in the wells after aspiration. Residual buffer can cause dilution of subsequent reagents. After the last wash step, it is a good idea to hit the plate several times over a piece of paper toweling to remove excess buffer. - How were reagents pipetted into wells?
In order to eliminate precision error, customers need to remember to pre-rinse all pipet tips used in the assay. We usually recommend that the customer draw up the liquid into the tip and aspirate it three times prior to addition into the well. Regular pipet calibration and maintenance is also essential to ensure that the tips fit properly and that the correct volumes are dispensed. Be sure reagents are not splashed between wells or outside of the wells during pipeting (especially if using repeater pipets).
Poor Standard Curve
- What was used as the standard diluent?
Diluents other than the supplied assay buffer may contain interfering substances that can affect the standard curve. - How was the precision of the standard curve?
If the %CV values for the standard curve signal values (e.g. optical density) are consistently above 5%, it may be a good idea to pay particular attention to pipetting technique. If the standard curve signal values were acceptable but the sample precision was not, the problem relates to the sample. Also, see recommendations under "Poor Precision". - Were the Blank and NSB values subtracted out?
If the net signal values (e.g. optical density) are not used, the signal values will appear higher than those presented in the sample data in the instruction manual. - How were the standard dilutions prepared?
It is important that test tubes of an appropriate size and material are used. Standard dilutions must be properly mixed (e.g. vortexed) while preparing the serial dilutions. It is also crucial that the standard dilutions be prepared and used within the time specified in the product specific instruction manual. Never store unused standard dilutions for a later use.
Edge Effects
- Where was the plate incubated?
Often times the conditions for ambient incubations can be less than ideal. If there is a draft in the area or the plate is incubated on a cold lab bench, this can lead to uneven color development. - If multiple plates were run, were they stacked on top of each other during incubation?
Multiple plates should only be incubated in a single layer. This will assure that no area of the plate is at a different temperature than any other. - If a non-ambient incubation was required, was the plate properly sealed?
Making sure that the plate sealer is tightly covering all of the wells will help to discourage uneven evaporation of the well contents, or condensation for colder incubation conditions.
