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Why not have a look at our ELISA Sample Preparation Guide, our ELISA Standard Curve Guide for competitive kits or non-competitive kits, or our Troubleshooting Guide?
Normal analyte concentrations should be estimated or determined by the end user from contemporary literature.Yes, running duplicates or triplicates of the standard and samples monitor assay precision and increase confidence in the assay results obtained.
No, the kits are made using stripwell microplates. This allows the user to analyse different numbers of samples at different times.
Each user should obtain their own standard curve. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result.
The stability of the kit is determined by the rate of activity loss. The loss rate is typically less than 5% within the expiration date under appropriate storage conditions. Please store the kit according to the storage instruction in the kit manual (different kits may have different storage instructions). This ensures appropriate kit performance.
For best results, we recommend using fresh samples. Alternatively, samples should be stored at -20 °C, -80 °C or in liquid nitrogen.
Abbexa ELISA kits have been optimized to provide the best possible results. Modifying the format or protocol may give inaccurate and wrong results.
Yes, provided the expected concentration falls within the kit's range, ideally mid-range. Please note that sample types other than those validated have not been tested.
It is recommended that only sample values that fall within the kit's dynamic range are used. Values outside the range of the standard curve are generally non-linear, which can lead to incorrectly extrapolated values. Samples that generate values higher than the highest standard should be repeated using a higher dilution factor. If sample values fall below the range of the assay, the sample is considered to be non-detectable.
Yes. These values are required to calculate the sample concentrations. They reflect performance changes from day to day and assay to assay and are required for reproducibility and troubleshooting.
Sample volumes are typically 50-100 μl, but can vary. Specific sample volumes are specified in the product manual. We recommend samples are diluted to mid-range of the kit for best results. Lower concentrations close to the lower end of the range may not be accurately detected, as the kit will lose range with the passing of time or if not stored at the appropriate temperature. This affects all ELISAs.
It is not recommended that you alter the volumes since all Abbexa ELISA kits are designed for optimal performance at the given volumes. Preferably, seek to either use more concentrated or diluted samples.
Our kits are freshly made for each order, so if you ordered and received the kits together, common components can be used. However, please note that QC testing is performed on these specific lots. It is never recommended to use your own components or components from other kits or vendors.
The sample may not contain the analyte. A matrix effect may be masking the detection. Ensure that the recommended dilution was followed as stated in the kit insert. If dilution was recommended, check to be sure that the dilution was performed properly. Over-dilution may cause the sample to fall below the range of the standard curve.
Both automated or manual washing are viable options. If you are using an automated plate washer, we recommend that the calibration be checked on a regular basis, and that the system is flushed with the Plate Washing Buffer prior to washing. The same is true for manual washing. A repeater or a wash bottle can also be used. The user should be careful to ensure that all of the contents are aspirated and the plate tapped dry on lint-free paper.
Reliable results can be obtained without a plate shaker, but the O.D.s observed will generally be lower than those obtained using a plate shaker.
The plate reader should be set to 450 nm. If wavelength correction is available, set to 540 or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plates. Readings made directly at 450 nm without correction may be higher and less accurateFor the ELISA assay, reading at dual wavelengths is done to correct for the optical density contributed by the plastic well, the lamp and optical fluctuations.
If I extract my sample, do I still need to follow the recommended dilutions given in the kit insert?
The amount of sample dilution needed after an extraction procedure will be affected by the effects of purification and concentration in the protocol used. The amount of dilution or concentration will have to be determined by the end user.
The amount of a given analyte may vary not only from species-to-species, but also between tissue and cellular sources. The best source of this information is the current literature that is easily accessed through the Internet at multiple scientific databases.
The optical density is affected by a number of physical conditions such as time and temperature. We suggest that you shorten or lengthen the final incubation with substrate solution to compensate.
Improper Washing:Check volume of washing buffer reservoir and make sure all recommended washing steps are being performed.
Contaminated Substrate: Make sure there is no contamination of the substrate with metal ions or oxidizing reagents. Keep the extra substrate solution separately during the ELISA substrate development time.
Substrate exposed to light: Exposure to light may result in a blue color of the substrate. Keep solutions in the dark (vial) until ready to dispense into the plate. If all wells are intensely and equally colored with no intensity gradient observed in the standard dilution series, then it may be necessary to observe the substrate reaction as the color is developing, in order to stop the reaction sooner.
Contaminated Substrate: Make sure there is no contamination of the substrate with metal ions or oxidizing reagents. Keep the extra substrate solution separately during the ELISA substrate development time.
Substrate exposed to light: Exposure to light may result in a blue color of the substrate. Keep solutions in the dark (vial) until ready to dispense into the plate. If all wells are intensely and equally colored with no intensity gradient observed in the standard dilution series, then it may be necessary to observe the substrate reaction as the color is developing, in order to stop the reaction sooner.
Most biological samples, whether derived from serum, plasma, or cell culture fluid, contain a complex mixture of different proteins and salts. The presence of these dissolved components in the sample wells may reduce the binding ability of the antibody and antigen. They may also cause a false signal due to nonspecific binding interactions of IgG present in the samples. This translates into a much longer time frame for equilibrium/end point binding conditions to be established and false results.
Samples may require dilution to fall within the functional range of the assay. Our sample diluent buffer formulation also reduces assay background noise associated with matrix effects and minimizes nonspecific binding interactions of IgG in the samples, thus optimizing the assay signal.
