Help & FAQs: ELISAs

  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can distilled water be used instead of deionized water to dilute kit diluents?
  • R&D Systems uses deionized water to dilute diluent concentrates. Distilled water has not been evaluated for impact on assay performance.
  • Can ELISA Kits be used with tissue homogenates (or other non-validated sample types)?
  • Unfortunately, R&D Systems has not routinely validated tissue homogenates as a sample type for ELISA kits. This does not mean that ELISA kits are not suitable for other sample types than we have tested; it means further investigation is needed. One will need to perform a spike and recovery study to determine if an unvalidated sample type will work with a particular kit. To perform a spike and recovery experiment, one should divide a sample into two aliquots. In one of the aliquots, the user should spike in a known amount of the kit standard. A dilution series is performed comparing the spiked versus the unspiked sample. Generally, samples with expected recovery and linearity between 80-120% are considered acceptable. This method can be used to validate any sample type that has not been evaluated by R&D Systems. For a more detailed spike and recovery protocol, please contact Technical Service.Note: Acceptable ranges should be determined individually by each laboratory. Additionally, Technical Service can help determine if a buffer component is not compatible with a given ELISA kit. Please see the Citations tab on the product webpage for peer-reviewed papers utilizing a wide range of sample types.
  • Can ELISA plates be stacked on top of each other during assay incubations?
  • To ensure consistent environmental conditions for all plates, it is recommended that ELISA plates are not stacked during incubations.
  • Can recombinant proteins sold as stand-alone reagents be used as standards in the DuoSet® ELISA Development Kits?
  • These products are not calibrated for use in an ELISA system, and must be calibrated versus an existing mass calibrated standard. Standards provided in ELISA kits have been calibrated to a master calibrator.
  • Can the standard curve be extended in either direction?
  • R&D Systems cannot support standard concentrations outside the validated standard curve. The standard curve range has been validated for reliable and accurate performance across multiple kit lots and operators. The lowest standard concentration is the lowest limit that R&D Systems can guarantee for reliable assay measurements. Adding a concentration above or below the standard curve may result in inconsistent signals or false positives.
  • Does Bio-Techne provide ELISA testing recommendations for inactivating samples thought to contain Covid-19 or other viruses?
  • Bio-Techne does not provide recommendations for inactivation of viruses in samples. Heat and chemical pre-treatments may cause conformational changes to most proteins. These proteins do not revert to their normal conformation without the help of molecular chaperones. It is recommended that investigators validate pre-treated samples with each ELISA.
  • Does R&D Systems add Heterophilic Blocking Reagents to Assay diluents and Calibrator Diluent provided in ELISA and immunoassay kits? Will you recommend treating samples with HBT reagents?
  • R&D Systems does not add HBT reagents to diluents, but instead formulates specialized diluents that are designed to alleviate false positives to achieve the most accurate results. Our diluents are designed to minimize interfering factors such as binding partners, soluble receptors, HAMA, and rheumatoid factor. With R&D Systems immunoassays, it is not necessary to add heterophilic blocking reagents to the samples. 
  • Does the addition of assay diluent need to be accounted for in sample dilutions and calculation of the final analyte concentrations?
  • No, the use of assay diluent for sample dilutions is not a factor when calculating the final concentrations of analytes. This is because both standards and samples are mixed with the same volume of diluent and sample values are measured against the standard curve.
  • Have R&D Systems ELISA kits been evaluated for cross-reactivity with different species?
  • When available, recombinant proteins for different species are evaluated in an ELISA kit for cross-reactivity and interference. R&D Systems cross-reactivity test results are reported in the Specificity section of the product datasheet. ELISA kits may have been used in testing different species samples and can be found in the citations tab on the product specific webpage.
  • How are cell lysates prepared for use in Quantikine® ELISA kits?
  • For those Quantikine® ELISAs where cell or tissue lysate is a validated sample type, sample preparation instructions for lysate are included in the product insert.  Components in lysate and lysis buffer can impact immunoreactivity, so if lysate is not a validated sample type, care must be taken in sample preparation and validation.   
  • How is an ELISpot assay different from an ELISA assay?
  • Both assays employ the sandwich ELISA format utilizing capture and detection antibodies. The traditional ELISA is designed measure the concentration of analyte in biological samples such as serum, plasma, and conditioned medium. In contrast, the ELISpot assay provides information about the relative number of cells secreting the specific analyte. In an ELISpot assay the cells are added to the plate and the immobilized capture antibody in the immediate vicinity of the cell binds the secreted protein of interest.The sites of cytokine secretion are visualized as spots in the bottom of the well.
  • How many samples can be assayed in a Parameter™ ELISA Kit?
  • The Parameter ELISA Kits will generally run a 7-point standard curve, non-specific binding wells, and 39 samples in duplicate. This may vary slightly by kit so please refer to each datasheet for details.
  • If the Calibrator Diluent appears turbid or discolored, is it ok to use?
  • Calibrator Diluents may be formulated with animal serum or a protein base, and will be indicated in the reagent description in the Materials Provided section. Diluent lots may differ in appearance, including color and turbidity, due to the inherent variability of these biological components. Each lot is QC tested to ensure the kit passes performance specifications prior to sale. 
  • The protocol requires a 0.12" orbit shaker set to a speed setting of 500 RPM. Are there alternatives to this size and/or speed setting?
  • The ELISA protocol was validated using a 0.12" orbit shaker at 500 RPM. This is similar to a shaker used for any Luminex assay. If the shaker orbit is different but has similar speed capability, please contact Technical Service for additional recommendations. R&D Systems recommend ThermoFisher model # 4625, Microtiter Plate Shaker.
  • What are R&D Systems' recommendations for reducing %CVs and improving assay precision?
  • There are various techniques that can be adapted to improve assay accuracy and precision. These include reverse pipetting (rather than forward), avoiding vortexing or shaking during the reconstitution of lyophilized reagents, and the use of either a squirt bottle, auto plate washer, or manifold dispenser rather than pipettes for plate washing. If concerns remain, please contact Technical Service for further assistance.
  • What are the dimensions of the ELISA microplates included in R&D Systems ELISA kits?
  • The plates included in R&D Systems ELISA kits meet standard footprint dimensions for microplates, 127.76 mm x 85.48 mm x 14.22 mm (length x width x height).
  • What is a competitive ELISA?
  • In the competitive immunoassay approach, also termed "labeled analyte technique", there exists a competition between the endogenous unlabeled antigen and an exogenous labeled antigen for a limited amount of antibody binding sites. Therefore, a decreasing signal indicates higher concentrations of the analyte being measured.
  • What is a sandwich ELISA?
  • A sandwich ELISA uses an immobilized capture antibody specific for the analyte of interest in a sample. After the analyte is bound to the immobilized antibody, a labeled secondary antibody specific for the analyte is used for detection. The analyte is "sandwiched" between the two antibodies. The sandwich ELISA is extremely sensitive, and the values obtained are quantitative when compared with a standard curve.
  • What is R&D Systems doing to reduce its use of packaging from non-renewable resources?
