Yes. Please contact firstname.lastname@example.org and provide lot information of the kit and its components.
It is best to follow the recommended protocol without unnecessary delays. However if you wish to delay then it may be better to delay the procedure at the blocking step (after coating with the capture antibody). You can add 200 μl of blocking buffer in the wells and either the plates can be kept overnight at 4°C (minimal or no loss of signal) or frozen at -20°C for few days (it may have some impact on the signal). Delaying the procedure at the first step (coating with capture antibody beyond 16-20 hrs at 4°C) is not advisable because it may lead to high background.
It is possible. Generally, coating for 16-20 hours at 4°C is recommended. Longer incubation time may increase the amount of capture antibody bound to the plates and this may also increase the background noise.
We don’t recommend this. Sensitivity of a kit depends on the individual components and their collective validation as a kit and it will not change by adding extra points to the standard curve.
While this may be possible, you may end up with a plateau in signal at higher concentrations of the standard. It is generally recommended to use the concentration range recommended.
This is not recommended. The ELISA MAX™ reagents are optimized for a particular set lot, and they are not recommended for applications other than ELISA.
Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected. Recombinant standards used are different. First of all, different kits may use recombinant proteins expressed and purified using different method. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold difference in term of immunoreactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for cytokine immunoreactivities.
Each BioLegend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) and protocols etc all affect the final assay performance.
Since the antibodies are validated in house for ELISA, it is empirical to find out if the antibodies work for applications such as flow cytometry. We recommend you use flow cytometry validated antibodies for this purpose.
No. It is not recommended because the ELISA protein standards are not sterile, may contain other carrier proteins, and not tested for bioactivity. Therefore this material is not bioassay grade.
No. Tissue Culture grade plates are designed for cell culture purpose and do not typically have high binding capacity. We recommend using high protein binding plates such as Nunc Maxisorp™ plates (Cat# 423501).
Almost all of BioLegend’s ELISA products can be used for tissue/ cell extract/homogenate samples as long as the samples are prepared in such a way that they are compatible with immune-reactivity:
• Tissues/cells should be lysed or homogenized in a neutral pH buffer that contains no denaturing chemicals (such as urea, thiourea, SDS).
• No or minimal levels of detergent (SDS, Triton X-100 etc).
• No excessive ionic strength (salt concentration greater than physiological ionic strength).
• The buffers should contain sufficient protease inhibitor cocktails to preserve the target proteins from proteolytic degradation by enzymes released from cells.
Theoretically you can but we don't recommend it and we also can't guarantee the performance of the kit as indicated in the product manual. Antibodies may not recognize or show poor binding towards the protein standard if the immunogen protein used to generate these antibodies is different in terms of structure or sequence. It is therefore empirical to find out if it works. It is best to acquire the ELISA kit components from one source.
It is recommended that for accurate results samples be stored aliquoted for one-time use only. However it is empirical to find out if reusing samples work for a particular analyte as it will depend on sample stability.
No, ELISA MAX™ Deluxe sets no longer come with plates, but Coating Buffer and Assay Diluent (for blocking and dilutions) are included in the ELISA MAX™ Deluxe Sets. Plates can be ordered separately (Cat. No. 423501), and instructions on coating the plates are in the sets’ product manuals.
Dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.
• Increase incubation times (1st incubation, detection, avidin-HRP or TMB substrate)
• Shake plates during incubation steps
• Make sure that the standard is completely reconstituted before use.
• Increased washing and soaking in between washings to further decrease background.
• If possible read the plate at 570-590 nm for background subtraction.
• Improve duplicate CV% by controlling pipetting error, washing with bigger volume of washing buffer, etc
• Use a 5-PL or 4-PL curve-fitting method for better calculation at the lower end of the curve. This is usually done with a better curve-fitting software, rather than the linear curve fitting.
It depends on how your samples are analyzed whether in duplicate or triplicate. For example you can run 80 samples with no replicates or 40 samples in duplicates and so on.
If coated plates cannot be used immediately, they should be sealed and stored overnight at 4°C.
If you are using the same clone for both the detection and capture antibodies, the epitope may already be occupied by one of the antibodies and prevent binding of the other. You should always choose different clones for your capture and detection antibodies.
The Wash buffer is the same for all the current LEGEND MAX™ kits. All the part numbers on the Wash Buffer bottles in these kits should be the same. For ELISA MAX™ Deluxe and ELISA MAX™ Standard sets, we provide a recipe for the wash buffer on each kit’s technical data sheet. This recipe is the same for all ELISA MAX™ sets.
We typically use a stabilizer for pre-coated plates. The washings were designed to remove these components before you start the assay. If you do not do the washings, the effect on assay performance is negligible.
For our LEGEND MAX™ and ELISA MAX™ Deluxe formats we recommend following the prescribed protocol. We can’t guarantee kit performance once fixed protocols are changed. Below are just general suggestions for our ELISA MAX™ Standard format.
a) Control background noise: For example, increase number of washings and soaking times in between washes.
b) Control assay precision: For example, be more careful and more consistent in your pipetting, use fresh paper towels for tapping plates to avoid contamination of avidin-HRP from dirty paper towels and increase washing volumes.
c) Increase incubation times: However this usually also increases background so the assay sensitivity may not necessarily increase.
d) Concentrate your sample if possible.
e) Use a five parameter logistic curve fitting method, which can accurately calculate sample concentration at the lower end of the standard curve. In many cases, samples indeed contain very low to non-detectable levels. No matter how you manipulate the assay you may still not be able to obtain detectable concentrations.
This is dependent on the targets being detected and the biological processes. Customers are advised to study the difference between serum and plasma for the targets of interest and decide on the sample type to be used for quantification. Depending on targets, there may be a difference in concentrations of the targets between serum and plasma. The most important factor in preparing plasma or serum samples is consistency in preparation to ensure precise measurements. In general, plasma/serum samples should be free of particulate matters, contain no excess lipids, and have no hemolysis.
These types of contaminants will contribute to background, and adversely affect the precision of the assay. The key is to prepare the sample the same way each time. That is, centrifuging samples at the same speed, for the same time, removing the serum or plasma immediately after centrifugation and aliquoting and freezing the samples in the same time. Avoid repeated freezing and thawing cycles.
No, we don’t recommend it.
That information is proprietary. However the clonality (polyclonal or monoclonal) and host species details may be provided upon request.
LEGEND MAX™ kits with Precoated plates:
ELISA MAX™ Deluxe:
ELISA MAX™ Standard:
Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. For LEGEND MAX™ kits refer to the respective ELISA manual for more information.
For ELISA MAX™ Standard format the sensitivity should match the ELISA MAX™ Deluxe version if BioLegend components are used. As for individual LEGEND MAX™ and ELISA MAX™ Deluxe formats the sensitivity values are mentioned in the manual for each kit.
BioLegend's LEGEND MAX™ Kits are guaranteed for 3 months from the date of receipt. ELISA MAX™ sets are guaranteed for 12 months from the date of receipt. For lot-specific expiration date, refer to the box label on each Kit or Set.