FAQs

In your LEGEND MAX™ ELISA Kits, there is a step that calls for washing the plates before adding sample. What is the purpose of this step?

We typically use a stabilizer for pre-coated plates. The additional washing step is designed to remove these components before you start the assay. If you do not perform the washing, the effect on assay performance is negligible.

What antibody clones are used in your ELISA Kits?

Information regarding the antibody clones used in our kits is proprietary. However, the clonality (polyclonal or monoclonal) and host species of the antibodies may be provided upon request.

Should I use serum or plasma samples for my ELISA experiment?

This is dependent on what targets you are analyzing and the specifics of your study. Ideally, we advise determining the difference between serum and plasma for the targets of interest and deciding on the sample type to be used for quantification. Depending on the 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 at the same time. Avoid repeated freezing and thawing cycles.

My sample may have very low levels of analyte. What methods can I use to improve detection?

For our LEGEND MAX™ and ELISA MAX™ Deluxe formats we recommend following the provided protocol. We cannot guarantee kit performance if there are deviations from the protocol. Below are general suggestions if you are using our ELISA MAX™ Standard format:

• Control background noise by increasing the number of washings and soaking time between washes.
• 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 and increase washing volumes.
• Increase incubation times. Be aware that this typically also increases background so the assay sensitivity may not necessarily increase.
• Concentrate your sample, if possible.
• Use a five-parameter logistic curve fitting method to more accurately calculate sample concentration at the lower end of the standard curve.

In many cases, the sample may simply contain very low to non-detectable levels of the analyte. No matter how you manipulate the assay, you may still not be able to obtain detectable concentrations.

We ran out of capture/detection antibody in our ELISA kit. Can we use a standalone/single antibody to replace it?

No, we do not recommend using standalone reagents to replace kit components and cannot guarantee the performance of the kit if you replace or combine reagents from different kits/manufacturers.

How many samples can I run with your kit?

This will depend on whether your samples are analyzed in duplicate or triplicate. For example, after adding the standards, you can run 80 samples with no replicates or 40 samples in duplicate and so on.

How should the plates be stored after coating?

If coated plates cannot be used immediately, they should be sealed and stored overnight at 4°C.

I have multiple LEGEND MAX™ ELISA kits that I want to run simultaneously. Can I use the same wash buffer for all the kits?

The wash buffer provided in all our LEGEND MAX™ kits is the same and the part numbers on the wash buffer bottles in these kits should be identical. 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.

I am using the biotin and purified formats of the same antibody clone to try to set up my own ELISA, but I am having no success.

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. We recommend choosing different clones for your capture and detection antibodies.

Where can I find troubleshooting tips for ELISA assays?

Please consult our ELISA troubleshooting guide.

When should I use LEGEND MAX™ ELISA kits with pre-coated plates?

LEGEND MAX™ Kits are ready-to-go, designed for ease-of-use, and minimize requirements for coating the ELISA plates and preparing buffer dilutions. We recommend LEGEND MAX™ Kits for users who are new to ELISAs.

What kind of coating buffer should I use for my ELISA?

In general, we recommend using PBS (pH 7.4) or carbonate buffer (pH 9.5). The exact coating buffer you choose may be dependent on the cytokine being detected.

Why do we recommend not using azide in ELISA buffers?

Azide is an irreversible inhibitor of HRP.

What is the sensitivity of your ELISA kits?

If you are using LEGEND MAX™ or ELISA MAX™ Deluxe format, please consult the individual kit’s manual for more information on sensitivity. We do not provide this information for the ELISA MAX™ Standard Sets but in theory, it should match that of the ELISA MAX™ Deluxe format if you use all of BioLegend’s reagents.

What is the shelf-life of BioLegend ELISA products?

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 a lot-specific expiration date, refer to the box label on each product.

Can I use the recombinant standard provided with the kit for bioassay?

No, we do not recommend using the recombinant protein standard for any other applications. The protein standards included in the kit are calibrated for use as an ELISA standard and have been not tested for bioactivity. Additionally, ELISA protein standards may contain other carrier proteins and have not undergone the same sterility testing as our bioactive recombinant proteins.

Can I use tissue culture grade sterile plates for ELISA?

No. Tissue culture plates are designed for cell culture purposes and do not typically have high protein-binding capacity. For ELISAs, we recommend using high protein-binding plates such as Nunc Maxisorp™ plates (Cat. No. 423501).

Can I use the capture and detection antibody provided as part of an ELISA Kit for other applications such as flow cytometry staining?

The antibodies used in our ELISA Kits have been validated for their use in ELISA assays and we cannot guarantee that they will work in other applications, like flow cytometry. Instead, we recommend you use flow cytometry validated antibodies for these experiments.

Can I use your ELISA kits for my tissue samples?

In general, BioLegend’s ELISA products can be used for tissue or cell extracts/homogenates as long as the samples are prepared in such a way that they are compatible with immunoreactivity. For this, we recommend using the following guidelines:

• 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 (such as SDS, Triton™ X-100).
• 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.

Can samples such as serum be reused?