  • Environmental stewardship is important to R&D Systems and its employees. R&D Systems is ISO 14001:2015 Certified and as part of this we are continuously reviewing our sustainability practices and "green" options. First and foremost, the energy expended to ship back the styrofoam box is more detrimental to the environment than having the facility re-use or recycle it. Our stance is to encourage our customers to implement a recycling program locally. At R&D Systems we have chosen to reduce the use of styrofoam as much as possible by: doing extensive stability testing in order to determine which products can be shipped with minimal packaging at ambient temperatures, converting the use of non-recyclable packaging materials to recyclable plastics, cardboard, or biodegradable materials, and continuing to investigate alternatives to dry ice shipments, the use of re-usable containers, and gel packs that allow for smaller styrofoam containers. Employees were key to initiating a recycling program at our facilities. Internally, we recycle paper, plastic, cardboard, aluminum, and glass.
  • What is the expected range of OD values for the highest standard concentration?
  • Many factors can influence the high standard OD value. Signal can vary according to operator technique for the standard curve preparation, washing method, incubation variability, and plate reader calibration. R&D Systems shares typically observed standard curve ODs in the assay insert. The maximum assay signal is expected to measure between 1.0 and 3.5 OD. For added confidence in assay performance, we offer QC Immunoassay Controls for our Quantikine and Parameter brand ELISAs.
  • What is the half-life of a cytokine in serum/plasma/cell culture supernates/cell-cultured media?
  • R&D Systems does not determine the half-life of cytokines in samples such as serum, plasma, cell culture supernates, or cell-conditioned media. The general recommendation is to use the sample immediately or aliquot into single-use volumes and store the samples frozen. Multiple freeze-thaw cycles should be avoided to prevent protein degradation.
  • What is the purpose of wavelength correction? Do I need to use 540 or 570 nm?
  • The purpose of wavelength correction is to correct for optical imperfections in the plate. R&D Systems generally recommends in ELISA protocols to set wavelength correction to 540 nm or 570 nm. Wavelength between 540 - 690 nm can be used as correction. Readings made directly at 450 nm for ELISA assays may be higher and less accurate.
  • What should be done if an ELISA kit contains a broken and/or spilled vial(s)?
  • Please contact Technical Service immediately if a kit contains a broken or spilled vial. If available, a replacement component will be sent (lot-matched if needed). If the assay is in process, alternative solutions can be discussed. R&D Systems has not evaluated the effects of stopping an ELISA at any point within the protocol and cannot guarantee assay performance if delays occur. 
  • Why are my wells green after adding the Stop Solution?
  • This happens when the substrate in the well does not completely mix with the stop solution. After addition of the stop solution, tap the plate gently or place on a shaker until the mixture in the wells turns yellow. To prevent this from happening, we recommend touching the pipette tip to the side of the well at a 45 degree angle to the plate and quickly dispensing into the well. This angle and speed helps disperse the Stop solution throughout the well.
  • Why do some DuoSet ELISAs have different standard curve ranges than the Quantikine ELISA Kits for the same analyte?
  • These ELISAs may use different reagents that can result in different sensitivity. The DuoSet ELISAs are development assays, validated for testing cell culture supernatants. Additional optimization may be performed by the end user to achieve greater sensitivity and validate more complex sample types. The Quantikine ELISA kits are often validated for multiple sample types, and the reagent formulations have been thoroughly optimized to achieve the greatest sensitivity.
  • Why doesn't the assay range extend to the stated sensitivity?
  • Sensitivity is the lowest measurable value that is statistically not equal to zero. It is calculated based on the signal of the background and the inherent variability of the assay. It is commonly determined by taking the mean O.D. plus two standard deviations from 20 zero replicates. This value is converted into analyte concentration from the standard curve. The low standard is the lowest possible point at which R&D Systems feels confident that the value is in the linear portion of the standard curve and, therefore, quantifiable.
  • Why is a 4-PL curve fit needed to generate the standard curve?
  • R&D Systems validates ELISA Kits using a 4-parameter logistic (4-PL) curve-fit using ELISA analysis software. Alternatively, using Microsoft Excel, the data may be linearized by plotting the log of the standard concentrations versus the log of the standard O.D., and the best fit line can be determined by linear regression. This procedure will produce an adequate but less precise fit of the data.
  • Why is the sensitivity value lower than the standard curve range?
  • R&D Systems defines sensitivity as the minimal detectable dose that can be reliably distinguished from the assay background. This is calculated by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. The standard curve range has been validated for reliable and accurate performance across multiple kit lots and operators. The lowest standard concentration is the lowest limit that R&D systems can guarantee for reliable assay measurements. Sample values extrapolated below the lowest standard concentration may not be accurate.
  • Why is the wash buffer yellow?
  • R&D Systems wash buffers contain ProClin® 300. ProClin 300 is a preservative used in diagnostic and research reagents that is clear to pale yellow in color. The yellow color may become more pronounced over time and occurs more rapidly at room temperature than at 2-8 °C. This color change has no impact on the preservative or the performance of the assay. 
  • Why is there a brown precipitate in my wells after addition of the stop solution?
  • This is due to incomplete washing after the HRP-labeled detection antibody (or streptavidin-HRP) incubation. When HRP is present during the substrate and subsequent stop solution additions, an orange-brown or brown precipitate is observed. This may be remedied by the addition of a 30 second soak on each wash step followed by complete removal of all liquid in the wells.
  • Why is there a discrepancy in mass values when using a recombinant retail protein as a QC control?
  • There are several reasons why mass values calculated by ELISA versus those based on the mass reported on the vial label differ. First, a large dilution is required to place the recombinant protein on the ELISA standard curve. Typically this is a dilution from μg/mL to pg/mL. Any dilution step can introduce inaccuracy and the larger the dilution step, the greater the potential for error. Any pipetting error or mis-calibrated pipette can result in apparent over- or under-recovery. Second, R&D Systems immunoassays have been developed to measure a level of protein captured by one antibody and detected by a second antibody. This measurement is calibrated to standards established when the kit was initially developed. The protein determination of these initial standards are the Master Calibrators against which all new standards are formulated. This provides R&D Systems immunoassay kits with consistency between manufacturing lots. In general, a +/- 25% recovery of the amount stated on the vial when using an ELISA to determine a protein concentration is expected. There may be slight differences in the immunologically recognizable mass between lots of protein, so the apparent concentration provided on the vial may vary from lot-to-lot when measured in the ELISA. If recombinant retail proteins are being used to make controls, it is better to value assign the mass based on measurement in ELISA and not use the mass on the vial when setting control levels.
  • Why is there a sample dilution necessary in this ELISA Kit?
  • There are primarily two reasons for dilutions. In some assays most samples read above the standard curve, thus requiring a dilution for analyte levels to fall within the range of the assay. A second reason for dilution is to limit interference due to factors in complex matrices.
  • Why is there high variability (i.e., poor reproducibility) between sample replicates?
  • The two main reasons for high variability in an ELISA are inconsistent pipetting and ineffective wash techniques. Reverse pipetting is recommended for optimal precision, and when an automated washer or handheld vacuum manifold are unavailable, squirt bottle washing is recommended. Please see the ELISA protocol videos for a demonstration of recommended techniques.
  • Why must I use polypropylene tubes for standard dilutions in certain assays?
  • Certain proteins or analytes will bind to glass and polystyrene, but do not readily bind to the polypropylene tubes.
  • Can fetal calf serum (FCS) be used instead of heat-inactivated goat serum for diluting the detection antibody?
  • The use of heat-inactivated goat serum is important to keep background low and improve the signal-to-noise ratio of the assay. The use of FCS is not recommended for this purpose.

