Please find below many questions and answers about ELISA kits, if you still can’t find an answer to your question then please either sent us email at: firstname.lastname@example.org or tell us about it through our contact us section.
1. How should one store samples?
We would always recommend using fresh samples whenever possible (often these give the best results). Alternatively, samples can be stored at – 20°C, – 80°C or in liquid nitrogen (depends on the sample).
2. Can sample types be used that are not recommend in the kit insert?
In most cases yes, providing the expected concentration is within the kit’s range (ideally this should be mid-range). It is important to note that the sample types mentioned in the protocol insert have been validated and confirmed to work under those specified conditions. Other sample types have not been tested and therefore there is no guarantee whether they will work or not. We will not be held responsible if other sample types are used and this will also make the returns policy invalid.
3. What is the storage temperature for the kit?
All our kits usually have a recommended storage temperature of 2-8°C. However, there are some components of the kit that may require – 20°C storage. Please refer to the product protocol insert for further details. It is important to follow the recommended correct storage conditions for all the reagents as per instructed in the insert, this will ensure achieving best performance for the kit.
4. Can one modify the protocol insert?
We would not recommend this, since our ELISA kits are optimised to provide the best possible results under these conditions. Modifying the protocol or format could comprise on the quality of the results obtained. It could even give rise to wrong or inaccurate results.
5. Can components from different kits be used?
It is possible to use common parts from kits that have been manufactured from the same lot/batch (but not recommended), since QC testing is performed on specific lots. However, each kit is prepared freshly for each order and the kit components have be optimised for that kit alone. We would never recommend the use of components from other kits, other supplier kits and even own components.
6. What sample volumes and concentration can one use?
Sample volumes are usually between 50-100μl, however, this can vary from assay to assay. It is recommended to dilute samples to the mid-range of the kit in order to achieve the best results. Also, be aware that there is a possibility that low concentrations may not be detected accurately that are close to the lower end of the range, since the range of the ELISA assay is known to be lost over time or if the kit is not stored appropriately. Do not alter sample volumes that are recommended in the insert because these have been designed for optimal performance. Instead, it is advised to seek to use either more diluted or concentrated samples.
7. What to do with samples that generate values outside the dynamic range of the assay?
Samples that generate values which are higher than the highest standard need to be repeated using a higher dilution factor. For sample values that fall below the range of the assay, these are considered to be non-detectable. Values outside the range of the standard curve are usually non-linear and because of this property it is not possible to extrapolate a value correctly using this curve. We recommend only sample values that fall within the assay’s dynamic range are used.
8. Does one need to run all the samples at the same time?
No, because most kits are using strip-well microplates, this allows the use of single strips at a time which can be used to analyse different number of sample at different times.
9. Does a blank and standards have to be run every time?
Yes, since these values are required in order to calculate the concentration of the samples and for reproducibility. This is important because it could reflect changes in performance from assay to assay and day to day.
10. Do duplicate samples and standards need to be run?
Yes, this is important in monitoring the assay’s precision and increases the confidence in the results that are obtained from the assay. We would recommend that a minimum of duplicates but ideally triplicates (or more) should be run.
11. How can one obtain reproducible results?
Any variations in the incubation time or even the temperature, operator, washing and pipetting techniques being used and the age of the kit can all lead to many variations being created. These can directly affect the results that are obtained and it is also highly recommended that each user obtains their own standard curve.
12. Standards working fine but no signal from samples?
This could be due to many reasons. The simple fact might be that the sample may not contain the analyte think you are testing. The recommended dilution was not followed correctly, where over dilution of a sample may cause it to fall below the range of the standard curve, thereby making it undetectable. There could also be a matrix effect which may be masking the detection.
13. What is sample matrix effect?
Most biological samples are made up of a complex mixture of many different proteins and salts. The presence of these dissolved components is known to reduce the binding ability of antigens and antibodies. This leads to much greater time frame required for end point binding or equilibrium conditions being established. False signals are also known to be generated because of non-specific binding interactions of IgG present in the sample. The formulation of sample diluent buffer can be used to reduce the assay background noise which is associated with matrix effects and also minimise non-specific binding of IgG thereby helping to optimise the assay signal. For this reason alone many samples may need dilution in order to fall within the functions range of the assay.
14. Is a plate shaker required?
It is highly recommended since it is known that the O.D.s values that are obtained are usually much lower when compared to when a shaker is used.
15. What are the recommended processes to carry out washing steps?
Both manual and automated washing procedures are viable options. We recommend calibration is performed on a regular basis and systems are flushed prior to washing. Care must also be taken to aspirate all of the contents and lint-free paper used when the plate is tapped dry.
16. What are the recommended dilutions if a sample has been extracted?
In this case, it is advised not to follow the recommended dilution stated in the kit manual, since the amount of sample dilution required following an extraction procedure will be affected by the effects of the extraction and purification steps. The amount of concentration or dilution will need to be determined by the end user.
17. How is wavelength correction carried out?
The plate reader must be set to 450nm and if wavelength correction is available, then this needs to set to 540 or 570nm. This subtraction will correct any optical imperfections that may be present in the plate. If however, this is not available in the reader then one needs to subtract readings at 540nm or 570nm from the reading at 450nm. The main reason why readings at dual wavelengths is carried out is to correct for optical density that may be contributed by the plastic wells, the lamp or any other optical fluctuations. If readings are only directly made at 450nm without any correction that this may results in higher and less accurate results being obtained.
18. What are the main causes of getting high background signals?
A major cause is having a contaminated substrate which contains oxidising agents or metal ions. Improper washing is another well-known cause, it is vital to check volume of washing buffer reservoir and to follow exactly the recommended washing procedure mentioned within the protocol insert. Exposing the substrate to light can result in turning the substrate into its measuring blue colour and this can also lead to a high background signal.