We do not recommend reusing samples. For accurate results, samples should be aliquoted and stored for one-time use only. If you decide to reuse a sample, you would need to perform additional validation experiments to ensure you are able to get accurate readings. This would be influenced by a number of factors including sample stability.

Can I use your matched ELISA antibody pairs with a protein standard from another company?

If the antibodies were provided as part of a kit, we do not recommend combining reagents from different ELISA Kits. We cannot guarantee the performance of the kit if you combine reagents from multiple kits or manufacturers. If you have purchased individual antibodies and are developing your own ELISA, it may be possible but would need to be tested. Antibodies may not recognize or show poor binding toward the protein standard if the immunogen used to generate those antibodies is different from the protein standard in terms of sequence or structure.

At what step can I delay my ELISA procedure for a few days? Can I freeze my plates for later use?

It is best to follow the recommended protocol without any additional delays. However, if you wish to pause the experiment, we would recommend delaying the procedure at the blocking step (after coating with the capture antibody). You can add 200 μL of blocking buffer in the wells and keep the plates 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.

I ran out of some ELISA kit components, can I them buy separately?

It may be possible to purchase some kit components individually. Please contact our technical service group and provide lot information of the kit and its components to determine whether this is feasible.

Can I mix reagents from different ELISA MAX™ Sets or use them for other applications?

This is not recommended. The ELISA MAX™ reagents are optimized for a particular lot, and they have not been quality tested for applications other than ELISA.

Can coating with capture antibody be carried out for longer than overnight?

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 increase the background noise.

Can I add an extra standard at the higher end of the standard curve in order to calculate higher concentration of the analyte?

We don’t recommend this practice. 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.

Can I add an extra standard at the lower end of the standard curve to enhance the sensitivity of my assay?

While this may be possible, you may end up with a signal plateau at higher concentrations of the standard. It is generally recommended to use the concentration range recommended in the user manual.

Does phenol red in the medium interfere with an ELISA assay?

No, phenol red in cell culture media will not interfere with the readings from an ELISA assay.

For some of your ELISA kits, why do my serum samples require dilution with assay buffer?

In some cases, dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.

How can I obtain better signal and sensitivity for my ELISA assay?

• Increase incubation times (with samples, detection antibodies, or avidin-HRP or TMB substrate). Please note, this may also increase the background in your assay and may not increase sensitivity.
• Shake plates during incubation steps.
• Ensure 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 450-570 (or closest) nm for background subtraction.
• Improve duplicate CV% by controlling pipetting error or washing with larger volume of washing buffer.
• Use a 5-PL or 4-PL curve-fitting method, rather than a linear curve fitting. This is usually performed with a curve fitting software and provides better calculation at the lower end of the curve.

What is the difference between your ELISA Kits and Sets?

The primary differences between these kits are the reagents included within the kit or set.

LEGEND MAX™: Fully validated and ready-to-use kits. They contain all required reagents, including a 96-well strip plate pre-coated with capture antibody.

RAPID MAX™: cut assay time down to less than 90 minutes. Each kit is fully validated and contains a 96-well strip plate that has been pre-coated to immobilize the capture antibody.

ELISA MAX™ Deluxe: A more cost-effective option that includes most buffers, pre-titrated antibodies, recombinant standard and substrate. Plates are not included in this kit and must be purchased separately.

ELISA MAX™ Standard: Provides the basic components to complete an ELISA including a recombinant protein standard, capture and detection antibodies, and Avidin-HRP. This set requires some optimization and is perfect for those that want to stretch their budget by providing their own buffers and plates.

Do the ELISA MAX™ Deluxe Sets come with plates?

No, ELISA MAX™ Deluxe Sets do not come with plates. Coating Buffer and Assay Diluent (for blocking and dilutions) are included in these sets. Plates can be ordered separately (Cat No. 423501), and instructions for coating the plates are in the sets’ product manuals.

Can I use different components from different companies for my ELISA?

Different manufacturers often use different antibody clones in their kits. The specificity of the antibodies partially dictate how much signal is being detected.

Additionally, different kits may use recombinant protein standards that were expressed and purified using different methods. For example, recombinant proteins expressed in E. coli can show different immunoreactivities primarily due to refolding inconsistences. Kit standards are produced and calibrated against different references between manufacturers, making it difficult to compare components from different kits. Each BioLegend ELISA Kit was developed and validated with reagent concentrations and protocols optimized for analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) and protocols will affect the final assay performance.

Why are my sample’s cytokine concentrations different using LEGEND MAX™ kits vs other ELISA kits?

It is very difficult to obtain exactly the same sample concentrations at pg/ml levels when comparing different cytokine kits from different vendors, in part, because suppliers may use different reagents (including antibody clones) that have different immunoreactivity toward the target protein. It is not uncommon that the cytokine concentration may vary a few-fold between kits.

In general, particularly for those cytokines in pg/mL ranges, it is more important to have matching biological trends instead of matching absolutely concentrations. This is to say, the same cytokine measured by different kits from different vendors should show the same pattern of biological changes for relevant treatments.

What is the expected concentration of a particular analyte in biological samples (e.g. in serum samples of naÏve animals or normal humans)?

Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. If you are using a LEGEND MAX™ Kit, please refer to the respective manual for more information.