Cell-Based Fluorometric ELISAs

  • How does R&D Systems recommend normalizing the data obtained using the Cell-Based ELISAs?
  • R&D Systems® Cell-Based ELISAs permit the simultaneous detection of two proteins in the same microplate well without the need for lysate preparation. Kits come in two formats: phospho-protein kits contain antibodies to measure both the phosphorylated and the total protein, while total protein kits contain antibodies to the protein of interest and a second housekeeping protein. Both formats allow for the normalization of the target protein in different wells. To normalize the data obtained in the Cell-Based ELISA, R&D Systems recommends the following procedure:At the end of the assay, the ELISA plate is read at 450 nm and at 600 nm.Take the average of the control wells and subtract from all wells read at each wavelength (subtract the average control reading at 450 nm from all readings at 450 nm and the average control reading at 600 nm from all readings at 600 nm). Use these numbers to continue the normalization.Normalize the 450 nm reading by setting one of the readings to 1 and normalizing the rest accordingly. For example, if the 450 nm readings are 58386 and 55357, 61201 and 53658 for 2 sets of duplicates, choose 55357 as 1 and then 58386/55357=1.055 so the 58386 reading becomes 1.055 normalized. Also, 61201/55357=1.106 and 53658/55357=0.969.Use these normalized 450 nm numbers to normalize the 600 nm numbers. For example, if corresponding 600 nm values were 709 and 612, 6426 and 6045 for the 2 sets of duplicates, then take 709/1.055= 672, 612/1=612; 6426/1.106= 5810 and 6045-0.969=6236. Now take the average of the 650 nm readings for each duplicate: (672+612)/2=642+-30; 5810+6236)/2=6024+-214. Use these values for further data analysis.Note: Separate treatments do not need separate normalizations for total protein.

DuoSet ELISA Development Systems

  • After opening the vials, should the DuoSet ELISA antibodies and standard be stored at the reconstitution concentration or at the working concentration (after dilution)?
  • The DuoSet antibodies and standard should be reconstituted as specified on the Certificate of Analysis. The reconstitution concentration is what is recommended for storage. After dilution to the working concentration, the antibody or standard should be immediately used, and any excess volume should be discarded. Storage at the working concentration is not recommended.
  • Are DuoSet ELISAs compatible with detergents?
  • R&D Systems only validates the buffer system recommended in each individual product datasheet. A small amount of detergent may be tolerated by the capture antibody and antigen, but detergents have not been evaluated for assay impact. Additional validation testing is recommended to verify assay performance with modified buffer systems.
  • Are the same antibody pairs for each analyte used in the Quantikine versus DuoSet ELISA kits? 
  • The identity of the matched antibody pairs used in the Quantikine and DuoSet ELISAs is considered proprietary. Quantikine and DuoSet ELISA kits are designed separately and for different sample types, so optimization and validation are not the same. The lab chooses the antibody pair that has demonstrated optimal performance for each analyte.
  • Can DuoSet ELISA plates be stored once plate coating is complete?
  • R&D Systems has validated the DuoSet plate preparation method as written in the product datasheet - plates are coated overnight and assayed the next day. Longer incubation of the Capture Antibody plate coating (step 1 of the Plate Preparation) is not recommended. For short-term (24 hour storage), stop at the blocking step (Step 3 of the Plate Preparation Protocol) and store the plate wet in the Reagent Diluent blocking buffer overnight at 4 degrees Celsius. Equilibrate the plate to room temperature the next day and proceed with assay protocol. Blocking longer than 24 hours is not recommended. For additional information and guidance, please contact Technical Service.
  • Can ELISA plates be stacked on top of each other during assay incubations?
  • To ensure consistent environmental conditions for all plates, it is recommended that ELISA plates are not stacked during incubations.
  • Can I extend the time required for the plate preparation protocol?
  • Our standard protocol for plate coating is for overnight incubation only. Generally, coated plates can be blocked for up to 24 hours.
  • Can recombinant proteins sold as stand-alone reagents be used as standards in the DuoSet® ELISA Development Kits?
  • These products are not calibrated for use in an ELISA system, and must be calibrated versus an existing mass calibrated standard. Standards provided in ELISA kits have been calibrated to a master calibrator.
  • Can serum/plasma samples be analyzed with the DuoSet® ELISA Development System?
  • The DuoSet ELISAs are developed and validated with cell culture supernatants. Additional optimization by the end user of the DuoSet ELISA buffer and assay conditions may be necessary for validating serum and plasma. Spike/recovery and linearity experiments are recommended to determine whether sample values reported from unvalidated sample types are accurate. Please contact Technical Service for a Spike/Recovery and Linearity protocol. Consult the R&D Systems ELISA Guide for recommendations in designing ELISAs to measure protein levels in complex matrices, such as serum or plasma.
  • Could R&D Systems provide assay parameters such as sensitivity, inter and intra-assay precision, recovery and linearity for Duoset assays?
  • A Duoset ELISA is an experimental ELISA sytem and only core reagents for the assay (Capture antibody, Detection antibody, Standard and Streptavidin-HRP) are supplied in the kit. Several ancillary reagents needed for the assay are chosen by the end user.  Given this, parameters such as sensitivity, inter and intra-assay precision, recovery and linearity could vary to some extent at user's end and should be assessed by the user if needed.  The above parameters are provided in the fully validated Quantikine kits.
  • Do any of the provided components in the Duoset kit contain Azide?
  • No azide is added to any of the components supplied in the DuoSet. Neither the capture nor the detection antibody contain any preservatives.
  • I am getting very low or no color development in my DuoSet® ELISA Development System. What is wrong?
  • Many things can contribute to low or no color development including the following common reasons.BSA Grade: It is important to choose a high quality grade BSA that is fatty acid free and/or 98% pure. This reduces the amount of globulins, binding proteins, or other proteins that can interfere in the assay. R&D Systems recommends using the same BSA used in development of the DuoSet ELISA (Catalog # DY995;  Catalog # DY997), or Millipore Probumin® Diagnostic Grade BSA (Millipore; Catalog # 82-045).Refer to package insert for selection of the appropriate Diluent Concentrate or contact Technical Service.Reagent Temperature:  Assay reagents that are not at room temperature before use, or even an excessively cool room, can cause suppressed signal.Choice of Microplate: ELISA Grade Microplates Plates designated as "high-binding ELISA" plates are required. Use of plates other than ELISA grade (such as tissue culture grade) are not recommended because they may not bind a sufficient amount of the capture antibody. We offer the same 96-well clear plates used in the Quantikine® ELISA Kits (Catalog # DY990) and black plates found in QuantiGlo® ELISA Kits (Catalog # DY991).Treatment of Reagents: Inadequate time allowed for complete solublization (5-10 minutes with gentle agitation is suggested) or inappropriate storage of reagents can affect color development.
  • Is it possible to use reagents from a different assay lot if it is the same part number?
  • All of our assays have lot-matched reagents which have been QC tested for optimal performance. Interchanging component lots may impact the assay performance.
  • What animal serum is recommended for Reagent Diluent optimization for validating serum and plasma samples in DuoSets?
  • It is recommended to start with FBS for Reagent Diluent optimization.
  • What are R&D Systems' recommendations for manually washing an ELISA plate?
  • If an automated plate washer is unavailable, we advise washing with a squirt bottle. It is necessary to thoroughly flush the wells with wash buffer and for the plate to be fully decanted after washing. Overflow of wash buffer into surrounding wells is not a concern for assay performance. Washing with a multichannel pipette is not advised because it lacks sufficient force.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a DuoSet® ELISA Development System?
  • The DuoSet ELISA Development System has enough Capture Antibody, Detection Antibody, Protein Standard, and Streptavidin-HRP to create about fifteen 96-well plates. A sample protocol and reagents recommended for use with this system is supplied. Our DuoSet ELISA Development Systems are designed primarily for the high-throughput screening of cell culture supernatants, although researchers experienced in ELISA development have been able to adapt them for samples with complex matrices such as serum and plasma.
  • What is a matrix effect?
  • A matrix effect describes an inaccurate result due to a substance that interferes with the assay. Interference can be specific due to soluble receptors or other binding proteins competing for the antigen; interference can be semi-specific due to factors or substances such as heterophilic antibodies, autoantibodies, complement, and rheumatoid factors that interfere with the antibody binding causing false positive or negative results. Non-specific interference is due to protein concentration, ionic strength, pH, and other serum components that can affect the equilibrium or kinetics of binding between antigen and antibodies.
  • What is reverse pipetting?
  • Reverse pipetting is a technique recommended for viscous solutions. Press the pipette plunger past the first stop, before gently drawing up the solution. Press the pipette plunger to the first stop to dispense the measured volume.
  • What is the differences between Quantikine Kits and Duoset Kits?  
  • Quantikine kits are fully optimized and validated for the sample types listed in each specific protocol booklet. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product.Duoset Kits, in contrast, are ELISA development kits. These contain only vials of the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP. These kits are usually validated for cell culture supernatant samples only and require further development and validation for accurate measurement in more complex samples such as serum and plasma.The DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.
  • What is the recommended method for reconstitution of the lyophilized reagents?
  • For optimal recovery, equilibrate the vial and reconstitution buffer to room temperature. Add the volume indicated on the datasheet or Certificate of Analysis, and gently mix. Avoid vigorous vortexing that can cause foaming and protein denaturation.
  • Which ELISA plate is recommended for DuoSet® ELISA Development Kits?
  • R&D Systems offers clear (Catalog # DY990) and black (Catalog # DY991) microtiter plates, and plate sealers (Catalog # DY992). For added convenience, EvenCoat™ goat anti-mouse IgG microplates are available (Catalog # CP001; Catalog # CP002). EvenCoat microplates are clear, pre-blocked, 96-well polystyrene microplates coated with goat-derived antibody specific for the Fc region of mouse IgG. These plates may be used as a solid support for most sandwich ELISAs utilizing a mouse IgG capture antibody and a non-mouse IgG detection antibody. Other applications include competitive ELISA, IgG isotyping, and hybridoma screening/selection. EvenCoat Microplates may also be used with most DuoSet® ELISA Development Kits. Check your DuoSet ELISA datasheet to see if it can be used with EvenCoat Microplates. If there is no indication that it has been tested, please contact our Technical Service Department for more information.  
  • Why did this kit ship at a different temperature than the recommended storage temperature?
  • Kits may ship at a temperature different that the recommended storage temperature. We have validated shipping conditions for each assay to ensure stability and guarantee performance when the kit is used within the expiration. Upon receipt, immediately store it at the recommended temperature.
  • Why do some DuoSet ELISAs have different standard curve ranges than the Quantikine ELISA Kits for the same analyte?
  • These ELISAs may use different reagents that can result in different sensitivity. The DuoSet ELISAs are development assays, validated for testing cell culture supernatants. Additional optimization may be performed by the end user to achieve greater sensitivity and validate more complex sample types. The Quantikine ELISA kits are often validated for multiple sample types, and the reagent formulations have been thoroughly optimized to achieve the greatest sensitivity.
  • Why does the capture antibody vial in the DuoSet Kit appear to be empty?
  • Although there is always a slight possibility that a vial was not properly dispensed, this is a very rare occurrence and it would be extremely unusual to have received an empty capture antibody vial. There are multiple reasons why you may not observe a lyophilized pellet in your product. The two most common reasons for this observation are:The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.The capture antibody is formulated with a excipient to help protect the antibody during the lyophilization process and improve its long-term stability.  Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance. Our extensive testing over many years has shown that this phase shift does not affect the performance or stability of the product as long as care is taken to ensure the complete solubilization of the antibody prior to transferring to another vessel.
  • Can fetal calf serum (FCS) be used instead of heat-inactivated goat serum for diluting the detection antibody?
  • The use of heat-inactivated goat serum is important to keep background low and improve the signal-to-noise ratio of the assay. The use of FCS is not recommended for this purpose.

DuoSet ELISA Development Systems - 5/15 Plate Configuration

DuoSet IC (Intracellular) ELISA Development Systems

  • Are ancillary kits available to purchase for DuoSet IC (Intracellular) ELISAs?
  • R&D Systems does not offer complete ancillary kits for DuoSet IC ELISAs. DuoSet ELISA Ancillary Reagent Kit 2 (Cat. # DY008B) contains many reagents needed to perform DuoSet IC ELISAs. However, some specialized buffers are required. Please see each kit's datasheet for buffer recommendations and/or recipes. Some of these specialized buffers may be formulated starting with Catalog #DYC001 or Catalog #DYC002.
  • I am getting very low or no color development in my DuoSet® ELISA Development System. What is wrong?
  • Many things can contribute to low or no color development including the following common reasons.BSA Grade: It is important to choose a high quality grade BSA that is fatty acid free and/or 98% pure. This reduces the amount of globulins, binding proteins, or other proteins that can interfere in the assay. R&D Systems recommends using the same BSA used in development of the DuoSet ELISA (Catalog # DY995;  Catalog # DY997), or Millipore Probumin® Diagnostic Grade BSA (Millipore; Catalog # 82-045).Refer to package insert for selection of the appropriate Diluent Concentrate or contact Technical Service.Reagent Temperature:  Assay reagents that are not at room temperature before use, or even an excessively cool room, can cause suppressed signal.Choice of Microplate: ELISA Grade Microplates Plates designated as "high-binding ELISA" plates are required. Use of plates other than ELISA grade (such as tissue culture grade) are not recommended because they may not bind a sufficient amount of the capture antibody. We offer the same 96-well clear plates used in the Quantikine® ELISA Kits (Catalog # DY990) and black plates found in QuantiGlo® ELISA Kits (Catalog # DY991).Treatment of Reagents: Inadequate time allowed for complete solublization (5-10 minutes with gentle agitation is suggested) or inappropriate storage of reagents can affect color development.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a DuoSet® IC ELISA Development System?
  • The DuoSet IC (Intracellular) ELISA Development System contains the basic components required for the development of a sandwich ELISA (or activity assay) to measure intracellular proteins in cell lysates. Each kit has sufficient capture antibody, detection antibody, standard, and Streptavidin-HRP to create two, five, or fifteen 96-well plates. R&D Systems supplies a sample protocol and reagents recommended for use with this system.
  • Why did this kit ship at a different temperature than the recommended storage temperature?
  • Kits may ship at a temperature different that the recommended storage temperature. We have validated shipping conditions for each assay to ensure stability and guarantee performance when the kit is used within the expiration. Upon receipt, immediately store it at the recommended temperature.
  • Why does the capture antibody vial in the DuoSet Kit appear to be empty?
  • Although there is always a slight possibility that a vial was not properly dispensed, this is a very rare occurrence and it would be extremely unusual to have received an empty capture antibody vial. There are multiple reasons why you may not observe a lyophilized pellet in your product. The two most common reasons for this observation are:The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.The capture antibody is formulated with a excipient to help protect the antibody during the lyophilization process and improve its long-term stability.  Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance. Our extensive testing over many years has shown that this phase shift does not affect the performance or stability of the product as long as care is taken to ensure the complete solubilization of the antibody prior to transferring to another vessel.

DuoSet IC (Intracellular) Phospho-site Specific ELISA Development Systems

  • Can the Phospho control in my DuoSet® IC (Intracellular) Phospho-specific ELISA kit be diluted to make a standard curve?
  • The Phospho control has not been validated for use as standard curve material and further dilutions may not yield linear signal development. The concentration recommended in the kit insert has been verified to function well as a positive control in order to compare treated to non-treated/control samples.
  • I am getting very low or no color development in my DuoSet® ELISA Development System. What is wrong?
  • Many things can contribute to low or no color development including the following common reasons.BSA Grade: It is important to choose a high quality grade BSA that is fatty acid free and/or 98% pure. This reduces the amount of globulins, binding proteins, or other proteins that can interfere in the assay. R&D Systems recommends using the same BSA used in development of the DuoSet ELISA (Catalog # DY995;  Catalog # DY997), or Millipore Probumin® Diagnostic Grade BSA (Millipore; Catalog # 82-045).Refer to package insert for selection of the appropriate Diluent Concentrate or contact Technical Service.Reagent Temperature:  Assay reagents that are not at room temperature before use, or even an excessively cool room, can cause suppressed signal.Choice of Microplate: ELISA Grade Microplates Plates designated as "high-binding ELISA" plates are required. Use of plates other than ELISA grade (such as tissue culture grade) are not recommended because they may not bind a sufficient amount of the capture antibody. We offer the same 96-well clear plates used in the Quantikine® ELISA Kits (Catalog # DY990) and black plates found in QuantiGlo® ELISA Kits (Catalog # DY991).Treatment of Reagents: Inadequate time allowed for complete solublization (5-10 minutes with gentle agitation is suggested) or inappropriate storage of reagents can affect color development.
  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • What is a DuoSet® IC ELISA Development System?
  • The DuoSet IC (Intracellular) ELISA Development System contains the basic components required for the development of a sandwich ELISA (or activity assay) to measure intracellular proteins in cell lysates. Each kit has sufficient capture antibody, detection antibody, standard, and Streptavidin-HRP to create two, five, or fifteen 96-well plates. R&D Systems supplies a sample protocol and reagents recommended for use with this system.
  • Why does the capture antibody vial in the DuoSet Kit appear to be empty?
  • Although there is always a slight possibility that a vial was not properly dispensed, this is a very rare occurrence and it would be extremely unusual to have received an empty capture antibody vial. There are multiple reasons why you may not observe a lyophilized pellet in your product. The two most common reasons for this observation are:The lyophilized product has become dislodged from the bottom of the vial during shipping/handling and is dispersed on the vial wall or cap. Tapping the vial firmly on the bench or a quick spin in a centrifuge may bring the lyophilized product back to the bottom of the vial.The capture antibody is formulated with a excipient to help protect the antibody during the lyophilization process and improve its long-term stability.  Over time, this excipient can absorb moisture and the product appearance may change from a typical lyophilized appearance to a transparent, film like appearance. Our extensive testing over many years has shown that this phase shift does not affect the performance or stability of the product as long as care is taken to ensure the complete solubilization of the antibody prior to transferring to another vessel.

ELISA Controls

Parameter Colorimetric ELISA / Assay Kits

QuantiGlo Chemiluminescent Sandwich ELISAs

Quantikine Colorimetric Sandwich ELISAs

  • Will your Quantikine ELISA kit be able to detect my target analyte in normal samples?
  • Samples which read within the standard curve are able to be quantified.  In some cases, normal samples have levels of the target analyte that are below the detection limit of the kit. Check the "Sample Values" section in the kit booklet to find the sample values R&D Systems obtained from apparently healthy individuals. It is also recommended to review literature to detirmine if there is an established normal range for your target.  There are also Quantikine High Sensitivity kits available for some cytokines, that are known to be present in very low amounts in normal samples. However, it is still important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples, when using the High Sensitivity Quantikine kits.
  • Are control sets available for Human Quantikine® ELISA kits?
  • R&D Systems offers tri-level control sets for the Human Quantikine ELISA Kits (colorimetric), Quantikine HS ELISA kits (high sensitivity), and QuantiGlo® ELISA kits (chemiluminescent). Catalog numbers and pricing for control sets can be found in the "Supplemental Products" tab on the product webpage.
  • Are Quantikine® ELISA Kit Wash Buffers interchangeable?
  • Wash buffer can be interchanged between like kits provided they have the same part number.Note: The Wash Buffer from a Quantikine ELISA Kit CANNOT be used in a Quantikine HS (High Sensitivity) ELISA Kit. The phosphate contained in the Quantikine ELISA Kit Wash buffer will interfere with the NADPH driven amplification of signal in the Quantikine HS ELISA kits. Additional bottles of Wash Buffer are available.
  • Are the same antibody pairs for each analyte used in the Quantikine versus DuoSet ELISA kits? 
  • The identity of the matched antibody pairs used in the Quantikine and DuoSet ELISAs is considered proprietary. Quantikine and DuoSet ELISA kits are designed separately and for different sample types, so optimization and validation are not the same. The lab chooses the antibody pair that has demonstrated optimal performance for each analyte.
  • Are there any suggestions on how to collect and handle non-validated sample types such as CSF?
  • We are not able to provide guidance for clinical sample collection.  Generally, we recommend looking to literature for published methodology and preparing samples in a manner that minimizes freeze-thaw cycles.
  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can ELISA plates be stacked on top of each other during assay incubations?
  • To ensure consistent environmental conditions for all plates, it is recommended that ELISA plates are not stacked during incubations.
  • Can I stop a Quantikine® ELISA kit or Parameter™ assay at any point, extend an incubation time, or change the suggested incubation temperature? 
  • R&D Systems has optimized the assays for both incubation times and temperatures. Each kit has only been validated for the protocol described in the kit datasheet. We cannot guarantee the performance of our kits when the protocol has been altered in any way. 
  • Can the reagents from Quantikine®, QuantiGlo®, Parameter™, or Surveyor™ IC ELISA Kits be interchanged?
  • Assay Diluent(s), Calibrator Diluent(s), and Substrate may be interchanged if they have the same part number AND lot number. R&D Systems does "whole kit QC" which means that we cannot support the use of reagents from other lots or sources being substituted into an assay. Plates and Conjugate cannot be interchanged under any circumstance.
  • How are ELISA kit results converted from mass to units?
  • For those analytes of which there is a WHO standard, an equation for converting to units is provided in the kit insert. Please note that these units are determined in bioassay, and are not mass calibrated.
  • How many samples can be assayed in a Human Quantikine® ELISA Kit?
  • Most Quantikine ELISA Kits will run the standard curve and 40 samples in duplicate. Please refer to the datasheet for details on each kit.
  • How many samples can be assayed in a non-human Quantikine® ELISA Kit?
  • Each plate in the non-human Quantikine ELISA Kit will run the standard curve, control, and 39 samples in duplicate per plate. Some of these ELISA kits come with two plates. Please see the datasheet for details.
  • How should cell culture supernate samples be diluted if there is not sufficient Calibrator Diluent? 
  • The kits are supplied with enough calibrator diluent to ensure the vast majority of samples fall within  the indicated assay range if the protocol is followed as stated in the booklet. Cell culture supernate samples can be diluted in two steps: an initial dilution in cell culture medium and a final dilution into Calibrator Diluent. Alternately, please contact Technical Service to inquire if additional Calibrator Diluent is needed. 
  • In the Specificity section of the product datasheet, you claim that data from your Quantikine ELISA directly correlates with an immunoprecipitation/Western blot. IP & Western blots aren't quantitative, so how can these be compared?
  • This data is meant to show that samples that are showing positive signal in IP/Western blot are also detectable as positive signal when ran in the ELISA, and vice versa. As far as directly comparing the two, IP/Western blots have a different dynamic range than ELISA, and we can only consistently see difference in IP/Western blot intensity when the ELISA signal is 4-5 fold different. A smaller-fold difference is unlikely to be apparent in the IP/Western blot.
  • Is it possible to use reagents from a different assay lot if it is the same part number?
  • All of our assays have lot-matched reagents which have been QC tested for optimal performance. Interchanging component lots may impact the assay performance.
  • Quantikine® ELISA kits often contain multiple diluents. Which diluent is used to dilute samples? What is the correct use of the assay diluent?
  • In general, the calibrator diluent should be used to make standard curve dilutions and any needed sample dilutions. If there are multiple calibrator diluents in a kit, the kit booklet will describe which one is correct for each sample type. Diluting both the standards and the samples with the same diluent allows them to be in a similar matrix while incubating in the plate.The assay diluent is only used at the beginning of the protocol where a specific amount is added to the plate prior to adding samples, standards, or controls. There is excess assay diluent provided for this step, and there is no other use for the assay diluent. The assay diluent allows for the introduction of components into the sample incubation and will prevent other substances present in the samples from interfering with the assay.
  • Should Quantikine Immunoassay Controls be assayed neat or diluted?
  • Quantikine Immunoassay Controls should be reconstituted as specified on the product datasheet and assayed neat. Do not dilute even if samples require dilution.
  • The Assay Diluent, RD1, supplied in my Quantikine® ELISA Kit looks like it contains a precipitate. Is this OK?
  • Due to saturating amounts of some buffer components, some of the RD1 Assay Diluents contain a light to heavy precipitate. In these instances, it is normal and it will be noted in the specific protocol booklet. If it is not noted in the protocol booklet, please contact Technical Service.
  • What are R&D Systems' recommendations for manually washing an ELISA plate?
  • If an automated plate washer is unavailable, we advise washing with a squirt bottle. It is necessary to thoroughly flush the wells with wash buffer and for the plate to be fully decanted after washing. Overflow of wash buffer into surrounding wells is not a concern for assay performance. Washing with a multichannel pipette is not advised because it lacks sufficient force.
  • What are the shipping conditions for the optional ELISA kit controls?
  • The shipping method for the control depends on the target analyte.  Some Quantikine® ELISA controls ship at ambient temperature, some ship on cold packs, and some ship on dry ice. 
  • What is a matrix effect?
  • A matrix effect describes an inaccurate result due to a substance that interferes with the assay. Interference can be specific due to soluble receptors or other binding proteins competing for the antigen; interference can be semi-specific due to factors or substances such as heterophilic antibodies, autoantibodies, complement, and rheumatoid factors that interfere with the antibody binding causing false positive or negative results. Non-specific interference is due to protein concentration, ionic strength, pH, and other serum components that can affect the equilibrium or kinetics of binding between antigen and antibodies.
  • What is included in a Quantikine® ELISA Kit?
  • Quantikine ELISA Kits are a complete kit consisting of a precoated microplate, Conjugated Detection Antibody, Standard, Diluents, Substrate, Stop Solution, Wash Buffer, and plate sealers. They are fully validated ELISAs for the sample types listed in the specific datasheet. They have been exhaustively tested for superior quality.
  • What is R&D Systems recommendations for lysis buffer to evaluate tissue homogenate or cell lysate samples in an ELISA kit?
  • When tissue homogenates or cell lysates are validated sample types, R&D Systems provides the necessary Lysis or Extraction Buffers in the ELISA kits. If tissue homogenates or lysates are not validated, R&D Systems offers Triton X-100 based (Cat. # DYC001) and NP-40-based (Cat. # DYC002) Sample Diluent Concentrates that can be modified according to research need. The most commonly used buffers are RIPA and NP-40 which are both suitable for whole-cell lysate/membrane bound proteins. Additional Cell Lysis Buffers are offered in our catalog.
  • What is reverse pipetting?
  • Reverse pipetting is a technique recommended for viscous solutions. Press the pipette plunger past the first stop, before gently drawing up the solution. Press the pipette plunger to the first stop to dispense the measured volume.
  • What is the difference between the Quantikine® and QuantiGlo® ELISA Kits?
  • Quantikine ELISA kits are a colorimetric assay that require a standard microplate reader with the appropriate wavelength filters for reading and wavelength correction. The QuantiGlo ELISA Kits have a Luminol-based substrate that is read by the intensity of light emitted or relative light units (RLU). Therefore, the QuantiGlo kits must be read on a luminometer. In general, the QuantiGlo ELISA has a wider dynamic range than the standard Quantikine ELISA. Please see the datasheet for specifications.
  • What is the difference between the traditional Quantikine® ELISA Kits and the High Sensitivity Quantikine HS ELISA Kits?
  • The High Sensitivity (HS) versions of Quantikine ELISA Kits are complete kits generally used when very low levels of the cytokine are expected. Quantikine HS Kits incorporate an amplification system designed to enhance the signal accompanying low analyte concentrations. It is strongly recommended to do a literature search to determine which kit range would best suit your needs.
  • What is the differences between Quantikine Kits and Duoset Kits?  
  • Quantikine kits are fully optimized and validated for the sample types listed in each specific protocol booklet. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product.Duoset Kits, in contrast, are ELISA development kits. These contain only vials of the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP. These kits are usually validated for cell culture supernatant samples only and require further development and validation for accurate measurement in more complex samples such as serum and plasma.The DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.
  • What is the recommended method for reconstitution of the lyophilized reagents?
  • For optimal recovery, equilibrate the vial and reconstitution buffer to room temperature. Add the volume indicated on the datasheet or Certificate of Analysis, and gently mix. Avoid vigorous vortexing that can cause foaming and protein denaturation.
  • What is the stability of supplemental ELISA kit controls (Quantikine, Quantikine HS, or QuantiGlo ELISA kits)?
  • Controls are assigned an expiration date of 3 months from date of receipt. They are to be used once and discarded. If the lyophilized controls are stored properly, it is possible that they will remain stable for an extended period of time, although we have not conducted extended stability testing. The controls have not been tested for stability after reconstitution.
  • Why did this kit ship at a different temperature than the recommended storage temperature?
  • Kits may ship at a temperature different that the recommended storage temperature. We have validated shipping conditions for each assay to ensure stability and guarantee performance when the kit is used within the expiration. Upon receipt, immediately store it at the recommended temperature.
  • Why is a different reconstitution volume required for the kit standard?
  • Reconstitution volume is established during manufacturing to ensure consistent mass calibration between different lots of standard. Refer to the standard vial label for the reconstitution volume.

Quantikine HS High Sensitivity Colorimetric Sandwich ELISAs

  • Will your Quantikine ELISA kit be able to detect my target analyte in normal samples?
  • Samples which read within the standard curve are able to be quantified.  In some cases, normal samples have levels of the target analyte that are below the detection limit of the kit. Check the "Sample Values" section in the kit booklet to find the sample values R&D Systems obtained from apparently healthy individuals. It is also recommended to review literature to detirmine if there is an established normal range for your target.  There are also Quantikine High Sensitivity kits available for some cytokines, that are known to be present in very low amounts in normal samples. However, it is still important to check the standard curve range and the sample values section to determine if the kit is likely to detect the target in your samples, when using the High Sensitivity Quantikine kits.
  • Are control sets available for Human Quantikine® ELISA kits?
  • R&D Systems offers tri-level control sets for the Human Quantikine ELISA Kits (colorimetric), Quantikine HS ELISA kits (high sensitivity), and QuantiGlo® ELISA kits (chemiluminescent). Catalog numbers and pricing for control sets can be found in the "Supplemental Products" tab on the product webpage.
  • Are Quantikine® ELISA Kit Wash Buffers interchangeable?
  • Wash buffer can be interchanged between like kits provided they have the same part number.Note: The Wash Buffer from a Quantikine ELISA Kit CANNOT be used in a Quantikine HS (High Sensitivity) ELISA Kit. The phosphate contained in the Quantikine ELISA Kit Wash buffer will interfere with the NADPH driven amplification of signal in the Quantikine HS ELISA kits. Additional bottles of Wash Buffer are available.
  • Are the same antibody pairs for each analyte used in the Quantikine versus DuoSet ELISA kits? 
  • The identity of the matched antibody pairs used in the Quantikine and DuoSet ELISAs is considered proprietary. Quantikine and DuoSet ELISA kits are designed separately and for different sample types, so optimization and validation are not the same. The lab chooses the antibody pair that has demonstrated optimal performance for each analyte.
  • Can a partial Quantikine® ELISA plate be used?
  • The Quantikine® ELISA plates have removable strips of wells. Unused wells may be removed from the plate, returned to the foil pouch containing the desiccant pack, and stored at 2-8° C for up to one month.
  • Can ELISA plates be stacked on top of each other during assay incubations?
  • To ensure consistent environmental conditions for all plates, it is recommended that ELISA plates are not stacked during incubations.
  • Can I stop a Quantikine® ELISA kit or Parameter™ assay at any point, extend an incubation time, or change the suggested incubation temperature? 
  • R&D Systems has optimized the assays for both incubation times and temperatures. Each kit has only been validated for the protocol described in the kit datasheet. We cannot guarantee the performance of our kits when the protocol has been altered in any way. 
  • Can the reagents from Quantikine®, QuantiGlo®, Parameter™, or Surveyor™ IC ELISA Kits be interchanged?
  • Assay Diluent(s), Calibrator Diluent(s), and Substrate may be interchanged if they have the same part number AND lot number. R&D Systems does "whole kit QC" which means that we cannot support the use of reagents from other lots or sources being substituted into an assay. Plates and Conjugate cannot be interchanged under any circumstance.
  • How are ELISA kit results converted from mass to units?
  • For those analytes of which there is a WHO standard, an equation for converting to units is provided in the kit insert. Please note that these units are determined in bioassay, and are not mass calibrated.
  • How many samples can be assayed in a Human Quantikine® ELISA Kit?
  • Most Quantikine ELISA Kits will run the standard curve and 40 samples in duplicate. Please refer to the datasheet for details on each kit.
  • How should cell culture supernate samples be diluted if there is not sufficient Calibrator Diluent? 
  • The kits are supplied with enough calibrator diluent to ensure the vast majority of samples fall within  the indicated assay range if the protocol is followed as stated in the booklet. Cell culture supernate samples can be diluted in two steps: an initial dilution in cell culture medium and a final dilution into Calibrator Diluent. Alternately, please contact Technical Service to inquire if additional Calibrator Diluent is needed. 
  • Is it possible to use reagents from a different assay lot if it is the same part number?
  • All of our assays have lot-matched reagents which have been QC tested for optimal performance. Interchanging component lots may impact the assay performance.
  • Quantikine® ELISA kits often contain multiple diluents. Which diluent is used to dilute samples? What is the correct use of the assay diluent?
  • In general, the calibrator diluent should be used to make standard curve dilutions and any needed sample dilutions. If there are multiple calibrator diluents in a kit, the kit booklet will describe which one is correct for each sample type. Diluting both the standards and the samples with the same diluent allows them to be in a similar matrix while incubating in the plate.The assay diluent is only used at the beginning of the protocol where a specific amount is added to the plate prior to adding samples, standards, or controls. There is excess assay diluent provided for this step, and there is no other use for the assay diluent. The assay diluent allows for the introduction of components into the sample incubation and will prevent other substances present in the samples from interfering with the assay.
  • Should Quantikine Immunoassay Controls be assayed neat or diluted?
  • Quantikine Immunoassay Controls should be reconstituted as specified on the product datasheet and assayed neat. Do not dilute even if samples require dilution.
  • The Assay Diluent, RD1, supplied in my Quantikine® ELISA Kit looks like it contains a precipitate. Is this OK?
  • Due to saturating amounts of some buffer components, some of the RD1 Assay Diluents contain a light to heavy precipitate. In these instances, it is normal and it will be noted in the specific protocol booklet. If it is not noted in the protocol booklet, please contact Technical Service.
  • What are R&D Systems' recommendations for manually washing an ELISA plate?
  • If an automated plate washer is unavailable, we advise washing with a squirt bottle. It is necessary to thoroughly flush the wells with wash buffer and for the plate to be fully decanted after washing. Overflow of wash buffer into surrounding wells is not a concern for assay performance. Washing with a multichannel pipette is not advised because it lacks sufficient force.
  • What are the shipping conditions for the optional ELISA kit controls?
  • The shipping method for the control depends on the target analyte.  Some Quantikine® ELISA controls ship at ambient temperature, some ship on cold packs, and some ship on dry ice. 
  • What is a matrix effect?
  • A matrix effect describes an inaccurate result due to a substance that interferes with the assay. Interference can be specific due to soluble receptors or other binding proteins competing for the antigen; interference can be semi-specific due to factors or substances such as heterophilic antibodies, autoantibodies, complement, and rheumatoid factors that interfere with the antibody binding causing false positive or negative results. Non-specific interference is due to protein concentration, ionic strength, pH, and other serum components that can affect the equilibrium or kinetics of binding between antigen and antibodies.
  • What is included in a Quantikine® ELISA Kit?
  • Quantikine ELISA Kits are a complete kit consisting of a precoated microplate, Conjugated Detection Antibody, Standard, Diluents, Substrate, Stop Solution, Wash Buffer, and plate sealers. They are fully validated ELISAs for the sample types listed in the specific datasheet. They have been exhaustively tested for superior quality.
  • What is reverse pipetting?
  • Reverse pipetting is a technique recommended for viscous solutions. Press the pipette plunger past the first stop, before gently drawing up the solution. Press the pipette plunger to the first stop to dispense the measured volume.
  • What is the difference between the traditional Quantikine® ELISA Kits and the High Sensitivity Quantikine HS ELISA Kits?
  • The High Sensitivity (HS) versions of Quantikine ELISA Kits are complete kits generally used when very low levels of the cytokine are expected. Quantikine HS Kits incorporate an amplification system designed to enhance the signal accompanying low analyte concentrations. It is strongly recommended to do a literature search to determine which kit range would best suit your needs.
  • What is the differences between Quantikine Kits and Duoset Kits?  
  • Quantikine kits are fully optimized and validated for the sample types listed in each specific protocol booklet. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product.Duoset Kits, in contrast, are ELISA development kits. These contain only vials of the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP. These kits are usually validated for cell culture supernatant samples only and require further development and validation for accurate measurement in more complex samples such as serum and plasma.The DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.
  • What is the recommended dilution for cell culture supernate samples in the High Sensitivity Quantikine ELISA Kits?
  • The High Sensitivity Quantikine  ELISA kits are not validated for use on cell culture supernate samples because cell culture supernate sample levels are expected to be high and easily detectable by the corresponding standard Quantikine ELISA. The expected values may be so high in cell culture supernate samples that the dilution you would perform to get levels within the standard curve range would change the matrix of the assay, and the sample values would not be accurate. Additionally, it may be difficult to choose the correct dilution factor to get a high value sample to fall within the narrow standard curve range of a high sensitivity kit. The high sensitivity kits may be useful for cell culture supernate samples with very low expected levels of the target, but may require sample validation when used for this purpose.
  • What is the stability of supplemental ELISA kit controls (Quantikine, Quantikine HS, or QuantiGlo ELISA kits)?
  • Controls are assigned an expiration date of 3 months from date of receipt. They are to be used once and discarded. If the lyophilized controls are stored properly, it is possible that they will remain stable for an extended period of time, although we have not conducted extended stability testing. The controls have not been tested for stability after reconstitution.
  • Why did this kit ship at a different temperature than the recommended storage temperature?
  • Kits may ship at a temperature different that the recommended storage temperature. We have validated shipping conditions for each assay to ensure stability and guarantee performance when the kit is used within the expiration. Upon receipt, immediately store it at the recommended temperature.
  • Why is a different reconstitution volume required for the kit standard?
  • Reconstitution volume is established during manufacturing to ensure consistent mass calibration between different lots of standard. Refer to the standard vial label for the reconstitution volume.

Quantikine IVD In Vitro Diagnostic Colorimetric ELISAs

QuicKit Colorimetric Sandwich ELISAs

  • Can ELISA plates be stacked on top of each other during assay incubations?
  • To ensure consistent environmental conditions for all plates, it is recommended that ELISA plates are not stacked during incubations.
  • Is it possible to use reagents from a different assay lot if it is the same part number?
  • All of our assays have lot-matched reagents which have been QC tested for optimal performance. Interchanging component lots may impact the assay performance.
  • What is a matrix effect?
  • A matrix effect describes an inaccurate result due to a substance that interferes with the assay. Interference can be specific due to soluble receptors or other binding proteins competing for the antigen; interference can be semi-specific due to factors or substances such as heterophilic antibodies, autoantibodies, complement, and rheumatoid factors that interfere with the antibody binding causing false positive or negative results. Non-specific interference is due to protein concentration, ionic strength, pH, and other serum components that can affect the equilibrium or kinetics of binding between antigen and antibodies.
  • What is reverse pipetting?
  • Reverse pipetting is a technique recommended for viscous solutions. Press the pipette plunger past the first stop, before gently drawing up the solution. Press the pipette plunger to the first stop to dispense the measured volume.
  • What is the recommended method for reconstitution of the lyophilized reagents?
  • For optimal recovery, equilibrate the vial and reconstitution buffer to room temperature. Add the volume indicated on the datasheet or Certificate of Analysis, and gently mix. Avoid vigorous vortexing that can cause foaming and protein denaturation.
  • Why did this kit ship at a different temperature than the recommended storage temperature?
  • Kits may ship at a temperature different that the recommended storage temperature. We have validated shipping conditions for each assay to ensure stability and guarantee performance when the kit is used within the expiration. Upon receipt, immediately store it at the recommended temperature.
  • Why is a different reconstitution volume required for the kit standard?
  • Reconstitution volume is established during manufacturing to ensure consistent mass calibration between different lots of standard. Refer to the standard vial label for the reconstitution volume.

Supplemental ELISA Products

  • What grade BSA is recommend for use in ELISA and ELISpot Development Systems?
  • The grade of the BSA used has been found to be a critical component for running a successful ELISA. BSA with a minimum purity of 98% is recommended. R&D Systems recommends using the same BSAs used in development of the DuoSet; Catalog # DY995 Reagent Diluent Concentrate 2, Catalog # DY997 Reagent Diluent Concentrate 1. Millipore Probumin® Diagnostic Grade BSA (Millipore, Catalog # 820451) is another example.
  • Which ELISA plate is recommended for DuoSet® ELISA Development Kits?
  • R&D Systems offers clear (Catalog # DY990) and black (Catalog # DY991) microtiter plates, and plate sealers (Catalog # DY992). For added convenience, EvenCoat™ goat anti-mouse IgG microplates are available (Catalog # CP001; Catalog # CP002). EvenCoat microplates are clear, pre-blocked, 96-well polystyrene microplates coated with goat-derived antibody specific for the Fc region of mouse IgG. These plates may be used as a solid support for most sandwich ELISAs utilizing a mouse IgG capture antibody and a non-mouse IgG detection antibody. Other applications include competitive ELISA, IgG isotyping, and hybridoma screening/selection. EvenCoat Microplates may also be used with most DuoSet® ELISA Development Kits. Check your DuoSet ELISA datasheet to see if it can be used with EvenCoat Microplates. If there is no indication that it has been tested, please contact our Technical Service Department for more information.  

Surveyor IC (Intracellular) ELISAs